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The Journal of Biological Chemistry

TABLE OF CONTENTS
2011 COMPENDIA COLLECTION: Recent Advances in Pathogenic

Human Viruses
3 Recent Advances in Pathogenic Human Viruses. H. Smith
53 Identification of Interactions in the E1E2 Heterodimer of
and Charles E. Samuel
Hepatitis C Virus Important for Cell Entry. Guillemette Maurin,

Judith Fresquet, Ophelia Granio, Czeslaw Wychowski,
Francois-Loc Cosset, and Dimitri Lavillette
7 Inhibitors of Histone Deacetylases. CORRELATION BETWEEN
S ISOFORM SPECIFICITY AND REACTIVATION OF HIV TYPE 1 (HIV-1) FROM

LATENTLY INFECTED CELLS. Kelly Huber, Genevie`ve Doyon, Joseph Plaks,
Elizabeth Fyne, John W. Mellors, and Nicolas Sluis-Cremer
65 Identification of Cis-Acting Elements in the 3-Untranslated
15 Host Protein Ku70 Binds and Protects HIV-1 Integrase from
S Region of the Dengue Virus Type 2 RNA That Modulate

Translation and Replication. Mark Manzano, Erin D. Reichert,
Stephanie Polo, Barry Falgout, Wojciech Kasprzak, Bruce A. Shapiro,
and Radhakrishnan Padmanabhan
Proteasomal Degradation and Is Required for HIV Replication.

Yingfeng Zheng, Zhujun Ao, Binchen Wang, Kallesh Danappa Jayappa,
and Xiaojian Yao
29 Impaired Infectivity of Ritonavir-resistant HIV Is Rescued by
S Heat Shock Protein 90AB1. Pheroze Joshi and Cheryl A. Stoddart
41 A Chimeric HIV-1 Envelope Glycoprotein Trimer with an

79 Structural Characterization of the Crimean-Congo
Embedded Granulocyte-Macrophage Colony-stimulating

Factor (GM-CSF) Domain Induces Enhanced Antibody and T
Cell Responses. Thijs van Montfort, Mark Melchers, Gozde Isik,
Sergey Menis, Po-Ssu Huang, Katie Matthews, Elizabeth Michael,
Ben Berkhout, William R. Schief, John P. Moore, and Rogier W. Sanders
S Hemorrhagic Fever Virus Gn Tail Provides Insight into Virus

Assembly. D. Fernando Estrada and Roberto N. De Guzman
S Online version of this article contains supplemental material.

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PROLOGUE
This paper is available online at www.jbc.org

2011 by The American Society for Biochemistry and Molecular Biology, Inc.
Printed in the U.S.A.
Recent Advances in Pathogenic Human Viruses*

H. Smith
From the American Society for Biochemistry and Molecular Biology, Rockville, Maryland 20852
Edited and curated by Charles E. Samuel1

From the Department of Molecular, Cellular, and Developmental Biology and the Bimolecular Sciences and Engineering Program, University of California,
Santa Barbara, California 93106
ularly delighted to offer meeting delegates the following compendium of recent research findings in virology, presented by
authors from around the world within the pages of The Journal
of Biological Chemistry (JBC). These current research papers
touch on a variety of topics related to viral disease in humans
and promise to provide avenues for better understanding and
combating viral pathogens. Beyond their clinical implications,
these papers also represent the light that virology inevitably
casts on processes essential to the wide spectrum of interests
that define modern biochemistry and molecular biology.
Written histories of virology research frequently start with

the year 1796, when English physician Edward Jenner used vaccinia-laden pus, collected from the cowpox lesions of a milkmaid, to inoculate a young patient against smallpox. Most
accounts are quick to acknowledge that prophylactic inoculation did not begin with Jenner but had in fact been practiced
centuries beforehand, in China and other regions, and that the
use of cowpox lesions had been used even in Jenners day in
Europe. However, the details of Jenners experiments were both
spectacular and well told. For example, after treating an 8-yearold patient with the cowpox inoculum, Jenner challenged the
patient by intentionally inoculating him with smallpox. By carefully recording his work, Jenner resonated with the Enlightenment ideals of the scientific method. Ever since, Jenners
records have provided a convenient milestone in the development of modern antiviral therapies.
Virologys historical importance to basic molecular biology
and the disciplines crucial role in health care around the globe
have been very much on the minds of the organizers of Recent
Advances in Pathogenic Human Viruses, a meeting sponsored
in part by the American Society for Biochemistry and Molecular Biology, this month in Guangzhou, China. Attendance at the
meeting by virologists from around the world not only reflects
the unprecedented therapeutic opportunities to be developed
from studies of viruses but also attests to the worldwide risks
posed by viral pathogens in our global age. A single case of a
deadly viral disease appearing in the English countryside today,
or in a small Chinese village, or anywhere else in the world, has
ramifications for global health that were unimaginable in Jenners day. Virology today must be considered along dimensions
that cross previously recognized borders, including those
drawn between countries, scientific disciplines, political and
economic ideologies, and even animal species.
Organization of the meeting on Recent Advances in Pathogenic Human Viruses in Guangzhou also reflects a deliberate
sentiment that the benefits and risks of virology-related
research are met best through collaborations that are also built
to global proportions. The American Society for Biochemistry
and Molecular Biology, founded through a shared sense of
excitement for scientific discovery more than a hundred years
ago, is proud to support the collegial spirit that has brought
international scientists together in Guangzhou. We are partic-
Its All Very Retro

Groundbreaking investigation into the molecular biology of
the retroviruses, culminating in the early 1970s, established
that RNA could be reverse-transcribed into DNA. This finding, made in laboratories focusing on the nucleic acid metabolism of avian viruses, broke the central dogma of molecular
biology that had been pronounced by Francis Crick in the
1950s. Cricks declaration that the expression of genetic information within all cellular organisms flows in a single-direction
synthetic pathway, from DNA to RNA to protein, reflected the
basic conceptual framework by which biologists had begun to
unlock mechanisms of gene regulation. This framework had
been built in large part through the work of basic virologists.
Similarly, it was a framework that was recast through the work
of virologists such as David Baltimore and Howard Temin.2
The discovery of reverse transcriptase, which revolutionized
molecular biology, originated from studies of basic retroviral
biology. In the field of retrovirology, the discovery brought
credibility particularly to Howard Temins thesis for the existence of a proviral intermediate for Rous sarcoma: that is, for the
existence of a DNA form of the viral genome that became
spliced into the host genome as a normal phase of viral infection. Temins proposal thus echoed Dulbeccos identification of
the proviral intermediate in the replication cycle of the polyoma
(DNA) virus.2 The elucidation of reverse transcriptase, along
with other elements of the HIV-1 molecular machinery that
drive the retroviral life cycle, has paved the way for therapeutic
approaches that have alleviated greatly the worldwide burden
of illness caused by HIV-1. In addition, whereas drugs that
inhibit the HIV-1 reverse transcriptase, integrase, and protease
have become important in the clinic, host factors that affect
* Translated into Chinese by Xing Guo, University of California, San Diego.

To cite articles in this collection, use the citation information that appears in
the upper right-hand corner of the first page of the article.
1
To whom correspondence should be addressed. Tel.: 805-893-3097; Fax:
805-893-5780; E-mail: samuel@lifesci.ucsb.edu.
The Nobel Prize in Physiology or Medicine 1975 was awarded jointly to

David Baltimore, Renato Dulbecco, and Howard Martin Temin for their
discoveries concerning the interaction between tumour viruses and the
genetic material of the cell. See http://nobelprize.org/nobel_prizes/medicine/laureates/1975/. Accessed June 14, 2011.
PROLOGUE: Recent Advances in Pathogenic Human Viruses

cation in distinct ways, protecting the integrase from degradation and mediating the genesis of viral nucleic acid intermediates upon cell entry.
Aiding and Abetting: Cell Proteins That Enable HIV Drug
ResistanceThe integrase is not the only viral enzyme to collude with specific cellular proteins in the service of HIV replication. As the JBC paper from Joshi and Stoddart (3) shows,
mutant forms of the HIV protease that fail to promote full maturation of the capsid protein (CA) can nevertheless attain enzymic functionality and restore viral replication by associating
with HSP90AB1, a cellular heat shock protein. This cell protein-virus protein collusion, moreover, depends on the activation status of the infected T cell: rescue of protease function by
HSP90AB1 occurs only in T cells that have been activated.
These results have fascinating ramifications for drug development, as pharmacological inhibition of HSP90AB1 prevents the
rescue of impaired virus, and most significantly, the mutant
proteases at the center of Joshi and Stoddarts experiments (3)
are typical of HIV that has become resistant to the HIV antiprotease drug ritonavir (Norvir). Admittedly, the actual mechanism of viral rescue (e.g. chaperone activity) by HSP90AB1 is
not yet clear, but CA conformation and interaction with other
host factors have been implicated in the postentry stage of HIV
infectivity. In any event, the authors illustrate the consummate
molecular behavior of HIV in usurping cellular functions.
Engineering of Anti-HIV Vaccines That Carry Cytokine
SignalsThe development of antiretroviral drugs that target
intracellular viral enzyme activity has been particularly crucial
to clinical efforts as effective HIV vaccines have remained elusive. Indeed, one of the hallmark challenges in combating AIDS
has come from attempts to marshal viral immunogenicity by
exploiting the envelope glycoprotein complex (Env). In their
provocative report, van Montfort et al. (4) directly address HIV
immunogenicity, hypothesizing that a vaccine component that
could carry a direct signal of immune activationthat is, a sort
of chemokinemight better alert host defense systems against
the invading virus. The authors hypothesize that if the overall
antigenic message can be amplified, the host might mount a
better defense. To investigate, the authors have constructed a
chimeric molecule that consists of the Env protein (in triplicate)
along with the immunostimulatory domain of the granulocytemacrophage colony-stimulating factor (GM-CSF). Upon injection into mice, the chimeric construct enhances both humoral
and cellular responses, compared with injection of Env alone.
The improved immunogenicity appears to reflect cytokine signaling, as the chimera retains GM-CSF activity in vitro.
Whether other cytokines may prove effective as immunostimulatory components when chimerically partnered with Env
remains to be seen.
retroviral infection have also entered center stage in the effort

to understand and combat HIV-1 infection. Four of the papers
in this compendium attest to the diversity of HIV-1 targets.
Going Pro-ViralThe proviral stage of the HIV-1 life cycle
poses a great challenge in treating infection because the viral
genome, once integrated into host DNA, can remain quiescent
throughout therapeutic regimens that otherwise clear the
blood of virus. Latent infection is thus a constant threat despite
the success of combinatorial drug strategies. HIV-1 latency is
the issue at hand in the JBC paper from Huber and colleagues
(1), who explore the therapeutic potential of pharmacologically
manipulating epigenetic regulation of HIV-1 proviral elements.
Specifically, the recruitment of histone deacetylase (HDAC) to
the long terminal repeats (LTRs) of the HIV-1 genome has been
linked experimentally to the induction of HIV-1 latency, leading Huber et al. (1) to question whether specific isoforms of
histone deacetylase might function differentially to maintain
proviral DNA in the form of quiescent chromatin. Indeed, in T
cells isolated from aviremic HIV-1-infected individuals undergoing combination antiretroviral therapy, HDAC inhibitors
have been able to induce chromatin relaxation as well as the
activation of viral genes. Huber et al. (1) use a number of known
inhibitors of HDAC activity to characterize nine distinct
HDAC isoforms in Jurkat cells with regard to HDAC inhibition
kinetics, association of HDAC isoforms with provirus-containing chromatin, and the effectiveness of distinct HDAC inhibitors in activating latent virus in vitro. In this way, the authors
report that the inhibition of HDAC3 is essential for activating
the provirus but that HDAC1, although amenable to inhibition,
is not a suitable target in terms of therapeutic HDAC inhibition
as reflected by activity in Jurkat T cells. The investigators thus
establish the importance of isoform-specific targeting in any
attempt to add HDAC inhibitors to combination antiretroviral
therapy. Moreover, as there are distinct cellular reservoirs that
may support latent virus, the authors offer their pharmacological method of profiling HDAC activities as a relatively direct
means of surveying HDAC isoform-specific activities in other
cell types.
Binding and Hijacking: HIV-1 IntegraseAlthough inhibitors of the HIV-1 integrase have reached clinical trials, with one
inhibitor having been approved for prescription, the roles of
cellular proteins in regulating the viral integrase are not fully
understood. Researchers have thus not yet tapped the therapeutic potential of inhibiting viral replication by blocking any of
the dozens of interactions that occur between the viral enzyme
integrase and cellular proteins. One such cellular protein is
Ku70, which participates in nonhomologous end-joining DNA
repair (a process implicated in retroviral infection) and has
been identified, in the form of a p70/p80 dimer, as an autoantigen in systemic lupus erythematosus. The current JBC paper
from Zheng et al. (2) adds a new dimension to Ku70 functionality in HIV-1 replication, capitalizing on the recent identification of Ku70 as a deubiquitinating enzyme. The authors establish that the C terminus of the integrase binds to N-terminal
sequences within Ku70 and that Ku70 promotes the deubiquitination of the integrase. Moreover, integrase mediates the incorporation of Ku70 into progeny virus. The Ku70 that is thus
hijacked upon virion assembly ultimately promotes viral repli-
Flaviviridae: Hepatitis C Virus and Dengue Virus

The Flaviviridae family includes scores of viruses, several of
which are important pathogens in humans. The genomes of
Flaviviridae viruses consist of single-stranded RNA of positive
polarity but, unlike the case of the retroviruses, engender no
DNA intermediate. Two of the JBC papers included in this
compendium concern the biology of Flaviviridae. The first
deals with envelope glycoproteins of the hepatitis C virus (of the
4

vitro assays and immunofluorescence to compare the mutational effects of RNA elements upon viral replication and translation. The authors markedly refine previous speculations concerning many secondary structures of the viral genome, and
they offer stringent evidence for regulatory roles of at least five
oligonucleotide sequences that cooperate differentially,
depending upon whether the ()-ssRNA genome is directing
translation or RNA-dependent RNA synthesis. The latter process, moreover, appears to utilize certain genomic RNA elements during ()-RNA synthesis but not ()-RNA synthesis,
whereas the former process (i.e. translation) may utilize elements in a context-dependent manner, depending on whether
translation occurs by canonical (i.e. 5-cap-dependent translation) or noncanonical mechanisms. The implications for controlling dengue viral infections and basic molecular biology are
unclear, but the virology of the system is compelling when
viewed in terms of mechanistic intricacies that are both elegant
and complex.
Hepacivirus genus), which remains a pathogen of global importance. The second focuses on the RNA genome of the dengue
virus (a Flavivirus), which is endemic to tropical and subtropical regions, causing tens of millions of infections per year.
Hepatitis C Virus: Cell Entry and Envelope Proteins E1 and E2
Since discovery of the hepatitis C virus, in 1989, investigations
into the two HCV envelope glycoproteins (E1 and E2) have
been hampered by technological difficulties associated with
carrying the virus in culture and by the high degree of sequence
variability manifest in both E1 and E2. Each of the two envelope
proteins contains a large glycosylated N-terminal ectodomain
and a C-terminal transmembrane anchor, and despite the challenges of assessing discrete infective stages of HCV in cell culture, specific roles for each protein in cell receptor binding and
cell fusion have been suggested (5). One of the characteristics of
the E1 and E2 proteins, which was quite remarkable when
reported in JBC well over a decade ago, is that discrete mutations that are confined to the transmembrane domain of either
protein can affect heterodimerization and virus replication (6).
In their current JBC paper, Maurin et al. (5) exploit the naturally occurring variability of E1 and E2 sequences to elucidate
the structural basis of intra- and intersubunit interactions that
enable E1E2 heterodimers to subserve cell entry in the infectivity cycle. In particular, the authors have identified combinations of E1 and E2 variants that can be coexpressed to produce
stable heterodimers that are nevertheless nonfunctional. By
applying site-directed mutagenesis to such inactive heterodimers, moreover, the authors have determined sequences
and residues, from both envelope proteins, that function concertedly to culminate in viral infectivity. In this way, the authors
establish structure-function relationships that go beyond
merely descriptive terms such as transmembrane sequence or
ectodomain. Indeed, discrete interactions within the E1 subunit of the heterodimer, as well as interprotomeric interactions
that emanate from discrete residues within the E1 transmembrane sequence, prove essential to entry of the virus into the
cell. Ultimately, the authors indicate, functional mechanisms of
viral replication and infection are effected through subtle facets
of the E1E2 interaction related to cell entry.
Dengue Virus: Structure and Function of the Viral Genome
Manzano et al. (7) focus their attention on the structure of the
ssRNA genome of the dengue virus. Typical of Flaviviridae
genomes, the dengue virus RNA is of positive polarity (()RNA), meaning that it can serve directly as message for the
translation of viral proteins. The genome also serves as a template for the RNA-dependent RNA polymerase activity of the
viral nonstructural protein 5 (NS5), whereby a negative RNA
strand is synthesized to serve as the template for production of
viral progeny genomes. In this way, replication of the viral
genome is intimately tied to the translation of viral proteins,
with both processes regulated by means of structural elements
that arise through intramolecular associations within the
ssRNA genome. One important element, or group of elements,
is the 3-untranslated region of the genome.
Manzano et al. (7) have employed a high-performance computer algorithm that duly considers the stabilities and probabilities of RNA folding intermediates as related to the core region
of the 3-untranslated region. In addition, the authors use in
The Gn Protein of the Crimean Congo Hemorrhagic Fever

Virus
Two members of the Bunyaviridae provide focus for the JBC
paper contributed by Estrada and De Guzman (8), who provide
atomic resolution, in solution, for the cytoplasmic portion of
envelope glycoprotein Gn of the Crimean Congo hemorrhagic
fever (CCHF) virus (of the genus Nairovirus). By comparison to
their previous assessment of the similar protein in a member of
the Hantavirus genus (also in JBC (9)), the authors offer insights
into the role of the Gn protein in CCHF virion assembly. The
100-residue cytoplasmic tail (in the respective proteins from
both viruses) contains two zinc finger-like CCHC sequences,
and the investigators establish by NMR that the four defining
residues of the two Cys-Cys-His-Cys motifs participate in functional zinc coordination. The two CCHC motifs, moreover,
interact to form a very stable compact structure, such that the
two zinc fingers appear, according to solution dynamics, to
behave as a single entity. Results for the Gn protein, moreover,
indicate that the zinc finger functionality is distinctive in further respects. First, the -fold that typifies the CCHC motif
is supplemented, to the N-terminal side of the second such
sequence, by a third (3) helix, so that the structure ultimately
consists of four -folds and three helices. The distribution of
charged residues in the Gn 43 structure, furthermore, is highly
suggestive of a function, typically associated with zinc fingers, in
interacting with RNA: multiple basic residues define one face of
the globular structure, ideal for interactions with nucleic acid,
whereas acidic residues tend to cluster on the opposing hemisphere of the structure. Indeed, the researchers confirm that the
electrophoretic mobility of CCHF viral RNA is significantly
retarded through interactions with the cytoplasmic tail of Gn. The
authors thereby offer a model in which the native integral Gn protein is envisaged to orchestrate the assembly of lipid envelope and
nucleoprotein components into progeny virions.
Conclusion
The seven papers presented in this compendium touch on
a variety of issues related to the basic biology of distinct
viruses. Many of the observations can be related to potential
5

Chem. 286, 1772217735
3. Joshi, P., and Stoddart, C. A. (2011) J. Biol. Chem. 286, 2458124592
4. van Montfort, T., Melchers, M., Isik, G., Menis, S., Huang, P.-S., Matthews, K., Michael, E., Berkhout, B., Schief, W. R., Moore, J. P., and Sanders, R. W. (2011) J. Biol. Chem. 286, 22250 22261
5. Maurin, G., Fresquet, J., Granio, O., Wychowski, C., Cosset, F.-L., and
Lavillette, D. (2011) J. Biol. Chem. 286, 2386523876
6. Op De Beeck, A., Montserret, R., Duvet, S., Cocquerel, L., Cacan, R., Barberot, B., Le Maire, M., Penin, F., and Dubuisson, J. (2000) J. Biol. Chem.
275, 31428 31437
7. Manzano, M., Reichert, E. D., Polo, S., Falgout, B., Kasprzak, W., Shapiro,
B. A., and Padmanabhan, R. (2011) J. Biol. Chem. 286, 2252122534
8. Estrada, D. F., and De Guzman, R. N. (2011) J. Biol. Chem. 286,
21678 21686
9. Estrada, D. F., Boudreaux, D. M., Zhong, D., St. Jeor, S. C., and De Guzman,
R. N. (2009) J. Biol. Chem. 284, 8654 8660
clinical opportunities, whereas others shed light not only on

viral processes of metabolism and replication, but also on
fundamental cell functions. The Journal of Biological Chemistry is proud to be a part of the unfolding history of virology,
and we continue to welcome important research findings
from virologists from around the world. We hope that participants of the Recent Advances in Pathogenic Human
Viruses will enjoy reading the stories told in the papers of
this compendium, assembled especially for the international
meeting in China.
REFERENCES
1. Huber, K., Doyon, G., Plaks, J., Fyne, E., Mellors, J. W., and Sluis-Cremer,
N. (2011) J. Biol. Chem. 286, 2221122218
2. Zheng, Y., Ao, Z., Wang, B., Jayappa, K. D., and Yao, X. (2011) J. Biol.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 25, pp. 2221122218, June 24, 2011
2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Inhibitors of Histone Deacetylases

CORRELATION BETWEEN ISOFORM SPECIFICITY AND REACTIVATION OF HIV TYPE 1
(HIV-1) FROM LATENTLY INFECTED CELLS
S
Received for publication, August 30, 2010, and in revised form, April 29, 2011 Published, JBC Papers in Press, April 29, 2011, DOI 10.1074/jbc.M110.180224
Kelly Huber1, Genevie`ve Doyon1, Joseph Plaks, Elizabeth Fyne, John W. Mellors, and Nicolas Sluis-Cremer2
From the Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine,
Pittsburgh, Pennsylvania 15261
Deacetylation of histone proteins at the HIV type 1 (HIV-1)
long terminal repeat (LTR) by histone deactylases (HDACs) can
promote transcriptional repression and virus latency. As such,
HDAC inhibitors (HDACI) could be used to deplete reservoirs
of persistent, quiescent HIV-1 proviral infection. However, the
development of HDACI to purge latent HIV-1 requires knowledge of the HDAC isoforms contributing to viral latency and the
development of inhibitors specific to these isoforms. In this
study, we identify the HDACs responsible for HIV-1 latency in
Jurkat J89GFP cells using a chemical approach that correlates
HDACI isoform specificity with their ability to reactivate latent
HIV-1 expression. We demonstrate that potent inhibition or
knockdown of HDAC1, an HDAC isoform reported to drive
HIV-1 into latency, was not sufficient to de-repress the viral
LTR. Instead, we found that inhibition of HDAC3 was necessary
to activate latent HIV-1. Consistent with this finding, we identified HDAC3 at the HIV-1 LTR by chromatin immunoprecipitation. Interestingly, we show that valproic acid is a weak inhibitor of HDAC3 (IC50 5.5 mM) relative to HDAC1 (IC50 170
M). Because the total therapeutic concentration of valproic
acid ranges from 275 to 700 M in adults, these data may explain
why this inhibitor has no effect on the decay of latent HIV reservoirs in patients. Taken together, our study suggests an important role for HDAC3 in HIV-1 latency and, importantly,
describes a chemical approach that can readily be used to identify the HDAC isoforms that contribute to HIV-1 latency in
other cell types.
tion must include compounds that purge the latent viral reservoirs thereby rendering them susceptible to cART.
HIV-1 can be maintained in a latent state by multiple different mechanisms that inhibit virus gene expression after integration into the cellular DNA (6 8). For example, epigenetic
modifications at or near the HIV-1 5-long terminal repeat
(LTR) can induce chromatin condensation that diminishes the
accessibility of the HIV-1 promoter to transcription factors. In
this regard, it has been well documented that different transcription factors can recruit histone deacetylase (HDAC)
enzymes to the HIV-1 LTR where they promote chromatin
condensation by deacetylating the -amino groups of lysine residues in histone tails (9 14). Eleven distinct zinc-dependent
HDAC isoforms have been identified in humans. These can be
classified into four families, namely class I (HDAC13 and -8),
IIa (HDAC4, -5, -7, and -9), IIb (HDAC6 and -10), and IV
(HDAC11), which differ in structure, enzymatic function, subcellular localization, and expression patterns (15). To date,
multiple studies have demonstrated that recruitment of
HDAC1 to the HIV-1 LTR by different DNA-binding complexes is sufficient to induce viral latency (9 14). However,
HDAC2 and HDAC3 can also bind to the HIV-1 LTR and may
also play an important role in viral latency (12, 16, 17).
Treatment of latently infected HIV-1 cell lines and/or
CD4() T cells from aviremic HIV-1-infected individuals on
cART with HDACI can lead to chromatin relaxation and induction of viral transcription (reviewed in Ref. 6). Therefore, HDACIs are considered as potential therapeutic agents for purging
the latent viral reservoir in HIV-1-infected individuals. However, the active site structures of the HDAC family are largely
conserved, and many HDACIs exhibit activity against multiple
HDAC isoforms. For example, suberoylanilide hydroxamic acid
(SAHA, vorinostat), an activator of latent HIV-1 expression
(18 20), is a nonselective HDACI that inhibits both class I and
class II HDAC isoforms (21). Because HDACs exert crucial
roles in numerous biological processes, including cell cycle, cell
differentiation, and survival (15), simultaneous inhibition of
multiple HDAC isoforms will likely reduce their therapeutic
window by promoting undesirable side effects and/or toxicity.
Accordingly, the development of HDACI for an HIV-1 curative
strategy requires knowledge of the HDAC isoforms contributing to viral latency and the development of inhibitors targeting
these isoforms. In this study, we use a chemical approach that
correlates the isoform specificities of HDACI with their abilities to reactivate latent HIV-1 expression to identify the HDAC
isoforms responsible for HIV-1 latency in Jurkat J89GFP cells.
Combination antiretroviral therapy (cART)3 can effectively

reduce plasma HIV-1 to undetectable levels. However, upon its
interruption, there is usually a rapid rebound of viremia (1).
This viremia is thought to arise from latently infected reservoirs
such as memory CD4() T cells or CD34() multipotent
hematopoietic progenitor cells (25). Therefore, any long term
therapeutic strategy targeted toward eliminating HIV-1 infec-
The on-line version of this article (available at http://www.jbc.org) contains

supplemental Fig. 1 and Tables 1 and 2.
1
Both authors contributed equally to this work.
2
To whom correspondence should be addressed: S817 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261. Tel.: 412-648-8457; Fax: 412-648-8521;
E-mail: nps2@pitt.edu.
3
The abbreviations used are: cART, combination antiretroviral therapy;
HDAC, histone deacetylase; HDACI, HDAC inhibitor; SAHA, suberoylanilide
hydroxamic acid; EGFP, enhanced green fluorescent protein; FW, forward;
REV, reverse; HIV-1, HIV type 1.
7
Inhibition of HDAC3 Required for Reactivation of Latent HIV-1

tamine, 100 units/ml penicillin, and 100 g/ml streptomycin.
CD8()-depleted peripheral blood mononuclear cells were isolated from fresh whole blood (100 ml) of HIV-negative individuals, as described previously (38). To determine HDACI cytotoxicity, 1 104 cells were plated in 96-well plates with varying
concentrations of drug. Following a 24-h incubation period, cell
viability was measured using either the 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyltetrazolium bromide (Roche Applied Science) or CellTiter 96 proliferation (Promega, Madison, WI)
assay. The concentration of HDACI that decreased cell viability
by 50% (i.e. 50% cytotoxic concentration (CC50)) was calculated
by regression analysis using SigmaPlot software.
Reactivation of Latent HIV-1 by HDACIJ89GFP cells are a
Jurkat T-cell line that contains a stably integrated, full-length
HIV-1 provirus (strain 89.6) with an enhanced green fluorescent protein (EFGP) reporter incorporated into the viral
genome (22). The viral genome in these cells is transcriptionally
silent. However, upon stimulation with tumor necrosis factor
or HDACI, viral transcription was activated, and viral expression can be measured by EGFP production. We chose this cell
line model of HIV-1 latency because the absence of viral expression was not due to mutations in either the Tat-TAR axis (e.g.
the ACH2 cell line (34) and the U1 promonocytic cell line (36))
or in the 5-LTR (e.g. the JK cell line (35)). The J89GFP cells
were maintained in RMPI 1640 medium supplemented with
10% FBS, 0.3 mg/ml L-glutamine, 100 units/ml penicillin, and
100 g/ml streptomycin. 5 105 cells/ml cells were plated in
6-well plates with varying concentrations of HDACI for 6 72 h.
The PI3K and Akt inhibitors wortmannin and Akt inhibitor IV
(AI4) were used at concentrations of 100 nM and 10 M, respectively. The cells were then washed in PBS, fixed in 4% paraformaldehyde, and stored at 4 C until analysis. Reactivation of latent
HIV-1 was determined by quantifying the percentage of EGFPpositive cells using a FACScan flow cytometer with FACSDiva
software (BD Biosciences).
DNA Microarray Analyses5 105 J89GFP cells were
treated with 200 nM SAHA, oxamflatin, scriptaid, and apicidin
for 24 h. Control experiments included J89GFP cells grown in
the absence of HDACI and Jurkat cells infected with HIV-1
(multiplicity of infection of 1) for 24 h. Total cellular RNA was
extracted from these cells using the RNeasy Plus RNA extraction kit (Qiagen Inc.) according to the manufacturers protocol.
RNA quantification, quality assessment, and DNA microarray
analyses were carried out by PhalanxBio, Inc. (Palo Alto, CA),
using the Human Whole Genome OneArrayTM microarray.
Each treatment condition and control were assessed in duplicate biological replicates, and all samples were run in duplicate
technical replicates on the arrays. Data analysis was performed
by PhalanxBio, Inc., using Rosetta Resolver software.
siRNA KnockdownsiRNAs targeting HDAC1, HDAC2, and
HDAC3, as well as a control scrambled sequence control
siRNA, were purchased from Qiagen (SA Biosciences). The
J89GFP cells were transfected with 60 nM siRNA using the
Neon Transfection System from Invitrogen, according to
the manufacturers protocol. The efficiency of gene knockdown
was assessed by determining mRNA levels (described below)
and by Western blot analyses of protein expression.
The results from this study suggest that potent inhibition of

HDAC3 may be important for reactivation of latent HIV-1.
EXPERIMENTAL PROCEDURES
MaterialsThe HDACI 4,5:8,9-dianhydro-1,2,6,7,11pentadeoxy-D-threo-D-ido-undeca-1,6-dienitol (depudecin),
suberoyl bis-hydroxamic acid, cyclo[(2S)-2-amino-8-oxodecanoyl-1-methoxy- L -tryptophyl-L-isoleucyl-(2R)-2-piperidinecarbonyl] (apicidin), cyclo-( D -Pro- L -Ala- D -Ala- L -2amino-8-oxo-9,10-epoxydecanoic acid) (HC toxin), (2E)5-[3-(phenylsulfonylamino)phenyl]-pent-2-en-4-ynohydroxamic acid (oxamflatin), 6-(1,3-dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-hexanoic acid hydroxyamide (scriptaid), sodium
butyrate, sodium 4-phenylbutyrate, SAHA, valproic acid, and
[(R)-(E,E)]-7-[4-(dimethylamino)phenyl]-N-hydroxy-4,6-dimethyl-7-oxo-2,4-heptadienamide] (trichostatin A) were obtained from Enzo Life Sciences (Plymouth Meeting, PA). 4-Dimethylamino-N-(6-hydroxyamino)-6-(oxohexyl]-benzamide
(CAY10398) and N-phenyl-N-(2-aminophenyl) hexamethylenediamide (CAY10433) were obtained from Cayman Chemical Co. (Ann Arbor, MI). Sodium 1-naphthoate was obtained
from TCI America (Portland, OR). Droxinostat was purchased from Sigma. Wortmannin was obtained from Sigma.
The AKT inhibitor IV was obtained from EMD Biosciences
(Gibbstown, NJ). The phospho-AKT antibody and AKT antibody were obtained from Cell Signaling Technology (Boston). The -actin antibody was obtained from Abcam (Cambridge, MA). DNA oligonucleotide primers were synthesized
by Integrated DNA Technologies (San Diego). The recombinant purified HDAC isoforms, the Fluorogenic HDAC assay
kit, and the HDAC assay substrates were purchased from
BPS Bioscience (San Diego). The J89GFP cells were a kind
gift from Dr David Levy.
HDAC Activity AssaysThe lysine deacetylase activity of
HDAC19 was assessed using the fluorogenic HDAC assay
(BPS Bioscience) according to the manufacturers instructions.
The HDAC3 used in this assay was complexed with human
nuclear receptor co-repressor 2 (NCOR2; amino acids 395
489), which is an activating co-factor of this HDAC isoform
(31). All assays were carried out under steady-state conditions,
and the assay read-out was optimized for linearity both as a
function of time and enzyme concentration. Inhibition assays
were carried out in 384-well plates. The assay volume was 25 l
and contained 0.1 mg/ml BSA, 20 M substrate, and varying
concentrations of the HDACI. All HDACI were dissolved in
DMSO. The final concentration of DMSO did not exceed 5.0%
(v/v). The formation of the fluorescent product was measured
using a SpectraMax M2 plate reader (Molecular Devices). The
excitation and emission wavelengths were 360 and 450 nm,
respectively. The concentrations of HDACI required to inhibit
50% of the deacetylase activity of an HDAC isoform (i.e. IC50)
were calculated by regression analysis using SigmaPlot software
(Systat Software, Inc., San Jose, CA).
HDACI CytotoxicityJurkat cells were maintained in RMPI
1640 medium supplemented with 10% FBS (Atlanta Biologicals), 0.3 mg/ml L-glutamine, 100 units/ml penicillin, and 100
g/ml streptomycin. HeLa and 293T cells were maintained in
DMEM medium supplemented with 10% FBS, 0.3 mg/ml L-glu8

Quantitative Analysis of Gene TranscriptsRNA was
extracted from treated cells using RNeasy Plus RNA extraction
kit (Qiagen Inc., Valencia, CA) according to the manufacturers
protocol. RNA was quantified using a Nanodrop 2000, and
200 400 ng of total RNA was used in each reaction. RNA was
amplified using the QuantiTect SYBR Green RT-PCR kit
(Qiagen Inc., Valencia, CA) and the DNA Engine Opticon
system (Bio-Rad). Initiated HIV-1 transcripts were detected
using primers TAR-FW (5-GTTAGACCAGATCTGAGCCT3) and TAR-Rev (5-GTGGGTTCCCTAGTTAGCCA-3).
Elongated HIV-1 transcripts were detected using primers
TAT-FW (5-ACTCGACAGAGGAGAGCAAG-3) and TATREV (5-GAGTCTGACTGTTCTGATGA-3). HDAC1 was
quantified using primers HDAC1-FW (5-CCAGTATTCGATGGCCTGTT-3) and HDAC1-REV (5-TGTACAGCACCCTCTGGTGA-3). HDAC2 was quantified using primers
HDAC2-FW (5-ATAAAGCCACTGCCGAAGAA-3) and
HDAC2-REV (5-TCCTCCAGCCCAATTAACAG-3). HDAC3
was quantified using primers HDAC3-FW (5-TGGCTTCTGCTATGTCAACG-3) and HDAC3-REV (5-TCTCTGCCCCGACTTCATAC-3). -Actin mRNA copies were used as
the normalization control (10) and were quantified using the
primers -actin-FW (5-GTCGACAACGGCTCCGGC-3)
and -actin-REV (5-GGTGTGGTGCCAGATTTTCT-3).
Relative gene expression levels were calculated using the
C(T) method (23).
Chromatin Immunoprecipitation (ChIP) Assays2 106
J89GFP cells were fixed with 1% formaldehyde for 10 min at
room temperature. ChIP assays were carried out using the EZChIP assay kit (Millipore Billerica, MA). Immunoprecipitation
was performed using 5 g of antibody (Invitrogen) against
HDAC13 or -8. Rabbit immunoglobulin G serum (5 g, Santa
Cruz Biotechnology Inc., Santa Cruz, CA) was used to control
for nonspecific immunoprecipitation of DNA. Forward
(LTRB primer 5, 5-AGGTTTGACAGCCGCCTA-3) and
reverse (LTRB primer 3, 5-AGAGACCCAGTACAGGCAAAA-3) primers specific for a 203-bp region in the HIV-1
LTR that encompasses the NF-B-binding site LTR were used
to detect a specific interaction between an HDAC isoform and
HIV-1 DNA, as described previously (10).
apicidin, and trichostatin A) were able to stimulate HIV-1

expression by more than 5% in the J89GFP cells, as measured by
flow cytometry analysis of EGFP expression. In this regard, it
should be noted that apicidin, scriptaid, oxamflatin, and SAHA
exhibited similar CC50 values and were tested in the J89GFP
cells at identical concentrations, therefore allowing for a direct
comparison of their ability to reactivate latent HIV-1 expression. Interestingly, valproic acid and sodium butyrate were
found to be relatively weak inhibitors of HDAC1 (IC50 175
M), but each elicited a different effect in the J89GFP cells as
follows: 1 mM valproic acid reactivated HIV-1 expression in
only 4.3% of cells; 1 mM sodium butyrate reactivated HIV-1
expression in 66.4% of cells. Taken together, these HDACI
screening studies show that inhibition of HDAC1 is not sufficient to reactivate HIV-1 expression in J89GFP cells.
Inhibition of HDAC3 Correlates with the Reactivation of
Latent HIV-1Based on the data described above, we next carried out in-depth analyses on the HDACIs apicidin, oxamflatin,
scriptaid, and SAHA (Fig. 2). Each of these HDACIs exhibit
similar potency against purified HDAC1 and similar cytotoxicity (CC50) values in Jurkat cells (Table 1). However, at concentrations of inhibitor ranging from 0 to 500 nM, apicidin and
oxamflatin reactivated HIV-1 expression in the J89GFP cells in
a dose-dependent manner, whereas SAHA and scriptaid elicited no effect (Fig. 2A). A time course experiment demonstrated
that this lack of activity was not due to an early or late EGFP
peak that was missed at the 24-h time point used in the doseresponse experiments (Fig. 2B). Because the EGFP expression
quantitated in Fig. 2, A and B, only provides information on the
translated protein, we also used quantitative RT-PCR to assess
the formation of HIV-1 RNA transcripts (Fig. 2C). Consistent
with the flow cytometry analyses, oxamflatin and apicidin significantly increased the abundance of elongated HIV-1 transcripts, but not initiated transcripts, compared with untreated
J89GFP cells. By contrast, scriptaid and SAHA did not significantly increase the formation of either initiated or elongated
HIV-1 transcripts.
We also carried out cDNA microarray analyses to determine
the magnitude and the extent of global gene expression changes
observed in the J89GFP cells after 24 h of treatment with 200 nM
oxamflatin, scriptaid, SAHA, or apicidin. Control experiments
included untreated J89GFP cells and Jurkat cells infected with
HIV-1 for 24 h. The cDNA microarray data are provided in
supplemental Tables 1 and 2 and Fig. 1. SAHA and scriptaid
were found to significantly (p 0.01) up- or down-regulate 3
and 1% of all genes compared with untreated cells, respectively.
One study reported that SAHA altered regulation in at least
22% of genes in CEM cells; however, a much higher concentration of drug (2.5 M) was used which caused 50% of cell death
after 24 h (30). Oxamflatin and apicidin resulted in gene expression changes in 11 and 8% of all genes compared with
untreated J89GFP, respectively. These higher gene expression
levels compared with scriptaid and SAHA could be due to inhibition of different HDAC isoforms (see below) and/or the expression
of HIV-1 proteins in the J89GFP cells. Microarray DNA analyses
revealed that 2.7% of all genes displayed altered expression
changes in HIV-1-infected Jurkat cells compared with uninfected
cells. Nevertheless, these data indicate that SAHA and scriptaid
RESULTS AND DISCUSSION

Potent Inhibition of HDAC1 Is Not Sufficient to Reactivate
Latent HIV-1Previous studies have demonstrated that
recruitment of HDAC1 to the HIV-1 LTR by different DNAbinding complexes is sufficient to induce viral latency (9 11,
13, 14). Accordingly, we hypothesized that a potent inhibitor of
HDAC1 would reactivate HIV-1 expression in the J89GFP cell
line model of viral latency. Initially, we screened 16 structurally
diverse HDACI (Fig. 1) for the following: (i) their inhibitory
activity against recombinant purified HDAC1; (ii) their cytotoxicity (CC50) in Jurkat cells; and (iii) their ability to reactivate
HIV-1 expression in J89GFP cells (Table 1). For point iii, the
highest possible sub-cytotoxic concentration of HDACI (determined from the cytotoxicity assessments) was used. Of the 16
HDACI tested, 6 (apicidin, HC toxin, scriptaid, oxamflatin,
SAHA, and trichostatin A) exhibited potent activity (IC50 100
nM) against purified HDAC1. Of these, only three (oxamflatin,
9
FIGURE 1. Chemical structures of HDACI used in this study.
TABLE 1
In vitro activity against HDAC1, cytotoxicity in Jurkat cells, and reactivation of latent HIV-1 in J89GFP cells by structurally diverse HDACI
HDACI
IC50 against HDAC1

M
HC toxin
Apicidin
Oxamflatin
Scriptaid
SAHA
TSA
M344
CAY10398
MC1293
CAY10433
SBHA
Depudecin
Sodium 1-naphthoate
Valproic acid
Sodium butyrate
Sodium 4-phenylbutyrate
a
0.000154
0.000299
0.003959
0.006421
0.0137
0.0169
0.0941
1.7780
4.245
9.36
4.54
25.33
200.6
171
175
162
Reactivation
dosea
Cytotoxicity CC50
M
% inhibition of HDAC1 at
reactivation doseb
% EGFP-positive J89GFP
cells (after 24 h)
99.7
99.7
99.2
98.7
97.3
74.7
51.5
21.9
54.1
51.6
95.6
3.8
4.75
85.4
85.1
86.1
1.3
40.6
31.2
3.2
2.5
17.2
3.1
0.9
2.7
5.2
57.5
1.3
1
4.3
66.4
2.5
0.05
10.0
6.0
6.0
10.0
0.10
0.5
1.0
10.0
1000
100
5.0
10
10,000
10,000
10,000
0.005
0.5
0.5
0.5
0.5
0.05
0.1
0.5
5
10
100
1
10
1000
1000
1000
The highest nontoxic concentration of HDACI (determined from the cytotoxicity assays in Jurkat cells) was administered to the J89GFP cells to determine the inhibitors
ability to reactivate latent HIV-1.
Maximum possible inhibition of HDAC1 at the concentration of inhibitor used in the reactivation experiments in J89GFP cells is shown (the actual inhibition in the
J89GFP cells is likely to be significantly less due to inefficient cellular uptake and nonspecific protein binding).
were taken up into the J89GFP cells and induced gene expression
changes. However, at the concentrations tested, they lacked the
ability to induce expression of latent HIV-1.
To gain insight into the mechanisms by which oxamflatin

and apicidin, but not scriptaid and SAHA, reactivated latent
HIV-1, we determined the in vitro inhibitory activity of each of
10

the HDACI against recombinant purified HDAC1 and -2, the
3-NCOR2 complex, and -4 9 (Table 2). In general, each of the
HDACI exhibited little or no activity against the class II HDAC
isoforms, although scriptaid exhibited excellent activity against

HDAC6 (IC50 34 nM) and oxamflatin activity against HDAC6
(IC50 390 nM) and HDAC7 (IC50 840 nM). By contrast, all
four of the HDACI were found to be very potent inhibitors of
the class I HDAC isoforms 1, 2, and 8 (IC50 20 nM). Interestingly, oxamflatin and apicidin, both of which induced HIV-1
outgrowth in the J89GFP cells, also potently inhibited the
HDAC3-NCOR2 complex (IC50 10 nM). By contrast, scriptaid
(IC50 320 nM) and SAHA (IC50 600 nM) were 100-fold
less active against the HDAC3-NCOR2 complex. Based on our
IC50 calculations, a 200 nM dose of apicidin and oxamflatin
would inhibit 95% of the deacetylase activity of HDAC13
and -8. (In the J89GFP cells, these values would likely be significantly less due to inefficient inhibitor uptake and nonspecific
protein binding.) By contrast, a 200 nM dose of scriptaid and
SAHA would inhibit 95% of HDAC1, -2, and -8 but would
only inhibit 25 and 18% of the deacetylase activity of HDAC3,
respectively. These values for HDAC3 would increase to 45 and
36%, respectively, at a dose of 500 nM. Taken together, these
data provide strong evidence that potent inhibition of HDAC3
is required to reactivate the expression of HIV-1 in the J89GFP
cells. The data also suggested that increasing the concentrations of scriptaid and SAHA to allow inhibition of HDAC3
would result in the activation of latent HIV-1 in the J89GFP
cells. Indeed, in Fig. 2D we show that 2 M scriptaid and SAHA
promote activation of latent HIV-1 in the J89GFP. At this concentration, the total inhibition of HDAC3 approaches 76 and
70% for scriptaid and SAHA, respectively. To further assess the
importance of HDAC3 inhibition in the reactivation of latent
HIV-1 infection, we assessed the ability of droxinostat to reactivate latent HIV-1 expression in the J89GFP cells. Previous
studies reported that this HDACI selectively inhibited HDAC3
and -8 but not HDAC1 and -2 (37). Indeed, we found that droxinostat is a reasonably potent inhibitor of recombinant HDAC3NCOR2 complex and HDAC8 (IC50 2.0 and 3.0 M, respectively) but shows only weak activity against HDAC1 and -2
(IC50 63 and 250 M, respectively) (Table 2). Of note, droxinostat was found to reactivate latent HIV-1 expression in a
dose-dependent manner (Fig. 3). At a 40 M concentration of
droxinostat, HDAC3 and -8 would be inhibited by 95%,
whereas there would be only partial inhibition of HDAC1 (38%)
and HDAC2 (14%). Taken together, these studies provide addi-
FIGURE 2. Reactivation of latent HIV-1 in J89GFP cells by oxamflatin, apicidin, scriptaid, and SAHA. A, J89GFP cells were treated with varying concentrations of HDACI (0 500 nM), and the percentage of cells expressing EGFP
was quantitated after 24 h by FACs. Error bars represent S.E. from at least three
independent experiments. B, J89GFP cells were treated with 200 nM HDACI,
and the percentage of cells expressing EGFP was quantitated at different time
intervals (0 75 h) by FACs. C, increase in HIV-1 RNA transcripts in cells treated
with 200 nM HDACI for 24 h as measured by quantitative RT-PCR. Error bars
represent S.E. from at least three independent experiments. The fold increase
in transcript relative to untreated cells is indicated above each bar for all
four HDACI. D, J89GFP cells were treated with varying concentrations of
HDACI (0 2 M), and the percentage of cells expressing EGFP was quantitated after 24 h by FACs. Error bars represent S.E. from at least three
independent experiments.
TABLE 2
In vitro activity of HDACI against class I and class II HDAC isoforms
IC50 against HDAC isoforms (nM )
Apicidin
Oxamflatin
Class I
HDAC1
HDAC2
HDAC3
HDAC8
0.30 0.15a1
1.2 0.80
0.98 0.22
0.26 0.09
3.96 0.87
0.16 0.11
10.3 1.2
0.37 0.15
0.64 0.09
1.4 0.74
607 93
14.5 1.1
Class II
HDAC4
HDAC5
HDAC6
HDAC7
HDAC9
50,000
50,000
50,000
50,000
50,000
3800 1100
50,000
390 73
840 39
50,000
14,000 1500
50,000
34 9
2200 350
50,000
HDAC isoform
a
b
Scriptaid
Data represent the mean S.D. from three replicate experiments.

ND, not determined.
11
SAHA
Droxinostat
Valproic acid
13.7 0.15
62.0 0.15
869 0.15
6.8 0.15
63,000
250,000
2000
5000
171,000
634,000
5,500,000
756,000
50,000
50,000
5500 760
50,000
50,000
NDb
ND
ND
ND
ND
ND
ND
ND
ND
ND
FIGURE 3. Reactivation of latent HIV-1 in J89GFP cells by the HDAC3-specific inhibitor droxinostat. The chemical structure of droxinostat is shown.
The number of J89GFP cells expressing EGFP was quantitated after 24 h by
FACs. Error bars represent S.E. from two independent experiments.
TABLE 3
Cytotoxicity of HDACI in different cells
CC50 (M)
a
b
HDACI
Jurkata
HeLaa
293Ta
Apicidin
Oxamflatin
SAHA
Scriptaid
10.0 0.1
5.9 0.1
10.0 0.1
6.1 0.3
11.3 1.7
9.4 0.8
11.3 0.3
8.9 1.8
12.2 1.4
6.0 1.0
14.1 0.2
5.8 0.2
CD8()-depleted
PBMCb
0.1
0.3
7.5
0.7
Data represent the mean S.D. from three replicate experiments.

Data represent the mean from two independent replicate experiments.
tional evidence that HDACI with specificity toward HDAC3

can reactivate latent HIV-1 expression in J89GFP cells.
Several studies have recently demonstrated that oxamflatin,
apicidin, scriptaid, and SAHA can reactivate latent HIV-1 in
different cell lines and/or in resting CD4() T cells from aviremic patients (18, 19, 20, 32). In each of these studies relatively
high concentrations (500 nM) of inhibitor were used. Of note,
each of these HDACI display significant toxicity in cell lines
(CC50 values range from 5 to 10 M) and in CD8()-depleted
peripheral blood mononuclear cells (CC50 values range from
0.1 to 7.5 M) (Table 3). In this regard, the small therapeutic
window of these HDACI highlights one potential limitation for
their inclusion in therapeutic combinations targeted toward
the eradication of HIV-1.
HDAC3 Resides at the HIV-1 LTR in J89GFP CellsThe data
described above provide strong evidence that inhibition of
HDAC3 is important for the activation of latent HIV-1. However, only two studies have identified this HDAC isoform at the
HIV-1 LTR (16, 17). Accordingly, we performed ChIP assays in
the J89GFP cells using antibodies specific for the class I HDAC
isoforms (Fig. 4A). We detected a strong signal for HDAC1 and
-3 indicating that both of these HDAC isoforms resided at the
HIV-1 LTR. We also detected a weak signal for HDAC2 suggesting that this isoform may also be present at the HIV-1 LTR
FIGURE 4. Role of HDAC13 in maintaining HIV-1 latency. A, ChIP assays

identify HDAC13, but not HDAC8, at the HIV-1 LTR in J89GFP cells. Two different HDAC1 antibodies (a and b) were used in the ChIP assays. B, quantitative real time PCR experiments show enrichment (versus rabbit IgG) of HDAC1
and -3 at the HIV-1 LTR in J89GFP cells that is lost upon treatment with apicidin. PCR data were normalized by quantification of the GAPDH promoter in
the input samples. C, mRNA expression of HDAC13 after knockdown by
siRNA. HDAC1* and HDAC2* reports on the mRNA levels in experiments in
which both HDAC1 and -2 were knocked down simultaneously. An siRNA with
a scrambled sequence was used as a control. D, reactivation of latent HIV-1 in
J89GFP cells after knockdown of HDAC1 or -2 or HDAC1 and -2. The number of
J89GFP cells expressing EGFP was quantitated after 24 h by FACs. Error bars
represent S.E. from three independent experiments.
in J89GFP cells. HDAC8 did not associate with the HIV-1 LTR.
These findings are consistent with a recent study by Keedy et al.
(17), who also reported that HDAC13 resided at the HIV-1
LTR in J89GFP cells. Of note, this study also reported that
HDAC8 is primarily sequestered in the cytoplasm and not the
nucleus in J89GFP cells (17). Quantitative real time PCR experiments confirmed the presence of HDAC1 and -3, but not
HDAC2, at the HIV-1 LTR (Fig. 4B). Importantly, the occupancy of HDAC1 and -3 at the HIV-1 LTR in the J89GFP cells is
lost upon treatment with apicidin (Fig. 4B) or oxamflatin (data
not shown). To further assess the role of HDAC13 in maintaining HIV-1 latency, we knocked down their gene expression
12

by siRNA (Fig. 4C). The magnitude of the siRNA-mediated
gene silencing was confirmed by quantitative PCR analyses of
mRNA levels (Fig. 4C) and by Western blot analysis (data not
shown). Knockdown of HDAC3 resulted in significant and substantial cell death that prevented subsequent analyses of HIV-1
latency. Interestingly, the knockdown of either HDAC1 or -2,
or a combination of HDAC1 and -2, did not result in the reactivation of latent HIV-1 expression in the J89GFP cells (Fig. 4D).
These data provide strong supporting evidence that inhibition
of HDAC1 and -2 is insufficient to reactivate latent HIV-1
expression.
Reactivation of Latent HIV-1 Expression by Apicidin and
Oxamflatin Is Partially Dependent on the PI3K/Akt Signaling
PathwayPeterlin and co-workers (20) have shown that SAHA
activates HIV-1 expression in latently infected cells via the
PI3K/Akt pathway. To determine whether this pathway is also
activated by apicidin and oxamflatin, we first assessed Akt
phosphorylation levels in J89GFP cells treated with TNF- (a
positive control) or with 500 nM apicidin, oxamflatin, SAHA, or
scriptaid (Fig. 5A). Increased Akt phosporylation was observed
following treatment with apicidin but not oxamflatin, SAHA, or
scriptaid. Because the concentration of SAHA used in this
experiment (500 nM) is not sufficient to reactivate latent HIV-1
expression in the J89GFP cells, it was not unexpected that Akt
was not activated. To further determine the impact of apicidin
and oxamflatin on activation of the PI3K/Akt signaling pathway, we used PI3K (wortmannin) and Akt (Akt inhibitor IV
(AI4)) inhibitors. Indeed, Akt and PI3K inhibitors decreased
but did not completely eliminate both apicidin and oxamflatininduced viral replication (Fig. 5B). Importantly, these inhibitors
had no significant effect on the basal levels of HIV-1 production
(Fig. 5B). These results suggest that the latent HIV-1 reactivation activity of both apicidin and oxamflatin is intertwined with
activation of the PI3K/Akt signaling pathway.
Valproic Acid Is a Weak Inhibitor of HDAC3In 2004,
Ylisastigui et al. (24) demonstrated that treatment of resting
CD4() T cells of aviremic patients with valproic acid induced
histone acetylation and promoted virus outgrowth. An initial
proof-of-concept study in which four volunteers infected with
HIV added oral valproic acid to their cART regimen for 3
months reported a significant decline in the frequency of resting CD() T-cell infection (25). However, several follow-up
studies found that valproic acid does not reduce the size of
latent HIV reservoir (26 29). Interestingly, we find that valproic acid is a weak inhibitor of the HDAC3-NCOR2 complex
(IC50 5.5 mM, Table 2) and that high concentrations of inhibitor (1 mM) are required in J89GFP cells to activate HIV-1
expression (Fig. 6). The total and free therapeutic concentrations of valproic acid in adults range from 275 to 700 M and
from 27 to 100 M, respectively. These therapeutic concentrations would be insufficient to inhibit HDAC3 in vivo. Accordingly, our data may explain why valproic acid has no effect on
the decay of latent HIV reservoirs in patients.
ConclusionsThis study demonstrates that HDAC3 resides
at the HIV-1 LTR in J89GFP cells and that inhibition of this
HDAC isoform is required for the activation of latent HIV-1
expression by HDACI. Interestingly, Archin et al. (33) recently
demonstrated that an HDACI (MRK12) specific for HDAC1
FIGURE 5. Activation of the PI3K/Akt signaling pathway by apicidin and

oxamflatin. A, Western blot analysis of Akt phosphorylation in J89GFP cells
treated with TNF- (positive control) or with 500 nM apicidin, oxamflatin,
SAHA, or scriptaid. Phosphorylated Akt and -actin were detected using
monoclonal antibodies specific for these proteins. B, J89GFP cells were
treated with 500 nM HDACI with or without wortmannin (100 nM) or the Akt
inhibitor IV (AI 4, 10 M). The number of J89GFP cells expressing EGFP was
quantitated after 24 h by FACs. Error bars represent S.E. from three independent experiments.
and -2 was unable to reactivate latent HIV-1 in J89GFP cells and

in resting CD4() T-cells from aviremic patients. By contrast,
an inhibitor (MRK13) specific for HDAC13 promoted virus
outgrowth in both assay systems (33). Taken together, these
studies suggest that potent inhibition of HDAC3 should be an
important criterion in the development of HDACI for HIV-1
curative strategies. Unfortunately, neither our study nor that of
Archin et al. (33) could address whether inhibition of HDAC3
alone is sufficient to induce virus outgrowth or if inhibition of
HDAC1 and/or -2 is also required. The identification and
development of inhibitors specific for HDAC3 may address this
question.
Finally, it is likely that there are several reservoirs of latent
HIV-1 infection in aviremic patients on cART. For example,
resting CD4() T-cells and CD34() multipotent hematopoietic progenitor cells have both been identified as reservoirs of
latent HIV-1 infection (25). The HDAC isoforms recruited to
the HIV-1 LTRs in these different cell types may be different. In
this regard, the chemical approach described in this study can
readily be used to identify the HDAC isoforms that contribute
to HIV-1 latency in other cell types. The primary advantages of
this approach include the ability to rapidly conduct studies in
cell types that cannot be easily transfected with siRNA (or
13

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FIGURE 6. Reactivation of latent HIV-1 in J89GFP cells by varying concentrations (0 10 mM) of valproic acid. The percentage of J89GFP cells
expressing EGFP was quantitated after 24 h by FACs.
shRNA) molecules or in patient-derived tissues or cells where

there may be insufficient material to carry out genetic studies.
Importantly, our chemical approach also provides an immediate assessment of the therapeutic potential of HDACI to reactivate latent HIV-1 expression in different cell types and/or
tissues.
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14
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 20, pp. 1772217735, May 20, 2011
Host Protein Ku70 Binds and Protects HIV-1 Integrase from

Proteasomal Degradation and Is Required for HIV Replication*
Received for publication, September 13, 2010, and in revised form, March 28, 2011 Published, JBC Papers in Press, March 29, 2011, DOI 10.1074/jbc.M110.184739
Yingfeng Zheng1, Zhujun Ao2, Binchen Wang, Kallesh Danappa Jayappa3, and Xiaojian Yao4
From the Laboratory of Molecular Human Retrovirology, Department of Medical Microbiology, Faculty of Medicine, University of
Manitoba, Winnipeg, Manitoba R3E 0J9, Canada
(IN).5 During HIV-1 integration, IN catalyzes the insertion of
newly reverse-transcribed 10-kb viral DNA into the host
genome. In addition, IN plays important roles in other viral
replication steps, such as reverse transcription, the nuclear
import of preintegration complexes (PICs), and chromatin targeting. By interaction with the host chromatin-tethering factor
LEDGF/p75, IN preferentially targets viral DNA into transcriptionally active sites in the host genome to optimize the transcription and translation of its gene products (13). Cellular
proteins are recruited to assist IN to accomplish integration
from different pathway, including nuclear import, shielding IN
from proteasomal degradation, integration site selection, and
gap repair (4). Recently, considerable interest has been focused
on the functional interaction between IN and host cellular proteins in the hope of disrupting their interactions, thereby blocking HIV-1 replication. In an attempt to identify host cellular
partners for IN, several research groups have identified a number of IN cofactors using the yeast two-hybrid system, coimmunoprecipitation (co-IP) assays, or in vitro reconstitution of the
enzymatic activity of salt-stripped PICs (511). A recent study
by Studamire et al. (5) found that 12 cellular proteins, including
Ku70, could bind to the INs of both the Moloney murine leukemia virus (MMLV) and HIV-1 through screening with a yeast
two-hybrid system. However, whether these cellular cofactors
are associated with HIV-1 IN during HIV replication and their
functional relevance remain unknown.
Ku70 is an evolutionarily conserved protein; it is found ubiquitously in eukaryotes and some prokaryotes, such as Archaea
and Bacteria (1214). It is well known as a DNA repair protein
and is part of the nonhomologous end-joining (NHEJ) pathway. Ku70 has also been implicated in many cellular processes, including antigen-receptor gene rearrangement, mobile
genetic element biology, V(D)J recombination of immunoglobulins, telomere maintenance, DNA replication, transcription,
cell cycle control, and apoptosis (13, 15). As a DNA repair protein, Ku70 can bind to any double-stranded DNA irrespective
of sequence specificity or end configuration, including 5 overhangs, 3 overhangs, or blunt ends (for a review, see Ref. 15).
Ku70 can also bind specific DNA sequences to affect gene transcription (16). For most biological functions in which Ku70 participates, Ku functions as a heterodimer consisting of Ku70 and
Ku80, named according to their respective molecular masses of
HIV-1 integrase (IN) is a key viral enzymatic protein acting

in several viral replication steps, including integration. IN
has been shown to be an unstable protein degraded by the
N-end rule pathway through the host ubiquitin-proteasome
machinery. However, it is still not fully understood how this
viral protein is protected from the host ubiquitin-proteasome
system within cells during HIV replication. In the present
study, we provide evidence that the host protein Ku70 interacts with HIV-1 IN and protects it from the Lys48-linked
polyubiquitination proteasomal pathway. Moreover, Ku70 is
able to down-regulate the overall protein polyubiquitination
level within the host cells and to specifically deubiquitinate
IN through their interaction. Mutagenic studies revealed that
the C terminus of IN (residues 230 288) is required for IN
binding to the N-terminal part of Ku70 (Ku70(1 430)), and
their interaction is independent of Ku70/80 heterodimerization. Finally, knockdown of Ku70 expression in both virusproducing and target CD4 T cells significantly disrupted
HIV-1 replication and rendered two-long terminal repeat circles and integration undetectable, indicating that Ku70 is
required for both the early and the late stages of the HIV-1 life
cycle. Interestingly, Ku70 was incorporated into the progeny
virus in an IN-dependent way. We proposed that Ku70 may
interact with IN during viral assembly and accompany HIV-1
IN upon entry into the new target cells, acting to 1) protect IN
from the host defense system and 2) assist IN integration
activity. Overall, this report provides another example of how
HIV-1 hijacks host cellular machinery to protect the virus
itself and to facilitate its replication.
Integration is an obligatory step in the life cycle of all of the

retroviruses and is performed by the viral enzyme integrase
* This
work was supported by Canadian Institutes of Health Research

(CIHR) Grants HOP-81180 and HBF 103212 and the Leaders Opportunity
Fund Award from the Canadian Foundation of Innovation (to X.-J. Y.).
1
Recipient of studentships from the Manitoba Health Research Council/
Manitoba Institute of Child Health (MHRC/MICH) and the CIHR International Infectious Disease and Global Health Training Program.
2
Recipient of a postdoctoral fellowship from the CIHR International Infectious Disease and Global Health Training Program.
3
Recipient of studentships from MHRC/MICH.
4
Recipient of the Basic Science Career Development Research Award from
the Manitoba Medical Service Foundation. To whom correspondence
should be addressed: Laboratory of Molecular Human Retrovirology, Dept.
of Medical Microbiology, Faculty of Medicine, University of Manitoba, 508
745 William Ave., Winnipeg R3E 0J9, Canada. Tel.: 204-977-5677; Fax: 204789-3926; E-mail: yao2@cc.umanitoba.ca.
15
The abbreviations used are: IN, integrase; PIC, preintegration complex; IP,
immunoprecipitation; WB, Western blot; MMLV, Moloney murine leukemia
virus; NHEJ, nonhomologous end-joining; Ub, ubiquitin; VSV-G, vesicular
stomatitis virus G; p.i., postinfection; PL, ProLabel; aa, amino acids; MOI,
multiplicity of infection.
Ku70 Binds and Protects HIV-1 Integrase

70 and 80 kDa. Two regions of Ku70 amino acids 1115 and
430 482 are responsible for its heterodimerization with Ku80
(17). Successful HIV-1 integration requires gap repair between
viral DNA and host genome, which is believed to be performed
by host DNA repair enzymes (18). Two different host DNA
repair pathways have been suggested to fill in the gap during
HIV-1 infection: the NHEJ and DNA damage-sensing pathways
(19 21). The NHEJ pathway begins with the recruitment of the
Ku70/80 heterodimer, followed by the catalytic subunit of
DNA-dependent protein kinase or DNA-PKcs, Xrcc4, and
DNA ligase IV. Studies have shown that the NHEJ pathway is
important for retroviral transduction or infection and for the
cell survival of infected or transduced cells (20, 2225). For
example, HIV-1-based vector transduction or infection was
markedly reduced in cells deficient in Ku80, DNA-PKcs, Xrcc4,
or ligase IV (22, 24). Moreover, NHEJ activity is required for
two-long terminal repeat (2-LTR) circle formation, and Ku70
has been detected in MMLV PICs (24, 26 28). Ku80 was also
shown to suppress HIV transcription by specifically binding to
a negative regulatory element within the LTR (29). All of these
observations suggest that Ku70 or the K70/80 heterodimer may
be involved in HIV-1 infection by affecting multiple steps of the
viral replication cycle, such as integration. In addition, a novel
deubiquitinating enzymatic activity of Ku70 was recently
described in which Ku70 has a regulatory effect on Bax-mediated apoptosis by decreasing the ubiquitination of Bax and
blocking Bax from proteasomal degradation (30). However,
whether Ku70 also exerts a deubiquitinating effect on other
identified binding partners of Ku70 and how Ku70 interacts
with the ubiquitin-proteasome pathway to deubiquitinate protein substrates are still unclear.
In this study, we investigated the interaction between Ku70
and HIV-1 IN and the potential roles of Ku70 during HIV-1
replication using cell-based coimmunoprecipitation and short
hairpin RNA (shRNA)-mediated knockdown approaches.
Interestingly, our results provide evidence that Ku70 is able to
protect HIV-1 IN from Lys48-linked polyubiquitination and
degradation by down-regulation of the overall protein polyubiquitination level within the host cells and by specific IN
deubiquitination through its binding to IN. Moreover, our
study showed that Ku70 depletion in both virus-producing and
target cells drastically inhibited HIV-1 replication and blocked
2-LTR formation and integration in the real-time PCR analysis.
Our data also showed that, mediated by HIV-1 IN, Ku70 was
incorporated into the progeny virus. All of these results suggest
that Ku70 may interact with IN during viral assembly and
accompany HIV-1 IN into newly infected cells to assist IN
integration activity and protect IN from host-mediated
degradation.
standard calcium phosphate precipitation technique was used,

as described previously (31).
Plasmids and ReagentsTo achieve high level IN expression,
a codon-optimized IN (INopt) cDNA was synthesized and
cloned into the pUC57 vector (GenScript Co., Ltd.). To construct pAcGFP-INopt, the INopt fragment was excised from
pUC57-INopt with BamHI and cloned in frame at the 3 end of
the pAcGFP1-C vector (Clontech) with the same restriction
enzyme. To construct pAcGFP-INwt/mut, each of the INwt/
mut coding sequences, including 1230, 1250, 1270,
50 288, 112288, and K186A/R187A, was amplified by PCRbased mutagenesis and subcloned into the pAcGFP1-C vector
(Clontech) in frame with the GFP coding sequence at the BglII
and BamHI restriction sites (32). Plasmids IN-YFP and MAYFP were described previously (33, 34). Untagged human fulllength Ku70 cDNA in the pCMV6-XL5 vector was purchased
from OriGene Technologies Inc. To construct SVCMV-T7Ku70, a Ku70 cDNA without a start codon was amplified and
cloned into the SVCMVin-T7 vector at the BamHI and NotI
restriction sites. The T7-Ku70 truncation mutants (1263,
1 430, 226 609, and 430 609) were obtained using the same
strategy. The nucleotide sequences of the mutagenic oligonucleotides are as follows: 5Ku70-BamHI, 5-TAGCCGGATCCTCAGGGTGGGAGTCATATTA-3; 3Ku70-NotI, 5-TATATGCGGCCGCTCAGTCCTGGAAGTGCTT-3; Ku70 263-NotI,
5-AGATGCGGCCGCCTAGAGCTTCAGCTTT-3; Ku70
430-NotI, 5-TATGCGGCCGCTCATGGAGGAGTCACCTGAAT-3; Ku70 226-BamHI, 5-TATGGATCCGATGAGGACCTCA-3; Ku70 430-BamHI, 5-ATATGGATCCCCAGGCTTCCAGCT-3. HA-tagged ubiquitin (HA-Ub) and mutants
HA-UbK48R and HA-UbK63R were described previously (35).
The HIV-1 proviruses pNL4.3-GFP, HxBru, and HxBru-IN-HA
were described earlier (32, 36). For single cycle HIV virus,
RT/IN/Env gene-deleted NL4.3lucBglRI provirus and
CMV-Vpr-RT-IN were described previously (34, 37, 38). CMVVpr-RT has two stop codons TAGTGA in place of the first six
nucleotides in IN sequences, and the sequence was confirmed
by sequencing. To construct CMV-Vpr-RT-IN-ProLabel (VprRT-IN-PL) plasmid, a two-step-based PCR method was used.
ProLabel tag sequence was amplified from the pProLabel-C
vector from ProLabelTM Detection Kit II (Clontech) and
inserted after the IN sequence in the CMV-Vpr-RT-IN plasmid
with the IN stop codon and ProLabel start codon removed. The
following primers were used: RT NheI 5, 5-GCAGCTAGCAGGGAGACTAA-3; R-RT-IN-ProLabel pstI 3, 5-GTCGACTGCAGAATTCGAAGCTTATTC-3; IN-ProLabel 5, 5-AGACAGGATGAGGATAGCTCCAATTCACTG-3; IN-ProLabel
3, 5-CAGTGAATTGGAGCTATCCTCATCCTGTCT-3.
Antibodies and ReagentsThe rabbit anti-GFP polyclonal
antibody (Molecular Probes Inc.), the rabbit anti-HA antibody
(Sigma), and mouse anti-T7 antibody (Novagen) were used
for immunoprecipitation. The antibodies for Western blot
(WB) were as follows: the mouse monoclonal anti--actin antibody (Abcam), mouse anti--tubulin (Sigma), mouse antiKu70 (Abcam), mouse anti-Ku80 (Abcam), horseradish
peroxidase (HRP)-conjugated anti-GFP antibody (Molecular
Probes), HRP-conjugated anti-HA antibody (Miltenyi Biotec),
and HRP-conjugated anti-T7 antibody (Novagen). As the sec-
Cell Lines and TransfectionHuman embryonic kidney
293T and HeLa cell lines were cultured in Dulbeccos modified
Eagles medium (DMEM) supplemented with 10% fetal calf
serum (FCS) and 1% penicillin/streptomycin. Human CD4
C8166 T-lymphoid cells were maintained in RPMI 1640
medium supplemented with 10% FCS and 1% penicillin/streptomycin. For the transfection of 293T cells and HeLa cells, the
16

and Protein A-Sepharose overnight. The IN-bound proteins
were detected by WB using anti-Ku70 or anti-T7 antibodies.
The same nitrocellulose membrane was then stripped and
probed with anti-GFP antibody to detect GFP-INwt/mut or
GFP expression. Meanwhile, 5% of the transfected cells were
lysed in 0.5% Nonidet P-40, and the lysates were used to detect
the expression of GFP-INwt/mut and Ku70 by WB using their
corresponding antibodies.
To examine the IN/Ku70 interaction in HIV-1-infected cells,
HIV-1 (HxBru or HxBru-IN-HA)-infected C8166 T cells were
lysed with 0.25% Nonidet P-40 and immunoprecipitated with
anti-HA antibody followed by WB with anti-Ku70 antibody to
detect IN-bound Ku70.
Detection of Ubiquitination of IN in the Absence or Presence of
Ku70To determine the ubiquitination level of HIV-1 IN in
the absence and presence of Ku70, 293T cells were cotransfected with GFP-IN and HA-Ub wild type or mutants K48R and
K63R with and without T7-Ku70wt or T7-Ku70(1 430). After
48 h, cells were lysed in 199 medium containing 0.25% Nonidet
P-40 and a protease/inhibitor mixture and immunoprecipitated with anti-GFP antibody. Then the precipitated complexes
were run on a 10% SDS-polyacrylamide gel and analyzed for the
presence of HA-Ub by WB with HRP-conjugated anti-HA antibody. Simultaneously, GFP-IN was detected by immunoblotting the same membrane with HRP-conjugated anti-GFP antibody. And protein band intensity was quantified using Quantity
One 1-D analysis software (Bio-Rad).
Virus Production and InfectionTo study the effect of
Ku70-KD on HIV-1 replication, equal amounts (quantified by
HIV-1 p24 antigen) of pNL4.3-GFP virus were used to infect
Ku70-KD or empty vector-transduced C8166 T cells for 2 h;
cells were then washed and cultured in a 37 C incubator. At
different time points, viral replication levels were monitored by
the measurement of p24 levels using an HIV-1 Gag-p24 ELISA.
To test the infectivity of progeny virus produced from the
Ku70-KD cells, empty-vector and Ku70-KD C8166 T cells were
infected with the same amounts of pNL4.3-GFP. Progeny
viruses were collected by ultracentrifugation after 4 days of
infection, and equal amounts of viruses (quantified by HIV-1
p24 antigen) were used to infect empty vector or Ku70-KD
C8166 T cells. Viral infection was examined at 3 days postinfection by monitoring HIV p24 levels in the supernatant.
Quantitative Real-time PCR1.5 106 stable C8166 T cell
lines with Ku70-KD or empty vector-transduced were infected
with the pNL4.3-GFP virus as described above. Heat-inactivated virus (70 C for 30 min) was used as a negative control for
infection. After 4 h of infection, cells were washed and cultured
in fresh RPMI medium. At 24 h postinfection, cells were harvested and washed with PBS twice. DNA was isolated using a
QIAamp blood DNA minikit (Qiagen). The total levels of
HIV-1 DNA, 2-LTR circles, and integrated DNA were quantified following the same procedure in an Mx3000P real-time
PCR system (Stratagene) as described (32).
Virus Composition and Incorporation of Cellular Protein into
HIV-1 VirionTo examine the viral protein compositions, the
pNL4.3-GFP viruses from empty vector-transduced and
Ku70-KD C8166 T cells were pelleted through a 20% sucrose
cushion at 35,000 rpm for 1.5 h at 4 C. Then equal amounts of
ondary antibodies, the ECLTM HRP-conjugated donkey antirabbit IgG and sheep anti-mouse IgG were purchased from
Amersham Biosciences. The WB detection ECL kit was purchased from PerkinElmer Life Sciences (Boston, MA). Nonidet
P-40 was from Roche Applied Science. Proteasome inhibitor
MG-132 and puromycin were obtained from Calbiochem. Subtilisin was purchased from Sigma.
Transient and Stable Knockdown of Ku70 in 293T, HeLa
Cells, and C8166 T CellsTo test the effect of Ku70 levels on
the stability of IN, siRNA targeting human Ku70 (GenBankTM
accession number NM_001469) was used to transiently knock
down Ku70 expression in 293T cells and HeLa cells using the
LipofectamineTM RNAiMAX transfection reagent (Invitrogen). The sense primer for this siRNA is 5-GAUCCAGGUUUGAUGCUCAtt-3, targeting Ku70 nucleotides 1094 1112.
In parallel, a scrambled siRNA (Invitrogen) was used as a negative control (siNC). After 5 nM siKu70 or siNC oligonucleotide
was transfected into cells for 12 h, cells were transfected again
with 5 nM siKu70 to maximize knockdown efficiency.
To produce a stable Ku70-knockdown (KD) 293T and
C8166 CD4 T cell line, lentivirus-like particles harboring
Ku70 shRNA were produced by cotransfecting the shRNA
pLKO.1 vector containing shRNA targeting the Ku70 mRNA
(5-CCGGCGACATAAGTCGAGGGACTTTCTCGAGAAAGTCCCTCGACTTATGTCGTTTTTG-3 (Oligo ID:
TRCN0000039608; purchased from Open Biosystems)),
packaging plasmid 8.2, and vesicular stomatitis virus G
(VSV-G) expressor into the 293T cells. After 48 h, shRNA
pLKO.1 vector particles were pelleted by ultracentrifugation
(32,000 rpm at 4 C for 1 h) and used to transduce cells for
48 h, followed by selection with 2 g/ml puromycin for 1
week. Ku70-KD efficiency was determined by WB analysis
with anti-Ku70 antibody. Endogenous -actin was used to
normalize sample loading. The pLKO.1 vector without the
shRNA sequence (empty vector) was introduced into cells by
the same method as a negative control.
Direct Immunofluorescence AssayTo test the effect of
Ku70-KD level on the expression of IN, HeLa cells were first
transfected with Ku70-specific siRNA oligonucleotides or nontargeting random siRNA (siNC) for 48 h and further transfected with GFP-INopt for another 48 h with or without
MG-132 (10 M) treatment. GFP fluorescence-positive cells
were imaged by microscopy under a 20 objective lens (Carl
Zeiss).
Coimmunoprecipitation Assay in 293T Cells and in HIV-1infected C8166T CellsTo detect the interaction between
GFP-INwt/mut and T7-Ku70wt/mut and to identify their
mutual binding regions, the cell-based co-IP assay was performed as described previously (37). Briefly, GFP or GFP-INwt/
mut plasmid was cotransfected with pCMV-Ku70 or T7-Ku70,
respectively, into 293T cells for 48 h. To increase GFP-IN stability, 10 M MG-132 was added 12 h prior to cell lysis for co-IP.
Then 90% of the transfected cells were lysed in 0.25% Nonidet
P-40 prepared in 199 medium containing a mixture of protease
inhibitors (Roche Applied Science) and clarified by centrifugation at 14,000 rpm for 30 min at 4 C. Supernatant was precleared with Protein G-agarose on a rotator for 2 h at 4 C and
subsequently subjected to IP with a rabbit anti-GFP antibody
17

tion. Noticeably, our results revealed that T7-Ku70 overexpression significantly increased IN expression (Fig. 1A, lanes 1 and
2). However, the coexpression of Ku70 with another HIV-1 protein, MA (MA-YFP), did not change the MA expression level
(Fig. 1A, lanes 3 and 4). This suggests that Ku70 is able to
increase IN expression. Alternatively, Ku70 could protect the
IN protein from degradation (40).
To further test whether endogenous Ku70 could exert the
same activity and whether it is due to a protective effect, we first
knocked down the Ku70 expression using specific siRNA in
293T (Fig. 1B) or HeLa cells (Fig. 1C) and checked the level of
GFP-IN expression by WB or fluorescence microscopy (Fig. 1,
B and C). To increase IN expression under normal conditions,
we used a pAcGFP-IN with a codon-optimized IN sequence
(GFP-INopt). The results showed that IN expression in
Ku70-KD cells was significantly decreased when compared
with IN expression in siNC cells (Fig. 1, B (compare lanes 1 and
2) and C (compare A1A3 and B1B3)). Intriguingly, in the
presence of the specific proteasome inhibitor MG-132 (10 M),
IN expression in Ku70-KD cells was remarkably increased,
reaching levels similar to those in siNC-transfected 293T and
HeLa cells (Fig. 1, B (compare lanes 4 and 3) and C (compare
C1C3 and D1D3)). Thus, these results clearly indicate that
Ku70 is able to protect IN from proteasomal degradation.
Ku70 Is Able to Interact with HIV IN in both 293T Cells and
HIV-1-infected T CellsProvided that Ku70 is able to protect
HIV-1 IN from proteasomal degradation, we next tried to
reveal the molecular mechanisms underlying this effect.
Because Ku70 has been implicated as an HIV-1 IN cofactor (5),
it is possible that IN could escape from the host proteasomal
degradation machinery by directly interacting with Ku70.
Therefore, we further investigated the interaction between HIV
IN and host protein Ku70 under more physiological conditions
by using a co-IP approach in 293T cells and HIV-1-infected
CD4 C8166 T cells.
First, a CMV-Ku70 expressor and GFP or GFP-INwt plasmid
were cotransfected into 293T cells. In order to prevent IN degradation, MG-132 (10 M) was added to the cells at 12 h before
cell lysis. The cells were then lysed and immunoprecipitated
with anti-GFP followed by WB using anti-Ku70. We found that
GFP-IN, but not GFP, was able to pull down Ku70 (Fig. 2A).
Given that both Ku70 and IN are DNA-binding proteins, we
added DNase I in the cell lysate during the co-IP assay and
found that GFP-IN still bound to Ku70 with DNase I treatment,
suggesting a direct protein-protein interaction (data not
shown). Considering that the protein overexpression system
might not necessarily reflect normal functional binding of
IN/Ku70, we also tested the authentic interaction of IN/Ku70 in
HIV-1-infected cells (Fig. 2B). To do this, C8166 CD4 T cells
were infected with HIV-1 HxBru or HxBru-IN-HA viruses. In
the provirus HxBru-IN-HA, an HA tag was inserted at the C
terminus of IN (33), allowing us to pull down HIV-1 IN-associated cellular proteins by using anti-HA antibody in the co-IP
assay. At 7296 h postinfection, the C8166T cells were lysed,
and the cell lysates were subjected to a co-IP assay to detect
IN-bound endogenous Ku70 in the infected cells. The results
showed that Ku70 was coprecipitated with IN-HA from
HxBru-IN-HA-infected cells but not from mock C8166T cells
viruses (normalized by p24 values) were lysed with 4 Laemmli

buffer and directly loaded onto an SDS-PAGE gel and analyzed
for IN and p24 expressions using their corresponding antibodies. The reverse transcription activity from the purified viruses
was analyzed by a reverse transcription assay using a commercial RT assay kit (Roche Applied Science) according to the manufacturers instructions.
To detect the presence of Ku70 in the HIV-1 particles, 15
106 CD4 C8166 T cells were mock-infected or infected with
pNL4.3-GFP for 3 days. Then supernatants from both cell cultures were ultracentrifuged at 35,000 rpm for 1.5 h through a
20% sucrose cushion. The pellets were dissolved in the same
volume of radioimmune precipitation assay buffer and mixed
with 20% (v/v) TCA, followed by precipitation on ice for 30 min
and acetone washing. Protein precipitates were dissolved in 4
Laemmli buffer and directly loaded onto a 10% SDS-polyacrylamide gel. Virus-associated Ku70 and p24 were then examined
by WB using the corresponding antibodies.
Subtilisin treatment of purified HIV-1 virions. The subtilisin
assay was performed according to the protocol as described
(39). The Vpr-RT-IN or Vpr-RT expressor was cotransfected
with VSV-G and NL4.3lucBglRI to produce single cycle IN
and IN virus. The viruses were first ultracentrifuged through
20% sucrose at 35,000 rpm for 2 h and then mock-treated or
treated with 0.1 mg/ml of subtilisin (Sigma) for 20 h at a 37 C
incubation. Subtilisin was inactivated by phenylmethylsulfonyl
fluoride. Virus was then repelleted as described above, lysed in
radioimmune precipitation assay buffer, and loaded onto SDSpolyacrylamide gel followed by WB. Blots were sequentially
probed with anti-Ku70, anti-IN, and p24 antibodies.
ProLabel Detection AssayTo test the effect of Ku70 on IN
during HIV infection, VSV-G pseudotyped HIV single cycle
virus containing ProLabel tag fused to the C terminus of IN was
generated to quantify IN expression under HIV infection.
NL4.3lucBglRI was cotransfected with Vpr-RT-IN-PL and
VSV-G expressor into 293T cells to generate VSV-G pseudotyped HIV-1 single cycle IN-PL virus. The viruses were used
to infect shKu70-KD or empty vector-transduced C8166T cells
for 3 h. The cells were washed three times and kept in fresh
medium and then lysed with lysis/complementation buffer at
8 h p.i. IN-ProLabel activity in the cell lysate was measured
according to the manufacturers instructions from the assay kit
(ProLabelTM detection kit II, Clontech).
Statistical AnalysisThe statistical significance was calculated using Students t test, and a p value of 0.05 was considered significant.
RESULTS
Cellular Protein Ku70 Protects HIV-1 IN from Proteasomal
DegradationAs a part of the NHEJ machinery, the host protein Ku70 has been shown to participate in HIV integration and
in the circularization of unintegrated viral DNAs (25, 27). Surprisingly, based on the results of a yeast two-hybrid assay, a
recent study indicated that HIV-1 IN may bind to Ku70 (5),
suggesting a direct association between HIV-1 IN and Ku70. To
further investigate this viral/host protein interaction, we coexpressed Ku70 (T7-tagged Ku70) and HIV-1 IN (IN-YFP) in
293T cells and analyzed their interaction after 48 h of transfec18
FIGURE 1. Effects of Ku70 on the stability of HIV-1 IN. A, 293T cells were cotransfected with IN-YFP or MA-YFP and T7-Ku70wt for 48 h. Expression of IN-YFP
(lanes 1 and 2) and MA-YFP (lanes 3 and 4) were detected by IP with rabbit anti-GFP antibody and anti-GFP-HRP antibody in a WB (top panel). An aliquot of cells
(about 5%) was collected and checked for T7-Ku70wt and -actin expression (middle and bottom panels). B, proteasome inhibitor MG-132 treatment restores
IN expression in Ku70-KD 293 T cells. After transfection with 5 nM siKu70 or the siNC for 48 h, 293T cells were transfected with optimized IN (GFP-INopt) for
another 48 h. The control and KD cells were treated with or without 10 M MG-132 for 12 h prior to harvesting. The expression of GFP-INopt was detected by
direct loading of protein samples onto a 10% SDS-polyacrylamide gel using HRP-conjugated anti-GFP antibody in a WB (lanes 1 4), and the same membrane
was probed with anti--actin antibody to assess protein loading. Ku70 knockdown efficiency was detected by mouse anti-Ku70 antibody in WB (lanes 5 and 6),
and -tubulin was used as the protein-loading control in each sample. C, the effects of Ku70 on HIV-1 IN expression were confirmed in HeLa cells by direct
fluorescence. HeLa cells were treated with 5 nM siKu70 or siNC for 48 h prior to transfection with GFP-INopt. After another 48 h, GFP-positive HeLa cells without
MG-132 (A1A4 and B1B4) or with 12-h treatment of 10 M MG-132 (C1C4 and D1D4) were examined by fluorescence microscopy.
cotransfected with GFP-IN or the expressor into 293T cells.

Based on a previous mutational analysis indicating that the
minimum region for DNA binding within the Ku70 core region
was estimated to be around aa 263 430, 1263 and 1 430
mutants were constructed; Ku70(226 609) was shown to be
defective for DNA-PK activity and DNA binding in the same
study (43). The Ku70(430 609) mutant was sufficient for the
heterodimerization of Ku70/80 but lost DNA end binding ability in the two-hybrid analysis (44). In parallel, 293T cells
cotransfected with T7-Ku70 and GFP plasmids were used as a
negative control. The results showed that T7-Ku70wt and deletion mutant T7-Ku70(1 430) were coimmunoprecipitated
with GFP-IN (Fig. 3B, top panel). Interestingly, T7-Ku70(1
430) displayed a higher binding affinity for IN than T7-Ku70wt
(Fig. 3B, top panel, compare lane 4 with lane 2). Because the
N-terminal truncation (aa 1263) of Ku70 did not interact with
IN (Fig. 3B, top panel, lane 3), whereas T7-Ku70(1 430)
showed a strong binding affinity, the aa 263 430 region of
Ku70 is probably necessary but not sufficient for IN interaction.
However, another Ku70 mutant, residues 226 609, which also
encodes the aa 263 430 region, failed to interact with GFP-IN
(Fig. 3B, top panel, lane 5), suggesting that another important
binding domain might exist within the N terminus (residues
1226) of Ku70. Therefore, both the N-terminal domain and
the core domain of Ku70 are suggestive of binding surface for
IN.
Ku70 forms a heterodimer with Ku80, a heterodimerization
that has been shown to contribute to many cellular processes. For example, the heterodimerization of Ku70/80 is
or HIV HxBru-infected C8166 T cells (Fig. 2B, top panel). The

same immunoblot was reprobed with anti-HA to detect the
immunoprecipitated level of IN-HA (Fig. 2B, second panel).
Similar levels of endogenous Ku70 and viral Gag-p24 were
detected in HxBru- and HxBru-IN-HA-infected cells (Fig. 2B,
third and fourth panel). Together, these results clearly indicated that the DNA repair protein Ku70 is an authentic, newly
described host cofactor for IN.
To delineate the Ku70-binding domain of IN, five previously
described IN N-terminal or C-terminal deletion mutants,
including GFP-IN(1230), GFP-IN(1250), GFP-IN(1270),
GFP-IN(50 288), and GFP-IN(112288), and one substitution
mutant, GFP-IN K186A/R187A (32, 37, 38), were used to test
Ku70 binding ability (Fig. 2C). The results revealed that GFP
and GFP-IN(1230) did not bind to Ku70 (Fig. 2D, lanes 1, 3,
and 8), whereas other truncated GFP-IN mutants (residues
1250, 1270, 50 288, and 112288) and the point mutant
K186A/R187A still retained their Ku70-binding ability (Fig. 2D,
lanes 2, 4 7, and 9). K186A/R187A, a well characterized oligomerization-defective mutant of IN (41, 42), still bound Ku70,
suggesting that multimerization of IN is not required for
IN/Ku70 interaction (Fig. 2D, lane 9). Overall, our analysis suggested that the C-terminal half of IN (IN(112288)) is sufficient
to bind to Ku70.
The Ku70 Truncated Mutant Ku70(1 430) Interacts with
HIV IN but Cannot Form a Heterodimer with Ku80To define
the IN-binding region within Ku70, expressors for four
T7-tagged Ku70 deletion mutants (residues 1263, 1 430,
226 609, and 430 609) were constructed (Fig. 3A) and
19
FIGURE 2. IN interacts with Ku70 in mammalian cell lines and in HIV-1-infected CD4 T-lymphocytes, and the interaction is through the C
terminus of IN. A, interaction of Ku70 with GFP-IN in 293T cells. GFP or GFP-IN was coexpressed with CMV-Ku70 as indicated into 293T cells for 48 h, and
cells were treated with the proteasome inhibitor MG-132 (10 M) for 12 h prior to co-IP analysis. Co-IP was performed using rabbit anti-GFP antibody for
immunoprecipitation and WB using anti-Ku70 to detect IN-bound Ku70 (top panel). GFP-IN and untagged pCMV-Ku70 were detected with the corresponding antibodies to assess the expression levels of IN and Ku70 (bottom panels of lanes 1 and 2). B, C8166 CD4 T cells were mock-infected or infected
with HIV-1 HxBru or HxBru-IN-HA, as indicated. Cells were collected at 72 h postinfection, and the cell lysates were immunoprecipitated with anti-HA
antibody and immunoblotted with anti-Ku70 antibody to detect IN-associated Ku70 in HIV-1-infected T cells (top panel). The same membrane was then
reprobed with anti-HA antibody to detect IN-HA expression (second panel). The same amount of cells was assessed for Ku70 expression prior to co-IP,
serving as the immunoprecipitation input (third panel). The p24 levels in the infected cells were checked with anti-p24 antibody (bottom panel).
C, schematic representation of the GFP-IN wild type, five IN deletion mutants, and point mutant K186A/R187A. D, the C terminus of IN is required for
IN/Ku70 interaction. GFP or various GFP-IN wild type/mutants were cotransfected with T7-Ku70 (lanes 1 8) into 293T cells, and their interactions were
studied by a co-IP assay. Cells were treated with MG-132 at a concentration of 10 M for 12 h prior to co-IP analysis. IN-bound Ku70 was detected by IP
with anti-GFP antibody and WB with anti-Ku70 antibody (top panel). Total cell lysates were analyzed for GFP-INwt/mutants and Ku70 expression (middle
and bottom panels). Input, 5% of total cell extract).
Ku70 Protects IN from Degradation by Reducing the Total

Ubiquitination Level in the Host Cells and by IN/Ku70(1 430)
BindingThe results above showed that Ku70 binds and protects IN from proteasomal degradation; however, the detailed
mechanisms underlying the degradation of IN through the
ubiquitination-proteasome pathway remain unclear. Proteins
tagged with ubiquitin can be monoubiquitinated or polyubiquitinated. The polyubiquitination chains are formed between
the C-terminal residue glycine 76 of ubiquitin and any other
internal lysine within the ubiquitin molecule (lysine 6, 11, 27,
29, 33, 48, or 63) through an isopeptide bond (46, 47). The two
most important polyubiquitination chains are the Lys48- and
Lys63-linked chains, with the Lys48-linked polyubiquitination
chain recognized by the 26 S proteasomal pathway for degradation and the Lys63-linked polyubiquitination chain implicated
in postreplicative DNA repair (48). In analysis of the ubiquitinproteasome pathway involved in IN degradation, HA-Ub
essential for activating DNA-PK and DNA repair and important for their nuclear translocation (43, 45). One might ask
whether Ku80 is also involved in the IN/Ku70 interaction or
if IN interacts with Ku70 indirectly through Ku70/80 heterodimerization. To address this question, we performed a
co-IP assay in which T7-Ku70wt or T7-Ku70(1 430) was
transfected into 293T cells. At 48 h post-transfection, cell
lysates were immunoprecipitated with anti-T7 antibody followed by WB with an anti-Ku80 antibody. According to
our previous findings, T7-Ku70wt was able to pull down
endogenous Ku80 (Fig. 3C, lane 2, top panel). Interestingly,
the deletion mutant T7-Ku70(1 430), which efficiently
binds IN, could not form a heterodimer with Ku80 (Fig. 3C,
lane 3, top panel). This result indicates that T7-Ku70(1
430) binding to IN was independent of Ku80. Thus, the heterodimerization of Ku70/80 may not be required for
IN/Ku70 interaction.
20
FIGURE 3. The N terminus (aa 1 430) of Ku70 binds IN, and IN/Ku70 interaction is independent of the heterodimerization of Ku70/80. A, schematic
diagrams depict the different T7-Ku70wt/mut constructs used in the domain-mapping experiments. The full length of T7-Ku70 is shown at the top, as indicated.
B, interaction of GFP-IN with T7-Ku70wt/mut. GFP or GFP-INwt was cotransfected with T7-Ku70wt/mut in 293T cells for 48 h. MG-132 (10 M) was added to the
cells 12 h prior to cell lysis to enhance protein expression. The co-IP assay was performed to map the IN binding region in Ku70 using anti-GFP antibody IP, and
a WB using anti-T7 antibody was performed to detect IN-bound T7-Ku70wt/mut (top). GFP, GFP-IN, and T7-Ku70wt/mut expression was checked by immunoblotting with anti-GFP or anti-T7 antibodies, respectively (middle and bottom panels). C, T7-Ku70(1 430) cannot form a heterodimer with Ku80. 293T cells were
mock-transfected or transfected with T7-Ku70wt and the aa 1 430 truncation mutant for 48 h. Heterodimerization of Ku70/80 was determined by co-IP with
anti-T7 antibody and WB using mouse anti-Ku80 antibody to detect T7-Ku70-bound endogenous Ku80 (top panel). About 5% of the cell lysates (input) were
checked for the expression of T7-Ku70wt/mut by WB using anti-T7 antibody (bottom panel).
for degradation were accumulated due to the defective Lys48linked polyubiquitination proteasome degradation pathway.
However, similar levels of HA-Ubwt, HA-UbK48R, and
HA-UbK63R were detected in the cell lysates (Fig. 4A, bottom
panel). The highest IN expression was detected in the
HA-UbK48R expression cells, clearly suggesting that GFP-IN is
degraded though the Lys48-linked polyubiquitination proteasomal degradation pathway.
To further investigate how Ku70 affects IN stability, we studied the ubiquitination level of IN in the presence of both
T7-Ku70 and HA-Ub. Because T7-Ku70(1 430) showed a
strong binding affinity with IN (Fig. 3), we also included this
T7-Ku70 deletion mutant to determine if the interaction of
Ku70/IN plays a role in the stability and ubiquitination of IN. As
expected, GFP-IN expression in the cells transfected with
T7-Ku70wt and T7-Ku70(1 430) deletion mutants was 1.47
0.11- and 1.78 0.24-fold increased compared with cells transfected with the empty vector (Fig. 4B, top panel; compare lanes
3 and 4 with lane 2). The total ubiquitin expression level
detected in the whole-cell extract was dramatically reduced in
the presence of wild-type Ku70 (Fig. 4B, lane 3, third panel).
Consistently, the co-IP data showed that ubiquitinated INbound proteins also remarkably reduced by 4.58 0.51-fold in
the presence of wild-type Ku70 (Fig. 4B, lane 3, second panel).
Interestingly, T7-Ku70(1 430) was still able to protect IN (Fig.
mutants K48R and K63R were included in our study, having

mutations of Lys48 and Lys63 to arginine that were expected to
disrupt Gly76-Lys48 and Gly76-Lys63 polyubiquitination chain
formation, respectively. First, HA-Ubwt or mutant HAUbK48R and HA-UbK63R were cotransfected with the IN
expressor (GFP-IN) into 293T cells. At 48 h post-transfection,
cells were lysed in 0.25% Nonidet P-40 and subjected to a co-IP
assay using anti-GFP antibody to pull down GFP-IN, followed
by WB with anti-GFP and anti-HA antibodies to detect HAUb-tagged IN-associated proteins (Fig. 4A, top and middle panels). Simultaneously, HA-Ub expression levels in the cells were
also checked (Fig. 4A, bottom panel). The data showed that
GFP-IN protein levels in the HA-UbK48R overexpression cells
were much higher than in HA-Ubwt- and HA-UbK63R-transfected 293T cells (Fig. 4A, top panel; compare lane 3 with lanes
2 and 4). Similarly, the ubiquitination level of IN-associated
protein expression was the highest in the HA-UbK48R-transfected sample (Fig. 4A, middle panel). The HA-tagged ubiquitination signal (Fig. 4A, middle panel) was from a pool of ubiquitinated IN and unknown IN-bound cellular proteins. The
observation of increased levels of ubiquitinated IN-associated
proteins in HA-UbK48R-cotransfected cells was expected
given the facts that the immunoprecipitate input or GFP-IN
(Fig. 4A, top panel) was the highest and that all of the IN-associated proteins subjected to the ubiquitin-proteasome system
21
FIGURE 4. IN is degraded through the Lys48-linked polyubiquitination proteasomal pathway, and Ku70 protects IN by reducing the overall ubiquitination level in the cells and partially blocking ubiquitination of IN and its bound cellular proteins. A, IN is degraded through Lys48-linked polyubiquitination. 293T cells were mock-transfected or cotransfected with GFP-IN and HA-Ubwt, HA-UbK48R, or HA-UbK63R for 48 h, as indicated. The ubiquitination
levels of IN and IN-associated proteins were checked by IP with anti-GFP antibody and anti-HA antibody in a WB (middle panel). The same membrane was
reprobed with anti-GFP antibody to detect GFP-IN expression in each sample (top panel). About 5% of the cells prior to co-IP were lysed in 0.5% Nonidet P-40,
and the protein contents were subjected to WB to detect total HA-Ub levels in the cells using anti-HA antibody (bottom panel). B, Ku70 protects IN from
degradation by targeting the cellular ubiquitin-proteasome pathway and reducing ubiquitin binding to IN. 293T cells were mock-transfected (lane 1) or
cotransfected with GFP-IN, HA-Ubwt and T7 vector, or T7-Ku70wt and T7-Ku70(1 430) (lanes 2 4). The co-IP assay was done at 48 h post-transfection using
anti-GFP antibody to pull down IN and its associated proteins and using immunoblotting with anti-HA antibody to determine the ubiquitination level of IN and
its associated proteins (second panel). The same membrane was reprobed with anti-GFP antibody to examine GFP-IN expression (first panel). Simultaneously,
equal amounts of total cellular proteins (about 5% of the total cell lysates) were resolved on a SDS-PAGE gel and immunoblotted with anti-HA and anti-T7
antibodies to determine the expression levels of the transfected protein expressors (third and fourth panels). -Actin was used as a loading control (bottom
panel). C, reduction of ubiquitin level by Ku70 is independent of the Lys48- and Lys63-linked polyubiquitination proteasomal pathway. 293T cells were
transfected with HA-Ubwt/mut with T7-Ku70 or T7 vector for 48 h. Cells were lysed and analyzed for HA-Ub expression using anti-HA antibody in a WB. On the
same membrane, -actin was used as a protein-loading control. D, endogenous ubiquitin levels were increased in Ku70-down-regulated cells. C8166 T cells
were transduced with empty vector or lentiviral vector expressing an shRNA against human Ku70 and selected with 1 g/ml puromycin. After 1 week of
selection, equal amounts of control or stable Ku70-KD cell lines were collected and lysed. The expression levels of ubiquitin, Ku70, and -actin were assessed
by WB using specific antibodies.
We then tested whether Ku70 affects specific lysine-linked

polyubiquitination proteasomal degradation pathways. The
T7-Ku70 expressor was cotransfected with HA-Ubwt, HAUbK48R, or HA-UbK63R into 293T cells. The results showed
that, in the presence of Ku70, overall HA-Ub expression was
still greatly reduced although Ub cannot form Lys48- or Lys63linked polyubiquitin chains (Fig. 4C). This observation was
confirmed by the fact that the down-regulation of Ku70
increased ubiquitin levels in the cells. We established a stable
Ku70-KD CD4 C8166 T cell line by transducing C8166 CD4
T cells with a lentiviral vector carrying Ku70 shRNA. In parallel,
the control cell line was transduced with an empty lentiviral
vector. After puromycin selection, control cells and Ku70-KD
cells were checked for Ku70 knockdown efficiency by WB (Fig.
4B, first and third panels, lane 4) and significantly reduced the
level of HA-tagged ubiquitination signal in IN-bound proteins
in the presence of T7-Ku70(1 430) by 1.18 0.19-fold (Fig. 4B,
second panel; compare lane 4 with lane 2; the percentage is
normalized by total HA-ubiquitin level in the third panel),
although it did not affect the overall ubiquitin level in the cells
(Fig. 4B, third panel; compare lane 4 with lane 2). These results
thus indicate that although T7-Ku70(1 430) lacks the activity
to reduce total ubiquitin level, as wild-type Ku70 does, it can
protect IN by specifically reducing the ubiquitination of IN and
its associated proteins. Based on these results, we conclude that
Ku70 protects IN through two mechanisms; Ku70 reduces total
ubiquitination level in the cells and reduces the ubiquitination
of IN and IN-bound cellular proteins through their interaction.
22
FIGURE 5. Differential replication kinetics in Ku70-KD C8166 T cells with different titers of viral infection. A and B, HIV-1 replication kinetics in Ku70-KD
or empty vector-transduced C8166 T stable cell lines at a high MOI of 0.5 (A) or a low MOI of 0.05 (B). Lentiviral shRNA targeting Ku70 or empty vectortransduced C8166 T stable cell lines were infected with different doses of pNL4.3-GFP virus for 2 h. At subsequent time intervals, the supernatants were
collected, and viral replication was monitored by measuring HIV-1 p24gag levels. The data shown are the means and S.D. values (error bars) of p24 values from
duplicate wells in one infection assay and are representative of three independent experiments. C, Ku70-KD inhibited the infectivity of progeny virus.
pNL4.3-GFP viruses produced from empty vector-transduced or shKu70-KD C8166 T cells were normalized by p24 and used to infect both empty vectortransduced and shKu70-KD C8166 T cells. Viral replication was monitored by p24 ELISA at 3 days postinfection. D, Ku70-KD impaired 2-LTR formation and
integration of proviral DNA. Stable Ku70-KD or empty vector C8166T cells were infected with the same amount of pNL4.3-GFP viruses produced from either
empty vector-transduced C8166T cells or shKu70-KD cells for 24 h. The cellular genomic DNA was extracted and quantified for HIV-1 late RT, 2-LTR, and
integrated DNA by real-time PCR. Infection with 70 C heat-inactivated virus served as a negative control (bar 1). Data are representative of two independent
experiments performed in duplicate, shown as mean S.D. The statistical significance is denoted as follows: *, p 0.05; **, p 0.01.
compared with the control cells (Fig. 5A). However, when

Ku70-KD cells were infected with a lower MOI of 0.05, viral
infection was undetectable by measuring HIV p24 levels up to
11 days. Nonetheless, in the control cells, viral replication
peaked at day 9 (Fig. 5B). Interestingly, Ku70 knockdown completely inhibited low MOI HIV-1 infection but only reduced
high MOI viral infection by 50%. This could be due to the fact
that the infection of a large amount of viruses produced from
normal T cells may at least partially overcome the shortage of
Ku70 inside the target cells and establish an efficient first cycle
of replication in Ku70-KD cells. If this is the case, we reasoned
that viruses produced from Ku70-KD cells should have a significantly lowered infectivity compared with the virus produced
from the normal cells. To test this possibility, we infected empty
vector-transduced and Ku70-KD C8166 T cells with the same
amounts of pNL4.3-GFP virus (normalized by p24 ELISA)
produced from either empty vector or Ku70-KD C8166 T cells.
At 3 days postinfection, virus-laden supernatants were harvested and checked for p24 antigen production by p24 ELISA.
4D, bottom panel). These cells were then used to detect endogenous ubiquitin levels. The results showed that the ubiquitin
levels were higher in the Ku70-KD cells than in the empty vector-transduced cells (Fig. 4D, top panel). Taken together, we
demonstrated here that Ku70 is able to protect HIV IN from
degradation by down-regulating cellular ubiquitin levels and
simultaneously preventing the ubiquitination of IN and its
associated cellular proteins.
Ku70 Knockdown Impairs HIV-1 ReplicationBecause Ku70
is able to bind HIV IN and protects IN from degradation, we
next tested whether and how Ku70 contributes to HIV-1 replication. To do so, the empty vector-transduced and Ku70-KD
C8166 T cells were infected with pNL4.3-GFP viruses at an
MOI of 0.5 (Fig. 5A) or 0.05 (Fig. 5B) for 2 h, and the viral
replication kinetics were monitored. The infection was examined at different time intervals by harvesting virus-laden supernatant and checking for HIV-1 p24 antigen release. Fig. 5A
shows that at the MOI of 0.5, HIV infection in Ku70-KD C8166
T cells was reduced by 50% at 4 and 5 days postinfection
23

or Ku70-KD cells were lysed and subjected to an RT assay
(Roche Applied Science). There was no significant difference in
viral RT activity observed between normal and Ku70-KD cells
(Fig. 6A, bottom panel). All of these data suggest that Ku70-KD
does not affect the processing of Gag/Gag-Pol or virus
maturation.
The observation that the Ku70-KD phenotype resulted in
defective progeny virus (Fig. 5) implies that Ku70 might be
packaged into the progeny virus particles affecting HIV-1 replication. To test this hypothesis, we checked for the presence of
Ku70 in the HIV virion. Briefly, the C8166 T cells were mockinfected or infected with HIV pNL4.3-GFP at an MOI of 1 for
2 h. Virions were isolated 4 days after infection on a 20% sucrose
gradient by ultracentrifugation (35,000 rpm) for 1.5 h. Virions
were lysed in radioimmune precipitation assay buffer and precipitated in 20% TCA to concentrate their protein contents
before WB analysis. Simultaneously, the infected or uninfected
C8166 T cells were lysed and analyzed by WB. Note that Ku70
was detected in the virions prepared from infected cells but not
in the mock-infected cells (Fig. 6B, top panel). Additionally, the
presence of p24 in the virions and the cells was detected by
immunoblotting with anti-p24 antibody on the same membrane (Fig. 6B, top panel).
To verify that Ku70 incorporation into HIV-1 virion is
mediated by IN, the VSV-G pseudotyped HIV-1 single cycle
infection system was used. Vpr-RT-IN or Vpr-RT was
transcomplemented with VSV-G and RT/IN/Env gene-deleted
NLlucBglRI provirus into 293T cells to produce IN and
IN virus (Fig. 6C). The viruses were collected and ultracentrifuged through 20% sucrose at 35,000 rpm for 2 h. Because subtilisin treatment can effectively remove the proteins (either
microvesicles or exosomes) outside the virions of purified
HIV-1 virions (51), the IN and IN virus were treated with 0.1
mg/ml subtilisin to remove potential contamination outside
the virion. As shown in Fig. 6B (bottom panel), the presence of
Ku70 is significantly higher in IN virus than IN virus without
subtilisin treatment (Fig. 6B (top panel), compare lane 2 with
lane 3). However, when virions were treated with subtilisin,
Ku70 was evidently detected in IN virus but not IN virus
(lanes 5 and 6, top panel). IN and p24 expression in both viruses
were monitored by blotting with anti-IN and anti-p24 antibodies. Meanwhile, Ku70 expressions in mock-transfected or
transfected cells were assessed (Fig. 6B, bottom panel). Taken
together, these results suggest that, mediated by IN, Ku70 is
incorporated into the HIV-1 particle.
The above results (Figs. 1 and 6B) suggest that virus-associated Ku70 might stabilize IN or protect it from degradation in
the infected cells. To investigate the protective effects of Ku70
on IN during viral infection, we assessed IN expression in the
presence or absence of Ku70 during VSV-G pseudotyped
HIV-1 single cycle infection. To do so, we have constructed
Vpr-RT-IN-PL plasmid with a ProLabel tag in the C terminus of
IN, which enables us to quantify IN expression by measuring
ProLabel activity (Fig. 6C). Vpr-RT-IN-PL is cotransfected with
VSV-G and NLlucBglRI provirus into 293T cells to produce
single cycle IN-PL virus (Fig. 6C). C8166 T cells were first
infected with the same amount of normal IN-PL virus or virus
produced from shKu70-KD 293T cells (normalized by p24
Consistent with the above results, when cells were infected with
virus produced from empty vector-transduced cells (empty
vector virus or E-virus), there was only a 2-fold difference in
Ku70-KD cells compared with its infection in empty vector
cells (Fig. 5C, compare bars 1 and 2). However, when empty
vector-transduced cells were infected with either E-virus or
Sh-virus produced from Ku70-KD cells, there was an
5-fold reduction of Sh-virus infection (Fig. 5C, compare
bars 1 and 3). Strikingly, Sh-virus infection in Ku70-KD cells
exhibited more severe impairment, with a 16-fold reduction
in viral infectivity compared with the E-virus infection in
empty vector-transduced cells (Fig. 5C, compare bars 1 and
4). Overall, this group of results indicates that down-regulation of Ku70 in both HIV-producing and target cells significantly impairs viral replication.
In order to investigate which early step(s) of viral replication
is blocked by Ku70-KD, infected C8166 T cells as described
above (Fig. 5C) were harvested at 24 h p.i., and DNA was isolated and assessed for late reverse transcription products (late
RT), 2-LTR circles, and integrated DNA by quantitative PCR
(32, 38). The results revealed that late RT products in the
Ku70-KD C8166T cells did not show significant reduction
when compared with normal empty vector-transduced cells
(p 0.05; Fig. 5D, left, compare bar 3 with bar 2), whereas late
RT product was about 50% reduced compared with Ku70-KD
cells infected with Sh-virus (p 0.05; Fig. 5D, left, compare bar
4 with bar 2). Strikingly, 2-LTR and integrated DNA in
shKu70-KD cells infected with either normal E-virus or Shvirus were undetectable under current assay conditions (p
0.01; Fig. 5D, middle and right panels, bars 3 and 4). 2-LTR
circle formation is indicative of nuclear import (49), but it also
requires proper circularization by host DNA repair enzymes
(27, 50). For example, depletion of NHEJ pathway components
(Ku80, XRCC4, and LigaseIV) resulted in undetectable or
reduced 2-LTR formation (27, 50). The undetectable 2-LTR
circle formation in Ku70-KD cell infection with both normal
E-virus and Sh-virus could be due to insufficient DNA circularization by Ku70-KD in the target cells (Fig. 5D, middle panel,
bars 3 and 4). All of these results indicate that the presence of
Ku70 is required for an efficient viral reverse transcription and
necessary for viral integration.
Host Protein Ku70 Is Incorporated into Viral Particles and
Stabilizes IN ExpressionBecause virus produced from
Ku70-KD cells cannot infect C8166 cells efficiently, we first
checked whether Ku70 knockdown may affect HIV-1 maturation. The progeny viruses produced from empty vector-transduced cells and Ku70-KD C8166 T cells were normalized by
p24 value and loaded onto an SDS-polyacrylamide gel. Simultaneously, infected C8166 T cells were lysed and also subjected
to the same analysis. Next, anti-p24 and anti-IN antibodies
were used to check for the presence of p24 and IN in the virions
and infected cells. We did not detect any difference in the
p24/IN ratio in the E-virus- and Sh-virus-infected cells (Fig. 6A,
top panel). This suggests that the virus Gag-pol processing
remained unaffected in the progeny virus produced from
Ku70-KD cells. To examine the RT activity in the virions produced in Ku70-KD cells, the same amounts of viruses (normalized by p24 ELISA) from either empty vector-transduced cells
24
FIGURE 6. Ku70 incorporation into HIV-1 particles and its effects on HIV-1 replication. A, knockdown of Ku70 does not affect the p24/IN profile in the
virions or virus-producing cells. HIV pNL4.3-GFP virus produced from empty vector-infected and shKu70-KD C8166 T cells were pelleted through a 20% sucrose
cushion and dissolved in RPMI 1640. Viral particles with the same amounts of p24 and equal amounts of infected cells were lysed, separated by SDS-PAGE, and
immunoblotted with anti-IN and anti-p24 antibodies (top panel). The data shown represent two independent experiments. The same amount of pNL4.3-GFP
virus (normalized by p24) produced from empty vector-transduced and shKu70-KD C8166 T cells was analyzed for HIV RT activity with a reverse transcriptase
assay kit (Roche Applied Science). The data are shown as the amount of RT in each well, reported in pg/well, representing the means and S.D. values (error bars)
from viruses produced in two independent experiments (bottom panel). B, top panel, Ku70 is present in the HIV-1 virion. C8166 T cells were infected with
pNL4.3-GFP virus or uninfected. After 4 days of infection, supernatants were centrifuged through a 20% sucrose cushion at 35,000 rpm for 2 h at 4 C. Pellets
were dissolved in radioimmune precipitation assay buffer and subjected to a TCA precipitation assay. Protein contents in the viruses and the cells were
analyzed by WB using anti-Ku70 and anti-p24 antibodies to determine the presence of various proteins. Bottom panel, Ku70 incorporation into HIV-1 virion is
dependent on IN. The Vpr-RT-IN or Vpr-RT expressor was cotransfected with VSV-G and NL4.3lucBglRI to produce single cycle IN and IN virus. The viruses
were subjected to a subtilisin resistance assay as described under Experimental Procedures. Virus-associated Ku70, IN, and p24 were analyzed by WB.
Endogenous Ku70 expressions in the transfected 293T cells were checked by blotting with anti-Ku70 antibody. C, top panel, schematic structure of RT/IN/Envdeleted HIV-1 provirus NL4.3lucBglRI and of the Vpr-RT-IN, Vpr-RT, and Vpr-RT-IN-PL fusion proteins. NL4.3lucBglRI and Vpr-RT-IN were described earlier
(34, 69), and Vpr-RT without IN expression has two stop codons TAGTGA after the last nucleotide of the RT sequence. Vpr-RT-IN-PL was obtained through
removal of the IN stop codon and insertion of the ProLabel gene after IN of Vpr-RT-IN plasmid as described under Experimental Procedures. Bottom panel,
NL4.3lucBglRI was cotransfected with Vpr-RT-IN-PL and VSV-G expressor into 293T cells to generate VSV-G-pseudotyped HIV-1 single cycle IN-PL virus. 2
106 shKu70-KD or empty vector-transduced C8166 T cells were infected with of IN-PL virus (2000 pg of p24) produced from shKu70-KD or normal empty
vector-transduced 293T cells for 3 h. The cells were washed three times, and half of the cells were collected. The rest of cells were kept in RPMI medium and
harvested at 8 h p.i. All of the cells were then lysed and measured for ProLabel activity by the POLARstar OPTIMA multidetection microplate reader. The results
are representative of three experiments, shown as mean S.D. RLU, relative light units.
cells were infected with virus produced from Ku70-KD 293T

cells, at 8 h p.i., the IN level was reduced to 34%, as compared
with that at 3 h p.i. (Fig. 6C, compare bar 6 with bar 5). All of
these results together suggest that Ku70 present in the progeny
virus and in the target cells contributes to stabilizing IN in the
early stage of HIV-1 replication.
ELISA) and washed thoroughly at 3 h p.i., and half amounts of

the cells were collected. The rest of the cells were harvested at
8 h p.i. All of the cells were lysed and measured for IN-ProLabel
activity. The result showed that when C8166 T cells were
infected with virus produced from normal 293T cells, the IN
level remains unchanged after 8 h p.i., as compared with that at
3 h p.i. (Fig. 6C, bottom panel, compare bar 2 with bar 1). When
Ku70-KD C8166 T cells were infected with virus produced
from normal 293T cells, the IN level remained 77% after 8 h p.i.,
as compared with that at 3 h p.i. (Fig. 6C, bottom panel, compare bar 4 with bar 3). Remarkably, when Ku70-KD C8166 T
DISCUSSION
We studied the interaction between the cellular DNA repair
protein Ku70 and HIV-1 IN and the potential roles of Ku70 in
HIV-1 replication. By using a cell-based co-IP assay, we dem25

its associated proteins through IN/Ku70(1 430) binding.
Indeed, the IN-binding peptide Ku70(1 430) was able to increase IN expression and reduce the ubiquitination of IN (and
its associated proteins) although it does not have any deubiquitinating activity (Fig. 4B, first and second panels, compare
lane 4 with lane 2). This suggests that Ku70 specifically binds IN
and possibly masks ubiquitin attachment site(s) in IN to reduce
the ubiquitination of IN, thus protecting IN from degradation
(Fig. 4B, top panel). This scenario seems very likely because the
attachment of ubiquitin to protein substrates, conducted by
ubiquitin ligases E3 during the last step, is via the covalent binding of the C-terminal Gly76 of ubiquitin to the -amino group of
an internal lysine residue in the substrate protein (59), and the
C terminus of IN(230 288) is known to be enriched in lysine
residues (e.g. Lys240, Lys244, and Lys264). Thus, in a future study,
it would be intriguing to investigate which lysine residue(s) in
IN is specifically recognized by ubiquitin and subjected to proteasomal degradation.
We also provided solid evidence that HIV-1 IN directly interacts with Ku70 in Ku70/IN-overexpressing mammalian cells
and in T-lymphocytes during HIV-1 infection (Fig. 2). Indeed,
this finding is not surprising, considering that previous studies
using MMLV IN as bait in a yeast two-hybrid system were able
to fish out Ku70 (5), that Ku70 is present in MMLV PICs (27),
and that Ku70 associates with the IN of Ty elements in Saccharomyces cerevisiae, yeast retrotransposons with life cycles similar to retroviruses (60). We then extended our study to delineate the mutual binding interface of IN and Ku70 and found
that the C terminus of IN (aa 230 288) appears to be involved
in the interaction with Ku70. One interesting observation here
is that the Ku70 deletion mutant 1 430, which was able to bind
IN, cannot form the Ku70/80 heterodimer (Fig. 3). This result is
consistent with a previous finding from a two-hybrid analysis
that the C-terminal 20 kDa of Ku70 (aa 430 609) is essential
for Ku70/80 heterodimerization (44) and suggests that the
Ku70/IN interaction may be independent of Ku80. It has been
shown that Ku70 has unique functions independent of Ku80,
although many cellular functions in which Ku70 participates do
require Ku70/80 heterodimerization. For example, the antiapoptotic activity of Ku70 by inhibiting Bax-mediated apoptosis is
independent of Ku80 (61). One concern here is that, despite the
fact that the Ku70 N terminus (aa 1 430) can still mediate IN
binding, we cannot rule out the possibility that IN is able to bind
the Ku70/80 heterodimer or that IN binding to Ku70 may be
enhanced by Ku70/80 association in mammalian cells. Indeed,
several cellular functions of Ku70 require association with
Ku80, and this heterodimerization formation enhances the stability of each subunit (62, 63).
In a finding of major relevance, we further determined that
Ku70 is required for HIV-1 replication. HIV-1 infection was
significantly impaired when Ku70 expression was knocked
down in both producer and target cells (Fig. 5). Moreover, with
a low MOI infection, Ku70-KD C8166 T cells were more significantly blocked in viral replication than for a high MOI infection (Fig. 5, A and B). These results indicate that Ku70 affects
both the early and the late steps of the HIV-1 life cycle. In order
to pinpoint the effect(s) of Ku70 on the early stage, we carried
out real-time PCR to quantify late RT, 2-LTR circles, and inte-
onstrated that Ku70 interacts with HIV-1 IN in 293T cells and

HIV-1-infected CD4 T cells. Deletion analyses on IN and
Ku70 indicated that an IN region encompassing aa 1230 was
unable to bind to Ku70, whereas the N-terminal region of Ku70
(aa 1 430) still retained the IN binding ability, and that their
interaction is independent of Ku70/80 heterodimerization. We
further discovered a dual mechanism for Ku70 in protecting IN
from proteasomal degradation (i.e. by reducing overall protein
ubiquitination levels within the host cells and by specifically
reducing the ubiquitination of IN via their binding interaction).
Finally, the knockdown of Ku70 expression in both virus-targeting and virus-producing CD4 T cells significantly impaired
HIV-1 replication. More specifically, Ku70-KD resulted in
undetectable 2-LTR and integration levels in the early stage of
viral replication. Taken together, our current study suggests
that Ku70 is required for both the early and late stages of the
HIV life cycle.
A recent study has found that Ku70 is able to reduce the
ubiquitination of Bax in the regulation of apoptosis (30). This
study indicated that the presence of Ku70 is able to specifically
deubiquitinate Bax; however, whether Ku70 could affect total
ubiquitination level in the host cells remained unknown. Presently, we discovered that, besides reducing the ubiquitination
of Bax, Ku70 is able to universally down-regulate the ubiquitination of the entire complement of cellular proteins. The
mechanism underlying this down-regulation of protein ubiquitination levels by Ku70 is unclear. A prior in vitro study
revealed that Ku70, defined as a novel deubiquitination
enzyme, was able to hydrolyze polyubiquitin chains into
monoubiquitin units (30). In the cascade of the ubiquitin-proteasome pathway, deubiquitination enzymes remove ubiquitin
chains from protein substrates and recycle the polyubiquitin chain
into free ubiquitin, before or after the polyubiquitination chain is
recognized by the 19 S cap of the 26 S proteasome (52). Thus, if
Ku70 does exert an overall deubiquitinating activity on cellular
protein, an increased free ubiquitin level in the cells would be
expected. Unfortunately, under our experimental conditions, we
were not able to detect an increased monoubiquitin form of ubiquitin when Ku70 was overexpressed. It appears that the total pool
of monoubiquitin in the cells was reduced when Ku70 was overexpressed. Thus, how Ku70 interacts with the host ubiquitin-proteasome system to regulate the total pool of monoubiquitin in
the cells and down-regulate the polyubiquitination of proteins
remains an open question.
During the HIV life cycle, the host ubiquitin-proteasome system is repeatedly used by HIV-1 viral proteins, such as Vif, Vpr,
Vpu, and IN, to ensure viral replication, either by targeting host
restriction factors for degradation, such as Vif/APOBEC3G or
Vpu/BST2, or by protection from proteasomal degradation by
host cellular cofactors (e.g. IN is protected by LEDGF/p75, and
Vpr is protected by Cul4A-DDB1DCAF1 ubiquitin ligase) (53
58). Here, we provide another example of a host cellular cofactor of IN, the DNA repair protein Ku70, which protects IN from
host proteasomal degradation in the overexpression system
and under HIV-1 infection (Figs. 1, 4B (top panel), and 6C (bottom panel)). Interestingly, our results showed that, in addition
to down-regulating the ubiquitin pool within the cells, Ku70 is
also able to specifically reduce the ubiquitination level of IN and
26

was profoundly defective even when it was used to infect normal cells (Fig. 5C, bar 3). However, we also cannot exclude the
possibility that the presence of Ku70 is required for other events
during HIV-1 morphogenesis. This notion is strengthened by
the previous findings that, during infection, free ubiquitin is
incorporated into viral particles of HIV-1, simian immunodeficiency virus, MMLV, and equine infectious anemia virus (66)
and that pr55Gag is monoubiquitinated during assembly and
budding (67). In addition, proteasome inhibitor treatment
interfered with the assembly and budding of HIV progeny virus
and efficiently inhibited HIV infectivity (68). Thus, further
investigation is certainly needed to fully understand how Ku70
impacts HIV-1 replication, and a better understanding of the
interplay between HIV-1 IN and Ku70 during viral infection
will rationalize the design of specific inhibitors to target this
viral/cellular protein interaction and consequently inhibit
HIV-1 replication.
grated DNA under the same infection conditions as in Fig. 5C.

Not surprisingly, 2-LTR circle formation in the Ku70-KD cells
was undetectable, which is consistent with a previous report
that NHEJ pathway is required for 2-LTR circle formation (Fig.
5D) (27, 50). In addition, integration of viral DNA was also
abrogated by Ku70-KD, whereas reverse transcription measured by late RT products were reduced by around 50% in the
Ku70-KD cells infected with shKu70 virus produced from
Ku70-KD cells (Fig. 5D). Thus, Ku70 seems to be a multifaceted
player in the early stage of viral infection. Ku70 is a component
of the NHEJ pathway (15) that has been extensively investigated
for its multiple functions during retroviral transduction or
infection in previous studies. With respect to the effects of
Ku70 on the early stage of HIV replication, one mechanism
might be that Ku70 is actively involved in the gap repair of
integration intermediates introduced by HIV-1 IN through the
NHEJ pathway (20, 2225, 27, 64). It explains an undetectable
HIV integration event when Ku70 is absent during viral infection. The second possible mechanism, with a plausibility demonstrated in this study, is that Ku70 may protect IN from degradation before viral DNA integration by reducing overall
cellular ubiquitination and/or by specifically interacting with
IN in HIV-infected cells. This scenario was supported by studying the effect of Ku70 on IN-PL metabolism after viral entry in
the early stage of viral replication (Fig. 6C). Depletion of Ku70
in the target cells had reduced IN expression to 77% at 8 h p.i.
Remarkably, when Ku70-KD C8166 T cells were infected with
virus produced from Ku70-KD 293T cells, the IN level at 8 h p.i.,
was reduced to 34%, as compared with that at 3 h p.i. (Fig. 6C,
bottom panel, compare bar 6 with bar 5). These observations
imply that the presence of Ku70 in both viruses and cells is able
to protect IN to avoid host ubiquitin proteasomal degradation
in the PIC. At this point, it should be noted that two other
cellular proteins, hRad18 and LEDGF/p75, were also previously
reported to protect IN from proteasomal degradation (58, 65).
However, unlike these two IN cofactors, whose actions are
mainly through their physiological binding to IN, Ku70 instead
displays two different activities to protect IN from degradation.
To date, it has not been clear how HIV-1 IN can coordinately
recruit these cofactors to protect itself from the host proteasomal degradation machinery.
With regard to the effect of Ku70 on the late stage of the viral
life cycle, we originally hypothesized that Ku70-KD might affect
the Gag/Gag-Pol ratio because Ku70 might protect IN as an
early Gag-Pol polyprotein precursor during viral assembly. In
fact, the results shown in Fig. 6A demonstrate that Ku70 does
not affect Gag/Gag-Pol processing and maturation. Further
analysis of virion composition revealed that the host cellular
protein Ku70 is present in the HIV-1 particles themselves (Fig.
6B). This finding indicates that Ku70 is packaged into HIV-1
particles as early as its assembly stage and becomes part of the
HIV-1 PICs after the virus enters target cells. Within the PIC
and associated with IN, Ku70 might deploy two mechanisms to
contribute to the early stage of HIV replication: 1) protecting IN
from the host proteasomal degradation pathway and 2) assisting viral protein IN in a specific replication step(s), including
2-LTR formation and integration. Consistently, our data
revealed that the progeny virus produced from Ku70-KD cells
AcknowledgmentsWe sincerely thank Dr. Heinrich Gottlinger for

kindly providing HA-ubiquitin plasmids and Dr. Shigemi Matsuyama for valuable comments.
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28
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 28, pp. 2458124592, July 15, 2011
Authors Choice
Impaired Infectivity of Ritonavir-resistant HIV Is Rescued by

Heat Shock Protein 90AB1*
Received for publication, April 5, 2011, and in revised form, May 19, 2011 Published, JBC Papers in Press, May 20, 2011, DOI 10.1074/jbc.M111.248021
Pheroze Joshi and Cheryl A. Stoddart1

From the Division of Experimental Medicine, Department of Medicine, University of California, San Francisco, California 94110
Certain ritonavir resistance mutations impair HIV infectivity
through incomplete Gag processing by the mutant viral protease. Analysis of the mutant virus phenotype indicates that accumulation of capsid-spacer peptide 1 precursor protein in virus
particles impairs HIV infectivity and that the protease mutant
virus is arrested during the early postentry stage of HIV infection before proviral DNA synthesis. However, activation of the
target cell can rescue this defect, implying that specific host factors expressed in activated cells can compensate for the defect in
ritonavir-resistant HIV. This ability to rescue impaired HIV replication presented a unique opportunity to identify host factors
involved in postentry HIV replication, and we designed a functional genetic screen so that expression of a given host factor
extracted from activated T cells would lead directly to its discovery by rescuing mutant virus replication in nonactivated T cells.
We identified the cellular heat shock protein 90 kDa (cytosolic), class B member 1 (HSP90AB1) as a host factor that can
rescue impaired replication of ritonavir-resistant HIV. Moreover, we show that pharmacologic inhibition of HSP90AB1 with
17-(allylamino)-17-demethoxygeldanamycin (tanespimycin)
has potent in vitro anti-HIV activity and that ritonavir-resistant
HIV is hypersensitive to the drug. These results suggest a possible role for HSP90AB1 in postentry HIV replication and may
provide an attractive target for therapeutic intervention.
ysis of Gag and studies with the HIV maturation inhibitor

bevirimat (2) indicate that uncleaved CA-SP1 by itself is sufficient to impair viral replication. This is because CA maturation
structurally modifies the HIV core in preparation for host factor-mediated uncoating in the infected cell (3). Physical
changes in CA affect overall core stability and reduce viral
infectivity (4). Recent studies by Muller et al. (5) have shown
that even low amounts (5%) of Gag processing intermediates
can display a transdominant negative effect on HIV infectivity
with the maturation cleavage between CA and SP1 being of
particular importance for the negative effect. In a previous
study, we found that although HIV with RTV resistance PR
mutations is minimally impaired for replication in mitogenactivated peripheral blood T cells it is highly impaired for replication in human thymus both in the SCID-hu Thy/Liv mouse
model and in thymic organ culture (6). Our results suggest that
the unprocessed Gag molecules disrupt CA maturation (and
thus prevent proper HIV core uncoating) and that cellular activation is the intrinsic difference between thymocytes and mitogen-activated T cells in determining the replication capacity of
RTV-resistant HIV.
Successful core uncoating is the major rate-limiting step in
productive retroviral infection and is controlled largely by cellular proteins (4, 7). For example, the host factor cyclophilin A
(CypA) interacts with HIV CA to promote uncoating of the
viral core (8); alternatively, various retroviral cores can be inactivated through contact between CA and host restriction factor
Ref1, Lv1, or Trim5 (9). Studies by Yamashita et al. (10) provided evidence that the CA protein is a dominant viral determinant for infection of nondividing cells. More recently, these
authors showed that tripartite motif family proteins and CypA
modulate the ability of HIV to infect nondividing cells through
their interaction with the CA protein (11). Uncoating and subsequent steps in early stage HIV infection remain unclear but
are thought to occur through active recruitment of cytoplasmic
proteins by the viral CA core (12, 13), and functional gene
silencing screens have revealed that various cellular pathways,
enzyme complexes, and cytoskeleton proteins are involved
in each step leading to proviral DNA integration (14 17).
Although there was minimal overlap among the screens in candidate HIV dependence genes, the consensus from these studies was that host factors associated with postentry HIV replication are cell-specific but often localize to common cellular
pathways (18). These interesting observations led us to hypothesize that highly impaired replication of RTV-resistant HIV in
thymocytes, as opposed to minimal impairment in peripheral T
lymphocytes, is a result of differences in the cytoplasmic envi-
Certain mutations in HIV protease (PR)2 that confer resistance to ritonavir (RTV), in particular I54V and V82A, result in
virus particles with incompletely processed Gag and impaired
replication capacity in vitro and in vivo. In vitro analysis indicates that the loss in infectivity results from decreased proteolytic activity of the mutant HIV PR, and RTV-resistant virus
particles display an accumulation of uncleaved Gag precursor
molecules, specifically capsid-spacer peptide 1 (CA-SP1) and
nucleocapsid-spacer peptide 2 (NC-SP2) (1). Mutational anal-
* This work was supported, in whole or in part, by a National Institutes of

Health grant from NIAID under Contract N01-AI-70002. This work was also
supported by California HIV/AIDS Research Program Grant ID09-SF-051 (to
C. A. S.) and in part by the University of California, San Francisco AIDS
Research Institute and the Harvey V. Berneking Living Trust.
Authors ChoiceFinal version full access.
1
To whom correspondence should be addressed. Tel.: 415-206-8149; Fax:
415-206-8091; E-mail: cheryl.stoddart@ucsf.edu.
2
The abbreviations used are: PR, protease; RTV, ritonavir; CA-SP1, capsidspacer peptide 1; NC-SP2, nucleocapsid-spacer 2; CypA, cyclophilin A;
HSP90AB1, heat shock protein 90 kDa (cytosolic), class B member 1; CA,
capsid; NC, nucleocapsid; PBMC, peripheral blood mononuclear cells; PHA,
phytohemagglutinin; VSV-G, vesicular stomatitis virus G; TCID50, 50% tissue culture infectious dose; MA, matrix; IN, integrase; m.o.i., multiplicity of
infection; CC50, 50% cytotoxic concentration.
29
HSP90AB1 Involved in HIV Replication

ronment between the target cells rather than limitations in the

PR mutant.
The characteristic cone-shaped HIV core is formed exclusively from CA molecules that initially arrange into a dense
hexagonal lattice of uncleaved CA-SP1 subunits (19). In this
immature conformation, CA hexamers are stabilized from
below by SP1 bundles (20). Ultimately, proteolysis at the
CA-SP1 junction enables the densely packed CA subunits to
rearrange into distinct CA hexamers, which then polymerize
into a stable mature HIV core lattice. In addition, this morphological transition prepares the HIV core for postentry events by
exposing local motifs in CA to interacting host factors within
the infected cell (21).
Three-dimensional analysis of released virus particles has
revealed that the number of Gag molecules is double the number of CA molecules needed for formation of the cone-shaped
capsid shell, indicating that 50% of CA in the mature virion
may not be part of the capsid (22). High resolution images of
mature retroviral cores show a range of core polymorphisms,
which include amorphous shapes, multiple and nested cores,
and incompletely closed shells, suggesting that viable retroviral
cores do not have a uniquely specified structure; rather a range
of CA structures may be acceptable (23). Moreover, thin section
electron microscopy (EM) analysis reveals that virus particles
with minor amounts of intermediate Gag cleavage products
do not appear to block maturation because the cone-shaped
core is present in most particles. However, subtle alterations
in virion morphology that are not easily detected by EM may
be functionally relevant (5). Consequently, RTV-resistant
HIV particles might contain intact cores that assume a defective structure because of uncleaved CA-SP1, thereby precluding uncoating in thymocytes that lack the appropriate
CA-interacting host factors.
In this report, we confirm that uncleaved CA-SP1 severely
impairs RTV-resistant HIV replication in thymocytes and
nonactivated T cells and that activation of T cells dramatically reverses this defect. The PR mutant is arrested at the
postentry stage of uncoating in nonactivated cells, and this
defect presented an opportunity for identification of host
factors that rescue RTV-resistant HIV replication. Thus, we
designed a genetic screen so that expression of a given host
factor would lead directly to its discovery by virtue of its
ability to rescue a fluorescent RTV-resistant reporter virus.
Using the PR mutant fluorescent reporter, we sought to
identify host factors with the greatest positive effect on HIV
replication by sorting the most brightly fluorescent infected
cells. Through this screen, we identified the cellular heat
shock protein 90 kDa (cytosolic), class B member 1
(HSP90AB1) as a factor that independently rescues RTVresistant HIV replication. Moreover, we show that pharmacologic inhibition and RNA interference of HSP90AB1
blocks HIV replication in primary human T cells, underscoring the advantage of screening for HIV-interacting host
proteins that promote virus replication. The ability of
HSP90AB1 to rescue RTV-resistant HIV suggests that this
host factor is involved in early postentry virus replication
possibly by interacting with the HIV CA protein and represents a novel target for new antiviral therapies.
Cells and Viruses293T and P4-Magi cell lines were cultured at 37 C in Dulbeccos modified Eagles medium
(HyClone) supplemented with 10% fetal bovine serum (FBS)
(HyClone), and Jurkat E6-1, Jurkat E6-1 CypA/, CEM T4,
and Sup T1 were cultured at 37 C in RPMI 1640 medium
(Invitrogen) supplemented with 10% FBS. All cell lines were
obtained from the National Institutes of Health AIDS Research
and Reference Reagent Program. PBMC were obtained from
six HIV-seronegative donors and maintained in RPMI 1640
medium supplemented with 10% FBS, 1 g/ml PHA-P (Invitrogen), and 5% human IL-2 (Roche Applied Science) for 3 days
before inoculation. The infectious HIV proviral DNA construct
pNL4-3 was used in all studies, and the I54V, V82A, and A71V
mutations in HIV PR and A431V in the NC-SP2 cleavage junction were generated by oligonucleotide-directed single strand
mutagenesis. Briefly, the Gag-Pol region of pNL4-3 was excised
using the SwaI restriction endonuclease and cloned into the
pBluescript cloning vector (Stratagene). The I54V, A71V, and
V82A mutations were created individually or in combination
within the protease gene using the QuikChange II site-directed
mutagenesis kit (Stratagene), and similarly, the A431V mutation was introduced in the NC-SP2 cleavage junction. All mutations were verified by sequencing. The respective mutations
were rebuilt into the parental NL4-3 by transferring the SpeIAge I fragment from the intermediate cloning vector. The wildtype (WT) HIV-RFP and HIV PRI54V/V82A-RFP fluorescent
reporter virus constructs were created by inserting the coding
sequence of dTomato red fluorescent protein (RFP) (24)
between the env and nef regions of pNL4-3. Initially, the
BamHI-KpnI fragment was excised from pNL4-3 and cloned
into the pBluescript vector. The end of gp41 gene was amplified
by PCR to generate a BglII recognition site at the 3-end of the
amplicon, and the start of the nef gene was amplified with
primers terminating the PCR product in a KpnI recognition
sequence. The RFP coding sequence was excised from
dTomato by first digesting with EcoRI and subsequently blunting the 3-end with mung bean nuclease. The 5-start of RFP
was digested with BamHI and ligated to the 3-end of the gp41
PCR product, and the 3-end of RFP was blunt end-ligated to
the 5-start of the nef gene PCR product. The three-piece ligation product was excised from the intermediate pBluescript
cloning vector and rebuilt into the parental NL4-3 clone to
produce an infectious molecular clone with the RFP open reading frame beginning 14 nucleotides downstream of the gp41
termination codon and ending one nucleotide upstream of the
nef initiation sequence. The lentiviral green fluorescent protein (GFP) reporter system was used for VSV-G pseudotyping
experiments as described previously (25). The I54V and V82A
PR mutations were introduced in the psPAX2 Gag-Pol packaging construct by site-directed mutagenesis, and recombinant
virus particles were generated by transient transfection in 293T
cells using calcium phosphate (Invitrogen). The VSV-G envelope was derived from the pMD2G plasmid, and the HIV Env
was derived from the pDOL construct (National Institutes of
Health AIDS Research and Reference Reagent Program). Vector combinations used to generate virus particles included
30

cotransfecting 3 g of each envelope plasmid, 8 g of the packaging plasmid with and without the PR mutations, and 10 g of
the GFP reporter plasmid. Two days after transfection, the culture medium was clarified by filtration (0.45 m) and frozen in
aliquots under liquid nitrogen. The virus stocks were titrated as
described in the following section.
HIV Stock Virus Preparation and TitrationAll mutant and
WT virus stocks were generated by transient transfection of
293T cells using Lipofectamine 2000 (Invitrogen). Two days
after transfection, the culture medium was clarified by filtration
(0.45 m) and frozen in aliquots under liquid nitrogen. 50%
tissue culture infectious doses (TCID50) were determined with
serial half-log dilutions of each virus stock in quadruplicate
wells of PHA-activated PBMC (1 105 cells/well) and calculated as a reciprocal of the dilution at which 50% of the wells
contained detectable (50 pg/ml) HIV p24 measured by ELISA
(PerkinElmer Life Sciences).
Western Blot AnalysisVirus particles were concentrated
from clarified culture medium by ultracentrifugation through a
20% sucrose cushion in phosphate-buffered saline (PBS) at
130,000 g for 2 h at 4 C. The pellet was slowly dissolved in
Laemmli loading buffer (Sigma). High molecular weight viral
proteins were separated on 8 20% linear gradient polyacrylamide gels, and smaller viral proteins were resolved on 15% gels.
The viral proteins were electroblotted onto Immobilon PVDF
membrane (Millipore) and incubated individually with specific
primary antibodies. The following primary antibodies were
obtained from the AIDS Reagent Program: CA (catalog number
6521), MA (catalog number 4811), reverse transcriptase (RT)
(catalog number 11338), integrase (IN) (catalog number 7374),
PR (catalog number 4105), Nef (catalog number 709), Vif (catalog number 6459), Vpr (catalog number 3951), gp120 (catalog
number 2343), and gp41 (catalog number 7623). The NC and
p6 primary antibodies were obtained from the National Cancer
Institute AIDS Vaccine Program. Peroxidase-conjugated secondary antibodies (Pierce) were used, and immune complexes
were visualized by enhanced chemiluminescence (Pierce).
In Vitro Infectivity AssayP4-Magi indicator cells were
seeded in a 12-well plate and inoculated at a multiplicity of
infection (m.o.i.) of 0.005 with WT and mutant stock viruses.
At 48 h after inoculation, cells were stained with 5-bromo4-chloro-3-indolyl--D-galactopyranoside to detect infected
cells, and the percentage of blue cells was determined
microscopically.
In Vivo Infectivity AssayImplantation of fragments of
human fetal thymus and liver under the kidney capsule to create SCID-hu Thy/Liv mice and inoculation of the Thy/Liv
implants with HIV were carried out as described previously (6).
Implanted mice were inoculated with 1,000 TCID50 of HIV, and
Thy/Liv implants were collected after 21 days and processed for
viral RNA quantification by branched DNA assay and p24
ELISA. Protocols were approved by the University of California,
San Francisco Institutional Animal Care and Use Committee.
Infectivity Assay in Activated CellsJurkat E6-1 cells were
treated with increasing concentrations of phorbol 12-myristate
13-acetate (Sigma) for 24 h. Cells were resuspended in fresh
growth medium, inoculated with stock virus at an m.o.i. of
0.005, and cultured for 12 days. Culture supernatant was
assayed every 2 days for HIV p24 by ELISA. Additionally, Jurkat

E6-1 cells were activated with CD3- and CD28-coated magnetic
beads as recommended by the manufacturer (Dynal) for 24 h.
Cells were resuspended in fresh growth medium, inoculated
with stock virus at an m.o.i. of 0.005, and cultured for 8 days.
Culture supernatant was assayed for HIV p24 by ELISA.
Virus-Cell Fusion AssayThe BlaM-Vpr HIV fusion assay
was performed as described previously (26). WT HIV-BlaMVpr and HIV PRI54V/V82A-BlaM-Vpr chimeric viruses were
produced by triple transfection of 293T cells, titrated, and used
to inoculate Jurkat E6-1 cells at a range of m.o.i. values for 2 h at
37 C. Cells were then loaded with the CCF2-AM fluorogenic
substrate (Invitrogen) overnight at room temperature. Cells
were centrifuged and resuspended in PBS. The extent of viruscell fusion was determined by fluorescence of the cleaved
CCF2-AM using a three-laser BD FACSAria system (BD
Biosciences).
Quantitative Assay for Intracellular HIV Reverse TranscriptionWT HIV and HIV PRI54V/V82A virus stocks pretreated with DNase I and 10 mM MgCl2 for 1 h to remove contaminating plasmid DNA were used to inoculate Jurkat E6-1
cells at an m.o.i. of 0.005. Cells were collected at intervals after
inoculation, and total cellular DNA was extracted with the
DNeasy kit (Qiagen). The extent of proviral DNA synthesis was
quantified by real time PCR using the RU5 primers and probe
set to detect early reverse transcripts and the MH531-MH532
primers and LTR-P probe to detect full-length proviral DNA as
described previously (27, 28). Reaction mixtures included the
Ex Taq master mix (TaKaRa), 100 ng of DNA, a 200 nM concentration of each primer, and a 100 nM concentration of probe.
Thermal cycling conditions were 2 min at 50 C, 10 min at
95 C, and 40 cycles of 95 C for 15 s and 60 C for 60 s. Control
Jurkat E6-1 cells were pretreated with 50 M azidothymidine
for 4 h before inoculation.
Endogenous Reverse Transcriptase Assay and Total Viral
RNA ContentWT HIV and HIV PRI54V/V82A virus (500
TCID50) were pretreated with DNase I and 10 mM MgCl2 for 1 h
to remove contaminating plasmid DNA and concentrated as
described above. The concentrated virus was resuspended in 10
l of PBS and diluted with 40 l of endogenous RT buffer (5 mM
MgCl2; 50 mM NaCl; 10 mM dithiothreitol; 0.5 mM dATP,
dCTP, and dGTP; 50 mM Tris (pH 8); 0.015% Triton X-100; 10
M [-32P]TTP). The reaction mixtures were incubated at
37 C for 30 min and terminated with 0.5% SDS and 25 mM
EDTA. Samples were spotted on DE81 filters, washed three
times in 2 SSC, and dried, and 32P was quantified by liquid
scintillation counting. Control samples were treated with 25 M
nevirapine. Total RNA was extracted from the same concentrated virus samples using the QIAmp Viral RNA kit (Qiagen),
dissolved in an ice-cold solution of 10 mM NaOH and 1 mM
EDTA, and spotted on a nitrocellulose membrane. The membrane was prehybridized overnight at 42 C in hybridization
buffer (50% formamide, 5 SSC, 1 Denhardts solution, 50
mM NaHPO4 (pH 6.5), 250 g/ml denatured salmon sperm
DNA) followed by incubation with a random primed HIV-specific 32P-labeled probe (Stratagene) at 42 C for 16 h. The membrane was washed three times in 2 SSC and dried, and the
31

recovered by PCR amplification using primers specific to the
modified retroviral vector that flanked the cDNA sequence.
The extracted cDNAs were recloned into the modified pFB
vector and subjected to a second round of selection where only
cells with a high red fluorescence were sorted (0.01 0.1% of
total RFP-positive cells) after which cDNA inserts were amplified using a reduced number of PCR cycles to ensure even representation of each individual cDNA. PCR products were
recloned into the modified pFB retroviral vector plasmid, bacterial colonies representing single cDNAs were picked, and
transducing retroviral particles were generated individually as
described above. Jurkat E6-1 cells were transduced at an m.o.i.
of 0.1 with individual cDNA-expressing retroviral particles and
then inoculated with the HIV PRI54V/V82A-RFP reporter
virus as described above. The cells were assayed 24 h after inoculation by flow cytometry, and cDNA clones that supported
replication of the RTV-resistant reporter virus were identified
by expression of the RFP in infected cells. The DNA sequence of
selected cDNA clones was aligned with the NCBI Human
Genome Project database using the BLAST human sequence
search engine.
Cloning and Expression of HSP90AB1The full-length protein-coding sequence of HSP90AB1 was PCR-amplified from
the cDNA clone isolated through the genetic screen and
inserted immediately downstream of the FLAG sequence in
the pFB retroviral expression vector (Stratagene). In addition, the E42A and D88A mutations (31) were introduced in
the native HSP90AB1 sequence by oligonucleotide-directed
single strand mutagenesis. Transducing retrovirus stocks
carrying the native and mutant HSP90AB1 cDNAs were generated and titrated as described above. Jurkat E6-1 cells were
transduced with the cDNA-expressing retrovirus at an m.o.i.
of 0.1 and cultured for 24 h. The pFB-GFP reference virus
was included as a negative control and used to determine
recombinant protein expression. A fraction of the cells were
resuspended in fresh growth medium, inoculated with WT
and mutant stock viruses at an m.o.i. of 0.005, and cultured
for 8 days. Culture supernatant was assayed for HIV p24 by
ELISA. The remaining cells were lysed in CelLytic M solution (Sigma), and recombinant proteins were immunoprecipitated using anti-FLAG M2 affinity gel (Sigma). Immunoprecipitated protein from an equal number of transduced
cells was separated on a 10% polyacrylamide gel matrix and
subjected to Western blot analysis using the anti-FLAG
M5 clone antibody (Sigma) and anti-HSP90AB1 antibody
(Abcam) as described above. The endogenous expression
level of HSP90AB1 was assayed on 10 g of protein extracted
from nonactivated and CD3- and CD28-activated Jurkat
E6-1 cells. Cellular HSP90AB1 protein levels were quantified
on a Typhoon Trio imager (GE Healthcare). Equal protein loading was confirmed by probing for actin (Abcam).
In Vitro Antiviral Activity AssayAntiviral activity of 17-(allylamino)-17-demethoxygeldanamycin (Sigma) was assayed in
PHA-activated PBMC as described previously (32). Briefly,
PBMC were inoculated in bulk with each virus at an m.o.i. of
0.001 for 2 h, cells were washed, and 1 105 cells in 100 l were
seeded in triplicate wells of a 96-well plate. Wells were treated
with 100 l of serial half-log dilutions of the drug or with
amount of hybridized probe was measured by liquid scintillation counting.

Construction of Retroviral cDNA Expression LibraryTotal
RNA was extracted from CD3- and CD28-activated Jurkat E6-1
cells using the RNeasy kit (Qiagen) and subjected to consecutive rounds of mRNA purification using the Oligotex mRNA
purification kit (Qiagen). First strand synthesis was carried out
on 5 g of purified mRNA using a hybrid XhoI-oligo(dT)
primer (Stratagene), and the resulting cDNAs had 5-EcoRI and
3-XhoI cohesive ends for directional ligation into the retroviral
vector. The retroviral cDNA expression library was constructed using the pFB retroviral vector (Stratagene), and the
multiple cloning region was replaced with a PCR fragment
containing a Kozak sequence, ATG start codon, and FLAG
tag sequence followed by the EcoRI and XhoI recognition
sequences. Two additional constructs were generated by inserting A and AA nucleotides immediately before the EcoRI site to
ensure in-frame expression of the cDNA library. The coding
sequence for the GFP derived from pAcGFP1-1 (Clontech) was
inserted into the modified pFB vector using the EcoRI and XhoI
sites and served as a stuffer fragment to reduce the background
of parental vector in the cDNA library. Furthermore, the modified pFB-GFP construct was used as a reference virus to calculate the titer of the retroviral cDNA expression library (29). The
cDNA was ligated directionally into the modified pFB plasmid
by replacing the stuffer fragment, and the reaction products
were used to transform XL-10 GOLD Escherichia coli (Stratagene) by electroporation. The bacterial colonies were amplified
in semisolid 2 LB-agarose (Stratagene) to reduce the potential
for underrepresentation of particular clones due to overgrowth
of faster growing colonies.
Production and Titration of Retroviral cDNA Expression
LibraryTo generate a library of transducing viruses, 20 g
of the pFB cDNA expression plasmid was transiently transfected into the AmphoPack-293 packaging cell line (Clontech) using Lipofectamine 2000 (Invitrogen). The culture
supernatant was harvested 48 h after transfection, clarified
by low speed centrifugation, and filtered (0.2 m), and aliquots were stored under liquid nitrogen. Because the pFB
transducing vector does not carry a reporter, the titer of the
retroviral cDNA expression library was determined by a onestep quantitative RT-PCR using a calibrated RNA standard
curve generated with the pFB-GFP reference virus. The
m.o.i. of the pFB-GFP virus was determined by flow cytometry, and the titer of the retroviral cDNA-expressing library
was normalized to the RNA titer of the reference GFP-transducing virus (30).
Genetic ScreenJurkat E6-1 cells were transduced with the
cDNA-expressing retroviral library at an m.o.i. of 0.1 in multiple sets to ensure even representation of the cDNA library and
cultured for 24 h. Transduced cells were inoculated with the
HIV PRI54V/V82A-RFP reporter virus at an m.o.i. of 0.05
and cultured for an additional 24 h. Cells were resuspended in
PBS, and red fluorescent cells were sorted on a FACSVantage
DiVa cell sorting system (BD Biosciences). The RFP-sorted
cells were examined microscopically to confirm that all the cells
expressed red fluorescence, and total cellular DNA was
extracted with the DNeasy kit (Qiagen). The cDNA insert was
32
FIGURE 1. Impaired replication of RTV-resistant HIV results from uncleaved CA-SP1. A, schematic of the protein domain organization in the HIV Gag
polyprotein precursor. Proteolytic cleavage of the Gag polyprotein during HIV maturation results in the MA (p17), CA (p24), NC (p7), and p6 proteins. B, Western
blot analysis of Gag cleavage products from purified WT HIV and mutant virus particles carrying the indicated RTV resistance mutations in the protease. The
right panel shows PR mutant virus particles including compensatory substitutions in the NC-SP2 cleavage junction and the A71V mutation in HIV protease.
Insufficient cleavage of Gag is indicated by the presence of p25 (CA-SP1) and p8 (NC-SP2) protein bands. The Western blot is representative of three independent purified virus preparations. C, Western blot analysis of Pol polyprotein and Nef precursor protein cleavage products from wild-type and RTV-resistant
HIV particles. Complete cleavage of the Pol polyprotein is indicated by mature RT, IN, and PR proteins. Molecular mass of mature viral proteins is indicated on
the left, and the panel is representative of three independent purified virus preparations. D, triplicate wells of P4-Magi cells were inoculated with each virus at
an m.o.i. of 0.005 and stained for -galactosidase activity after 48 h. The columns represent mean number of infected blue cells from four (open circles)
independent experiments. E, SCID-hu Thy/Liv mice were inoculated with 1,000 TCID50 of each virus, and Thy/Liv implants were collected 21 days after
inoculation. Viral replication was assessed by branched DNA assay for HIV RNA and by ELISA for p24. The columns represent means, and open circles represent
individual animals from the same cohort.
RESULTS
medium alone, and supernatants were assayed at day 7 for p24

by ELISA. Parallel cellular toxicity of the drug was determined
on uninfected cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Sigma) on day 7. The 50% inhibition of virus replication (IC50) and 50% cytotoxic concentration (CC50) were calculated as the mean of three independent
assays. Similarly, the antiviral activity of an siRNA against
HSP90AB1 was assayed. The sense and antisense strands of the
siRNA were synthesized separately (Integrated DNA Technologies) with the following modifications: HSP90AB1 sense,
5-UUCAGGGCAUUGCUAUUUAdT*dT*-3;
HSP90AB1
antisense, 5-UAAAUAGCAAUGCCCUGAAdT*dT*chol-3;
scrambled sense, 5-GGUUAUAAUUUCGUUCGACdT*dT*-3; scrambled antisense, 5-GUCGAACGAAAUUAUAACCdT*dT*chol-3. * designates substitution of the phosphodiester bond with a phosphorothioate bond, and chol indicates
cholesterol conjugation. The two RNA strands were annealed
in equimolar amounts, and WT HIV- and HIV PRI54V/V82Ainfected PBMC were treated with quarter-log dilutions of the
siRNA duplex. Inhibition of viral replication and cellular toxicity were assayed after 7 days, and results represent the mean of
three independent assays.
RTV Resistance Affects Cleavage Efficiency of HIV Protease

Sequential cleavage of the Gag polyprotein by HIV PR occurs
during virus maturation and is essential for infectious virus particle formation (Fig. 1A). In our previous report, HIV gag and
pol sequences isolated from patients receiving RTV (and other
PR inhibitor) monotherapy revealed that the I54V and V82A
drug resistance mutations in HIV PR led to decreased viral fitness. We introduced the I54V and V82A mutations into the
PR-coding sequence of the NL4-3 clone of HIV and analyzed
individual viral proteins by Western blot. Cleavage of the MA
and p6 proteins was not affected by the RTV-resistant HIV PR;
however, a significant reduction in cleavage at the CA-SP1 and
NC-SP2 junctions was observed in the presence of both PR
mutations (Fig. 1B).
In vivo resistance to RTV occurs sequentially with the V82A
primary mutation located in the PR active site frequently preceding the I54V secondary mutation (33). This combination
confers greater resistance to RTV in vivo but decreases the in
vitro activity of HIV PR. Eventually, resistance to RTV in vivo
results from compensatory mutations within HIV PR and in
33

Gag target sites to counteract reduced activity of the mutant PR
(34, 35). We introduced the naturally occurring A431V compensatory mutation into the NC-SP2 cleavage junction (1); this
facilitated cleavage of NC-SP2 but did not alter partial cleavage
at the CA-SP1 junction by the RTV-resistant HIV PR. Furthermore, we introduced an additional substitution in PR (A71V)
commonly observed in patients failing RTV monotherapy (36)
and showed that this substitution restored enzymatic activity to
the RTV-resistant PR at both the CA-SP1 and NC-SP2 junctions (Fig. 1B). The HIV PR is also responsible for cleavage of
the Gag-Pol polyprotein and Nef precursor protein, and we
confirmed that the I54V and V82A PR substitutions did not
affect proteolysis of these additional substrates (Fig. 1C).
Uncleaved CA-SP1 (p25) but Not Uncleaved NC-SP2 (p8)
Impairs RTV-resistant HIVTo determine whether insufficient cleavage of CA-SP1 and NC-SP2 affected replication of
RTV-resistant HIV, we challenged P4-Magi indicator cells with
the different PR mutants at equal m.o.i. values (Fig. 1D). The
I54V PR mutation alone did not affect virus replication,
whereas significant replication impairment was observed with
the V82A mutation. The combined effect of I54V and V82A PR
mutations severely impaired RTV-resistant HIV replication.
The NC-SP2 compensatory substitution did not rescue infectivity of the PR mutant, however, suggesting that uncleaved
CA-SP1, but not NC-SP2, impairs replication of RTV-resistant
HIV. The compensatory A71V PR substitution rescued infectivity of RTV-resistant HIV, confirming that RTV resistance
impacts virus replication.
More importantly, we confirmed impaired replication of
RTV-resistant HIV in human thymus implants of SCID-hu
Thy/Liv mice. We inoculated SCID-hu Thy/Liv mice with
1,000 TCID50 of each virus. The Thy/Liv implants were collected 21 days after inoculation for quantification of HIV RNA
by branched DNA assay and p24 by ELISA. The in vivo pattern
of RTV-resistant HIV replication was similar to that seen in
vitro: the combination of I54V and V82A PR mutations
impaired replication of RTV-resistant HIV (HIV PRI54V/
V82A) in human thymocytes, and compensatory cleavage of
NC-SP2 did not rescue infectivity of the PR mutant (Fig. 1E).
Taken together, our results indicate that the presence of
uncleaved CA-SP1 is the primary cause for impaired replication
of RTV-resistant HIV in human thymocytes. This defect, however, does not adversely affect RTV-resistant HIV replication in
activated peripheral T cells. We speculate that, as a result of
uncleaved CA-SP1, the RTV-resistant HIV core lattice assumes
an alternate conformation that cannot undergo proper uncoating in thymocytes. If so, host factors up-regulated in activated
cells may make the difference between the presence and the
absence of efficient viral replication.
Impaired Replication of RTV-resistant HIV Is Rescued by Cellular ActivationOur earlier studies indicated that expression
of specific host factors in PHA-activated PBMC rescued the
impaired replication of RTV-resistant HIV. To validate this
hypothesis, we screened CD4 T cell lines for their ability to
show a differential capacity to support PR mutant virus replication in the presence or absence of activation. At base line,
Jurkat T cells failed to support HIV PRI54V/V82A virus replication when inoculated at the same m.o.i. as that of WT HIV.
Replication of this PR mutant was supported when Jurkat T

cells were first activated with phorbol 12-myristate 13-acetate,
and the amount of replication increased in a dose-dependent
manner (Fig. 2A). Replication of the HIV PRI54V/V82A mutant
increased dramatically in response to phorbol 12-myristate
13-acetate activation, indicating a direct influence of select host
factors on replication of RTV-resistant HIV. We confirmed the
role of activation on RTV-resistant HIV replication by activating Jurkat T cells through the T cell receptor using a mixture of
antibodies against CD3 and CD28 (Fig. 2B). In addition, activation of CEM T4 cells, Sup T1 cells, and human PBMC specifically through T cell receptor-ligand binding effectively rescued
RTV-resistant HIV replication (Fig. 2C). These results confirm
that host factors unique to activated human T cells override the
transdominant negative effects of uncleaved CA-SP1 and facilitate replication of RTV-resistant HIV.
Replication of RTV-resistant HIV Is Arrested after Entry
and before Reverse TranscriptionTo determine whether
uncleaved CA-SP1 prevented fusion of the RTV-resistant virus
to the target cell, we inoculated nonactivated Jurkat T cells with
HIV PRI54V/V82A-BlaM-Vpr chimeric virus using a fluorescence fusion assay (26). In comparison with WT HIV-BlaMVpr chimeric virus, we did not observe fusion defects, suggesting that replication of RTV-resistant virus is impaired at a
postentry stage of the virus life cycle (Fig. 2D). We also inoculated Jurkat T cells with WT HIV and HIV PRI54V/V82A and
quantified early and late proviral reverse transcripts over a 24-h
time course by real time PCR. The absence of both early and late
RTV-resistant proviral transcripts in nonactivated Jurkat T
cells suggested that RTV-resistant HIV infection was arrested
soon after entry, most likely during uncoating (Fig. 2E).
However, full-length RTV-resistant proviral transcripts were
detected in CD3- and CD28-activated Jurkat T cells (Fig. 2E),
indicating functional reverse transcription by the PR mutant. In
addition, HIV PRI54V/V82A virus was tested for endogenous
RT activity and the ability to package viral genomic RNA. No
significant differences in RT activity or viral RNA content were
observed (Table 1), confirming that RTV-resistant HIV is not
defective in RNA packaging or formation of a functional ribonucleoprotein-RT complex within the virion. Taken together,
these results suggest that defective uncoating of RTV-resistant
HIV cores in nonactivated T cells precludes reverse transcription of proviral DNA, resulting in dead-end infection. In contrast, cellular activation provides appropriate host factors that
facilitate uncoating of the PR mutant virus core, leading to productive proviral DNA synthesis of RTV-resistant HIV.
Pseudotyping of RTV-resistant HIV Does Not Rescue
Impaired ReplicationInfectivity of HIV can be influenced by
the route of entry, and pseudotyping with envelope proteins
from other viruses (e.g. VSV-G) enables some mutant virus to
bypass early postentry blocks (37). Using GFP reporter viruses,
RTV-resistant HIV was targeted for alternate entry through the
endosomal pathway by pseudotyping with the VSV-G envelope, but this failed to rescue infectivity to RTV-resistant HIV in
nonactivated Jurkat T cells (Fig. 3A). The pseudotyped RTVresistant HIV, however, responded predictably to cellular activation (Fig. 3A). In addition, we inoculated nonactivated Jurkat
T cells with PR mutant at increasing m.o.i. values to determine
34
FIGURE 2. Cellular activation rescues impaired replication of RTV-resistant HIV. A, Jurkat E6-1 cells treated with increasing concentrations of phorbol
12-myristate 13-acetate (PMA) for 24 h were inoculated at an m.o.i. of 0.005. The cells were cultured for 12 days, and results represent mean supernatant HIV p24
levels S.E. (error bars) from six independent experiments. B, Jurkat E6-1 cells were activated with a mixture of CD3 and CD28 antibodies for 24 h, inoculated
at an m.o.i. of 0.005, and cultured for 8 days. The columns represent mean supernatant HIV p24 levels from four (open circles) independent experiments. C, CEM T4,
Sup T1, and PBMC were activated with a mixture of CD3 and CD28 antibodies for 24 h, inoculated at an m.o.i. of 0.005, and cultured for 8 days. The columns represent
mean supernatant HIV p24 levels from four (open circles) independent experiments. D, wild-type HIV-BlaM-Vpr and HIV PRI54V/V82A-BlaM-Vpr chimeric viruses at a
range of m.o.i. values were used to inoculate nonactivated Jurkat E6-1 cells for detection of CCF2 substrate cleavage by flow cytometry. Each m.o.i. was analyzed in
triplicate, and the columns represent mean percentage of cleaved CCF2 from six (open circles) independent experiments. E, activated and nonactivated Jurkat E6-1 cells
were inoculated at an m.o.i. of 0.005, and cellular DNA was extracted over a 24-h period. Early and late proviral reverse transcripts were quantified by TaqMan real time
PCR in triplicate, and the results represent mean S.E. (error bars) of six independent experiments. AZT, azidothymidine.
TABLE 1
RT activity is expressed as counts/25 l of spotted reaction mixture.
Results represent the mean S.E. of six independent RT assays. Viral
RNA content was calculated from four independent RNA extractions,
and results represent the mean S.E. of eight spots (two per sample)
Virus
Endogenous
RT activity
Viral RNA content
Wild-type HIV
HIV PRI54V/V82A
Wild-type HIV nevirapine
HIV PRI54V/V82A nevirapine
cpm 104/500 TCID50

2,204 271
1,626 678
75 26
113 18
cpm 102/500 TCID50

387 56
426 84
Not determined
Not determined
was comparable with levels observed in activated Jurkat T cells,

indicating that the necessary host factor is not CypA (Fig. 3C).
Cellular Activation Does Not Repair Maturation Defect of
RTV-resistant HIVIn addition, we observed that cellular activation did not enhance maturation of RTV-resistant HIV by
facilitating CA-SP1 cleavage in virus particles released from
activated Jurkat T cells. We inoculated CD3- and CD28-activated Jurkat T cells with RTV-resistant HIV and harvested virus
particles from the culture supernatant. Western blot analysis of
the RTV-resistant HIV proteins confirmed that virus particles
released from activated T cells contained uncleaved CA-SP1
and NC-SP2 subunits (Fig. 3D) that were unable to infect nonactivated Jurkat T cells.
Genetic Screen to Identify HIV-interacting Host Factors
Given the above evidence, we reasoned that host factors
expressed in activated T cells interact with the uncleaved
CA-SP1 subunit to rescue RTV-resistant HIV replication. To
identify such factors, we designed a genetic screen using a
cDNA library generated from CD3- and CD28-activated Jurkat
T cells (Fig. 4A). The cDNA library was introduced into nonactivated Jurkat T cells using a modified retroviral vector, and
transduced cells were then inoculated with RTV-resistant HIV
carrying the gene for RFP inserted into the viral RNA genome
such that expression of the reporter would occur only upon
whether host restriction factors are responsible for impaired

replication of RTV-resistant HIV. A common feature of retroviral postentry blocks is that host restriction factors can be saturated by virus overload (38), but increasing the m.o.i. to 0.1
(equivalent to 500 ng of p24) did not rescue RTV-resistant HIV
replication in nonactivated Jurkat T cells (Fig. 3B).
CypA Does Not Rescue Replication of RTV-resistant HIV in
Activated T CellsTo determine whether overexpression of
CypA contributed to the rescue of RTV-resistant HIV in activated T cells, we activated CypA-null Jurkat T cells (CypA/)
(39) with antibodies against CD3 and CD28 and then inoculated the cells with RTV-resistant HIV. Replication of the PR
mutant in CD3- and CD28-activated CypA/ Jurkat T cells
35
FIGURE 3. Characteristics of impaired RTV-resistant HIV replication. A, WT HIV-GFP and HIV PRI54V/V82A-GFP reporter viruses at an m.o.i. of 0.1 with
HIV Env and WT HIV-GFP and HIV PRI54V/V82A-GFP reporter viruses at an m.o.i. of 0.01 pseudotyped with VSV-G Env were used to inoculate nonactivated and CD3- and CD28-activated Jurkat E6-1 cells. The columns represent mean GFP fluorescence from four (open circles) independent infection
experiments. B, nonactivated Jurkat E6-1 cells inoculated with HIV PRI54V/V82A virus at increasing m.o.i. values were cultured for 8 days. The columns
represent mean supernatant HIV p24 levels from four (open circles) independent experiments. C, CD3- and CD28-activated Jurkat E6-1 and Jurkat E6-1
CypA/ cells were inoculated at an m.o.i. of 0.005 and cultured for 8 days. The columns represent mean supernatant HIV p24 levels from four (open
circles) independent experiments. D, Jurkat E6-1 cells were activated with antibodies against CD3 and CD28 for 24 h, inoculated at an m.o.i. of 0.05, and
cultured for 8 days. Virus particles were purified from culture supernatants, and CA and NC viral proteins were detected by Western blot analysis. Results
are representative of three independently purified virus preparations.
Nonactivated Jurkat T cells transduced with the retrovirusbased cDNA library were cultured briefly to allow adequate
expression of the gene products and then inoculated with the
HIV PRI54V/V82A-RFP virus. Jurkat T cells infected by the PR
mutant reporter virus were sorted for bright red fluorescence
by flow cytometry. The host factor-expressing cDNAs were
PCR-amplified from genomic DNA of the sort-purified Jurkat
T cells using retrovirus-specific primers and subjected to an
additional round of selection in the genetic screen (Fig. 4C).
The second round of screening yielded a greater number of
RTV-resistant HIV-infected red fluorescent cells, and we
sorted the very brightest (0.01 0.1%) RFP-positive cells. Selection of brightly red fluorescent cells ensured isolation of host
factors that strongly interacted with RTV-resistant HIV and
reduced false-positive events that can occur with routine
manipulation of target cells. Host factor cDNAs were amplified
using a limited number of PCR cycles to ensure even representation of each clone. The PCR products were recloned into the
modified retroviral vector, and individual bacterial colonies,
representing single cDNA clones, were tested independently
with the HIV PRI54V/V82A-RFP virus. Through the second
round of selection we identified a cDNA clone, which aligned
with the human HSP90AB1 gene (GeneID 3326), that consistently supported RTV-resistant HIV replication when expressed in nonactivated Jurkat T cells.
Validation of HSP90AB1 in RTV-resistant HIV Replication
To confirm the influence of HSP90AB1 in RTV-resistant HIV
replication, we generated a full-length expression clone of the
host factor. We also introduced the inactivating E42A and
productive infection and integration of RTV-resistant HIV. We

predicted that transduced Jurkat T cells that supported productive infection of the RTV-resistant RFP HIV would express a
permissive host factor encoded by the unique cDNA present in
the infected cell. Infected cells with high RFP expression (i.e.
those enabling integration and replication of RTV-resistant
HIV) were enriched by fluorescence-activated cell sorting
(FACS), and the cDNA responsible for rescue of the replication-impaired RTV-resistant RFP reporter virus was extracted
and further characterized.
The WT and PR mutant reporter viruses were constructed so
that the highly sensitive RFP would be expressed independently
of the viral Nef protein in infected cells, and the presence of the
reporter gene did not affect virus particle formation (Fig. 4B).
Bright red fluorescence was detected 1218 h after inoculation
with the WT HIV-RFP reporter virus, and we were able to
reproduce the CD3 and CD28 antibody activation-dependent
rescue of RTV-resistant HIV with the HIV PRI54V/V82A-RFP
virus. The cDNA library was constructed for directional gene
expression under control of the retroviral promoter, and
expressed host factor proteins were preceded by an N-terminal
FLAG tag. The high complexity retrovirus-based cDNA library
was transfected into an amphotropic packaging cell line to generate a high titer transducing virus stock. The recombinant
virus stock was used to transduce nonactivated Jurkat T cells at
a low m.o.i. of 0.1 to limit the number of cDNAs expressed in
individual cells. We achieved 85% transduction efficiency of
the cDNA library in nonactivated Jurkat T cells and confirmed
protein expression using an anti-FLAG antibody.
36
FIGURE 4. Strategy of genetic screen. A, schematic of the genetic screen to identify host factors that rescue RTV-resistant HIV replication. B, Western blot
analysis of WT HIV-RFP (R1) and HIV PRI54V/V82A-RFP (R2) reporter virus proteins compared with the protein profile of WT HIV. Defective cleavage of Gag in the
HIV PRI54V/V82A-RFP reporter virus is indicated by the presence of p25 and p8 proteins. Panels represent individual viral proteins of the Pol polyprotein,
accessory gene products, and surface (gp120) and transmembrane (gp41) Env proteins in mature virus particles. Shown are representative data from one of
three independent experiments. C, Jurkat T cells infected with the HIV PRI54V/V82A-RFP reporter virus were sorted by flow cytometry and gated based on the
intensity of red fluorescence. In the second round of FACS, total RFP cells were gated into two populations, and cells with the highest fluorescence intensity
(0.01 0.1%) were sorted. SSC-A, side scatter area.
RTV-resistant HIV Is Hypersensitive to HSP90AB1 Inhibition

in VitroThe abundant HSP90AB1 chaperone protein mediates conformational maturation of several cellular proteins and
is believed to stabilize deregulated signaling proteins in oncogenesis. We examined the in vitro antiviral activity of 17-(allylamino)-17-demethoxygeldanamycin, a pharmacologic inhibitor of HSP90AB1 (40) that is currently in clinical trials to treat
metastatic solid tumors and lymphomas (41). 17-(Allylamino)17-demethoxygeldanamycin had potent activity against WT
HIV NL4-3 in PHA-activated PBMC, and RTV-resistant HIV
(as predicted by our hypothesis) was hypersensitive to the drug
with a mean IC50 7-fold lower than for WT HIV (0.024 versus 0.17
M, p 0.05 by paired Students t test) (Fig. 6A). We confirmed
that inhibition of HSP90AB1 in activated PBMC does indeed prevent HIV replication by using a cholesterol-conjugated siRNA to
knock down HSP90AB1 expression (Fig. 6B).
D88A mutations into the native HSP90AB1 sequence to prevent ATP binding and hydrolysis (31). Jurkat T cells transduced
with recombinant retrovirus carrying the native and mutant
forms of HSP90AB1 were inoculated with WT and RTV-resistant HIV, and the extent of virus replication was determined in
relation to HIV replication in CD3- and CD28-activated Jurkat
T cells (Fig. 5A). Replication of RTV-resistant HIV in Jurkat T
cells expressing the native HSP90AB1 protein was comparable
with mutant virus replication in CD3- and CD28-activated
cells, whereas the inactivated mutant form of HSP90AB1 failed
to rescue RTV-resistant HIV replication. Overexpression of
HSP90AB1 in Jurkat T cells did not adversely affect replication
of WT HIV, and to eliminate the possible influence of retrovirally
mediated gene transfer on RTV-resistant HIV replication, Jurkat T
cells were simultaneously transduced with a GFP-expressing retroviral vector. Furthermore, we immunoprecipitated the FLAGtagged recombinant proteins from an equal number of transduced
Jurkat T cells to ensure comparable expression of both native and
mutant HSP90AB1 proteins (Fig. 5B).
Cell activation through external stimuli often results in the
overexpression of several cellular proteins, and we accordingly
observed a mean 2-fold increase in HSP90AB1 protein production in CD3- and CD28-activated Jurkat T cells (Fig. 5C). Taken
together, these results suggest that up-regulation of HSP90AB1
is necessary for RTV-resistant HIV replication.
DISCUSSION
The emergence of drug resistance is not always beneficial
to HIV and sometimes comes at the cost of decreased viral
fitness. In the case of RTV-resistant HIV, the PR mutant has
highly impaired infectivity in thymocytes and nonactivated
T cells (1, 6). Nonetheless, we show here that loss of RTVresistant HIV fitness can be rescued in activated T cells,
suggesting that the variation in PR mutant virus replication
37
FIGURE 5. HSP90AB1 can rescue RTV-resistant replication. A, nonactivated Jurkat E6-1 cells transduced with retrovirus vectors expressing native and mutant
HSP90AB1 and GFP were inoculated with WT and RTV-resistant HIV at an m.o.i. of 0.005. Results are expressed as relative infectivity (%) of WT and RTV-resistant
HIV replication in CD3- and CD28-activated Jurkat T cells and represent mean supernatant HIV p24 levels S.E. (error bars) for six independent transduction
experiments. B, immunoprecipitation of recombinant proteins from transduced Jurkat E6-1 cells. Equal volumes of resolved proteins were probed with
anti-FLAG- and anti-HSP90AB1-specific antibodies. Results are representative of three independently immunoprecipitated preparations. C, protein lysate from
nonactivated and CD3- and CD28-activated Jurkat E6-1 cells probed with anti-HSP90AB1- and -actin-specific antibodies. Protein levels were quantified on a
Typhoon Trio imager, and the mean S.E. increase in HSP90AB1 expression in CD3- and CD28-activated Jurkat T cells was 2.0 0.5-fold. Results are
representative of three independently prepared cell lysates.
ant HIV replication. Although defective core stability resulting from uncleaved CA-SP1 is the favored explanation for
impaired infectivity of RTV-resistant HIV, we cannot ignore
the possibility that only half of the total CA molecules
assemble into the viral core. Consequently, RTV-resistant
HIV particles may contain an intact core irrespective of
uncleaved CA-SP1. Nevertheless, we show here that the
presence of uncleaved CA-SP1 in virus particles precludes
reverse transcription of the viral genome in thymocytes and
nonactivated T cells and that replication of RTV-resistant
HIV is arrested during the early postentry stage of the virus
life cycle.
The intent of this study was to identify HIV-uncoating host
factors by exploiting this easily reversible model of RTV-resistant HIV replication. A genetic screen was designed on the premise that RTV-resistant HIV replicates specifically in activated
T cells; therefore, expression of cellular genes from activated T
cells in nonactivated T cells should potentially support RTVresistant HIV replication. The search for host factors, carried
out under stringent conditions, revealed that HSP90AB1 can
rescue RTV-resistant HIV replication.
Identification of HSP90AB1 as a potential uncoating host
factor validated our selection strategy and reveals an exciting
avenue of HIV postentry modification by cellular chaperones. Mammalian HSP90AB1 is one of two cytosolic isoforms in the heat shock protein 90 family that collectively
regulate cell homeostasis by stabilizing a diverse clientele of
proteins (43). Heat shock protein 90 chaperones in particular have been linked to replication of several viruses where
they are essential for picornavirus capsid maturation and
activate the viral polymerase complexes of several RNA
viruses (44). A recent study by Vozzolo et al. (45) demonstrates that heat shock protein 90 interacts with the HIV
is a result of the target cell environment rather than limitations in the PR mutant. In this study, we used the model of
RTV-resistant HIV replication to identify host factors
expressed in activated T cells that rescue impaired replication of the PR mutant.
The temporal cleavage of Gag by HIV PR is critical for
virus maturation, and RTV resistance affects cleavage efficiency of mutant PR specifically at the Gag spacer peptide
junctions. As a result, uncleaved CA-SP1 accumulates in
virus particles and impairs RTV-resistant HIV replication in
human thymocytes and nonactivated T cells. A similar replication defect has been observed with the maturation inhibitor bevirimat, which binds to SP1 and prevents CA lattice
formation (42). Bevirimat causes a dose-dependent inhibition of CA-SP1 cleavage and severely impairs HIV infectivity
in human thymocytes (32). Furthermore, RTV-resistant HIV
is hypersensitive to the maturation inhibitor, indicating that
high levels of uncleaved CA-SP1 in a virus particle adversely
affect its ability to infect target cells.
The short SP1 peptide attached to the C terminus of CA
enables HIV cores to assemble into immature (SP1-present)
or mature (SP1-absent) lattices. The accumulation of uncleaved CA-SP1 in virus particles most likely affects stability
of the HIV core structure, which is rapidly degraded in the
infected cell. Alternatively, uncleaved CA-SP1 subunits
might transform the HIV core into an unusually stable structure that cannot undergo obligatory postentry modifications. However, cellular activation rescues RTV-resistant
HIV replication, suggesting that the PR mutant is infectious
but in need of specific uncoating factors that are underrepresented in human thymocytes and nonactivated T cells. In
contrast, activated cells express unique host factors that
compensate for uncleaved CA-SP1 and rescue RTV-resist38
FIGURE 6. RTV-resistant HIV is hypersensitive to HSP90AB1 inhibition in vitro. A, PHA-activated PBMC inoculated at an m.o.i. of 0.001 were treated with
serial half-log dilutions of 17-(allylamino)-17-demethoxygeldanamycin, and the IC50 was calculated after 7 days. The CC50 of the drug was determined by
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The graph represents one of three independent assays. The mean S.E. IC50 for WT HIV
was 0.17 0.03 M, the mean IC50 for the PR mutant was 0.024 0.001 M, and the mean CC50 was 1.9 0.26 M. B, PHA-activated PBMC inoculated at an m.o.i.
of 0.001 were treated with serial quarter-log dilutions of a cholesterol (chol)-conjugated siRNA duplex against HSP90AB1 (left) and a control scrambled siRNA
(right). The IC50 was calculated after 7 days, and the CC50 of the siRNA was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay.
The graph represents one of three independent assays. The mean S.E. IC50 for WT HIV was 2 0.06 nM, the mean IC50 for the PR mutant was 1.4 0.19 nM,
and the CC50 was 65 nM. No significant inhibition of WT HIV and PR mutant virus replication or cytotoxicity was observed with the cholesterol-conjugated
scrambled siRNA duplex.
the development of drug resistance and are therefore attractive

targets for antiviral therapy.
DNA promoter sequence and regulates viral gene expression. However, this study also suggests that heat shock protein 90 is involved in the early postentry stage of HIV replication. Furthermore, HSP90AB1 is intimately coupled with
the ubiquitin-proteasome pathway, and recent studies indicate that ubiquitin ligases and components of the proteasome play a structural role in HIV uncoating and reverse
transcription (46). Overall, a link between virus evolution
and chaperone dependence has been hypothesized where
cellular chaperones are proposed to counteract the destabilizing effects of viral capsid mutations that continuously
emerge in response to immune surveillance (47). Such a buffering mechanism would allow for greater flexibility in viral
protein structure. If this is the case, then up-regulation of
HSP90AB1 in activated T cells might compensate for the
transdominant negative effects of uncleaved CA-SP1 and
rescue RTV-resistant replication. Then again, the anti-HIV
effect of 17-(allylamino)-17-demethoxygeldanamycin and
the fact that RTV-resistant HIV is hypersensitive to
HSP90AB1 inhibition suggest an association between the
cellular chaperone and HIV core uncoating.
It remains to be determined whether HSP90AB1 directly
influences HIV uncoating or whether this cellular protein regulates other host factors required for viral replication. However, virus-host protein interactions by nature are refractory to
AcknowledgmentsWe thank Mary Beth Moreno, Jose Rivera, Sofiya

Galkina, Barbara Sloan, Valerie Stepps, and David Chung for expert
technical assistance.
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40
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 25, pp. 22250 22261, June 24, 2011
A Chimeric HIV-1 Envelope Glycoprotein Trimer with an

Embedded Granulocyte-Macrophage Colony-stimulating
Factor (GM-CSF) Domain Induces Enhanced Antibody and
T Cell Responses*
Received for publication, February 11, 2011, and in revised form, April 1, 2011 Published, JBC Papers in Press, April 22, 2011, DOI 10.1074/jbc.M111.229625
Thijs van Montfort, Mark Melchers, Gozde Isik, Sergey Menis, Po-Ssu Huang, Katie Matthews,
Elizabeth Michael, Ben Berkhout, William R. Schief, John P. Moore, and Rogier W. Sanders1
From the Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity
Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, 1105 AZ Amsterdam, The Netherlands, the
Department of Biochemistry, University of Washington, Seattle, Washington 98195, and the Department of Microbiology and
Immunology, Weill Medical College of Cornell University, New York, New York 10065
An effective HIV-1 vaccine should ideally induce strong
humoral and cellular immune responses that provide sterilizing
immunity over a prolonged period. Current HIV-1 vaccines
have failed in inducing such immunity. The viral envelope glycoprotein complex (Env) can be targeted by neutralizing antibodies to block infection, but several Env properties limit the
ability to induce an antibody response of sufficient quantity and
quality. We hypothesized that Env immunogenicity could be
improved by embedding an immunostimulatory protein
domain within its sequence. A stabilized Env trimer was therefore engineered with the granulocyte-macrophage colony-stimulating factor (GM-CSF) inserted into the V1V2 domain of
gp120. Probing with neutralizing antibodies showed that both
the Env and GM-CSF components of the chimeric protein were
folded correctly. Furthermore, the embedded GM-CSF domain
was functional as a cytokine in vitro. Mouse immunization studies demonstrated that chimeric EnvGM-CSF enhanced Env-specific antibody and T cell responses compared with wild-type
Env. Collectively, these results show that targeting and activation of immune cells using engineered cytokine domains within
the protein can improve the immunogenicity of Env subunit
vaccines.
Ideally, a vaccine should induce strong humoral and cellular

immune responses that are protective against the extensive
variety of HIV-1 strains that are now circulating. T cell-based
vaccines can partially control viral replication and reduce the
viral set point in animal models (5), but have not yet protected
humans in clinical trials (6, 7). HIV-1 Env2 gp120 subunit vaccines have induced antibody (Ab) responses that could inhibit
neutralization-sensitive laboratory strains in monkeys, but no
protection has been observed against primary HIV-1 strains (8,
9). Monomeric gp120 vaccines in humans also failed to induce
protective Ab responses (10, 11). A recent vaccine trial in Thailand using a recombinant canarypox virus expressing HIV-1
proteins followed by a gp120 protein boost showed a modest
reduction in HIV-1 acquisition (12).
Sterilizing protection can be achieved in nonhuman primates
by passively immunizing with neutralizing Ab (nAb). The infusion of nAb b12 directed against the CD4-binding site (CD4BS)
on gp120 inhibited vaginal infection with SHIV in a dose-dependent manner (13, 14). Other passive immunization studies
using broadly active nAb such as 2F5 or 2G12 also showed
protection against SHIV challenge (1518). Passive immunization with the b12 nAb 218 h after viral challenge did not block
HIV-1 infection, showing that the time window for nAb to provide protection against HIV-1 acquisition is limited (19). Thus,
preexisting nAb can provide sterilizing protection, but inducing them with current vaccines has been unsuccessful.
Formulating antigens in adjuvants can improve their immunogenicity. Commonly used adjuvants that stimulate the innate
immune system include agonists of pattern recognition receptors such as Toll-like receptors, as well as inorganic molecules,
emulsion surfactants, or other carrier molecules that have
immune stimulating properties (20). An alternative, more specific approach is the use of co-stimulatory host-derived proteins that activate specific immune cells. The adjuvant effects of
The HIV-1 pandemic continues to spread worldwide. It

would be desirable to have a protective vaccine, but despite
massive effort over the past 20 years such a vaccine remains
elusive. Live attenuated vaccines have provided the most robust
protection against viral challenge in animal models, but they are
considered unsafe for human use (13). Moreover, the immune
correlates of protection by live attenuated vaccines remain
undefined, hampering the design of safer vaccines (4).
* This work was supported, in whole or in part, by National Institutes of Health

Grants AI36082 and AI45463 (to J. P. M.). This work was also supported by
Grants 2008013 and 2009012 (to R. W. S.) and 2005021 (to B. B.) from the
AIDS Fund (Amsterdam) and by the International AIDS Vaccine Initiative.
1
Recipient of Veni and Vidi fellowships from the Netherlands Organization
for Scientific Research (NWO) and a Mathilde Krim fellowship from the
American Foundation for AIDS Research (amfAR). To whom correspondence should be addressed: Meibergdreef 15, 1100 DE Amsterdam, The
Netherlands. Tel.: 31-20-5665279; Fax: 31-20-6916531; E-mail: r.w.sanders@
amc.uva.nl.
41
The abbreviations used are: Env, envelope glycoprotein complex; Ab, antibody(ies); nAb, neutralizing antibody(ies); HIV, human immunodeficiency
virus; SHIV, simian-human immunodeficiency virus; GMR, GM-CSF receptor; BAFF, B cell activation factor; rhGM-CSF, recombinant human GM-CSF;
CD4i, CD4-induced; bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
Chimeric HIV-1 Env-GM-CSF Enhances Immunity

Research and Reference Reagent Program (ARRRP), Division of
AIDS, NIAID, National Institutes of Health. Monoclonal Ab
(mAb) 2F5 and 2G12 were a gift from Hermann Katinger
through the ARRRP. CD4-IgG2, sCD4, and anti-V3 gp120 mAb
PA-1 were gifts from Bill Olson (Progenics Pharmaceuticals,
Tarrytown, NY). mAb b12 was donated by Dennis Burton (The
Scripps Research Institute, La Jolla, CA). mAb 17b, 48d, 412d,
and 39F were gifts from James Robinson (Tulane University,
New Orleans, LA). Goat D7324 Ab was purchased from Aalto
Bio Reagents, Dublin, Ireland. Peter Kwong and John Mascola
(Vaccine Research Center, Washington) donated VRC01. AntiGM-CSF Ab (clone Ab54429) was purchased from Abcam
(Cambridge, UK). Recombinant human GM-CSF (rhGM-CSF)
was obtained from Schering-Plough (Brussels, Belgium).
Labeled mouse-specific T cell Ab TCR-APC (clone H57-597),
CD3-APC (clone 1452C11), CD4-PerCP (clone RM4-5), and
CD8-PE (clone 53-6.7) were obtained from BD Biosciences.
Mouse ImmunizationsPlasmid DNA amplified in DH5
bacteria was isolated using the EndoFree plasmid giga kit (Qiagen, Venlo, The Netherlands). Five outbred NMRI mice/construct were immunized on days 0, 14, 28, and 42 with 20 g of
DNA in the abdominal dermis using gene gun technology.
Blood samples were obtained on days 0, 14, 28, and 42. Immunizations were carried out by Genovac (Freiburg, Germany) at
the facilities of MfD Diagnostics (Wendelsheim, Germany). All
animals were kept according to DIN EN ISO 9001:2000 standards, the regulations of the German Welfare Act of May 19,
2006 (BGBI I S. 1206), the regulations of European Union
Guidelines 86/609/EWG of November 24, 2006, and the European Agreement of March 18, 1986 for the protection of animal
trials and other scientific studies using vertebrates (Act of
December 11, 1990 (BGBI II S. 1486)). All protocols dealing
with animal manipulations are in accordance with guidelines
published by the Federation of European Animal Science Association and the German Society of Laboratory Animal Science
and are carefully reviewed by the MfD Diagnostics animal care
committee. The study was approved by the Landesuntersuchungsamt (Koblenz, Germany), Permit 23 177-07/A 10-15-001.
Cells293T cells were maintained in Dulbeccos modified
Eagles medium (DMEM; Invitrogen) supplemented with 10%
heat-inactivated fetal calf serum (FCS; HyClone, Perbio, EttenLeur, The Netherlands), MEM nonessential amino acids (0.1
mM; Invitrogen), and penicillin/streptomycin (both at 100
units/ml). TZM-bl cells, which express the HIV-1 receptors
CD4, CCR5, and CXCR4, were cultured and maintained in
DMEM supplemented with 10% FCS, 0.1 mM MEM, and penicillin/streptomycin. These cells contain the luciferase gene
under control of the HIV-1 LTR promoter and were used to
measure HIV-1 neutralization. TF-1 cells, a gift from Paul
Coffer (Universiteit Utrecht), were cultured in RPMI 1640
(Invitrogen) with 10% FCS supplemented with 50 units of
rhGM-CSF/ml (Schering-Plough). Splenic lymphocytes were
maintained in RPMI 1640 (Invitrogen) supplemented with 10%
FBS, HEPES, glutamine, sodium pyruvate, penicillin/streptomycin, MEM, and 2-mercaptoethanol.
Env Production293T cells were transiently transfected (32)
with plasmids expressing recombinant Env using linear polyethylenimine (PEI) (molecular weight 25,000; Polysciences
such co-stimulatory molecules can be increased by covalently

linking them directly to the antigen of choice (2124).
One immune molecule that can serve as an adjuvant in vaccines is the granulocyte-macrophage colony-stimulating factor
(GM-CSF). This cytokine is produced by fibroblasts, activated
T lymphocytes, macrophages, and tumor, endothelial, mesothelial, and epithelial cells and functions as a hematopoietic
growth factor. GM-CSF is secreted during various types of
inflammation to stimulate proliferation and prevents apoptosis
of various immune cells (25), such as antigen-presenting cells
(APCs) (26). Its activity is mediated by the GM-CSF receptor
(GMR) that is composed of a specific -GMR domain and a
commonly shared c subunit that is also a part of the IL-3 and
IL-5 cytokine receptor. Signaling is mediated by the formation
of a hexameric complex consisting of two GM-CSF molecules
and the four receptor subunits. Ultimately, two of these GMCSF-GMR hexamers can dimerize to form a dodecameric complex. Depending on the GM-CSF concentration and the nature
of the ligand-receptor complex, GM-CSF can induce different
downstream signaling cascades. Low picomolar concentrations
of the cytokine promote cell survival, whereas higher GM-CSF
concentrations can stimulate cell proliferation.
Here we investigated whether GM-CSF embedded within
the HIV-1 trimeric gp140 can augment Env-specific immune
responses. The rationale for integrating GM-CSF within the
Env protein was to simultaneously activate immune cells that
are targeted by the antigen, resulting in a selective and specific
augmentation of the antigen-specific immune response. To do
this, the first and second variable loop domain (V1V2) of Env
was replaced by GM-CSF. The resulting chimeric EnvGM-CSF
was folded properly and had GM-CSF activity in vitro. Mice
immunized with the chimeric protein had stronger Env-specific Ab and T cell responses than those given unmodified Env
protein. Embedding the cytokine domains is therefore useful
for enhancing the immunogenicity of HIV-1 Env-based
vaccines.
Plasmid ConstructionThe pPPI4 plasmid (Progenics Pharmaceuticals, Tarrytown, NY) encoding stabilized SOSIP.R6IZ-H8 gp140 (Env) using optimized codons has been described
elsewhere (2731). The Env construct, based on the subtype B,
CCR5 primary isolate JR-FL, is shown in Fig. 1. We also used
plasmids encoding Env coupled to B cell activation factor (EnvBAFF) or CD40 ligand (Env-CD40L). These constructs were
generated by exchanging the C-terminal His tag for a codonoptimized mouse CD40L or BAFF sequence. Codon-optimized
DNA encoding either human GM-CSF (amino acids 26 139)
or mouse GM-CSF (amino acids 26 136) flanked by HindIII
and BmgBI restriction sites was synthesized (Mr. Gene GmbH,
Regensburg, Germany). The V1V2 domain of Env was
exchanged with the GM-CSF sequences using the HindIII and
BmgBI sites. The Env construct containing human GM-CSF
was used for Ab probing and in GM-CSF activity assays,
whereas the constructs containing mouse GM-CSF were used
to immunize mice.
ReagentsDC-SIGN-Fc was purchased from R&D Systems
(Minneapolis, MN). HIV-Ig was obtained through the AIDS
42

Europe GmbH, Eppelheim, Germany) or Lipofectamine 2000
transfection reagent according to the standard manufacturers
protocol (Invitrogen). Briefly, plasmid DNA was diluted in onetenth of the final culture volume of DMEM and mixed with PEI
(0.15 mg/ml final concentration). After incubation for 20 min,
the DNA-PEI mix was added to the cells for 4 h before being
replaced with normal culture medium. Env-containing supernatants were harvested 48 h after transfection and frozen in
aliquots.
gp140 Trimer ELISAEnv trimer ELISAs were performed as
described previously (28, 31). Supernatants containing Histagged Env gp140 proteins were diluted 1:3 in TBS, 10% FCS
and added for 2 h to preblocked nickel-nitrilotriacetic acid-His
Sorb 96-well plates (Qiagen). After three washes using TSM (20
mm Tris, 150 mm NaCl, 1 mm CaCl2, and 2 mm MgCl2), serially diluted polyclonal HIV-Ig, Env-specific mAb, DC-SIGNFc, or CD4-IgG2 in TSM, 5% BSA was then added for 2 h, with
or without 1 g/ml sCD4, followed by three washes with TSM,
0.05% Tween 20. HRP-labeled goat anti-human immunoglobulin G (0.2 g/ml, Jackson ImmunoResearch) was used as the
secondary Ab, and absorption at 450 nm was measured after the
colorimetric reaction was stopped using H2SO4.
gp120 ELISAAnti-gp120 Ab titers were measured by
ELISA (28). In short, Microlon 96-well plates (Greiner Bio-One,
Alphen aan den Rijn, The Netherlands) were coated with antigp120 Ab D7324 (10 g/ml). The plates were washed twice with
TBS, and residual protein-binding sites were blocked with 2%
skim milk powder (Sigma-Aldrich) in TBS for 30 min. gp120
from transiently transfected 293T cells was added to D7324coated wells for 2 h at room temperature. Mice serum serially
diluted in 20% sheep serum (Biotrading, Mijdrecht, Netherlands) and 2% skim milk powder in TBS was applied for 2 h.
gp120-specific mouse IgG was detected with HRP-labeled goat
anti-mouse IgG (Jackson ImmunoResearch) used at 1:5000 (0.2
g/ml) followed by colorimetric detection. End point titers
were calculated using GraphPad Prism, version 5.03, by determining the serum dilution at which the A450 signal was three
times above the A450 background signal obtained without mice
sera.
SDS-PAGE, Blue Native PAGE, and Western BlottingSDSPAGE and Western blotting were performed as described previously (28). Env was detected using the primary mAb, PA-1
(0.2 g/ml), and a secondary HRP-labeled goat anti-mouse IgG
(1:5,000 dilution) followed by Western Lightning ECL solution
(PerkinElmer Life Sciences). Blue native PAGE was carried out
as reported previously (28). Briefly, purified protein samples or
cell culture supernatants were diluted with an equal volume of
a buffer containing 100 mM MOPS, 100 mM Tris with HCL, pH
7.7, 40% glycerol, and 0.1% Coomassie Blue immediately prior
to loading onto a NuPAGE 4 12% bis-Tris gel (Invitrogen).
Typically, gel electrophoresis was performed for 2 h at 150 V
(0.07 A) using 50 mM MOPS, 50 mM Tris, pH 7.7, as the
running buffer.
HIV-1 NeutralizationTZM-bl cells, used to measure
HIV-1 infection, were cultured to 70 80% confluency in a
96-well plate. HIV-1 SF162 or JR-FL (derived from peripheral
blood mononuclear cell supernatants) was mixed and incubated for 1 h with serially diluted pooled sera from five Envwt or
five EnvGM-CSF immunized mice. The target cells were washed

once with PBS prior to the addition of virus (5 ng/ml CA-p24)
with or without mouse sera. The protease inhibitor saquinavir
(400 nM; Hoffmann-La Roche) was added to block replication
together with 40 g/ml DEAE-dextran (Sigma-Aldrich) in a
total volume of 200 l. The medium was removed 2 days postinfection, and the cells were washed once with PBS before lysis
with reporter lysis buffer (Promega, Madison, WI). Luciferase
activity was measured using a luciferase assay kit (Promega) and
a Glomax luminometer according to the manufacturers
instructions (Turner BioSystems, Sunnyvale, CA). All infections were performed in duplicate. The luciferase activity measured in uninfected cells was taken as background and subtracted from the signals derived from the experimental
samples.
V3 Peptide Competition AssayPooled sera from five Envwtor EnvmGM-CSF-immunized mice were incubated with a mix of
three peptides (Env V3-1, NNNTRKSIHIGPGRA; Env V3-2,
SIHIGPGRAFYTTGE; Env V3-3, GRAFYTTGEIIGDIR (30
g/ml each)) overlapping the V3 sequence of HIV-1 JRFL or
with an unrelated peptide (QAPKPRKQ; 90 g/ml) for 1 h at
room temperature. The peptide-serum mixtures were then
tested in the HIV-1 neutralization assay. To control for nonspecific inhibition by the peptides, we also performed neutralization assays using mAb b12 (6 g/ml) in the presence or absence
of the three V3 peptides.
GM-CSF Activity AssayTF-1 cells (50,000 in 50 l) were
seeded in a 96-well plate. Serially diluted supernatants from
Envwt, EnvhGM-CSF, mock transfected 293T cells, or rhGM-CSF
(75 units/ml) diluted in mock transfected medium were added
in quadruplicate. The cells were harvested on day 5, and the
number of living cells was measured by flow cytometry. The
number of living TF-1 cells from wells treated with mock
medium (15,000 cells/75 l) served as a background and was
subtracted from test values.
T Cell ResponsesThe number of splenic T cells from immunized mice was determined by LSR-II flow cytometry (BD Biosciences) using standard cell surface staining protocols. Data
were analyzed using FlowJo software. In short, prior to staining,
Fc receptors were blocked using an anti-mouse CD16/CD32
antibody (clone 2.4G2). Cell surface staining of TCR, CD3,
CD4, and CD8 was carried out in PBS supplemented with 10%
FBS. In vitro restimulation of T cells (CD4 and CD8 combined) in unfractionated splenocyte cultures was carried out by
culturing 5 105 cells/well with JR-FL gp120 (10 g/ml) in a
final volume of 200 l. Stimulation by an anti-CD3e antibody (2
g/ml, BD Biosciences, clone 1452C11; catalog number
553057) served as a positive control and stimulation by culture
medium as a negative control. Supernatants were collected
after 96 h at 37 C in 5% CO2 and stored at 80 C until further
analysis. The concentrations of IL-2, IL-4, IL-5, IL-10, IL-21,
and IFN in the supernatants were measured by a sandwich
ELISA according to the manufacturers instructions (OptEIA
mouse ELISA kits, BD Biosciences) with the use of a 3,3,5,5tetramethylbenzidine substrate kit (BD Biosciences). The assay
sensitivity limits were 3 pg/ml for IL-2 and IL-21, 8 pg/ml for
IL-4, 16 pg/ml for IL-5, and 30 pg/ml for IL-10 and IFN.
43
FIGURE 1. Design of chimeric EnvhGM-CSF. A, linear representation of EnvhGM, EnvV1V2, and EnvGM-CSF. Clade B JR-FL gp140 (amino acids 31 681) contains
several modifications that have been described elsewhere (31). The amino acid sequences of the Env-hGM-CSF junctions are shown. The linker residues are
indicated in italics and GM-CSF residues in bold type. A potential site for N-linked glycosylation is underlined. B, schematic of EnvhGM-CSF compared with Envwt
and EnvV1V2. The V1V2 loop present in Envwt was replaced by amino acids 26 139 of hGM-CSF or amino acids 26 136 of mGM-CSF.
44

Modeling Chimeric gp120hGM-CSF TrimersStructural models for the gp120-hGM-CSF within a trimeric spike were generated as follows. The trimeric configuration of gp120 in an
unliganded spike was obtained by fitting the b12-bound conformation of the gp120 HxBC2 core (PDB ID: 2NY7 (33)) into the
cryoelectron tomography density of unliganded HIV-1 BaL
strain using the program Chimera (34, 35). RosettaDesign (36,
37) was used to thread the sequence of JRFL core gp120 onto
the 2NY7 gp120 structure from residues 83 to 492. The structure of hGM-CSF (PDB ID: 2GMF; chain A, residues 9 122
(38)) was inserted at the V1V2 stem between residues 127 and
195 (gp120 2NY7 numbering) flanked by Gly-Ser-Gly linkers
on both sides. The conformations of the connecting segments
and the rigid body orientation of hGM-CSF relative to the
gp120 trimer were modeled using RosettaRemodel.3 Briefly, the
backbones for the connecting segments (2NY7 residues 112
127 and 195212 plus the GSG linkers) were generated using an
ab initio fragment insertion protocol (39), and cyclic coordinate
descent was used to maintain proper backbone connectivity
(40, 41), and rebuilt segments were further optimized by cyclic
coordinate descent refine (40, 41) and side-chain repacking.
Modeling of hGM-CSF on the gp120 V1V2 stem bound to the
b12 Fab fragment was carried out to determine functional steric
constraints in the model. Disulfide bonds in the V1V2 (2NY7
residues 119 205 and 126 196) were approximated by CC
distance constraints. The V3 loop was left truncated as in 2NY7.
Possible isoforms (1000) of EnvhGM-CSF were generated, and
models (100) with the lowest energy state were further filtered for (a) clash-free binding of b12, CD4 (D1D2) (33), or
VRC01 Fab (42) to the gp120 trimer or (b) maintenance of solvent exposure of the three glycosylation sites in hGM-CSF and
the V1V2 stem. The resulting set of models was grouped into
three classes on the basis of up, down, and side conformation. A
representative model of each class was chosen for the figures.
Statistical AnalysisStatistical significance is indicated in
Figs. 6 and 7 (*, p 0.05; **, p 0.01; *** p 0.001). The specific
statistical tests performed depended on the nature of the experiments and are indicated in the respective figure legends.
FIGURE 2. EnvGM-CSF is expressed as a trimer. Shown are reducing SDS-PAGE

(upper panel) and Blue native PAGE (lower panel) analyses of Envwt, EnvV1V2,
and EnvGM-CSF proteins secreted from transiently transfected 293T cells.
Recombinant purified JR-FL gp120 (50 ng) was included for comparison.
Chimeric EnvhGM-CSF Is Expressed Efficiently and Forms

TrimersTo determine whether the chimeric EnvhGM-CSF protein was produced and folded properly, we transiently transfected 293T cells with gp140 (Envwt), gp140 lacking the V1V2
domain (EnvV1V2) (31), and EnvhGM-CSF. The expression of all
constructs was determined using SDS-PAGE and Western
blotting (Fig. 2, upper panel). The expression of EnvhGM-CSF
was slightly lower than Envwt. The EnvhGM-CSF protein had an
apparent molecular mass of 150 kDa, Envwt of 140 kDa, and
EnvV1V2 of 120 kDa, consistent with the expected sizes (Fig. 2,
upper panel). The Envwt, EnvV1V2, and EnvhGM-CSF proteins
were all predominantly expressed as trimers when analyzed by
Blue native PAGE (Fig. 2, lower panel), showing that replacing
the V1V2 domain with hGM-CSF did not markedly affect
trimer formation or stability.
Chimeric EnvhGM-CSF Is Recognized by Neutralizing AntibodiesTo determine whether EnvhGM-CSF had an antigenic
structure comparable with Envwt, we used a trimer ELISA and
assessed its binding to a panel of Ab and receptor mimics (28).
Polyclonal Ig from pooled HIV-positive patient sera (HIV-Ig)
bound similarly to Envwt, EnvhGM-CSF, and EnvV1V2 (Fig. 3A).
The 2F5 mAb, directed to the gp41 epitope located far from the
GM-CSF insertion, also bound identically to the three proteins
(Fig. 3A). Similar findings were made using the human DCSIGN-Fc protein and the 2G12 mAb, which both recognize
oligomannose N-glycans on gp120 (Fig. 3B).
We then tested the conformation of Env epitopes closer to
the GM-CSF insertion. V3 mAb 39F recognized the three Env
proteins equally well, as did CD4-IgG2, a receptor mimic for
CD4 (Fig. 3C). nAb VRC01 and b12 against discontinuous
epitopes associated with the CD4-binding site (CD4BS) recognized EnvhGM-CSF (Fig. 3C), but the binding was subtly less efficient compared with that to Envwt (by 2-fold for VRC01 and
4-fold for b12). Thus, the CD4BS on EnvhGM-CSF is intact, but
the accessibility and/or conformation of the b12 and, to a lesser
RESULTS
Design of an HIV-1 Env Trimer with an Embedded GM-CSF
DomainTo generate a trimeric HIV-1 Env immunogen that
could be targeted to immune cells and simultaneously stimulate
immune activation, we deleted the V1V2 domain of gp120 and
replaced it with almost the complete sequence of the GM-CSF
cytokine. We used the stabilized SOSIP.R6-IZ gp140 protein
backbone, hereafter called Env (2730). The mouse or human
GM-CSF (mGM-CSF or hGM-CSF) sequence was inserted
after the second cysteine bridge in the V1V2 stem between
amino acids 127 and 195. To facilitate flexibility at the junctions
between GM-CSF and Env, Gly-Ser-Gly linkers were added to
the N and C termini of the GM-CSF sequence (Fig. 1, A and B).
In total, 120 and 116 amino acids were introduced for hGMCSF and mGM-CSF, respectively, at the expense of 65 amino
acids of the V1V2 domain.
P. S. Huang and W. R. Schief, manuscript in preparation.

45
FIGURE 3. Chimeric EnvhGM-CSF is recognized by conformational Env Ab and receptor mimics. ELISA analysis of the binding of Env variants to: HIV-Ig and
2F5 (A); glycan-dependent DC-SIGN-Fc and 2G12 (B); 39F (V3), CD4-IgG2, b12, and VRC01 (CD4BS) (C); and 17b, 48d, and 412d (CD4i) in the absence and
presence of soluble CD4 (sCD4) (D). Culture supernatant from mock transfected 293T cells was used as a negative control.
extent, VRC01 epitopes is subtly altered by the replacement of

the V1V2 domain by GM-CSF.
CD4 binding induces a conformational change in Env
involving the rearrangement of the V1V2 domain, which
exposes the coreceptor binding site and the overlapping
CD4-induced (CD4i) epitopes. In the absence of CD4, the
CD4i epitope mAb 17b, 48d, and 412d bound poorly to Envwt
and EnvhGM-CSF but efficiently to EnvV1V2, as expected (Fig.
3D, left panels). Adding soluble CD4 substantially increased
the binding of these mAb to EnvV1V2 and especially to
Envwt, but binding to EnvhGM-CSF was improved only modestly (Fig. 3D, right panels). Hence the presence of GM-CSF in
the V1V2 region either limits the accessibility of the CD4i epitopes
or blocks the conformational changes that expose them. Collectively, the Env conformational analyses show that the overall antigenic structure of EnvhGM-CSF is similar to that of Envwt, but some
epitopes located close to the insertion site are affected by the GMCSF insertion.
The GM-CSF Domain of Chimeric EnvhGM-CSF Is Functional

To probe the integrity of the GM-CSF domain, we performed
an ELISA using a conformation-dependent neutralizing GMCSF mAb (Fig. 4A). The EnvhGM-CSF protein was efficiently
recognized by this mAb, whereas Envwt and EnvV1V2 were not.
Hence the GM-CSF domain embedded within the Env backbone assumes a similar conformation as native GM-CSF.
The functional activity of the inserted GM-CSF domain was
tested using TF-1 cells, which require GM-CSF signaling for
proliferation. The number of living TF-1 cells was measured by
flow cytometry after a 5-day culture with 293T cell supernatant
containing the EnvhGM-CSF, Envwt, or EnvV1V2 protein. Supernatant from untransfected 293T cells was used as a negative
control, and rhGM-CSF protein diluted in 293T culture
medium was used as a positive control. TF-1 cells exposed to
EnvhGM-CSF medium proliferated in a dose-dependent manner,
as did cells stimulated with authentic rhGM-CSF (Fig. 4B). In
contrast, apoptosis was observed in TF-1 cell cultures treated
46

with Envwt, EnvV1V2, or the mock medium. The hGM-CSF
protein tethered to the Env backbone was not as potent as soluble rhGM-CSF at stimulating TF-1 cell proliferation. The
maximal response of EnvhGM-CSF, extrapolated from the curve

trend, was calculated at 32,000 living cells. rhGM-CSF had a
maximal response of 41,000 living TF-1 cells. Based on the
half-maximal response levels for the secreted EnvGM-CSF and
the authentic rhGM-CSF, we estimated the GM-CSF activity
of EnvhGM-CSF. The half-maximal stimulation response of
EnvhGM-CSF-containing supernatant was reached at a 3-fold
dilution, whereas the half-maximal response to rhGM-CSF
occurred at 1.9 units/ml. EnvhGM-CSF was therefore estimated to have a GM-CSF activity equivalent to 5.7
units/ml.
Model of an EnvhGM-CSF TrimerBased on the atomic structure of hGM-CSF (43), the structures of gp120 in complex with
b12, CD4 (33), or VRC01 (42), a gp120 trimer model (44), and
additional information provided by the antigenicity and functional data (Figs. 3 4), we generated a structural model of the
EnvhGM-CSF trimer. Because of the flexibility of the linkers, GMCSF can be modeled on the gp120 core in multiple orientations.
Three symmetric EnvhGM-CSF trimer views (down, side, and
up) are depicted in Fig. 5 to illustrate the diversity of potential low energy conformations. We note that hGM-CSF will
likely sample a range of conformations around and between
the down, side, and up conformations and that the orientation of GM-CSF may not be symmetrical in the three gp120
subunits. All of the models in Fig. 5 are compatible with
VRC01, b12, and CD4 binding. The model with hGM-CSF in
the upper position may be the most accurate, because in that
FIGURE 4. The GM-CSF domain of chimeric EnvhGM-CSF is functional.

A, ELISA analysis of the binding of a neutralizing, conformation-dependent
anti-hGM-CSF Ab. B, proliferation of TF-1 cells in response to culture supernatant containing EnvGM-CSF, Envwt, or rhGM-CSF. The numbers of cells present
after 5 days of culture in a volume of 75 l are given. Culture supernatant from
mock transfected 293T cells was used as a negative control (15,000 cells)
and deducted from the test values.
FIGURE 5. Three different models of EnvhGM-CSF trimers. hGM-CSF (magenta) is shown in the up (top panels), side (middle panels), and down (lower panels)
orientation attached to gp120 (cyan). The models were generated using Chimera (35), RosettaDesign (36), and RosettaRemodel as described under Experimental Procedures and rendered using PyMOL (version 1.3, Schrodinger, LLC).
47

EnvmGM-CSF Induces Enhanced Env-specific Ab Responses in
MiceTo test whether incorporating the GM-CSF protein
within Env would improve Env-specific immune responses, we
immunized mice with plasmids expressing Env, with or without
embedded murine GM-CSF, according to the schedule presented in Fig. 6A. In addition we immunized mice with plasmids encoding the Env protein (again, with or without embedded mGM-CSF) coupled to mouse BAFF (mBAFF) or mouse
CD40L (mCD40L). BAFF and CD40L are co-stimulatory molecules linked C-terminally to the Env to provide an additional
stimulus for immune activation. The mice were immunized on
days 0, 14, 28, and 42, and the gp120-specific IgG response was
measured by ELISA. The gp120-specific Ab titer for mice immunized with plasmids encoding Env-mBAFF or Env-mCD40L was
not significantly greater than found with Env (Students t test;
Fig. 6B). Thus, the Env-mBAFF and Env-mCD40L constructs
did not improve the immune response against Env whether
mGM-CSF was present or not. To increase the statistical power
of subsequent analyses, we pooled all of the mice groups that
received Env and all of the groups given Env containing embedded mGM-CSF, irrespective of the additional presence of
mBAFF or mCD40L sequences (Fig. 6C). Anti-Env Ab were
detected at low levels after the first boost on day 14. The end
point titers from Env- and EnvmGM-CSF-immunized mice were
significantly different after the second boost on day 28. The
corresponding titers for the EnvmGM-CSF group were 3-fold
higher than for the Env control group (471 versus 174, p 0.03).
The titer difference between Env and EnvmGM-CSF further
increased at day 42 (1357 versus 4918, p 0.0002). Collectively,
inserting GM-CSF within an Env immunogen enhanced the Ab
response to the Env component of the chimeric protein.
EnvmGM-CSF Induces Improved Env-specific T Helper
ResponsesGM-CSF is known to activate a variety of cells, but
not B cells, implying that the enhanced Ab response was probably mediated indirectly through other cell types. We therefore
investigated the Env-specific T cell response in spleen cells
from mice immunized with Env or EnvmGM-CSF, focusing on
the induction of cytokines that are important for B cell
responses. Spleens were harvested on day 56, and their cytokine
production profile was measured by restimulating the splenocytes with gp120 protein or anti-CD3 compared with a mock
stimulation.
gp120-induced IL-2 secretion was 37 pg/ml for Env-immunized mice and 54 pg/ml for EnvmGM-CSF mice (p 0.06) (Fig.
7A). IL-2 secretion in response to anti-CD3 Ab could not be
detected (Fig. 7B). IL-4 production by splenic T cells from the
EnvmGM-CSF group was 6-fold higher than the Env group (41
versus 7 pg/ml, p 0.0001; Fig. 7A), and IL-4 secretion induced
by the anti-CD3 Ab was also higher (147 versus 75 pg/ml, p
0.02; Fig. 7B). Env-specific IL-5 production was similarly
increased in the EnvmGM-CSF group compared with the Env
group (45 versus 14 pg/ml, p 0.02), whereas IL-5 secretion in
response to the anti-CD3 Ab was comparable. We could not
detect Env-specific IL-21 production, but production of this
cytokine in response to the anti-CD3 mAb was significantly
higher in the EnvGM-CSF-immunized mice than in the Env
group (63 versus 23 pg/ml, p 0.003). There were no differences in IFN or IL-10 production between the Env and
FIGURE 6. EnvmGM-CSF induces better anti-Env Ab responses in mice. A,

mouse immunization scheme. B, end point anti-gp120 IgG titers obtained
from mice immunized with different Env immunogens at day 42 as determined by ELISA. C, end point anti-gp120 IgG titers over the course of the
immunization experiment as determined by ELISA. The individual end
point titer is shown for each mouse. Note that both comparison groups
(Env versus Env containing embedded mGM-CSF) contain 15 mice. Within
these groups of 15, five mice were immunized with Env, Env-BAFF, or
Env-CD40L or, for comparison, with EnvGM-CSF, EnvGM-CSF-BAFF, or EnvGMCSF-CD40L. The mean end point titers of the 15 mice/group are shown
S.E. Statistical significance was determined using a one-tailed MannWhitney test, and significant p values are represented with asterisks: *, p
0.05; ***, p 0.001.
orientation the accessibility of the CD4i epitope is restricted

by GM-CSF, as observed with the CD4i Ab-binding data
(Fig. 3).
48
FIGURE 7. EnvmGM-CSF induces enhanced Env-specific T helper responses. Splenocytes were incubated with control media, gp120, or anti-CD3 stimuli for
96 h in vitro. Release of cytokines in the supernatant of gp120 (A)- or anti-CD3 (B)-stimulated splenocytes was measured. Cytokine responses from splenocytes
treated with media provided background values that were subtracted from the values obtained with anti-CD3 or gp120 stimulation. Statistical significance was
determined using a two-tailed Students t test, and significant p values are represented with asterisks: *, p 0.05; **, p 0.01; ***, p 0.001.
EnvmGM-CSF immunized mice in response to either gp120 or

anti-CD3 mAb restimulation.
Collectively, these results indicate that EnvmGM-CSF is better
than Envwt at inducing Env-specific T cell responses to supply T
help for B cells. The cytokine data are thus consistent with the
enhanced Ab responses.
EnvmGM-CSF Induces Enhanced Neutralizing Ab Responses
The mouse model is not an ideal system for monitoring the
induction of nAb responses against HIV-1 (45). Nonetheless,
we tested whether the sera from mice immunized with Envwt or
EnvmGM-CSF DNA constructs contained Ab with neutralizing
activity against HIV-1. Day 42 sera from 5 mice of each of the
Envwt and EnvmGM-CSF groups were pooled and tested in a single-cycle neutralization assay using TZM-bl reporter cells. Ab
neutralization was tested on the highly sensitive tier 1 isolate,
SF162, and the more resistant tier 2 isolate, JR-FL. The pooled sera
had no neutralization activity against JR-FL (data not shown).
Sera from both groups of mice were active against SF162, but the
50% titer was significantly higher for the pooled EnvmGM-CSF sera
(368 versus 110, p 0.0001; Fig. 8A). Although neutralization of
the sensitive SF162 has little relevance to the development of a
useful vaccine, the results confirm the outcome of the Ab assays
and show that replacing the V1V2 domain with GM-CSF
improves the Ab response to Env.
Neutralization of SF162 by immune sera is predominantly
mediated by V3-specific Ab (46). To assess whether EnvmGM-CSFimmunized mice also predominantly generate V3-directed nAb,
we performed a V3 peptide competition assay. Mice sera of day 42
were preincubated with an excess of three overlapping V3 peptides
to inhibit the neutralization by V3-directed nAb (Fig. 8B). Envwt
sera inhibited SF162 infection by 51%, and preincubation with V3

peptides restored infection to 100%, whereas the addition of an
unrelated control peptide did not restore infection. These results
show that SF162 neutralization by Envwt sera was mediated exclusively by anti-V3 specificities. In contrast EnvmGM-CSF sera
reduced SF162 infection by 72%, and adding V3 peptides only partially reversed the inhibition (to 59%), implying that nAb with
specificities other than V3 were present. As a control, we showed
that neutralization of SF162 by nAb b12 against the CD4BS was
not affected by the addition of the V3 peptides (Fig. 8B). Thus,
replacing the V1V2 domain with GM-CSF may divert the Ab
response away from V3 and toward other epitopes on Env that
may be more relevant to broadly active neutralization.
DISCUSSION
To improve the immunogenicity of HIV-1 Env vaccines, we
constructed a chimeric gp140 trimer in which the V1V2 region
of gp120 was replaced by the GM-CSF cytokine. We selected
GM-CSF because it has a well defined adjuvant activity, and
usage in humans has been shown to be safe. We found that the
chimeric EnvGM-CSF protein was more immunogenic than the
unmodified Env trimers in mice, with improvements in both Ab
and T helper responses.
Adjuvants can be more powerful when they are coupled
directly to antigen rather than when the two components are
supplied as a mixture. For example, conjugating oligodeoxynucleotides (CpG ODN) to a Gag protein enhances the magnitude and quality of Gag-specific T cell responses (21). In earlier
studies, the immune response was improved by linking HIV-1
Env antigens directly to co-stimulatory molecules such as C3d,
49

Although GM-CSF embedded into Env was functional, it did
not stimulate TF-1 cell proliferation to the same level as recombinant, soluble GM-CSF (Fig. 4). It is possible that, despite the
use of flexible linkers, GM-CSF binding to its receptor is partially blocked by proximal regions of Env. Alternatively, one
GM-CSF molecule may be able to bind to the GMR normally,
but the formation of a ternary dodecameric complex is affected,
limiting downstream signaling (5153). In theory, the binding
of Env to CD4 on TF-1 cells could counteract the proliferative
activity of GM-CSF (54). This explanation, however, seems
unlikely, because the addition of Envwt did not affect the activation of TF-1 cells by rhGM-CSF (data not shown).
The serum half-life of recombinant GM-CSF is short, varying
from 0.9 to 2.5 h (55, 56). This factor could adversely influence
the adjuvant effect of GM-CSF, particularly when the immunogen and cytokine are either mixed or transcribed from separate
plasmids. How fusion of Env with GM-CSF affects the half-life
of both proteins requires further study.
Chimeric proteins such as those described here have not
been described previously, but GM-CSF has been used as an
adjuvant in vaccine studies with a variety of results. Co-delivering GM-CSF in peptide-based vaccines, either as recombinant
protein or plasmid DNA, augments HIV-1-specific T cell
responses to various extents (5759). Co-delivery of rhesus
macaque GM-CSF expressed from DNA with Gag, Pol, and Env
DNA followed by an MVA boost results in higher avidity antiEnv Ab in rhesus macaques (60). Mice immunized with a bicistronic plasmid encoding gp120 and GM-CSF have stronger
gp120-specific CD4 T cell responses than mice immunized
with gp120 and GM-CSF on two separate plasmids (61). Hence,
the method of GM-CSF co-delivery affects the outcome of the
immune response. The timing of GM-CSF co-delivery also
influences the induction of the response. In one study, delivery
of a GM-CSF plasmid prior to vaccination with a plasmid
encoding a vaccine antigen yielded a Th2-type response,
whereas GM-CSF delivery after vaccination with the same
antigen resulted in a Th1-type response. Both Th1 and Th2
responses are generated when GM-CSF and antigen are administered simultaneously (62). Although Ab to recombinant
human GM-CSF protein (rhGM-CSF) arose when it was used
in a human vaccine trial, they caused neither adverse hematologic events nor a loss of GM-CSF function (57).
We initially chose to introduce GM-CSF as a co-stimulatory
molecule, but other molecules could be incorporated as well. A
number of cytokines can increase the immunogenicity of HIV
vaccines. Co-administration of murine IL-12 with HIV-1 Env
pDNA markedly enhances the cell-mediated immune response
in mice (59). Other cytokines such as IL-2, IL-4, IL-6, IL-10,
IL-15, and IL-18 can augment the humoral and/or cell-mediated immune responses to HIV and non-HIV antigens (63 68).
However, some limitations need to be recognized when
incorporating cytokines into the Env backbone. First, the length
and molecular weight of the inserted protein should probably
be similar to the replaced V1V2 region, which spans 60 to 80
amino acids, including 6 glycans (in total 20 kDa). Moreover,
the N and C termini of the folded cytokine preferably should be
close together such that the bulk of the protein protrudes away
from the Env protein. Another constraint is the oligomeric state
FIGURE 8. EnvmGM-CSF induces enhanced neutralizing Ab responses that

are partially dependent on V3. A, the percentage of SF162 infectivity in the
absence or presence of serial dilutions of pooled sera from the Envwt or
EnvmGM-CSF mice was measured in a single-cycle infectivity assay using
TZM-bl cells. Significance was determined using a two-way analysis of variance test. B, the percentage SF162 infectivity in the presence of pooled sera
diluted 20 was measured in the absence or presence of a mixture of three
overlapping V3 peptides or of an unrelated peptide. To assess whether the V3
peptide pool had nonspecific effects, we measured whether they affected
neutralization by b12.
tumor necrosis factor (TNF), Fms-like tyrosine kinase receptor-3 ligand, or Fas ligand (FasL) (23, 24, 47 49). The advantage
of chimeric adjuvant fusion proteins over chemically linked
adjuvant antigens complexes is that the former can be produced easily directly from DNA at a constant antigen to adjuvant ratio.
Instead of linking the co-stimulatory adjuvant molecules
such as FasL or TNF to the N or C termini of Env, we replaced
the V1V2 loop domain with the co-stimulatory GM-CSF molecule. The V1V2 loop was selected because this region can be
deleted from Env without compromising the structure and, to
some extent, the function of the protein (31, 50). Flexible linkers were added at the Env-GM-CSF junctions to enable the
cytokine to fold independently into a native structure and to
reduce the possibility of a steric clash between the two components of the chimera. The chimeric EnvGM-CSF protein was
expressed efficiently in 293T cells as a secreted trimer (Fig. 2).
Ab binding to most Env epitopes was very similar to that seen
with the corresponding, unmodified Env, with the exception of
the b12 and CD4i epitopes close to the GM-CSF insertion site,
which were affected modestly (Fig. 3).
50

of the cytokine; it is unlikely that dimeric molecules, such as
IL-10 or IL-12 (69, 70), could be embedded as a single functional unit within the trimeric Env complex. Taking these constraints into account, we have recently succeeded in replacing
the V1V2 region with a functional human IL-4.4
In conclusion, we have shown that embedding a cytokine
within an HIV-1 Env trimer improves the immunogenicity of
the Env moiety. This approach may be useful when considering
how to develop an HIV-1 vaccine aimed at inducing protective
humoral immunity.
16.
17.
18.
19.
20.
21.
AcknowledgmentsWe are grateful to Stephan Heynen for technical

assistance and to William Olson, Dennis Burton, James Robinson,
Paul Coffer, John Mascola, and Peter Kwong for reagents.
22.
23.
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52
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 27, pp. 2386523876, July 8, 2011
Identification of Interactions in the E1E2 Heterodimer of

Hepatitis C Virus Important for Cell Entry*
Received for publication, December 18, 2010, and in revised form, April 27, 2011 Published, JBC Papers in Press, May 9, 2011, DOI 10.1074/jbc.M110.213942
Guillemette Maurin, Judith Fresquet, Ophelia Granio, Czeslaw Wychowski, Francois-Loc Cosset1,2,
and Dimitri Lavillette1,3
From the Universite de Lyon, UCB-Lyon1, IFR128, INSERM, U758, and Ecole Normale Superieure de Lyon, Lyon F-69007 and
Molecular and Cellular Virology of Hepatitis C, Center for Infection and Immunity of Lille Inserm U1019, CNRS UMR8204, Universite
Lille Nord de France, Institut Pasteur de Lille, Lille F-59021, France
Several conserved domains critical for E1E2 assembly and
hepatitis C virus entry have been identified in E1 and E2 envelope glycoproteins. However, the role of less conserved domains
involved in cross-talk between either glycoprotein must be
defined to fully understand how E1E2 undergoes conformational changes during cell entry. To characterize such domains
and to identify their functional partners, we analyzed a set of
intergenotypic E1E2 heterodimers derived from E1 and E2 of
different genotypes. The infectivity of virions indicated that
Con1 E1 did not form functional heterodimers when associated
with E2 from H77. Biochemical analyses demonstrated that the
reduced infectivity was not related to alteration of conformation
and incorporation of Con1 E1/H77 E2 heterodimers but rather
to cell entry defects. Thus, we generated chimeric E1E2 glycoproteins by exchanging different domains of each protein in
order to restore functional heterodimers. We found that both
the ectodomain and transmembrane domain of E1 influenced
infectivity. Site-directed mutagenesis highlighted the role of
amino acids 359, 373, and 375 in transmembrane domain in
entry. In addition, we identified one domain involved in entry
within the N-terminal part of E1, and we isolated a motif at
position 219 that is critical for H77 function. Interestingly, using
additional chimeric E1E2 complexes harboring substitutions in
this motif, we found that the transmembrane domain of E1 acts
as a partner of this motif. Therefore, we characterized domains
of E1 and E2 that have co-evolved inside a given genotype to
optimize their interactions and allow efficient entry.
virus that belongs to the Hepacivirus genus of the Flaviviridae

family (1). The two surface glycoproteins, E1 and E2, are processed by signal peptidases of the endoplasmic reticulum from
a 3000-amino acid-long polyprotein encoded by the HCV
genome (2).
Because of difficulties in propagating HCV in cell culture,
many gaps remain in our understanding of the functions of E1
and E2. A major advance in the investigation of their functions
was the development of HCV pseudoparticles (HCVpp) consisting of native HCV envelope glycoproteins E1 and E2 assembled onto retroviral core particles (35). Extensive characterization of HCVpp showed that they mimic the early steps of the
HCV life cycle (6, 7). Furthermore, data obtained with HCVpp
can now also be confirmed with the developed cell culture system that allows efficient amplification of HCV (HCVcc) (8 10).
The E1 (31 kDa) and E2 (70 kDa) proteins are glycosylated in
their large N-terminal ectodomains and are anchored into the
membrane by their C-terminal transmembrane domains. E1
and E2 form a heterodimer stabilized by noncovalent interactions that is retained in the endoplasmic reticulum (11). This
oligomer was thought for a long time to be the prebudding form
of the functional complex (12), which is present at the surface of
HCV particles (13) and is involved in viral entry. Recent investigation of the E1E2 complex incorporated into HCVcc challenges this notion by proving the existence of large high molecular weight complexes stabilized by disulfide bridges (14).
HCV E2 is responsible for virion attachment to target cells
and can bind different receptors including several capture molecules, the CD81 tetraspanin, and the scavenger receptor BI
(for review, see Refs. 6 and 7). Recently, a three-dimensional
structural model of E2 has been proposed as a class II fusion
protein (15) based on the determination of its disulfide bonds
which suggested that it can act alone to mediate binding and
membrane fusion. However, both E1 and E2 appear to possess
domains implicated in fusion (16 19). Moreover, several antibodies directed against E1 are able to neutralize cell entry, presumably at a stage distinct from receptor binding (20 22).
Therefore, the role of E1 in HCV infection remains unclear.
The two transmembrane domains of the E1E2 heterodimer
were shown to be important for different functions and interactions between the two glycoproteins. Studies of mutations
occurring in conserved regions and analyses using cross-neutralizing antibodies have shown that these domains are
involved in ER retention, heterodimerization of E1E2 on the
Hepatitis C virus (HCV)4 is an important public health concern worldwide, as it is a major cause of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. HCV is an enveloped
* This work was supported by Agence Nationale pour la Recherche contre le

SIDA et les Hepatites Virales, the FINOVI foundation, and by the European
Research Council (ERC-2008-AdG-233130-HEPCENT).
1
Both authors contributed equally to this work.
2
To whom correspondence may be addressed: Enveloppes Virales et Ingenierie des Retrovirus, Unite de Virologie Humaine, Inserm U758, ENS de
Lyon, 46 allee dItalie, 69364 Lyon Cedex 07, France. Tel.: 33-4-72-72-87-26;
Fax: 33-472-72-81-37; E-mail: Flcosset@ens-lyon.fr.
3
To whom correspondence may be addressed: Enveloppes Virales et Ingenierie des Retrovirus, Unite de Virologie Humaine, Inserm U758, ENS de
Lyon, 46 allee dItalie, 69364 Lyon Cedex 07, France. Tel.: 33-4-72-72-87-26;
Fax: 33-472-72-81-37; E-mail: Dimitri.lavillette@ens-lyon.fr.
4
The abbreviations used are: HCV, hepatitis C virus; tmd, transmembrane
domain; HCVpp, HCV pseudoparticles; HCVcc, cell culture produced HCV;
FFU, focus forming unit.
53
E1 Domains Interplay during HCV Cell Entry

blocked in Tris-buffered saline (1 M, pH 7.4) with 5% milk powder and 0.1% Tween 20 (34). The blots were probed with appropriate primary and secondary antibodies (1:10,000-diluted
horseradish peroxidase-conjugated anti-mouse or anti-goat;
Dako) in Tris-buffered saline, 5% milk, 0.1% Tween 20. Bound
enzyme-labeled antibody was visualized using an enhanced
chemiluminescence kit (SuperSignal West Pico chemiluminescent substrate; Pierce). To perform immunoprecipitation
assays, the pelleted virions were lysed in immunoprecipitation
buffer (20 mM Hepes, pH 7.5, 1 mM EGTA, 1 mM EDTA, 150 mM
NaCl, and 1% Triton X-100), and the medium containing
HCVpp was precleared by overnight incubation with a 1:1 mixture of protein A- and protein G-Sepharose beads (Amersham
Biosciences) at 4 C. After a centrifugation at 13,000 g at 4 C
for 5 min, the supernatants were incubated with the conformation-dependent anti-E2 monoclonal antibody AR3A (35) for 2 h
at 4 C, and the immune complexes were precipitated using a
1:1 mixture of protein A- and protein G-Sepharose beads for 1h
at 4 C. The complexes were washed three times with immunoprecipitation buffer (50 mM NaCl, 50 mM Tris, pH 7.5, 10 mM
EDTA) and analyzed by SDS-polyacrylamide gel electrophoresis followed by Western blot using anti-E1 (IGH204) and
anti-E2 (H52) antibodies.
Infection AssaySupernatants containing HCVpp were harvested 36 h after transfection and filtered through 0.45-mpore-size membranes. Huh-7 target cells (4 104 cells/well in
24-well plates) were incubated with different dilutions of
HCVpp harboring the chimeric glycoproteins for 4 h at 37 C.
Supernatants were removed, and cells were incubated in complete medium for 72 h at 37 C. Cells were detached and analyzed by FACS Canto II (BD Biosciences) for GFP expression.
CD81 Pulldown AssaysE1E2-transfected cells were lysed in
pulldown lysis buffer (50 mM Tris HCl, pH 7.4, 150 mM NaCl, 20
mM Imidazole, 2 mM Tris(2-carboxyethyl)phosphine, and 1%
Triton X-100). Cell lysates were incubated or not with a recombinant protein containing the large extracellular loop of human
CD81 fused to a His6 tag (soluble CD81-LEL-His6) overnight at
4 C. Then, with nickel-nitrilotriacetic acid magnetic agarose
beads and the BioSprint 15 Work station of Qiagen, the lysates
were washed with NPI-20-T buffer (50 mM NaH2PO4, 300 mM
NaCl, 20 mM imidazole, 0.05% Tween 20, pH 8) and eluted with
a NPI-250-T buffer (50 mM NaH2PO4, 300 mM NaCl, 250 mM
imidazole, 0.05% Tween 20, pH 8). Eluted samples were incubated with non-denaturing buffer and analyzed by SDS-polyacrylamide gel electrophoresis followed by Western blot using
anti-E1 (IGH204), anti-E2 (H52), and anti-CD81 (JS81, BD Biosciences) antibodies.
Cell-Cell Fusion Assay293T donor cells (2.5 105 cells/
well seeded in six-well tissue culture dishes 24 h before transfection) were cotransfected using calcium phosphate reagent
with 10 ng of E1E2 chimeric heterodimers and 20 ng of an
HIV-1 long terminal repeat (LTR) luciferase reporter plasmid
(a kind gift of Francoise Bex, Institut de Recherches Microbiologiques Jean-Marie Wiame) (36). As a negative control, cells
were cotransfected with 10 ng of empty phCMV plasmid and 20
ng of the HIV-1-LTR-luciferase reporter plasmid. Twelve
hours later, transfected cells were detached with Versene (0.53
mM EDTA; Invitrogen) and reseeded at the same concentration
surface of the viral particles, and even fusion between viral and
cellular membranes (2328).
The aim of this study was to characterize interactions
between E1 and E2 and the cross-talk between these domains
for conformational changes during entry. We assume that such
domains of E1 and E2 will have co-evolved inside a given genotype to optimize their interactions and allow efficient entry. In
this report we identified non optimal intergenotypic heterodimers that we used to identify less conserved domains
involved in E1E2 interactions. We focused on E1E2 intergenotypic heterodimers between H77 (gt1a) and Con1 (gt1b) strains,
and we generated chimeras in E1 by substituting H77 for Con1
sequences and vice versa to restore optimal entry function. We
discovered that both the ectodomain and transmembrane domain
are involved in the cross-talk, taking part during the conformational changes required for entry. Interestingly we show that
the N terminus of E1, more precisely the AIL motif, and the
transmembrane of E1 H77 need to be homogenous, which is to
say from the same strain, to achieve optimal entry. This interaction is crucial for the entry of H77/JFH1 HCVcc chimera and
seems to be genotype-dependent, as these interactions are not
crucial for Con1. Thus, the specific interactions between E1 and
E2 vary between strains.
Cell LinesHuh-7 (29), Huh7.5 (30), and 293T (ATCC
CRL-1573) cells were grown in Dulbeccos modified Eagles
medium (Invitrogen) supplemented with 10% fetal bovine
serum (Perbio), 50 IU/ml penicillin, and 50 g/ml streptomycin
(Invitrogen).
Production of HCVppAll chimeric E1E2 heterodimers have
been constructed by PCR and/or digestion between genotype
1a strain H77 (31) (GenBankTM accession number AF009606)
and 1b strain Con1 (32) (GenBankTM accession number
AJ238799). All mutants were verified by sequencing. For infection assays and Western blots, HCVpp were produced as previously described (3) from 293T cells cotransfected with a
murine leukemia virus (MLV) Gag-Pol packaging construct, an
MLV-based transfer vector encoding the green fluorescent protein, and each of the E1E2 expression constructs. For Western
blotting and co-immunoprecipitation assays, the pseudoparticles were purified and concentrated from the cell culture
medium by ultracentrifugation at 82,000 g for 1 h 45 min at
4 C through 1.5 ml of a 20% sucrose cushion. Viral pellets were
suspended in phosphate-buffered saline (PBS) to concentrate
the viral particles 100-fold. As a control for infection assays and
co-immunoprecipitation assays, pseudoparticles devoid of viral
glycoproteins were produced in parallel.
Incorporation of E1E2 Glycoproteins onto Viral Particles
Viral pellets were subjected to Western blot analysis using a
mouse anti-HCV E1 antibody (IGH204) (Innogenetics), a
mouse anti-HCV E2 antibody (H52) (33), and a goat antiMLV-CA antibody (anti-p30; Viromed). Viral pellet samples
were mixed with 6 loading buffer (375 mM Tris-HCl, pH 6.8,
3% sodium dodecyl sulfate (SDS), 10% glycerol, and 0.06% bromphenol blue), and the samples were analyzed by electrophoresis in 12% polyacrylamide gels in the presence of 0.1% SDS.
After protein transfer onto nitrocellulose filters, the blots were
54

(105 cells/well) in 6-well plates. Huh-7-Tat indicator cells (4
105 cells/well), detached with EDTA and washed, were then
added to the transfected cells. After 24 h of cocultivation, the
cells were washed with PBS, incubated for 5 min in a pH 5
fusion buffer (130 mM NaCl, 15 mM sodium citrate, 10 mM
MES, 5 mM HEPES), and then washed 3 times with medium.
The luciferase activity was measured 24 h later using a luciferase assay kit according to the manufacturers instructions
(Promega).
HCVcc Infection; in Vitro Transcription, HCVcc Production,
Titration, and Viral Spread KineticsTo generate infectious
HCV RNAs, pJFH-1S1a-NS21a2a VPL, termed H77/JFH1,
and mutant were linearized at the 3 end by XbaI digestion and
were treated with Mung Bean nuclease. Purified linearized
DNAs were used as templates for in vitro transcription with the
RiboMAXTM (Promega Corp.). In vitro transcribed RNA was
used to electroporate Huh7.5 cells using Gene Pulser II apparatus (Bio-Rad) in an L3 laboratory, according to European safety
regulations, and cells were cultured under standard conditions.
Supernatant infectivity titers were determined as focus-forming units (FFUs) per ml. Huh7.5 cells were infected with different dilutions of culture supernatants. 2 days post-infection,
FFUs were visualized after NS5A immunostaining as described
previously (37). FFU calculations were based on counts of
NS5A-positive cells. For the kinetic assays, producer cells were
infected with a multiplicity of infection of 0.04. For virus spread,
Huh7.5 producer cells were split and analyzed by FACS Canto
II by NS5A immunostaining.
Quantitative Detection of HCV Core Protein by ELISAHCV
core protein was quantified using the Trak-C Core ELISA
(Ortho Clinical Diagnostics, Neckargemund, Germany) according to the manufacturers instructions.
sis on purified HCVpp indicated that the level of Con1 E1 incorporated onto HCVpp harboring intergenotypic (heterogeneous) complexes (chimera #2) was similar to the quantity of E1
incorporated onto infectious HCVpp harboring the wild type
(homogenous) Con1 complex. Similarly, H77 E2 was incorporated onto HCVpp harboring intergenotypic (heterogeneous)
complexes (chimera #2) in a similar quantity to the H77 E2
incorporated onto infectious HCVpp E1E2 wt (homogenous)
H77 (Fig. 1B). As with the in trans system, the expression of
Con1 E1/H77 E2 in cis (chimera #2) from a polyprotein precursor still led to the production of HCVpp with a decreased titer
compared with the wt homogenous combination or the H77
E1/Con1 E2 (chimera #4) mirror combination (Fig. 1D). Correlated with the biochemical analysis, these results indicated that
the reduced infectivity observed with the Con1 E1/H77 E2 heterodimer (chimera #2) was not related to alterations of expression and incorporation of E1E2 heterodimers but rather to a cell
entry defect (Fig. 1, B and D). Furthermore, compared with the
mean 2-log decrease in titer in trans, we obtained a 1-log
decrease in cis. This difference may be linked to the fact that the
folding of the heterodimer is not optimal when E1 and E2 are
expressed in trans as the folding of E1 and E2 are dependent on
each other. Therefore, we decided to make subsequent chimera
constructions in cis.
To further characterize the chimera Con1 E1/H77 E2, we
wondered whether its non-optimal infectivity was linked to
suboptimal recognition of the CD81 receptor. Using pulldown
assays with soluble CD81-LEL harboring a His6 tag, Western
blotting analyses indicated that an equal quantity of E1 and E2
was co-immunoprecipitated, indicating that CD81 binding was
not impaired in the different constructions. As a control, the
input of CD81 precipitated was similar in all cases and E1E2 was
not detected without soluble CD81 (Fig. 1C).
Both the Ectodomain and Transmembrane Domain of E1
Are Important for Heterodimer FunctionalityTo determine
which domains are involved in the reduction of infection of the
intergenotypic Con1 E1/H77 E2 heterodimer (chimera #2), we
constructed chimeric E1 by substituting either the ectodomain
or transmembrane domain (tmd) of H77 and Con1 strains,
respectively. Biochemical analysis indicated that the different
chimeric heterodimers had no alterations of expression and
incorporation of E1E2 heterodimers (Fig. 2A). To ensure that
the glycoproteins incorporated onto the particles were well
associated on a conformational heterodimer, we carried out
immunoprecipitation of E1E2 heterodimers from purified
HCVpp using the AR3A conformational anti-E2 antibody.
Western blotting analyses of precipitated complexes indicated
that the different constructions displayed well-folded and associated E1E2 heterodimers on particles in similar proportions
compared with the wild type heterodimers (Fig. 2B). To investigate further, pulldown assays with soluble CD81-LEL indicated that an equal quantity of E1 and E2 were co-immunoprecipitated, indicating that CD81 binding was not impaired in the
different constructions (Fig. 2C). Therefore, the entry properties of chimeric heterodimers are not linked to conformation or
assembly problems on the particles.
We next investigated the infectivity of the HCVpp harboring
the different E1E2 chimeras generated (Fig. 2D). The titers of
RESULTS
The Functionality of Intergenotypic E1E2 Heterodimers for
Entry Depends on the Compatibility of E2 with the E1 Genotype
To characterize the role of less conserved domains of the E1E2
heterodimer involved in cross-talk between either glycoprotein
during their conformational changes in entry, we analyzed
intergenotypic E1E2 complexes derived from E1 and E2 of different genotypes. The infectivity of HCVpp generated using E1
and E2 expressed in trans from individual plasmids indicated
that H77 E1 (gt1a) forms functional heterodimers when associated with E2 derived from all genotypes tested from 1b, 2a, 3, 4,
and 5 (Fig. 1A). Conversely, Con1 E1 (gt1b) does not form functional heterodimers when associated with E2 from H77 (gt1a)
or JFH1 (gt2a) strains. Our study was focused on combinations
using H77 and Con1 strains because of the use of antibodies
against E1 (IGH204) and E2 (H52), which recognized linear
epitopes on both strains. For a more natural expression context
in producer cells, we decided to compare the results of E1E2
intergenotypic heterodimer for H77 and Con1 expressed in cis.
These conditions allowed the production of equal amounts of
E1 and E2 in HCVpp producer cells. Moreover, the HCVpp
titers were increased 10-fold, improving the sensitivity of the
assay as has been previously described (3, 38, 39). Western blot
analysis on producer cells lysates did not indicate any difference
in expression level (Fig. 1B). Furthermore, Western blot analy55
FIGURE 1. Cell entry property of HCVpp harboring E1E2 intergenotypic heterodimers. A, shown is infectivity of HCVpp using E1 from H77 (gt1a) or Con1
(gt1b) strains and E2 from 6 different strains (gt1a strain H77, gt1b strain Con1, gt2a strain JFH1, gt3 strain UKN3.1.9, gt4 strain UKN 4.11.1, and gt5 strain UKN
5.14.4) expressed in trans. The infectious titers were deduced from the transduction efficiencies, determined as the percentage of GFP-positive viable cells. The
S.D. are the means of four experiments. B, expression and incorporation of E1E2 glycoproteins derived from different combinations of E1E2 heterodimers using
E1 or E2 from H77 (gt1a) or Con1 (gt1b) expressed in cis onto HCVpp are shown. The expression in cis of the E1E2 glycoproteins was verified by Western blot of
cell lysates of HCVpp producer cells using an anti-E1 antibody (IGH204), an anti-E2 antibody (H52), and an anti-capsid antibody (anti-p30, MLV-CA). The
incorporation of the E1E2 envelope was analyzed by Western blot of viral particle pellets. Chimeras #2 and #4 are the same as those described in D. C, shown
is binding to CD81. The binding of E1E2 heterodimers to CD81 was analyzed by pulldown assays with (CD81) or without (CD81) soluble CD81-LEL-His6
followed by Western blot using anti-E1 (IGH204), anti-E2 (H52), and anti-CD81 (JS81) antibodies. D, infectivity of the HCVpp derived from different combinations of E1E2 heterodimers using E1 or E2 from H77 (gt1a) or Con1 (gt1b) expressed in cis. The heterodimers are represented with schematic drawings. From
bottom to top: E1 ectodomain, the TMD of E1 (cross), E2 ectodomain and TMD of E2 (cross). Black represents the domains that are from Con1 strain, and domains
from H77 strain are in white. Infectious titers were determined as in A.
tmd of H77 E1 into the wt Con1 E1E2, the conclusion was

similar. Indeed, even though the H77 E1/Con1 E2 combination
(chimera #4) is optimal for entry, the introduction of either the
transmembrane domain (chimera #6) or the ectodomain (chimera #5) alone leads to a 6- and 10-fold reduction in titer,
respectively, compared with HCVpp harboring Con1 wt complex. Therefore, depending on the strain origin of E2, the fulllength ectodomain and tmd have different roles in heterodimer
functionality.
The Transmembrane Domain of E1 Is Essential for EntryAs
the ectodomain and tmd are important for entry, we first
decided to determine which amino acids in the transmembrane
domains were responsible for the non-optimal functionality of
chimeras #3 and #5 observed in Fig. 2. Based on sequence align-
the different constructs indicated that the ectodomain was

responsible for the loss of titer in the Con1 E1/H77 E2 combination (chimera #2). Indeed, compared with the titer of wild
type H77, the introduction of the ectodomain led to a 2-log
decrease (chimera #3). On the contrary, the substitution of the
transmembrane of H77 E1 with the one of Con1 (chimera #1)
did not modify the titer compared with the wild type H77. This
may suggest that the tmd does not play a role in the function of
intergenotypic heterodimer. However, when the titer of the
Con1 E1/H77 E2 combination (chimera #2) was compared with
the titer of the H77 harboring the sole ectodomain of Con1 E1
(chimera #3), there was a 1-log difference, which in this context
did indicate a role for the tmd. Interestingly, when the mirror
chimera was generated by introducing the sole ectodomain or
56

L359I, Y362F, I373V, or M375L did not increase the titer of
chimera #5 to the level of the fully infectious chimera #4 (Fig.
3B). We then generated different combinations of double and
triple substitutions. Infection assays using these HCVpp indicated that three amino acid mutations are necessary to restore
titer; L359I, I373V, and M375L (Fig. 3B). The expression and
incorporation on HCVpp of the different heterodimers were
verified by Western blot and were seen to be at equivalent levels
(data not shown). The fact that incorporation is not affected is
consistent with previous studies that demonstrated that these
amino acids were not implicated in heterodimerization (26). As
the transmembrane of Con1 seemed also to have a role in the
functionality of chimeric heterodimers, we introduced the
opposite substitution in the mirror chimeric heterodimer #3
(Fig. 3C). The H77 construct harboring the ectodomain of
Con1 (chimera #3) was only weakly infectious, whereas the H77
E2 when associated with the Con1 E1 (chimera #2) was less
affected (Fig. 3C), so we reintroduced Con1 amino acids into
the H77 tmd of chimera #3. We obtained comparable results
with heterodimers harboring the triple substitution I359L,
V373I, and L375M, increasing the titer of chimera #3 (Fig. 3C).
Therefore, regardless of the strain, the same three amino acids
are important for infectivity of the HCVpp harboring chimeric
heterodimers.
The N-terminal Motif Has a Genotype-dependent Role in
HCV Entry ProcessWe next wondered which region of the
H77 E1 ectodomain is needed for heterodimer functionality.
Based on the sequence alignment of H77 and Con1 E1, we
established different boundaries for the construction of chimeras with the 123 (chimera #7) or 36 (chimera #8) N-terminal
residues from Con1 in the H77 E1 (Fig. 3A). As for other chimeras, biochemical analysis indicated that expression, incorporation, conformation, and CD81 binding capacity were similar
for each construction (Fig. 4, AC). Infection assays using
HCVpp harboring these chimeric heterodimers indicated that
residues included in the 36 N-terminal amino acids of H77 were
necessary for optimal infection of H77 (Fig. 4D). The repertoire
of the residues in this N-terminal domain (data not shown)
indicated that two different motifs are particularly heterogeneous between the different genotypes; that is, the SNA/PNS
(208 210) and the MIM/AIL (219 221) motifs in Con1/H77,
respectively (Fig. 3A). To establish which of these motifs were
crucial for H77 heterodimer function in entry, we restored
these motifs in the non-optimal H77 chimeric heterodimer harboring the 36-N-terminal domain from Con1 (chimera #8). On
one hand, biochemical analysis indicated that the difference of
titer was not linked to a defect in expression, assembly, conformation, or binding to CD81 (Fig. 5, AC). On the other hand,
the results of infection assays indicated that the substitution of
the SNA motif to PNS was not able to restore the wild type titer,
whereas the substitution of the MIM motif to AIL was sufficient
to restore an infectivity closely similar to wt H77 (Fig. 5D). To
further verify this, we introduced a single mutation in the defective heterodimer (chimera #8) and showed that the M219A substitution was sufficient to restore optimal infectivity. On the
contrary, Leu-221 did not play a role in H77 heterodimer functionality (Fig. 5D). Therefore, Ala-219 seemed critical for the
functionality of the H77 heterodimer. To confirm this hypoth-
FIGURE 2. Properties of HCVpp harboring chimeric E1 in E1E2 heterodimers. A, expression and incorporation of E1E2 glycoproteins onto
HCVpp are shown. The expression and the incorporation of the chimeric heterodimers were verified by Western blot as described in Fig. 1B. B, folding of
E1E2 heterodimers is shown. The folding and heterodimerization of E1 and E2
glycoproteins on HCVpp were analyzed by co-immunoprecipitation of purified viral particles with the AR3A antibody, which recognizes a conformational epitope on E2, followed by Western blot of pellets using E1 (IGH204)
and E2 (H52) antibodies. C, shown is binding to CD81. The binding of E1E2
heterodimers to CD81 was analyzed by pulldown assays as described before
in Fig. 1C. D, infectivity of the HCVpp harboring chimeric E1 with H77 E2 or
Con1 E2 is shown. The infectious titers were deduced from the transduction
efficiencies, determined as the percentage of GFP-positive viable cells. The
S.D. represents the means of four experiments. The heterodimers are represented as previously described in Fig. 1D.
ment analysis of the transmembrane domain of H77 and Con1,

we found 4-amino acid differences at position 359, 362, 373,
and 375 (Fig. 3A). To analyze their respective contribution, we
first mutated each residue individually in the chimeric heterodimer Con1 harboring the ectodomain of H77 E1 (chimera
#5) leading to the reintroduction of the H77 amino acid in the
transmembrane domain. However, the single substitutions
57
FIGURE 3. Properties of HCVpp harboring mutations in the E1 transmembrane domain. A, shown is a comparison of E1 sequences from H77 and Con1
strains. In the sequence alignment, the differences are represented in bold. The point of chimerization is represented and denoted by the number referring to
the name of chimeras. The transmembrane domain (TMD) is boxed in gray. B, shown is infectivity of the HCVpp harboring mutations in the E1 tmd of chimera
#5, which reintroduce H77 amino acids. C, infectivity of the HCVpp-harboring mutations in the E1 tmd of chimera # 3, which reintroduce Con1 amino acids, is
shown. The infectious titers were deduced from the transduction efficiencies, determined as the percentage of GFP positive viable cells. The S.D. are the means
of four experiments. The heterodimers are represented as previously described in Fig. 1D.
infectivity of the H77 E1E2 MIM mutant could be due to a

defect in the membrane fusion process. To address this point,
we performed cell-cell fusion (syncytium) assays (16) whereby
293T donor cells, expressing a luciferase marker gene under the
control of the HIV-1 promoter, were cocultured with Huh-7Tat indicator cells, expressing the HIV-1 transactivator of
transcription (Tat) protein. Because the HIV-1 promoter
requires Tat for efficient expression, only fused cells should
express detectable levels of luciferase. Donor cells were transfected with expression plasmids encoding wild type or mutant
E1E2 glycoproteins. The results were in agreement with the
results of infection assays. Compared with wt H77, mutants
esis, we introduced a mutation in this motif in H77 E1E2 or

Con1 E1E2. As expected, the titer of HCVpp harboring H77
E1E2 with the MIM motif was decreased by 1 log compared
with wt H77 (Fig. 5E, black bar). On the contrary, the introduction of the AIL motif in Con1 E1E2 induced no decrease of titer
compared with wt Con1 (Fig. 5E, black bar). Altogether, these
results indicate that the non-optimal titer of HCVpp induced
by the introduction of the MIM motif or A219M substitution in
the H77 E1 N terminus is due to a genotype-dependent entry
defect.
Because our results indicated a normal capacity of the
mutant proteins to mediate CD81 binding (Fig. 5C), the loss in
58

and mutant Con1 E1E2 with AIL motif (Fig. 5E, gray bar). Thus,
the results of cell-cell fusion assays indicated that the mutant
H77 E1E2 with the MIM motif is impaired in its capacity to
mediate membrane fusion.
To confirm these results in replicative HCVcc, which affords
a more relevant model of assembly, we introduced this substitution into an H77/JFH1 HCVcc chimeric construct harboring
E1E2 from strain H77. Transcribed RNA was electroporated
into Huh7.5, and the titers of virus released into the cell culture
supernatants were measured 2 days post-electroporation. We
verified that the producer cells were electroporated at equivalent levels by NS5A immunostaining and FACS analyses to
ensure that the difference of titer was not caused by a different
level of electroporation and initial expression (Fig. 5F, white
bar). The titer of HCVcc harboring H77 with the MIM motif
was 10-fold lower than the titer of HCVcc carrying the wt H77
E1E2 (Fig. 5F, black bar). However, using core ELISA assays, we
showed that the quantity of core released into the cell culture
supernatant was similar, even slightly higher for HCVcc harboring the mutated MIM H77 E1E2 (Fig. 5F, gray bar). This
result strongly suggests that the difference in titers observed
between HCVcc harboring wt H77 E1E2 and mutated MIM
H77 is due to an entry defect rather than an assembly problem.
HCVcc harboring the mutated MIM H77 E1E2 has a reduced
titer linked to an inhibition of viral growth and production of
viral particles (Fig. 5G). Indeed, whereas H77 HCVcc propagate
in cell culture after low multiplicities of infection (0.04), as
shown by infectious titer in the supernatant (Fig. 5G, left panel),
by the percentage of infected cells measured with NS5A-positive cells (Fig. 5G, middle panel), or by the quantity of core
released into the supernatant (Fig. 5G, right panel), the HCVcc
harboring the mutated MIM H77 E1E2 displayed much slower
propagation rates. This is in agreement with the infectivity
defect of HCVcc after electroporation (Fig. 5F) and of HCVpp
(Fig. 5E). Altogether, these data indicate that the role in entry of
the N-terminal motif AIL/MIM is essential for the H77 strain.
The N-terminal Domain and the Transmembrane Domain of
H77 Interact for EntryHaving determined that three amino
acids are important in the E1 transmembrane and that the E1
N-terminal AIL/MIM motif is critical at least in H77 E1, we
decided to test the impact of the association of the full-length
heterogeneous transmembrane domain with the AIL/MIM
motif in the constructions described in Fig. 2. We, therefore,
substituted the AIL for MIM or MIM for AIL motif in the chimeric heterodimers that contain the ectodomain and/or transmembrane domain of E1 from the two different strains (Fig. 6).
Biochemical analysis indicated again that differences of titer
were not linked to expression, incorporation, or conformation
of E1E2 on HCVpp (Fig. 6, A and B). It should be noted, however, that the quantity of E2 and E1 co-immunoprecipitated
using the AR3A anti E2 antibody is slightly lower for chimera #4
and #4MIM, chimera #5 and #5MIM, Con1 and Con1AIL, and
chimera #6 and #6AIL, than for the other chimeras (Fig. 6B).
This difference might be due mainly to a different affinity of
AR3A antibody for E2 H77 and E2 Con1, leading to a weaker
co-immunoprecipitation of the chimera harboring a Con1 E2
than H77 E2 rather than major conformation differences
induced by mutations. A difference was also detected in the
FIGURE 4. Properties of HCVpp harboring E1E2 chimeric E1 ectodomain

in H77 heterodimer. A, expression and incorporation of E1E2 glycoproteins
onto HCVpp is shown. The expression and incorporation of the chimeric heterodimers were verified by Western blot as described in Fig. 1B. B, folding of
E1E2 heterodimers is shown. The folding and heterodimerization of E1 and E2
glycoproteins on HCVpp were analyzed by co-immunoprecipitation (Co-IP) as
described before in Fig. 2B. C, binding to CD81 is shown. The binding of E1E2
heterodimers to CD81 was analyzed by pulldown assays as described before
in Fig. 1C. D, infectivity of the HCVpp-harboring chimeric ectodomain of E1
with H77 E2 is shown. The infectious titers were deduced from the transduction efficiencies, determined as the percentage of GFP positive viable cells.
The S.D. are the means of four experiments. The heterodimers are represented as previously described in Fig. 1D and 3A.
H77 E1E2 with the MIM motif had reduced cell-cell fusion
activity (Fig. 5E, gray bar), concomitant with decreased infectious titers (Fig. 5E, black bar). On the contrary, no significant
differences in cell-cell fusion were measured between wt Con1
59
FIGURE 5. Properties of HCVpp and HCVcc-harboring mutations in the N-terminal chimeric E1. A, shown is expression and incorporation of E1E2
glycoproteins onto HCVpp. The expression and the incorporation of the chimeric heterodimers were verified by Western blot as described in Fig. 1B.
B, folding of E1E2 heterodimers is shown. The folding and heterodimerization of E1 and E2 glycoproteins on HCVpp were analyzed by co-immunoprecipitation (Co-IP) as described in Fig. 2B. C, shown is binding to CD81. The binding of E1E2 heterodimers on CD81 was analyzed by pulldown assays as
described in Fig. 1C. D, infectivity of the HCVpp harboring mutations in the N-terminal chimeric ectodomain of E1 with H77 E2 is shown. The infectious
titers were deduced from the transduction efficiencies, determined as the percentage of GFP-positive viable cells. The S.D. are the means of four
experiments. The heterodimers are represented as previously described in Fig. 1D. The white lines with an asterisk symbolize the mutation of the
different motifs. E, infectivity of HCVpp and cell-cell fusion assays with H77 and Con1 E1E2 heterodimers with mutations in the AIL/MIM motif are shown.
The infectious titers were deduced from the transduction efficiencies, determined as the percentage of GFP-positive viable cells. Cell-cell fusion assays
were represented as the percentage of fusion compared with the E1E2 H77 heterodimer. The S.D. are the means of four experiments. The heterodimers
are represented as previously described in Fig. 1D. The white or black lines with an asterisk symbolize the mutations of the motif. F, impact of the AIL motif
in H77/JFH1 HCVcc construct for entry is shown. Two days post-electroporation, infectivity (in black), quantity of core protein in the supernatant (in gray)
and level of electroporation (in white) were studied. Huh7.5 cells were infected with different dilutions of culture supernatants. Supernatant infectivity
titers were determined as FFUs/ml based on counts of NS5A-positive cells in focal immunoassay. Quantity of Core (in fmol/ml) in supernatant was
measured by ELISA Core for each construct. Levels of electroporation were detected by FACS by NS5A immunostaining of electroporated cells and are
represented as the percentage of positive cells. The S.D. are the means of four experiments. G, kinetic assays for H77/JFH1 HCVcc constructs are shown.
Wt H77 E1E2 is represented in black and mutated H77MIM in gray. Producer cells were infected with a multiplicity of infection of 0.04 on day 0 and
analyzed every 3 days over 9 days for infectious titers (left graph), virus spread (middle graph), and quantity of core in the supernatant (right graph).
Supernatant infectivity titers were determined as in F. For virus spread, Huh7.5 producer cells were split and analyzed by FACS by NS5A immunostaining
as in F. Quantity of core (in fmol/ml) in supernatant was measured by ELISA Core for each construct.
60
FIGURE 6. Properties of HCVpp harboring AIL or MIM mutations in chimeric E1E2 heterodimers for ectodomain or tmd of E1. A, expression and
incorporation of E1E2 glycoproteins onto HCVpp are shown. The expression and the incorporation of the chimeric heterodimers were verified by Western blot
as described in Fig. 1B. B, folding of E1E2 heterodimers is shown. The folding and heterodimerization of E1 and E2 glycoproteins on HCVpp were analyzed by
co-immunoprecipitation (Co-IP) as described in Fig. 2B. C, binding to CD81 is shown. The binding of E1E2 heterodimers on CD81 was analyzed by pulldown
assays as described in Fig. 1C. D, infectivity of the HCVpp harboring chimeric heterodimers is shown. The infectious titers were deduced from the transduction
efficiencies, determined as the percentage of GFP-positive cells. The S.D. are derived from the means of four experiments. The heterodimers are represented
as previously described in Fig. 1D. The white or black lines with a star symbolize the mutation of the different motifs.
MIM, Con1 and Con1AIL). However, for the construction containing the E1 transmembrane domain of H77, the titer was
improved when associated with AIL motif, which restored the
homogenous combination (compare chimera #3 and #3AIL,
chimera #6 and #6AIL, H77MIM and H77). The only exception
was the heterodimer H77 E1/Con1 E2 (chimera #4), which was
still fully functional when the MIM motif was present (Fig. 6D).
These results suggest a compensative interaction between H77
E1 ectodomain and Con1 E2 that could counteract the defective
interaction of the E1 transmembrane domain of H77 with the
Con1 MIM motif.
quantity of H77 E2 and Con1 E2 recognized by CD81, which

probably reflects the difference in CD81 binding of the two
genotypes as previously described (40). However, when the
quantity of E1 or E2 detected with the different combinations
was compared with the wt E1E2 combination harboring the
same E2 strain, no major differences were observed between the
different combinations (Fig. 6C).
Interestingly, when the constructs contained the transmembrane domain of Con1, the titers were the same regardless of
whether the MIM or AIL motif was present (compare chimera
#1 and #1MIM, chimera #2 and #2AIL, chimera #5 and #5
61

mera #5MIM and #5). However, the association of the H77 E1
ectodomain with Con1 sequence (chimera #5) was not functional regardless of the N-terminal motif. Therefore, in this
case, there is probably another interaction involved in the
defect of the infectivity that may involve E2 and/or another
domain in E1.
Similarly, we suggested that the compatibility between the
H77 E1 tmd and the N-terminal motif AIL is important for H77.
In this respect, when H77 E1 tmd is associated with the AIL
motif, the infectivity of HCVpp is optimal (chimeras #3AIL,
#6AIL, and H77). The exception to this observation is the intergenotypic heterodimer H77 E1/Con1 E2 (chimera #4), which is
functional with both motifs. There are, again, probably other
interactions involved in the optimization of infectivity.
As our theory does not apply for two chimeras (#4 and #5),
the analyses should take into account the origin of E2. Indeed,
for these two chimeras, the E2 is derived from Con1 (that does
not tolerate all E1). Because of these discrepancies with the
model of interaction between the N-terminal motif and the tmd
in E1 (which are validated by all the constructs harboring E2
H77), another level of interaction involving E2 should be
included. Our results also indicated different characteristics
between chimeras harboring H77 E2 or Con1 E2. Indeed, chimeras harboring Con1 E2 are less efficient for CD81 binding.
This differential characteristic for receptor binding depending
on E1E2 strains has already been studied. CD81 and scavenger
receptor BI can interact with E1E2 heterodimer differently
depending on HCV strains (40, 42 45). All these differences
underlined that E1E2 functionality for entry can differ between
genotypes and even strains. Hence, the understanding of the
precise molecular mechanisms of E1E2 during entry might be
difficult to generalize to all HCV strains.
Therefore, we propose that an interaction inside E1 is important for the optimal functionality of HCV envelope glycoproteins (see interaction 1 in Fig. 7), but another interaction
between E1 and E2 also appears to be important (see interaction
2 in Fig. 7). However, in our analysis, we were unable to more
precisely uncover this second interaction. One possibility will
be to generate and identify other intergenotypic or interstrain
heterodimers sharing the same N-terminal motif and focus on
characterizing the nature of E1 and E2 cross-talk.
The Cross-talk between the N Terminus and tmd Highlights a
New Role for Certain Amino AcidsIt was surprising to find a
need for compatibility and, therefore, maybe an interaction
between the N terminus and tmd of E1 for H77. Indeed, even
though the role of E1 is still poorly characterized and despite its
interaction with E2 not yet being fully understood, these two E1
domains were not expected to interact, as the E1 N-terminal
domain is localized to the intraluminal compartment and the
other three amino acids identified lie in the transmembrane
region. However, it can be speculated that the association of the
AIL motif with the H77 E1 tmd may lead to an optimal conformation of E1 at a certain stage of the biosynthesis and thus be
necessary for an optimal dialogue in the heterodimer E1E2 for
the entry steps.
Previous studies have indicated that the tmd of HCV glycoproteins exhibits unusual features, although interestingly, the
amino acids identified in the E1 tmd had not been previously
Altogether, these results strongly suggest that the interaction

of the transmembrane of E1 H77 and the AIL motif are necessary for entry but not for heterodimerization and CD81 binding. It must be noted that this interaction can be counteracted
by other potential interactions between E1 and E2.
DISCUSSION
To identify domains implicated in the HCV entry process
that have co-evolved in a given genotype, we generated intergenotypic E1E2 heterodimers. We identified intergenotypic
incompatibility between E1 and E2 that led to partial loss of
entry function in certain combinations, namely, when Con1 E1
is associated with E2 from H77 (as in chimera #2) or JFH1. Entry
defect was not due to the lack of heterodimerization or the lack
of CD81 interaction. Focusing on genotype H77 and Con1, we
determined that the contributions of the transmembrane
domain and ectodomain of E1 could not be separated, and we
identified 2 non-conserved regions that played a role in interaction within E1. More precisely, we identified the N-terminal
motif AIL/MIM (219 221) and 3 amino acids, previously not
considered important (359, 373, and 375) in the transmembrane domain, as necessary for optimal infectivity. Interestingly, the cross-talk between the N-terminal E1 motif (AIL/
MIM) and the transmembrane domain of E1 seems to be
important only for H77 E1E2 functionality. Indeed, HCVpp
harboring chimeras with Con1 E1 tmd resulted in the same
level of infection for both AIL/MIM motifs. However, most of
the HCVpp harboring chimeras with H77 E1 tmd gave a lower
titer when associated to the MIM (Con1) motif compared with
the AIL (H77) motif. Therefore, we showed that it is essential
for certain domains to be homogeneous (i.e. from the same
HCV strain) as they have co-evolved within a given HCV genotype to achieve interrelations for optimizing cell entry functions. Interestingly, our work is complementary to a recent
study (41) that used a similar strategy to identify that E1 JFH1
(gt2a) does not form functional heterodimers when associated
with E2 from H77 (gt1a). This study demonstrated intradomain
interactions within E2 but did not focus on E1.
Besides N-terminal and tmd Cross-talk in E1, E2 Is Involved
in Another InteractionDespite confirming a function of
cross-talk between E1 and E2 domains, we were also able to
make a detailed characterization of interactions inside E1. This
result is most likely linked to the fact that there was no reciprocity in the identified non-optimal combination. Indeed, whereas
Con1 E1 did not tolerate H77 E2 (chimera #2) or JFH1 E2 for
optimal infectivity, E1 from H77 tolerates all E2, including
Con1 E2 (chimera #4). This alone indicates that some aspects of
cross-talk between E1 and E2 are not identical, depending on
the genotypes and the E1 considered. However, it was surprising to observe that when only the ectodomain was exchanged,
the reciprocity was verified. Indeed, the Con1 E1 ectodomain
did not tolerate H77 sequences (chimera #3) and neither did the
H77 E1 ectodomain tolerate Con1 sequences (chimera #5).
However, the mechanism of non-functionality of these chimeras might be different.
Based on our results, we suggested that the tmd of Con1 can
tolerate both AIL/MIM motif (in Fig. 6D, compare chimera #2
and #2AIL, Con1 and Con1AIL, chimera #1MIM and #1, chi62

However, the role of the N-terminal domain of E1 is not
known. Previous studies have never identified this region as
important for E1E2 function. Indeed, only the tmd, juxtamembrane region (49), and the potential peptide fusion in E1 (16, 17)
have been studied in depth. Our studies demonstrate that the
AIL motif of E1 is important for the membrane fusion process
(Fig. 5E), which probably explains the suboptimal titer of
HCVpp (Fig. 5E) and the defect in proliferation of HCVcc
H77MIM mutant. The conformation and the role of this region
might give some clues about the role of E1 in E1E2 heterodimer
during entry.
The Understanding of E1 Role during Entry Could Lead to
New Therapeutic StrategiesDifferent strategies of vaccination have been considered, and some suggest the use of E1 as
antigen (50 52). These assays only lead to a poor number of
antibodies against E1, but some have the benefit of being able to
cross-neutralize HCV entry of many strains. The epitope on E1
that has been characterized for these neutralizing antibodies
and lies in the ectodomain of E1 (313327) is outside our N-terminal region and the potential fusion peptide. As our motif is
only important for H77, antibodies or peptides that could be
synthesized against this region might not be efficient at blocking entry of different strains. Our results indicate, as the functionality of E1 is strain-dependent, that it would be difficult to
find a clinical strategy that could prevent entry of HCV from
different genotypes by targeting particular domains of E1.
To find the second potential interaction between E1 and E2
(Fig. 7), it would be interesting to study other strains that are
not dependent on the AIL motif or have the AIL motif on both
sequences to find another domain of E1 important for entry
that could be more useful for different strains that could
lead to the development of new therapeutic strategies.
Thus, these studies led to the identification of E1E2 domains
that have co-evolved within a given HCV genotype and mediate
the cross-talk necessary for achieving cell entry functions. The
identified cross-talk is restricted to H77 and involves mainly E1.
However, other interactions need to be identified to complement the proposed model, and domain interactions between E1
and E2 need to be more precisely determined.
FIGURE 7. Schematic representation of potential interactions in E1E2 heterodimers necessary for entry. The transmembrane domains (TMD), the
ectodomains of E1 and E2, and the N-terminal motif (AIL) of E1 H77 are represented. Arrow 1 represents the interaction between the AIL motif and the
tmd of H77 essential for entry in H77 heterodimer. Arrow 2 represents the
potential compensatory interaction that could act between Con1 E2 and H77
E1 ectodomain and allow entry without interaction 1.
highlighted (25, 26, 46). The tmd of E1 and E2 are composed of

less than 30 amino acids forming two hydrophobic stretches
separated by a short segment containing one or two fully conserved charged residues (47). A study of the topology of the tmd
of HCV envelope glycoproteins has shown a reorientation of
the C termini of these domains, leading to a single membranespanning topology (23). It can be speculated that at a certain
point of synthesis and maybe also in later events, the amino acid
identified here in the tmd may be exposed intraluminal and
interact with the E1 N-terminal domain.
Another possibility stems from research showing that the
amino acid sequence in the tmd or the intracytoplasmic tail can
influence the conformation of the ectodomain (27, 48). Therefore, the amino acids identified in the H77 E1 tmd may have an
indirect role in the N-terminal domain by allowing a particular
conformation of the ectodomain and allowing the N-terminal
domain to act efficiently.
Studies of HCV envelope glycoproteins with heterologous
expression systems have shown that the tmd of these proteins
plays a major role in the assembly of the E1E2 heterodimer (25)
and its subcellular localization (46) and entry (26). Alanine
insertions within the tmds of HCV envelope glycoproteins have
identified the central regions of these domains as well as the
N-terminal part of the tmd of E1 as sequences involved in heterodimerization (25). Another study identified individual residues within the central region as well as the N-terminal part of
the tmd of E1 that participate in those interactions through a
tryptophan replacement scan of these regions (26). Therefore,
our studies attribute to the C-terminal half of the tmd a role in
entry that has never been shown before and has no link with
heterodimerization.
AcknowledgmentsWe thank Felix Rey and Thomas Krey for discussions at the early stage of the work. We thank S. Kabani for critical
reading of the manuscript. Production of soluble CD81-LEL was performed in the Protein Production and Analysis facility, IFR 128 Biosciences Lyon-Gerland (France). We are grateful to our co-workers
and colleagues for encouragement and advice.
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64
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 25, pp. 2252122534, June 24, 2011
Printed in the U.S.A.
Identification of Cis-Acting Elements in the 3-Untranslated

Region of the Dengue Virus Type 2 RNA That Modulate
Translation and Replication*
S
Received for publication, February 25, 2011, and in revised form, April 21, 2011 Published, JBC Papers in Press, April 22, 2011, DOI 10.1074/jbc.M111.234302
Mark Manzano, Erin D. Reichert, Stephanie Polo, Barry Falgout, Wojciech Kasprzak, Bruce A. Shapiro1,
and Radhakrishnan Padmanabhan2
From the Department of Microbiology and Immunology, Georgetown University School of Medicine, Washington, D. C. 20057,
the Center for Biologics Evaluation and Review, Food and Drug Administration, Bethesda, Maryland 20892, the Basic Science
Program, SAIC-Frederick, Inc., and the Center for Cancer Research Nanobiology Program, NCI-Frederick, National Institutes of
Health, Frederick, Maryland 21702
Using the massively parallel genetic algorithm for RNA folding, we show that the core region of the 3-untranslated region of
the dengue virus (DENV) RNA can form two dumbbell structures (5- and 3-DBs) of unequal frequencies of occurrence.
These structures have the propensity to form two potential
pseudoknots between identical five-nucleotide terminal loops 1
and 2 (TL1 and TL2) and their complementary pseudoknot
motifs, PK2 and PK1. Mutagenesis using a DENV2 replicon
RNA encoding the Renilla luciferase reporter indicated that all
four motifs and the conserved sequence 2 (CS2) element within
the 3-DB are important for replication. However, for translation, mutation of TL1 alone does not have any effect; TL2 mutation has only a modest effect in translation, but translation is
reduced by 60% in the TL1/TL2 double mutant, indicating
that TL1 exhibits a cooperative synergy with TL2 in translation.
Despite the variable contributions of individual TL and PK
motifs in translation, WT levels are achieved when the complementarity between TL1/PK2 and TL2/PK1 is maintained even
under conditions of inhibition of the translation initiation factor 4E function mediated by LY294002 via a noncanonical pathway. Taken together, our results indicate that the cis-acting
RNA elements in the core region of DENV2 RNA that include
two DB structures are required not only for RNA replication but
also for optimal translation.
bers, many of which are significant human pathogens (1). The

MBFV members are classified into three subgroups: DENV,
yellow fever virus, and Japanese encephalitis virus (JEV) (2, 3).
The four serotypes of DENV (DENV1 to -4) cause an estimated
50 million cases of infections, with 10% of those leading to
severe forms of the disease, dengue hemorrhagic fever and dengue shock syndrome (4 6).
The viral genome is a single-stranded RNA of positive ()
polarity containing 11 kilobases (10,723 nt for DENV2 New
Guinea C strain, GenBankTM accession number M29095 (7)).
The 3-end is non-polyadenylated, and the 5-end has a type I
cap structure (for a review, see Ref. 8). Flanking the single long
open reading frame are the 5- and 3-UTRs, which contain
conserved cis-acting RNA secondary structure elements
required for translation and replication (9 18). The viral RNA
is translated to form a polyprotein precursor, which is processed by host and viral proteases in the endoplasmic reticulum
membrane. Protein processing gives rise to three structural (C,
prM, and E) and seven nonstructural (NS) proteins: NS1, NS2A,
NS2B, NS3, NS4A, NS4B, NS5 in that order (for reviews, see
Refs. 8, 19, and 20) and references therein). According to the
current model for replication, assembly of the viral replicase
complex occurs in a cytoplasmic membrane organelle, followed
by negative ()-strand RNA synthesis starting at the 3-end of
the viral genome, resulting in a double-stranded replicative
form. NS3 and NS5 are multifunctional proteins, known to
physically and functionally interact as a complex and thought to
be components of the replicase complex in flavivirus-infected
cells (2123). The ()-strand is used as a template for synthesis
of progeny ()-RNA (24) (reviewed in Refs. 1 and 25).
The 5-UTRs of flavivirus RNAs are 100 nt in length and
fold to form stable secondary structures (16, 26, 27). The 5-terminal region within 160 nt forms two stem-loop (SL) structures, SLA and SLB (Fig. 1A), and is important for binding the
NS5 protein, the viral RNA-dependent RNA polymerase
(RdRP), in vitro (16). Mutations that disrupt this interaction
The dengue virus (DENV)3 is a mosquito-borne flavivirus

(MBFV) in the Flaviviridae family that consists of over 70 mem-
* This work was supported, in whole or in part, by National Institutes of Health

(NIH) Grants AI 57705 and AI 70791 (to R. P.); by NCI, NIH, Contracts NO1CO-12400 and HHSN261200800001E (to W. K.); and by the Intramural
Research Program of the NCI, NIH, Center for Cancer Research. This work
constitutes a partial fulfillment of the requirements for the degree of Doctor of Philosophy at Georgetown University School of Medicine for E. R. and
M. M.
S
supplemental Tables 1 and 2 and Figs. 13.
1
To whom correspondence may be addressed. E-mail: shapirbr@mail.nih.
gov.
2
To whom correspondence may be addressed. E-mail: rp55@georgetown.
edu.
3
The abbreviations used are: DENV, dengue virus; EMCV, encephalomyocarditis virus; IRES, internal ribosome entry site; MBFV, mosquito-borne flavivirus; JEV, Japanese encephalitis virus; nt, nucleotide(s); RdRP, RNA-depen-
dent RNA polymerase; UAR, upstream of AUG region; CS, conserved

sequence; RCS, repeat conserved sequence; DB, dumbbell structure; Rluc,
Renilla luciferase; MPGAfold, massively parallel genetic algorithm; hpt, h
post-transfection; qPCR, quantitative PCR; WNV, West Nile virus; cHP, capsid-coding region hairpin; TL, terminal loop; EMEM, Earles minimum
essential medium; PK, pseudoknot.
65
Cis-Acting RNA Elements in the Dengue Virus 3-UTR

in a sequence-dependent manner. In contrast, there seems to
be a differential role for TL1/PK2 and TL2/PK1 in translation.
The lack of functional similarity between TL1 and TL2 despite
their identical sequences within two DBs could be explained by
results of our analysis of RNA secondary structures, their stabilities, and frequency of occurrence computed by the massively parallel genetic algorithm (MPGAfold) (4751). The
results of our study, taken together, provide a new insight into
the role of the cis-acting elements in the CR region in translation and replication.
severely affected viral replication (16, 28). Downstream of the

5-UTR in the capsid-coding region is the capsid-coding region
hairpin RNA (cHP) (Fig. 1A), which was shown to play a role for
the efficient selection of the start codon for initiation of translation of the single polyprotein (29, 30). Moreover, it is also
required for DENV and WNV replication (29, 30). Recently,
another base-paired interaction between the motifs known as
the 5-3 downstream of AUG region, or DAR, was also identified to be required for viral RNA replication (31, 32).
The 3-UTR of flaviviruses exhibits a great deal of sequence
divergence and size heterogeneity but also contains conserved
sequences that are required for replication (9 14, 16, 17, 33). It
consists of the variable region, core region (CR), and terminal
stem-loop (3-SL) region (Fig. 1A).
The 3-SL region is formed within 100 nt from the 3-end
(34, 35), which has been shown to play a significant role in
replication (33, 36 39). The CR includes the 3-cyclization
sequence (3-CS1) containing a short motif that is complementary to the 5 cyclization sequence (5-CS) located in the capsid
coding region (Fig. 1A). A long range RNA-RNA interaction
between these complementary motifs resulting in circularization of the genome was postulated (40) and demonstrated by
atomic force microscopy (13). The physical and functional
RNA-RNA interaction between 5- and 3-CS1 was shown to be
essential for RNA synthesis in vitro (10, 41) and in replication of
subgenomic replicons or infectious clones in cultured mammalian cells (11, 12, 14, 42, 43). In addition to the 5- and 3-CS1,
two regions of complementarity in the 5- and 3-terminal
sequences, termed the upstream of AUG region (UAR), were
also identified as a requirement for cyclization and RNA replication (13, 17, 44). The 5-UAR is located upstream of the
5-CS, and the 3-UAR is downstream of 3-CS1 (Fig. 1A).
The CR exhibits relatively high sequence conservation
among members of MBFV and is believed to fold into well
defined secondary structures independent of other regions of
the 3-UTR (45, 46). Within this region, upstream from the
3-CS1, the CS2 is present in all three subgroups in the MBFV,
whereas the repeat conserved sequence 2 (RCS2) is present only
in JEV and DENV subgroups (40) (Fig. 1B).
The regions containing CS2 and RCS2 are part of two almost
identical dumbbell-like (DB) secondary structures that have
been postulated to form in DENV (Fig. 1B). It is thought that
this duplication of dumbbell-like structures arose because of
the repetition of CS2 and RCS2 sequences in the 3- and 5-DB,
respectively. The leftmost loops of the 5- and 3-DBs contain a
set of identical sequences (underlined in 5-GAAGCUGUA-3)
(45). Sequence analysis of different serotypes of DENV showed
that the internal five nucleotides in these loops (referred to as
TL1 and TL2) and the two complementary pentanucleotide
sequences downstream of each DB, PK2 (5-GCAGC-3) and
PK1 (5-ACAGC-3), are highly conserved, suggesting possible
base pair interactions between TL1/PK2 and TL2/PK1 to form
two pseudoknots (5- and 3-) (45).
The role of the CR region in flavivirus replication or translation is not understood at present. In this study, we sought to
examine the functional roles of the TLs and PKs by using a
Renilla luciferase (Rluc) reporter replicon in BHK21 cells. We
show here that all four motifs played a crucial role in replication
Cells and Primers
BHK-21 cells (ATCC, Manassas, VA) were maintained at
37 C with 5% CO2 in complete Earles minimum essential
medium (EMEM) (CellGro, Manassas, VA), containing high
glucose (1 g/liter) and L-glutamine, and supplemented with 10%
fetal bovine serum and antibiotics (Invitrogen). All primers
were obtained from Integrated DNA Technologies (Coralville,
IA) and resuspended in H2O. Primer sequences are listed in
supplemental Table 1.
Replicon Construction
DENV2 Rluc RepliconThe Rluc gene was amplified from
phRL-SV40 (Promega, Madison, WI) placing SalI sites on both the
5- and 3-ends using RlucF and RlucR primers (supplemental
Table 1). The resulting fragment was cloned into pGEMTEasy
(Promega). The resulting insert vector was used as a template
using RlucF and RlucIresR primers (supplemental Table 1) to create a fragment containing Rluc with a SalI site at the 5-end and
the 5-terminus of EMCV IRES at the 3-end. Next, using the
GFP-expressing replicon, pRS424GFPIresDEN2 (52), as a template and primers IresF and NS1BstEIIR, a fragment containing
the entire sequence of EMCV IRES followed by the 5-end of
NS1 up to the BstEII site was created. Overlap PCR was performed using the products from the first two PCRs and primers
RlucF and NS1BstEIIR. The entire fragment was cloned into
pGEMTEasy. The insert was subcloned into the replicon plasmid pRS424GFPIresDEN2 using SalI and BstEII (New England
Biolabs, Ipswich, MA) to create the DENV2 Rluc replicon.
GND RepliconA replication-defective replicon clone was
created by engineering a point mutation in the conserved GDD
motif of the RdRP domain in NS5 (nucleotides 95539561).
First, an AatII/ApaI restriction fragment from the C-terminal
region of NS5 to the ApaI site at the 3-UTR was isolated from
pRS424GFPIresDEN2 and subcloned into pBR322 vector.
The GDD 3 GND mutation was introduced by site-directed
mutagenesis using primers GNDF and GNDR (supplemental
Table 1). The fragment was released with PmlI and ApaI and
subcloned into XhoI-SacI sites of pBR322, which contained the
replicon sequence from the XhoI site in NS3 to the SacI site at
the end of the 3-UTR. The sequence between XhoI and SacI
was cloned into the WT DENV2 Rluc replicon to yield the GND
mutant.
Replicon Mutants
The 5- or 3-UTR fragments containing the desired mutations were first amplified by overlap extension site-directed
66

transcription. The GLGpA RNA was used as an internal control
for transfections of the WT and mutant replicons. GLGpA and
() templates for the in vitro RdRP assays were made using the
Ampliscribe T7 high yield transcription kit.
mutagenesis PCR using primers containing desired mutations

(supplemental Table 1) and were purified from agarose gel
using the Zymoclean gel DNA recovery kit (Zymo Research,
Irvine, CA). Three purified PCRs were combined in a single
tube and vacuum-dried. Twenty micrograms of WT replicon
were digested overnight with 40 units of SalI for 5-UTR
mutations or BbvCI for 3-UTR mutations. These were purified
by phenol/chloroform/isoamyl alcohol (25:24:1), ethanol-precipitated, and resuspended in 5 l of water. The linearized WT
or the mutant replicon plasmid was mixed with the vacuumdried mutant PCR fragments, and the mixture was introduced
into Saccharomyces cerevisiae YPH857 (50 l) made chemically
competent using the S.c. EasyComp Transformation Kit (Invitrogen) (53). The 5- or 3-UTRs of mutant replicons were
amplified from the colonies, and their sequences were verified
(MC Lab, South San Francisco, CA). Plasmid DNAs harboring
the mutations were recovered using the Zymoprep II yeast plasmid minipreparation kit (Zymo Research), and 2 l were used
to transform 20 l of MAX Efficiency Stbl2-competent cells
(Invitrogen). Replicon plasmids were propagated in Escherichia
coli from single colonies, and their sequences within 5- and
3-UTRs were again verified. Details of sequences for mutant
replicons used in this study are shown in supplemental Table 1
and Figs. 3A, 4A, and 5A.
RNA Transfection
For each reaction, 106 BHK-21 cells were resuspended in
100 l of Ingenio solution (Mirus Bio, Madison, WI), and 3 g
of replicon RNA and 0.1 g of GLGpA RNA were added. Transfections were performed using a Nucleofector II electroporator
(Amaxa Biosystems, Cologne, Germany) using program A031.
Following pulsing, cells were carefully transferred in a tube containing 1 ml of prewarmed complete EMEM. Cells were allowed
to recover at 37 C for 5 min and then transferred to another
tube with 3 ml of complete EMEM. Cells were plated in 8 wells
of a 48-well plate, each containing 400 l. The rest of the cells
were plated in a 6-well plate containing 3 ml of medium. Each
transfection experiment was done in quadruplicate and was
repeated at least three times using at least two different batches
of in vitro transcribed RNA.
Cap-independent Translation Assay
In vitro transcribed mutant RNAs were transfected into
BHK21 cells as described above. After transfection, cells were
allowed to recover in 1 ml of complete EMEM at 37 C for 5
min. 100 l was seeded in each of 10 wells of a 48-well plate
containing 100 l of medium. Half of the wells were used as
no-treatment controls, whereas the other half contained
LY294002 with a final concentration of 40 M (Cayman Chemical, Ann Arbor, MI). Cells were lysed at 2 h post-transfection
(hpt) and assayed as described below.
Templates for in Vitro RdRP Assay

DNA templates for in vitro transcription harboring the
desired mutations in a 719-nt template containing the first 230
nt from the 5-end and the last 489 nt of the genome (54) were
prepared by an overlap PCR amplification strategy as follows. In
the first PCR, the region spanning the 5-UTR and the NS5-3UTR junction was amplified using the pSY2 plasmid as the template and the primers pSY2 EcoRI and NS5-3-UTRr. In the
second PCR, the full-length 3-UTR was amplified from the
replicon mutants using the start of 3-UTR and 3-end DEN2
primers (see supplemental Table 1 for primer sequences). The
two fragments were joined together by an overlap PCR and
purified from the agarose gel.
Luciferase Assays
At the desired time points, cells were lysed with 60 l of 1
Renilla luciferase lysis buffer to determine the Rluc signal using
a kit (Promega). Three additional wells were also lysed at 2 hpt
with 1 Passive Lysis Buffer (Promega) to measure the GLGpA
Fluc activity. To ensure complete lysis, plates were put on an
orbital shaker for 30 min. Rluc activity was measured using a
Centro LB 960 luminometer (Berthold Technologies) by injecting 100 l of 1 Renilla luciferase substrate and reading for 10 s
after a 2-s delay. Fluc readings were similarly measured using
1 luciferase assay reagent (Promega).
In Vitro Transcription of RNA Templates

Approximately 50 g of WT and mutant replicons were linearized with EcoICRI and purified twice with phenol/chloroform/isoamyl alcohol and once with chloroform to remove
excess phenol. DNA was ethanol-precipitated and resuspended in 20 l of RNase-free water. Three microliters were
used to make RNA with the Ampliscribe SP6 high yield transcription kit (Epicenter Biotechnologies, Madison, WI). The
reaction mixtures (20 l) contained 3.13 mM GTP, 8 mM
m7G(5)ppp(5)G cap analog (New England Biolabs) and 80
units of RNase inhibitor (New England Biolabs) and incubated
for 3 4 h at 37 C. Following transcription, DNAse I (Epicenter) was directly added to the mixture and incubated for
another 1 h at 37 C. 0.5 l of each reaction was visualized on a
0.5% agarose gel to verify the integrity of the RNA prior to
storage or use. The plasmid encoding the rabbit -globin 5and 3-UTRs flanking the firefly luciferase gene (pGLGpA) (15)
(kindly provided by Dr. Theo Dreher of Oregon State University) was linearized by Acc65I digestion and used for the in vitro
Immunofluorescence
Following transfection, cells were plated into wells of a 2-well
chamber slides (BD Falcon). At 96 hpt, the culture medium was
removed, and the cells were washed with PBS. The cells were
fixed in 100% cold methanol and placed at 20 C for 30 min.
After fixing, the cells were blocked in 1 PBS with 1% nonfat
dry milk for 1 h with rocking. Cells were washed three times
with PBS and then incubated with rabbit anti-NS5 IgG (1:250)
in PBS for 2 h with rocking. After washing, cells were incubated
with goat anti-rabbit IgG conjugated to FITC (ICN, Solon, OH)
(1:250) for 2 h with rocking. The secondary antibody was
removed, and cells were washed six times in PBS, mounted
using ProLong reagent (Molecular Probes), and visualized
using an Olympus Fluoview FV300 laser confocal microscope.
67

idet P-40, pH 7) supplemented with 500 l of Protease Inhibitor
Mixture VII (Calbiochem) and lysozyme (Pierce) on ice for 30
min. The lysates were sonicated on ice for 20 min total processing time (15 s on, 45 s off) and were centrifuged at 18,000 g for
45 min. Supernatant was allowed to bind to TALON resin
(Clontech) in a 50-ml tube for 1 h in a cold room on a shaker.
The resin was washed five times with 10 ml of Buffer A (50
mM NaH2PO4, 0.3 M NaCl, 10% glycerol, pH 7). At the last wash,
the slurry was transferred and packed in a 0.8 4-cm Poly-Prep
chromatography column (Bio-Rad). The protein was eluted
with 10 ml of buffer A containing 150 mM imidazole. Fractions
of 500 l were collected, and the presence of protein was monitored by a Bradford assay on a 96-well plate. The eluted fractions containing the most protein were combined in a single
tube, and 20 units of SUMO protease I (Life Sensors, Malvern,
PA) was added to cleave the N-terminal histidine tag to produce
a full-length NS5 with an authentic N terminus. Digestion was
allowed to proceed overnight on ice in the cold room. The purified protein was dialyzed using Spectra/Por 6 dialysis tubing
(50,000 molecular weight cut-off; Spectrum Laboratories, Rancho Dominguez, CA) for 6 h in 1 liter of enzyme buffer (50 mM
Tris-Cl, 50 mM NaCl, 5 mM MgCl2, 40% glycerol, 1 mM fresh
dithiothreitol, pH 7.5).
Quantitative PCR
Early Time PointsTo determine the RNA stabilities of replicon RNAs, we transfected 6 g of each RNA by electroporation into 2 106 BHK-21 cells as described above, and the
transfected cells were plated into 24-well plates (2 105 cells/
well). At the desired time points (1, 2, 4, and 6 hpt), cells were
harvested. Total RNA was extracted using 250 l of TRIzol
reagent (Invitrogen) following the manufacturers protocol,
resuspended in 40 l of diethylpyrocarbonate-treated water,
and stored immediately at 80 C until used for qPCR analysis.
Each RNA sample was analyzed in duplicate wells of a 96-well
plate, and the assay was performed on the Applied Biosystems
7900HT fast real-time PCR system. Primers and probe were
targeted to amplify nucleotides 97259820 in the NS5 gene.
The qPCR reaction mixture (50 l) contained 100 ng of
extracted RNA, a 0.2 M concentration of primers qPCR NS5F
and qPCR NS5R (supplemental Table 1), 0.1 M qPCR NS5
probe (supplemental Table 1) (from Dr. Robin Levis, Food and
Drug Administration), 1 rTth EZ buffer, 300 M each dNTP,
3 mM Mn(OAc)2, and 5 units of rTth DNA polymerase (Applied
Biosystems). The reactions were subjected to 60 C for 30 min
for reverse transcription, followed by 94 C for 1 min, and PCRs
were amplified through 40 cycles of 94 C for 15 s and 60 C for
1 min. RNA was quantified by reference to RNA extracted from
a virus stock with a known titer.
96 hpt Time PointTransfected cells grown in 6-well plates
for 96 hpt were washed with PBS and lysed with 1 ml of TRIzol
reagent, and total RNA was purified following the manufacturers protocol. cDNA was synthesized from 1.5 g of total RNA
using the iScript cDNA synthesis kit (Bio-Rad). qPCR analysis
was done on a iQ5 multicolor real-time PCR detection system
(Bio-Rad) on 2 l of the cDNA reaction together with a 0.25 M
concentration of qPCR NS1F and qPCR NS1R primers (supplemental Table 1; primer sequences from R. Takhampunya) in a
20-l reaction using iQ SYBR Green Supermix (Bio-Rad). Each
data point was done in triplicate. Thermocycling conditions
were as follows: 95 C for 3 min, 35 cycles of 95 C for 20 s, and
30 s each at 55 C and 72 C. Measurements of SYBR Green
signal were done at the annealing step. RNA copy numbers
were determined using purified PCR products of the NS1
region of known concentrations. Values were further normalized from GAPDH qPCR results generated using the same protocol and cDNA batch but with primers qPCR GAPDHF and
qPCR GAPDHR (supplemental Table 1).
In Vitro RdRP Assay

Each 25-l RdRP reaction contained 150 ng of RNA template, 4 Ci of [-32P]GTP, 0.5 mM ATP, CTP, UTP, 12.5 M
GTP, and 20 nM NS5 in 50 mM HEPES, 10 mM KCl, 5 mM MgCl2,
2 mM MnCl2, pH 8. The reaction was incubated at 37 C for 30
min. The RdRP products were purified as described previously
(10) and resuspended in 20 l of diethylpyrocarbonate-treated
water. A 5-l aliquot was mixed with an equal amount of Gel
Loading Buffer II (Ambion) and was heat-denatured at 70 C for
10 min and then flash-cooled on ice for 5 min. Samples were run
on a 8 M urea, 4% polyacrylamide gel in 1 TBE buffer at 125 V
and visualized using a Storm 840 PhosphorImager (Amersham
Biosciences). Band intensities were quantified using ImageJ
version 1.45 software (55).
MPGAfold Analysis
For secondary structure prediction analysis, we used the
MPGAfold (4751, 56, 57) to fold a 719-nt subgenomic RNA
(minigenome) from the DENV2 (New Guinea C strain) containing
the 5-terminal 226 nt, 42 nt from the C-terminal coding region of
NS5, including the UAG termination codon, and the 451-nt
3-UTR derived from pSY2 (10). Previous studies have shown that
this RNA molecule contains the essential cis-acting elements
required for efficient translation (15, 29, 30, 58) as well as negative
strand RNA synthesis in vitro (10, 41, 54, 59). MPGAfold utilizes
the genetic operators of mutation, recombination, and selection in
parallel on a high performance computer. The program uses previously reported energy rules (60), including efn2 coaxial energy
calculations at run time, to drive a population (e.g. 16,384) of RNA
structures toward a set of structures that have high fitness (low free
energy). MPGAfold is capable of predicting RNA secondary structure folding dynamics, focusing on the formation of significant
final structures and folding intermediates (61 65). Thus, MPGAfold is especially suited for identifying and visualizing, in addition
Expression and Purification of Full-length NS5

E. coli Rosetta (DE3)pLysS cells transformed with pSUMODEN2NS5NHis (a gift from Dr. Craig Cameron of Pennsylvania
State University) were cultured in 2 liters of Luria-Bertani (LB)
growth medium supplemented with 25 g/ml chloramphenicol, 30 g/ml kanamycin (C25K30), and 0.1% glucose at 37 C
until mid-log phase (A600 0.5 0.6). The medium was replaced
with a fresh 2 liters of LB-C25K30 containing 1 mM isopropyl
1-thio--D-galactopyranoside. Cells were incubated for 48 h at
16 C.
After induction, cells were harvested and lysed with 30 ml of
lysis buffer (50 mM HEPES, 0.3 M NaCl, 10% glycerol, 1% Non68
FIGURE 1. A, schematic of the DENV2 Rluc reporter replicon. Construction of WT and mutant replicons are as described under Experimental Procedures.
Briefly, using a DENV2 (New Guinea C strain infectious clone in yeast shuttle vector pRS424 (53), we have replaced the structural proteins with Rluc and EMCV
IRES, retaining the first 75 nt of C containing the cHP and the 5-CS, and the last 73 amino acids of E for proper endoplasmic reticulum translocation of NS1.
Mutant replicons were made using this backbone for yeast recombination. VR, variable region. B, the secondary structure analysis of the core region of the
3-UTR showed two DB structures (5- and 3-DB) (45). In the left arm of both DBs are identical pentanucleotide sequences, TL1 (nt 10,474 10,478) and TL2 (nt
10,56210,566), which are predicted to base-pair with downstream sequences PK2 (nt 10530 10534) and PK1 (nt 1061710621), respectively, to form two
pseudoknot structures, 5- and 3- (45). On the right arm of the DBs are duplicated conserved sequences RCS2 and CS2. C, a minigenome of DENV2 RNA
sequence containing both 5- and 3-UTR sequences and other essential elements for RNA synthesis in vitro (10, 54) was used for analysis of secondary
structures by MPGAfold as described under Experimental Procedures. Based on this analysis, a stable best fit structure (E 226.2 kcal/mol) has only the
3-DB, whereas the appearance of the 5-DB is visible in metastable structures such as in D (225.6 kcal/mol). The frequency of occurrence of individual stems
among the final predicted structures is color-coded, which increases from left to right (shown in the bottom left).
required for efficient viral RNA translation (15, 29, 30, 58) and
viral RNA synthesis (10 12, 16, 41). In this study, we focused
on the role of 5- and 3-DBs and potential pseudoknot base
pair interactions within the CR of DENV2 RNA in translation
and replication, which is currently not understood. This knowledge is a prerequisite for a detailed analysis of the trans-acting
factors that interact with these elements in mediating their
effects in these processes.
Earlier RNA secondary structure prediction studies have
revealed two almost identical DB-shaped structures (5- and
3-DBs) in the CR of DENV2 3-UTR (45, 46, 67) (Fig. 1, A and
B). These structural predictions and phylogenetic analyses were
based on the 3-UTR sequences alone from RNA folding algorithms. However, in previous studies using a subgenomic RNA
of 719 nt and the viral replicase complex from DENV2-infected
mammalian cells or purified NS5, we showed that long range
interactions between 5- and 3-terminal regions of DENV2
RNA are important for RNA synthesis in vitro. For example, the
3-UTR itself is not an active template for RNA synthesis in
vitro by the viral polymerase unless the 5-UTR is also added in
trans or is present in the same RNA (10, 16, 41, 59). Therefore,
in this study, we used the minigenome, a subgenomic RNA
sequence containing both 5- and 3-terminal sequences, for
to highly stable RNA conformations with low free energy, metastable states that potentially occur in a folding pathway. Because
the algorithm is nondeterministic, 100 independent runs at a
16,384 population level were performed for each WT and mutant
RNA to determine the structure or structural components that
form a consensus. StructureLab (56, 66) and, more specifically, the
StemTrace (51) component of StructureLab were used to analyze
the results and to obtain the frequencies of structural motifs (supplemental Table 2).
Statistical Methods
Rluc readings were normalized against the initial amount of
RNA transfected and the corresponding Fluc signal. These
were expressed as a percentage of WT. Because our objective is
to determine the effect of each mutation on translation and
replication, the values for each group were compared with WT
using unpaired Students t test ( 0.01), using GraphPad
Prism version 5.03 (GraphPad Software, San Diego, CA).
RESULTS
The 3-DB Forms a Stable Secondary Structure, Whereas the
5-DB Occurs Mostly in RNA Folding Intermediates as Revealed
by MPGAfoldA number of studies have indicated that secondary structures in both 5- and 3-terminal regions are
69

Kinetics of the Rluc activity following electroporation of the
WT and GND replicon RNAs in BHK21 cells show a peak of
Rluc activity at 2 hpt, indicating translation of the input WT
and GND replicon RNAs (Fig. 2A). This activity declined over
time, which was then followed by a second increase of luciferase
activity in WT replicon up to 96 hpt due to translation of the
newly replicated RNA; this second burst of Rluc activity was not
observed in the GND mutant. This was confirmed by directly
quantifying replicon RNA at 96 hpt through qPCR (Fig. 2B) and
by immunofluorescent staining for detection of NS5 (Fig. 2C).
These results indicated that the WT and GND replicons could
be used in our analysis of the effects of cis-acting elements in
translation and replication by quantifying the luciferase activities at 2 and 96 hpt, respectively.
The Roles of Conserved TL and PK Motifs in RNA TranslationTo verify whether the RNA folding inequality and the
frequency of occurrence between the 5- and the 3-DBs based
on the MPGAfold analysis reflect differences in TL1 and TL2
functionalities (Fig. 1, C and D), we analyzed the effects of
mutations engineered to disrupt the potential base pair interactions among TL1/PK2 in the 5-DB and TL2/PK1 in the
3-DB (Fig. 1B). We first deleted the conserved sequence of 5 nt
in one or both TLs, creating mutants 5TL1, 5TL2, and
5TL15TL2 (Fig. 3A). The results at 2 hpt indicated that
5TL2 modestly affected translation (72% of WT, p 0.0001),
whereas deleting TL1 did not have any effect (Fig. 3B). However, when both were deleted in tandem, translation further
decreased to 40% of WT (p 0.0001), suggesting that TL1
exhibits a cooperative synergy with TL2 in enhancement of
translation of DENV2 RNA.
To further investigate the role of TL1 and TL2, we employed
a second approach to disrupt the TL1/PK2 and TL2/PK1 base
pair interactions. We introduced point mutations by flipping
the TL sequences (5-GCUGU-3 3 5-UGUCG-3), generating TL1Flip, TL2Flip, and TL1FlipTL2Flip (Fig. 3A). At 2 hpt,
TL1Flip was efficiently translated at levels similar to WT, confirming the results obtained with 5TL1 (Fig. 3B, leftmost
panel). In the TL2Flip mutant, translation was reduced to 80% of
WT (p 0.0006) and 33% in TL1FlipTL2Flip (p 0.0001). The
results obtained with Flip mutants thus confirm our results
obtained with the deletion mutants described above. Next, we
examined whether the reduction in translation of some of the
mutant RNAs is due to differences in RNA stabilities. RNA
stabilities of the WT and mutants were determined by qPCR.
The results showed that there was no appreciable change in
RNA stabilities of these mutants from that of the WT (Fig. 3C).
We sought to examine whether the putative partners of TL1
and TL2 would also have a similar effect on translation. Conserved sequences PK2 and PK1 were flipped to make PK2Flip
and PK1Flip. These mutations unexpectedly did not affect translation (Fig. 3B, second panel). Another set of PK2 and PK1
mutants, PK2mut and PK1mut (Fig. 3A), were constructed to
verify this result. Both translated efficiently like WT, PK2Flip,
and PK1Flip (Fig. 3B, rightmost panel). To eliminate the possibility that a mutation in one PK might be rescued by the
other non-mutated PK, we mutated both PK2 and PK1 in the
same replicon RNA (PK2mutPK1mut). Fig. 3B shows that
PK2mutPK1mut still translated as efficiently as WT.
secondary structure prediction analysis using the MPGAfold

algorithm. The MPGAfold algorithm (47, 50, 56, 57), starting
from a pool of randomly generated stem-loop structures from a
given RNA sequence, recombines these parent structures over
multiple generations until the population of RNA molecules
evolves and reaches a consensus RNA structure. Because of the
stochastic nature of MPGAfold, multiple independent runs
must be done to determine the overall consensus structure for
the given RNA.
Fig. 1C shows the best fit structure (226.2 kcal/mol) from
100 different runs of the 719-nt DENV2 WT RNA sequence.
The frequency of occurrence of each local structure is shown by
a color scheme. The appearance of the already identified and
functionally characterized DENV2 RNA secondary structures
like the SLA, SLB (16, 27, 28), cHP (29, 30), 3-SL (33, 35, 37, 68),
and 5-3-CS1 base pairing (10 12, 14, 40, 41, 59) confirms the
reliability of the MPGAfold-predicted RNA structures.
MPGAfold shows the formation of the 3-DB with 97% frequency of occurrence (Fig. 1C and supplemental Table 2). However, the sequences involved in the proposed 5-DB do not
readily form the expected DB structure; instead, its upstream
half, including TL1, is involved in a long stem-loop structure,
whereas the lower half folds into another stem-loop with a frequency similar to that of the 3-DB (90%) (Fig. 1C).
In some runs, we noticed that the same RNA did form both
5- and 3-DBs as an energetically close suboptimal structure
(Fig. 1D, 225.6 kcal/mol). The frequency of occurrence of the
5-DB among the final structures reached only 6%. However, in
an additional 62% of runs, it was found in intermediate
structures.
This difference in the folding behavior of the two DBs led us
to hypothesize that it is the 3-DB that plays a significant role in
the viral life cycle. MPGAfold analysis showed that TL2 is part
of an unpaired loop in the 3-DB in the best fit structure,
whereas TL1 is alternatively base-paired with upstream
sequences (Fig. 1B). Thus, despite their identical sequences of
TL1 and TL2, it is TL2 that is available to bind PK1 in the 3-DB
to form the putative 3-. This notion is consistent with
sequence analysis of different flavivirus 3-UTRs showing that
the exact sequence of TL2 is conserved in DENV, WNV, Murray Valley encephalitis virus, and JEV, whereas TL1 is only conserved within the DENVs (45).
Construction and Characterization of the DENV2 Rluc
RepliconTo study the effect of different RNA motifs on viral
translation and replication, we constructed a DENV2 New
Guinea C strain replicon expressing Rluc (Fig. 1A). This was
cloned into the yeast shuttle vector pRS424 directly following
the SP6 promoter, which was used for in vitro transcription.
The viral structural proteins were replaced by the Rluc gene
fused with an EMCV IRES element, which directs cap-independent translation of the nonstructural proteins, NS1NS5. Specific sequences from the N terminus of the capsid-coding
region and the last 73 amino acids of the envelope protein were
retained because these are required in cis for RNA replication.
For specific details of the construction, see Experimental Procedures. In addition, we constructed the replication-deficient
GND replicon by substitution of the highly conserved GDD 3
GND in the polymerase domain (69, 70).
70
FIGURE 2. Characterization of DENV2 replicon encoding Rluc reporter. A, the kinetics of luciferase expression of WT and a replication-deficient mutant
(GDD 3 GND) were followed at different time points post-transfection (hpt). 2 and 96 hpt were determined to be the measure of translation and replication,
respectively, of input RNA electroporated into BHK21 cells. RLU, relative light units. B, direct quantification of replicated RNA by quantitative RT-PCR. Quantitative RT-PCR was carried out by RNA extraction from DENV2 replicon-transfected BHK21 cells at 96 hpt as described under Experimental Procedures. C,
detection of DENV2 NS5 by immunofluorescence staining for NS5. Expression of viral proteins was confirmed by immunofluorescence for detection of DENV2
NS5 in BHK21 cells transfected with DENV2 replicon as described under Experimental Procedures. Cells transfected with infectious RNA of DENV2 were used
for a positive control (DENV i.c.). Error bars, S.E.
Interestingly, the restoration of the base pairing between the

TLs and PKs in TL2FlipPK1Flip and TL1FlipPK2FlipTL2FlipPK1Flip
having the 20 and 67% defect in translation, respectively, with nonviral sequences (Fig. 3A) restored translation back to WT levels
(Fig. 3B, second panel). Similar restoration of translation to WT
levels was observed in another set of mutants that swapped the
positions of TL2 and PK1 sequences (TL2 3 PK1/PK1 3 TL2 in
Fig. 3B, third panel).
Taken together, our evidence suggests that TL1 and TL2 play
an important role in translation with unequal contributions and
that their specific sequences are not required. Substitutions
with non-viral sequences can still maintain efficient translation
at WT levels provided that they retain base pairing with PK2
and PK1.
The TLs and PKs Are Critical for Viral ReplicationReplication of WT and mutant replicons was assayed by measuring the
luciferase signal at 96 hpt. The results indicated that replication
of all of the mutants was attenuated (Fig. 3, D and E). TL1 and
TL2 have different contributions to replication. A deletion or
substitution mutation of the TL1 motif resulted in a 70 80%
decrease in Rluc signal (Fig. 3D) and a 50% drop in RNA levels
(Fig. 3E). Mutation of TL2, on the other hand, had a much
greater effect, with replication at 10% of WT. Mutating both
sequences in one RNA generated a more severe phenotype. In
contrast to their phenotypes in translation, PK2 and PK1
mutants are defective in replication (Fig. 3, D and E).
We also sought to examine whether restoring the base pairing in
TL and PK mutants with non-viral sequences could rescue the
71
FIGURE 3. Mutational analysis of conserved putative pseudoknot elements in translation and replication. A, WT and mutant RNA elements in the 5- and
3-DBs are shown. B, the effects of various mutations in translation were examined by measuring the luciferase activities in BHK21 cells transfected with
replicon RNAs at 2 hpt. C, the RNA stabilities of selected mutants were examined directly by qPCR. PFU, plaque-forming units. D and E, the effects of various
mutations in replication were determined. Transfected replicon RNAs in BHK21 cells were incubated for 96 hpt, and both Rluc activities (D) and RNA levels by
qPCR were measured (E). Error bars, S.E. *, p 0.001 compared with WT.
suitable for translation. We hypothesized that if we increase the

strength of the 3- by increasing the number of base pairs,
translation efficiency could be enhanced. We chose to mutate
the nucleotides adjacent to TL2 that are also conserved and yet
are not predicted to participate in the 3- base pairing (45, 46)
and have not been functionally studied previously.
We generated various mutants that increased the number of
base pairs by 1 4 bp in the region surrounding TL2 or PK1 (Fig.
4A). Mutating the A immediately downstream or upstream of
TL2 (1 down or 1 up) while increasing the base pairing to 6
nucleotides has a dramatic effect on translation (45 or 60% of
WT, respectively; Fig. 4B). Mutations of other residues resulting in 79 bp overall had negative effects on translation except
for one mutant containing 7 continuous bp, which did not show
an appreciable effect (2 up in Fig. 4, A and B) (p 0.1118).
The data collectively suggest that the conserved nucleotides
adjacent to TL2 are also important for efficient translation.
In contrast to their phenotypes in translation, these mutants
exhibit a more pronounced defect in replication (Fig. 4C). It is
interesting to note that even one base change (1 down) can
result in a 90% reduction in RNA synthesis. The critical roles of
these nucleotides in translation and replication thus explain
why these sequences are conserved (45, 46).
defect in replication. Unlike in translation, the base pairing

between mutant TL and PK motifs did not restore RNA synthesis
(TL1FlipPK2Flip, TL2FlipPK1Flip, TL1FlipPK2FlipTL2FlipPK1Flip, and
TL2 3 PK1/PK1 3 TL2 in Fig. 3, D and E). Because PK1 overlaps
with the 3-CS1 (Fig. 1B), we sought to determine whether the
loss of replication was due to the decrease in base pairing
between the cyclization sequences. We introduced compensatory mutations in the 5-CS to restore the loss of base pairing
between the 5-3-CS1 in PK1Flip and TL2FlipPK1Flip (indicated
by -3nt). MPGAfold analysis showed that the frequency of
occurrence of the 5-3-CS1 base pairing is reduced as expected
in the PK1Flip mutant because of a reduction in the number of
base pairs between the cyclization sequences. This frequency
was restored in the PK1Flip-3nt and the TL2FlipPK1Flip-3nt
mutants (supplemental Table 2). These compensatory mutations for restoration of the 5-3-CS1 base pairing in these replicons did not improve their replication (Fig. 3, D and E), suggesting that specific sequences within the 5-3-CS1 are
important for replication.
The Conserved Nucleotides Surrounding TL2 Motif Also Play
a Role in Translation and ReplicationOur data in Fig. 3B indicate the possibility that the TL2/PK1 interaction to form the
putative 3- might be playing a role in stabilizing a structure
72
FIGURE 4. Effect of mutations of the conserved sequences in the vicinity of TL2. A, the conserved nucleotides surrounding the TL2 motif were mutated to
strengthen the TL2/PK1 base pairing. Bases in lowercase type denote mutation. B, translation of WT and mutant replicons was measured by Rluc activities at 2
hpt. C, replication of WT and mutant replicons was measured by Rluc activities at 96 hpt (dark gray) and by qPCR (light gray). Error bars, S.E. *, p 0.001 compared
with WT.
nism. This compound inhibits the phosphotidylinositol-3

kinase, which leads to the hypophosphorylation of 4E-BP1
(eIF4E-binding protein 1) (71). This form of 4E-BP1 is able to
sequester eIF4E, thus suppressing cap-dependent translation of
the majority of capped mRNAs in the compound-treated host
cells. We selected mutant replicons that would give a good representation of all of the mutants we have done so far: 5TL15TL2,
TL1FlipTL2Flip, TL1FlipPK2FlipTL2FlipPK1Flip, PK2mutPK1mut,
4 mut, and CS2mut. The mRNA containing the 5- and
3-UTR of the -globin gene flanking a firefly luciferase coding
sequence with an engineered poly(A) tail (15) was used as an
internal control for the transfections of WT and mutant Rluc
replicons. The time course of this control RNA was followed in
the presence and absence of LY294002. The addition of
LY294002 to the transfected cells inhibited the cap-dependent
translation of this control mRNA (Fig. 6A). However, WT replicon RNA was translated efficiently in the presence of
LY294002 (Fig. 6B, WT) (58). We consistently observed an
increase in the Rluc signal obtained from the translation of WT
replicon RNA in the presence of the LY294002 compared with
the signal obtained in the absence of the drug. Moreover, the 5and 3-DB mutants that showed deficient translation in the
absence of the drug LY294002 were still defective in the presence of the drug (Fig. 6B). However, the cap-dependent translation of the control RNA GLGpA (15) included in all transfec-
The CS2 Sequence, but Not Secondary Structure, Determines

Its Role for Its FunctionalityAs a part of our overall objective
to study the role of 3-DB in translation and replication, we
examined the contribution of the CS2 region within the 3-DB
in these two processes. One previous study done using WNV
replicon reported that the entire deletion of CS2 resulted in a
significant impairment of replication (12). In this study, we
sought to determine the role of CS2 by creating substitution
mutations without altering its secondary structure (CS2mut)
(Fig. 5A) as predicted by MPGAfold (data not shown). To determine which part of the CS2 structure or sequence is important,
we made three additional replicons that introduced mutations
in the 3-DB junction (Loop Amut), stem (Stemmut), and loop
(Loop Bmut) (Fig. 5A). All three subdomains affected both translation and replication to a similar extent (Fig. 5, B and C). The
combination of all three mutations did not have an additive
effect, suggesting that all subdomains most likely functioned as
one structural motif that was essential for CS2 function.
Cis-Acting Elements in the Core Region Are Involved in a Noncanonical Cap-independent TranslationIt was reported that
DENV2 RNA required both 5- and 3-UTR to initiate translation under conditions of inhibition of translation initiation factor 4E by the addition of LY294002 (58). We sought to determine whether the RNA elements within the 3-DB are required
for the viral RNA to be translated by a noncanonical mecha73

The CR Elements Do Not Affect ()-strand SynthesisTo
explore the step at which the conserved sequences in the CR act
during replication, we tested the efficiencies of RNA synthesis
in vitro of ()-strand RNA templates containing selected mutations using a full-length NS5 with authentic N and C termini.
The RdRP assay showed that all mutants produced ()-strand
efficiently with the exception of PK1Flip and TL2FlipPK1Flip (Fig.
7). This defect in replication was rescued by restoring the 5-3CS1 complementarity (PK1Flip-3nt and TL2FlipPK1Flip-3nt).
These are in contrast to our replicon data that showed that the
5-3-CS1 and CR sequences were required for replication (Fig.
3, D and E). This suggests that these nucleotides are required at
subsequent steps of replication.
DISCUSSION
Previous in silico analyses of various DENV2 3-UTR
sequences have predicted that the CR folds into two similar
DB-like structures, 5- and 3-DB (45, 46, 67). Our study is
unique in that we take into consideration the influence of
5-terminal sequences and structures on the folding of 3-UTR
by using the MPGAfold for predicting RNA secondary
structures.
Moreover, MPGAfold is able to capture metastable intermediates in an RNA folding pathway. These metastable intermediates as well as stable and best fit structures predicted by
MPGAfold have been shown to have biological relevance in a
number of previous studies (61 65, 72). Another advantage of
MPGAfold is that besides computing the free energy of the
RNA, it also provides us with information on the relative frequencies of occurrence of local secondary structural motifs.
Although MPGAfold reveals the formation of RNA secondary structural elements, such as SLA, SLB, cHP, 5-CS/3-CS1
cyclization, 3-DB, and 3-SL readily (Fig. 1C), the 5-DB and
5-3-UAR interaction is not seen in this stable structure. Surprisingly, the base pairing between 5- and 3-UAR elements
occurs only 8% of the time in the final structure with an additional 27% in the intermediate structures (supplemental Fig. 1
and supplemental Table 2). This is consistent with the observation that the 5-3-UAR binding occurs only after the 5-3-CS1
circularization has been established (73), suggesting that the
latter is the primary driving force in genomic end-to-end communication. This conclusion is also supported by a stem trace
plot of the minigenome RNA maturation in an MPGAfold run
(data not shown). However, there is strong experimental evidence that 5-3-UAR base pairing is essential for relieving the
repression of RNA synthesis by RdRP in vitro by 3-SL (74). The
3-SL includes a part of 3-UAR element, which upon a conformational change in the presence of the viral polymerase is likely
to form the duplex 5-3-UAR (74). Conformational changes
occurring within the 3-SL due to 5-3-terminal RNA-RNA
interaction as well as the NS5/RNA interactions by structure
probing and footprinting methods have also been reported for
WNV (75). Thus, these results in light of MPGAfold analysis
suggest that the 5-UAR forms part of a long stem loop and that
the 3-UAR is a part of the 3-SL in the best fit structure (Fig.
1C); the 5-3-UAR duplex, which occurs only in a metastable
structure (supplemental Fig. 1 and supplemental Table 2),
FIGURE 5. Effect of mutations in the conserved CS2 region of 3-DB.

A, subsections of the CS2 region were mutated such that the predicted secondary structure is conserved. Bases in lowercase type denote mutation.
Translation (B) and replication (C) of WT and mutant replicons transfected into
BHK21 cells were measured at 2 and 96 hpt, respectively. Replication measured by Rluc assay (dark gray) and directly by qPCR (light gray) is shown. Error
bars, S.E. *, p 0.001 compared with WT.
tion experiments was inhibited in the presence of LY294002

(Fig. 6C), indicating that the drug was active in these assays.
These results indicated that the motifs in the CR are required
for the noncanonical cap-independent translation of the viral
RNA when the cap-dependent translation is inhibited. Interestingly, restoration of base pairing between TK1/PK2 and TL2/
PK1 in the mutant, TL1FlipPK2FlipTL2FlipPK1Flip, brought
translation back to the WT level (Fig. 6B), suggesting that there
is a functional interaction between these motifs.
74
FIGURE 7. RdRP assay of ()-copies of 719-nt minigenomes containing CR

mutations. WT or mutant ()-RNAs (150 ng) were used as templates for
()-strand synthesis by 20 nM purified NS5 in the presence of [-32P]GTP. RNA
products were run on 4% 8 M urea-polyacrylamide gel, detected by a PhosphorImager. Band intensities were measured by ImageJ software and
expressed as a percentage of WT. The experiment was repeated five times.
Error bars, S.E. *, p 0.05 compared with WT.
could possibly be formed upon polymerase binding prior to

RNA synthesis.
The previously predicted RNA structures of the CR using
3-UTR sequences showed the formation of both the 5- and
3-DBs (45, 46, 67). However, the best fit structure produced by
MPGAfold after 100 independent runs contained only the
3-DB with a high frequency (Fig. 1C). The simultaneous
appearance of both DBs occurs mostly in suboptimal folding
intermediates in which the population of RNA structures contains 62% 5-DB (Fig. 1D), although among the final structures,
the 5-DB was predicted at a very low frequency (6%). Thus, the
long range interaction of the 5- and 3-termini is an important
determinant in RNA structure prediction of flaviviruses as
revealed in this study. In addition to the folding influence of the
5-terminal sequence that includes 5-CS, the rarity of formation of the 5-DB may also be explained by the nature of the
bases in its long arm (Fig. 1B). The base pairing in this arm is
interrupted midway by an internal loop, reducing the number
of stacked pairs to five. On the other hand, the 3-DB has eight
uninterrupted base pairs (Fig. 1B). The position of the break in
the 5-DB arm increases the potential for that structure to
breathe open and allow its sequences to interact with other
nucleotides.
Because most of the final structures show the 5-DB
sequences in an alternative stem motif, TL1 remains largely
inaccessible to the proposed PK2 interaction to form the 5-.
FIGURE 6. Noncanonical cap-independent translation of selected
mutants of the CR. A, the translation of the control reporter containing 5and 3-UTRs and the 3 poly(A) of the -globin gene flanking the Fluc gene
(GLGpA as described in Ref. 15) in the presence and absence of 40 M
LY294002 is shown. B, the translation of selected mutant replicons was measured in the presence or absence of 40 M LY294002, an inhibitor of cap-dependent translation. Rluc activity of WT replicon measured in the absence of
LY294002 was set as 100%. C, the control RNA GLGpA was co-transfected with
WT and mutant DENV2 replicon RNAs in each transfection experiment. The
translation of control RNAs measured as Fluc activity was plotted as a percentage of Rluc activity of WT replicon RNA-transfected cells in the absence of
LY294002. Error bars, S.E. *, p 0.001 compared with WT.
75

mentarity. This sequence-specific requirement of PK1 in the
3-CS1 and its complementary sequence in the 5-CS in replication is not yet understood. Moreover, the in vitro RdRP assays
using ()-strand templates containing WT and 5- and 3-DB
mutant sequences and purified full-length DENV2 NS5 polymerase (54, 59) revealed that the minus strand synthesis was
not affected by these mutations (Fig. 7). These results suggest
that the RNA elements in the 5- and 3-DBs are involved at a
subsequent step in viral replication.
Our results, taken together, suggest that TL1 and TL2 have
variable modulatory effects in translation. It is noteworthy that
the translational defects in TL2Flip, TL1FlipTL2Flip, and TL2 3
PK1 could be rescued by a compensatory mutation in PK2 and
PK1, although mutations in PK1 and PK2 motifs per se do not
show any effect. One possible explanation is that there are alternative base pair interactions in which TL1 or TL2 could participate when PK1 and PK2 are mutated (supplemental Fig. 2). For
example, TL2 could participate in an alternative base pair interaction with a motif within the top loop structure of the 3-SL
when PK1 is mutated.
More importantly, the ability of the viral RNA to switch to a
noncanonical cap-independent pathway in translation was also
restored in the mutant, TL1FlipPK2FlipTL2FlipPK1Flip (Fig. 6B),
supporting the model that there is a functional interaction
between these motifs that is important in translation. A previous study examined the folding of the 5- and 3-DB regions of
DENV4 3-UTR RNA by RNA probing analysis, which revealed
that PK2 and PK1 were susceptible to double strand-specific
RNase V1 digestion. However, their binding partners as well as
the effect of the 5-terminal region on the folding of the CR
were not investigated (79). A more thorough structural analysis
is warranted to determine the solution structure of the CR and
identify short and long range tertiary interactions between this
region and different elements in the genome. Recent advances
in structure probing have made it possible to analyze RNA
structures in the context of the full viral genome. The high
throughput selective 2-hydroxyl acylation analyzed by primer
extension has been used to determine the complete structure of
the HIV-1 genome (80). A similar method can be applied to
DENV to solve the secondary structure of the flaviviral genome
and identify the binding partners of the elements in the CR.
Mutations of the surrounding nucleotides of the TL2 loop by
increasing the number of base pairs affected translation (Fig. 4,
A and B), suggesting the possibility that increasing the putative
base pairing between TL2/PK1 destabilizes the 3DB structure.
Another possibility is that these four nucleotides also exhibit a
sequence-specific requirement for binding of a protein. CS2 has
a similar sequence-specific requirement for translation. It
seems that mutation of any subregion results in a similar 50%
decrease in translation (Fig. 5, A and B). Because the CS2
sequence is duplicated in the 5-DB as RCS2 (Fig. 1B), it is likely
that RCS2 may cooperate with CS2 in translation.
Previous studies revealed that some host proteins bind
within the 3-UTR, although the precise roles of these RNAprotein interactions in the virus life cycle are not fully understood at present. For example, EF-1 binds to the 3-SL of
WNV (81, 82) and DENV (83). The La protein and polypyrimidine tract-binding protein bind to the 3-UTR of DENV4 (83).
The stability of the 3-DB structure, however, enables TL2 to be

maintained as a single-stranded loop that can readily bind to
the PK1 and form the 3-. In addition, the number of base pairs
at the stalk of the 3-DB corresponds to roughly a half helix
turn, positioning TL2 nearer to PK1, suggesting that the 3-
formation is sterically feasible. The differences in the frequency
of formation of the DBs and the accessibility of TL2 for interaction with PK1, respectively, support the notion that the 3-DB
elements are more important for the virus. This prediction
agrees with the sequence alignment data showing that the
3-DB elements are present in the entire flavivirus genus,
whereas the 5-DB is only found in the DENV and JEV serological groups (40, 76). Moreover, the TL2 and PK1 are conserved
within the flaviviruses, whereas TL1 and PK2 sequences are
only conserved within the DENV serotypes (45). Our results
show that the contribution of TL1 to translation can only be
seen when TL2 is also mutated (Fig. 3B), suggesting that TL1
cooperates with TL2 in translation.
MPGAfold analyses for RNA folding were based on the minigenome, whereas the effects of mutations in translation and
replication were analyzed in the context of the luciferase-based
replicon RNAs (10,210 nt). Therefore, we sought to assess the
potential impact of the luciferase coding region on the structures in the 5- and 3-terminal regions that contain the RNA
elements of interest. Due to the computational constraints of
the MPGAfold algorithm, we surveyed the full replicon secondary structures with the aid of two fast dynamic programming
algorithms: Mfold (60, 77) and RNAfold (78). A common selfcontained subdomain (1184 nt in length) bordered by conserved interactions between the 5-end of the Rluc gene and the
3-end of the NS5 gene was identified by both algorithms (data
not shown). Independent folds of this subdomain by the two
dynamic programming algorithms and MPGAfold indicated a
potential for interactions between the 5-end of the Rluc gene
and the 5-DB sequences upstream of its head SL (data not
shown). The key structural motifs discussed here (SLA, UAR,
CS, 5-DB head, 3-DB, and 3-SL) were present in the structures predicted by the dynamic programming algorithms,
whereas the full 5-DB was still found in energetically close
suboptimal structures, suggesting that the interference of the
Rluc gene in the RNA folding pathway is minimal.
Our results of replication assays suggest that mutations of
these conserved motifs all affect replication. The replicationdeficient PK1Flip and TL1FlipPK2FlipTL2FlipPK1Flip mutations
impact mainly the frequency of occurrence of the structural
motifs rather than inducing major full secondary structure
alterations as seen by MPGAfold and yet function differently
(supplemental Fig. 3, A and B). Therefore, there seems to be a
strong sequence-specific requirement for their function in replication because these mutations result in attenuation (Figs. 3,
D and E, 4C, and 5C). This sequence specificity for replication is
most evident in the TL2FlipPK1Flip-3nt mutant because the restoration of the TL2-PK1 and 5-3-CS1 base pairs was not sufficient to rescue the lethal phenotype (Figs. 3, D and E). A similar conclusion was reported in which the 3-CS1 involving the
same nucleotides that constitute PK1 was mutated in the WNV
replicon (43). Their results showed that specific bases in the 5and 3-CS1 are important for replication, regardless of comple76

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The polypyrimidine tract-binding protein also interacts with

DENV NS4A protein, and this interaction is required for viral
replication (84). The host proteins containing the RNA recognition motif, such as TIAR and TIA-1, were shown to interact
with WNV 3-SL of ()-strand RNA and increase the efficiency
of WNV growth (85). On the other hand, the Y-box-binding
protein-1 binds to the 3-SL of DENV RNA and represses translation (86). Only the poly(A)-binding protein, which plays an
important role in host mRNA translation, was reported to bind
within the DENV CR (87).
There are a number of examples in which the RNA elements
within the 3-UTRs of positive strand RNA viruses play an
important role in translation. In the Turnip crinkle virus
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RNAs, there are different classes of cap-independent translation elements within the 3-UTRs that take over the function of
the 5-cap (for a review, see Ref. 88). Although DENV has a
5-cap for translation of its genome, when the cap-dependent
translation is limiting, a similar mechanism may take over
under conditions of inhibition of translation initiation factor 4E
required for cap-dependent translation, as reported previously
(58). The identification of the RNA elements within the CR that
seem to be required for both cap-dependent and a noncanonical cap-independent translation warrants further investigation
to identify host factors that mediate in this pathway.
AcknowledgmentsWe thank Alan Gee for help with early MPGAfold analysis, Dr. Sofia Alcaraz for initial assistance with transfections, and Dr. Masako Nomaguchi for cloning some DNA fragments
used in replicon construction. We also thank Dr. Theo Dreher (Oregon
State University) for the pGLGpA plasmid, Dr. Craig Cameron (Pennsylvania State University) for the pSUMO-DEN2NS5NHis and E. coli
Rosetta (DE3)pLysS cells, and Dr. Maja Maric (Georgetown University) for the use of the Amaxa Nucleofector.
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78
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 24, pp. 21678 21686, June 17, 2011
Structural Characterization of the Crimean-Congo

Hemorrhagic Fever Virus Gn Tail Provides Insight into
Virus Assembly*
S
Received for publication, December 25, 2010, and in revised form, April 6, 2011 Published, JBC Papers in Press, April 20, 2011, DOI 10.1074/jbc.M110.216515
D. Fernando Estrada and Roberto N. De Guzman1

From the Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045
The RNA virus that causes the Crimean Congo Hemorrhagic
Fever (CCHF) is a tick-borne pathogen of the Nairovirus genus,
family Bunyaviridae. Unlike many zoonotic viruses that are only
passed between animals and humans, the CCHF virus can also
be transmitted from human to human with an overall mortality
rate approaching 30%. Currently, there are no atomic structures
for any CCHF virus proteins or for any Nairovirus proteins. A
critical component of the virus is the envelope Gn glycoprotein,
which contains a C-terminal cytoplasmic tail. In other Bunyaviridae viruses, the Gn tail has been implicated in host-pathogen
interaction and viral assembly. Here we report the NMR structure of the CCHF virus Gn cytoplasmic tail, residues 729 805.
The structure contains a pair of tightly arranged dual zinc
fingers similar to those found in the Hantavirus genus, with
which it shares about 12% sequence identity. Unlike Hantavirus
zinc fingers, however, the CCHF virus zinc fingers bind viral
RNA and contain contiguous clusters of conserved surface electrostatics. Our results provide insight into a likely role of the
CCHF virus Gn zinc fingers in Nairovirus assembly.
according to lengths as the S, M, and L (for Small, Medium, and

Large) segments (4). The viral proteins are the nucleocapsid
protein, two membrane glycoproteins Gn and Gc (also referred
to as G1 and G2 in other Bunyaviridae) (5, 6), a nonstructural
protein (NSm) (7), and an RNA polymerase (4). In the mature
virion, the Gn glycoprotein contains a 176 residue ectodomain
followed by a 24 residue transmembrane region and terminates
in a long cytoplasmic tail consisting of 100 residues (5, 7).
Recent results from other related Bunyaviridae viruses suggest the role of the Gn tail in viral assembly. For example, alanine mutagenesis of the cytoplasmic tails of Uukuniemi virus
(genus Phlebovirus) (8) and Bunyamwera virus (genus Orthobunyavirus) (9) affect the ability of virus-like particles (VLPs) to
effectively incorporate ribonucleoproteins, thus intimating a
role for Gn tails in genome packaging. More recently, the Gn
tail of Puumala virus (genus Hantavirus) was shown to co-immunoprecipitate with the Puumala nucleocapsid protein (10).
These results suggest that the CCHF virus Gn tail plays an
equally important role in viral assembly of genus Nairovirus.
The sequence of the CCHF virus cytoplasmic tail is somewhat variable in Nairoviruses (24% identity) and even more so
when compared with other Bunyaviruses (12% identity with
Hantavirus Gn tails). However, one characteristic feature present in four of the five genera of Bunyaviridae is a conserved dual
C-X-C-X-H-X-C motifs of cysteine and histidine residues with
X representing any amino acid (Fig. 1). Others have suggested
that the high cysteine content of the CCHF virus Gn tail could
be due to extensive disulfide bonding (5). Recently, we reported
that the cysteines in the Andes hantavirus Gn tail fold into a
novel arrangement of back-to-back classical zinc fingers
(11). Despite low sequence identity between the Gn tail of Nairoviruses and Hantaviruses, the spacing of the dual CCHC
motif in the CCHF virus most closely resembles that of Hantaviruses, suggesting the presence of a similar dual zinc finger
structure. To test this hypothesis, we determined the NMR
structure of the CCHF virus Gn cytoplasmic tail from residues
729 805. We report here the first known atomic structure of
any protein component of the CCHF virus and demonstrate
that the high cysteine content of the Gn cytoplasmic tail is
partly due to the presence of dual, back to back -type zinc
fingers similar to those found in Hantaviruses. Unlike Hantaviral zinc fingers, however, the electrostatic surface of the CCHF
virus zinc finger reveals a clear distribution of conserved electrostatic charges. Moreover, we demonstrate using electrophoretic mobility shift assays (EMSA) that these conserved electrostatics may play a role in forming a surface for binding viral
Recent outbreaks of the Crimean Congo Hemorrhagic Fever

(CCHF)2 virus along with the reported ability of the virus to
transfer between humans have raised concerns of a widespread
pandemic (1). The virus is transmitted to humans by tick bite or
by direct handling of infected animal meat or blood (1, 2). Infection causes a hemorrhagic fever and myalgia resulting in mortality rates approaching 30% (13). The virus contains an antisense RNA genome divided into three segments, and named
* This work was supported in part by AHA 0755724Z, K-INBRE, and NIH P20
RR016475 (to R. N. D.) and the Madison and Lila Self Graduate Fellowship
(to D. F. E.).
The atomic coordinates and structure factors (code 2L7X) have been deposited in
the Protein Data Bank, Research Collaboratory for Structural Bioinformatics,
Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
NMR assignments were deposited at the Biological Magnetic Resonance Bank
(BMRB ID 17383).
S
supplemental Figs. S1S3.
1
To whom correspondence should be addressed: Dept. of Molecular Biosciences, University of Kansas, 1200 Sunnyside Ave., Lawrence, KS 66045. Fax:
785-864-5294; E-mail: rdguzman@ku.edu.
2
The abbreviations used are: CCHF, Crimean Congo Hemorrhagic Fever;
EMSA, electrophoretic mobility shift assay; GB1, the B1 domain of Streptococcus protein G; HSQC, heteronuclear single-quantum coherence spectroscopy; NOE, nuclear Overhauser effect; R1, longitudinal or spin-lattice
relaxation rate; R2, transverse or spin-spin relaxation rate; ZF1, first CCHC
zinc binding array, residues 736 756; ZF2, second CCHC zinc binding array,
residues 761780.
79
Crimean-Congo Hemorrhagic Fever Virus Gn Zinc Finger
FIGURE 1. Sequence alignment of the Gn tails of representative members of family Bunyaviridae. Bunyaviridae is comprised of five genera: Nairovirus,
Hantavirus, Orthobunyavirus, Tospovirus, and Phlebovirus. The conserved CCHC-zinc finger motifs (boxed) are present in four of the five genera, with Phlebovirus the lone exception. Another recurring feature is the clustering of conserved basic residues (in blue) in the vicinity of the CCHC-motifs. Notably, these basic
residues overlap with ZF2 in Nairovirus, Orthobunyavirus, and Tospovirus, but are located outside ZF2 in Hantavirus.
RNA. Together, these data provide insight into the role of the
Gn tail in Nairovirus assembly.
tein were pooled and dialyzed at 25 C overnight in buffer (50

mM Tris-HCl pH 8.0, 20 mM NaCl, 1 mM DTT, 1 mM ZnSO4)
with 0.16 mg recombinant TEV protease (13) per 10 ml of
fusion protein. The TEV digestion mixture was dialyzed back
into buffer A and passed again through a 40 ml Q column
(GE Healthcare). The GB1 tag (theoretical pI of 5.6) was
retained on the column while Gn729 805 (theoretical pI of
8.6) was present in the flow-through. The 50 ml flowthrough fraction was concentrated using Ultra-15 centrifugal filters (Amicon) and dialyzed in NMR buffer (10 mM
NaPO4 pH 7.0, 10 mM NaCl, 1 mM DTT, 0.1 mM ZnSO4). The
Gn729 805 construct retained three residues (Gly-His-Met)
cloning artifacts at the N terminus.
NMR SpectroscopyNMR data were acquired at 25 C using
a Bruker Avance 800 MHz spectrometer equipped with a cryoprobe, processed with NMRPipe (14), and analyzed with
NMRView (15). Backbone assignments were obtained from
two-dimensional 1H-15N HSQC (16) and three-dimensional
HNCA (17), CBCA(CO)NH (17), and HNCACB (18). Secondary structures were identified from the C, C, and H chemical shifts (19). Side chain assignments were obtained from
two-dimensional 1H-13C HMQC (20), three-dimensional
HBHA(CO)NH (21), and three-dimensional 13C-edited
HMQC-NOESY (22) (tmix 120 ms). The histidine ring nitrogen atoms coordinated to Zn2 ions were identified from twodimensional 15N HMQC (23) using a nitrogen sweep width of
160 230 ppm. NOE (nuclear Overhauser effect) crosspeaks
were identified from three-dimensional 15N-edited NOESY-
Protein Expression and PurificationVarious constructs of
the CCHF virus (strain SPU103/87) Gn cytoplasmic region
(spanning residues 719 819) were subcloned from a synthetic
gene (GenScript) into the expression vectors pDZ1 and pDZ3
(12), which expressed His6-tagged GB1 fusion proteins with
TEV protease cleavage sites. For NMR structure determination,
the soluble Gn construct spanning residues 729805 (Gn729 805)
was expressed and purified under native conditions following
the method reported previously for the Andes hantavirus zinc
finger domain (11). Briefly, 15N- and 15N/13C-labeled proteins
were expressed in Escherichia coli BL21(DE3) grown in 1 liter
M9 minimal media supplemented with 0.1 mM ZnSO4 before
and after induction. Cells were grown at 37 C, induced with 1
mM isopropyl--D-thiogalactopyranoside at A600 0.8, and cell
growth was continued in a 15 C shaker incubator overnight (to
a final A600 2.0). Cells were harvested by centrifugation,
resuspended in buffer A (20 mM Tris-HCl pH 8.0, 20 mM NaCl,
1 mM DTT, 0.1 mM ZnSO4), and lysed by sonication. Cellular
debris was removed by centrifugation, and to the supernatant
was added one-tenth volume of 1% polyethyleneimine (pH 8) to
precipitate the nucleic acids. Following centrifugation, the
supernatant was bound to a 40 ml of Q column (GE Healthcare)
and eluted with a 280 ml linear gradient of buffer B (20 mM
Tris-HCl, pH 8.0, 0.5 M NaCl, 1 mM DTT, 1 mM ZnSO4). For
TEV protease digestion, fractions containing the fusion pro80

HSQC (24) (tmix 120 ms) and three-dimensional 13C-edited
HMQC-NOESY (22) (tmix 120 ms).
Backbone 15N relaxation parameters were acquired on a 0.5
mM 15N-labeled sample in NMR buffer. The steady-state heteronuclear {1H}-15N NOE was acquired as a pair of two-dimensional datasets in an interleaved manner (where portions of
each two-dimensional spectrum were acquired sequentially
until both datasets were completed) (25). The first two-dimensional dataset contained a 3-s proton saturation (achieved with
a series of 120 pulses) whereas the second two-dimensional
dataset contained a 3-s delay. The heteronuclear {1H}-15N NOE
was calculated as the ratio of the intensities for each peak in the
two datasets. Each two-dimensional dataset was acquired with
2048 (1H) 128 (15N) complex points, 32 scans per point, and
a 5 s recycle delay. Error bars were estimated using the standard
deviation of the background signal of each spectrum. The 15N
backbone relaxation rates R1 and R2 were acquired as described
(26). The time delays used to determine R1 were 10, 60, 120,
240*, 400, 900, and 1100 ms, and the time delays used to determine R2 were 20, 40*, 50, 60, 70, 90, 100, 120, and 150 ms (asterisk denotes spectra acquired in duplicate to estimate reproducibility). Peak intensities were obtained from NMRView (15) and
fitted using GNUPLOT (27). Deviations from fitting were
reported as error bars. Because of peak overlap, residues 749,
787, 791, and 796 were not used in the analysis.
Structure CalculationThe protocol used for NMR structure calculation has been described previously (11). Briefly,
unique NOE distance restraints were classified into upper
bounds of 2.7, 3.5, 4.5, and 5.5 and lower bound of 1.8 based
on peak volumes. Backbone dihedral angles in the -helical
regions identified by the secondary C, C, and H chemical
shifts (19) were restrained to (60 20) and (40 20).
Initial structures were generated using CYANA (28), followed
by molecular dynamics and simulated annealing in AMBER7
(29); first in vacuo, then with the generalized Born (GB) potential. Initial structural calculations were performed in CYANA
without the Zn2 restraints to confirm that the zinc finger
domain will fold from NOE-derived restraints only. Once the
topology of the Zn2-coordinated residues were confirmed,
subsequent CYANA structure calculations used distance
restraints that imposed tetrahedral Zn2-coordination to Cys
and His residues (22). Iterative cycles of AMBER calculations
followed by refinement of NMR-derived restraints were performed until the structures converged with low restraint violations and good statistics in the Ramachandran plot. A family
of twenty lowest energy structures were analyzed using
PROCHECK (30) and molecular graphics were generated using
PYMOL (31). The surface electrostatic potentials were calculated using APBS (32) and visualized in PYMOL (31).
In Vitro TranscriptionA DNA oligonucleotide representing the M genomic segment panhandle was assembled by PCR
primer extension and used for in vitro transcription. In vitro
transcription was carried out following manufacturers protocol (MAXIscript Kit, Ambion). Briefly, a 20-l reaction was
carried out for 1.5 h (37) and terminated by adding 2 l 0.25 M
EDTA and heating to 90 followed by rapid cooling on ice. Reaction mixtures were then treated with DNase I and subjected to
ethanol precipitation. The RNA transcripts were resuspended
in RNase-free ddH2O and analyzed for purity on a native 12%

acrylamide gel stained with SYBR Green II dye (Invitrogen).
RNA Binding AssaysTo assess protein-RNA binding by gel
electrophoresis, RNA transcripts were incubated on ice for 15
min with increasing amounts of either CCHF virus Gn729 805
or Andes hantavirus G1543599 in binding buffer (30 mM
NaPO4, 30 mM NaCl, pH 7.4). Samples were mixed with onehalf volume 50% glycerol and loaded onto a native 12% acrylamide Tris borate gel. The gel was run in a cooling water bath at
90 V for 1 h in Tris borate buffer, pH 8.3 and visualized by
staining with SYBR Green II dye. For nucleic acid size determination, each gel included a 100 bp DNA ladder (NEB, NO467S).
CD SpectroscopyCD spectra were collected in triplicate at
25 on a JASCO J-815 Spectro-polarimeter using a scanning
speed of 50 nm/min. Protein concentrations were kept at 1 M
in buffer (10 M NaPO4, 10 M NaCl, 0.1 mM ZnSO4). EDTA
and ZnSO4 titrations were applied to the same sample.
RESULTS
Protein Expression and PurificationOur previous work
with Hantavirus glycoprotein cytoplasmic tails indicates
expression of the tail is toxic to E. coli (11). Therefore, all constructs of the CCHF virus Gn cytoplasmic tail were expressed as
GB1 fusion proteins. The GB1 tag contained His6 for nickel
affinity purification and a TEV protease cleavage site to recover
the native Gn zinc finger domain. The fusion protein was
expressed in soluble form in E. coli, purified under native conditions, and digested with TEV protease to obtain the Gn zinc
finger domain. Longer constructs comprising the entire predicted cytoplasmic tail (Gn719 819) expressed as insoluble
inclusion bodies. Gn729 819, which was missing the first ten
residues following the transmembrane region, expressed as soluble protein but with low yield. Gn729 805 represented the longest construct containing the conserved C-X2-C-X1112-HX3-C (where X is any amino acid) that also expressed in high
enough yield to give high resolution NMR data.
Zn2 Is Required for Proper FoldingTo examine the reliance of Zn2-coordination on the proper folding of the CCHF
virus Gn tail, we recorded the two-dimensional 15N HSQC of
the Gn729 805 in the presence of 4 mM EDTA (Fig. 2A). The
spectrum in the presence of EDTA is collapsed between ppm
values of 6.5 and 8.6, whereas the folded spectrum in the
absence of EDTA is well dispersed between 6.5 and 9.3 ppm.
Narrowing of the spectrum suggests a loss of tertiary structure
upon removal of Zn2, indicating the requirement for Zn2
binding in folding of the domain. A similar titration using circular dichroism (CD) spectroscopy demonstrates that the presence of EDTA causes a downward spectral shift, indicating a
transition toward an unfolded protein (Fig. 2B). Here we also
demonstrate that the addition of Zn2 ion back into the sample
recovers the trace of the original native spectrum. Therefore,
Zn2 is required for proper folding of the CCHF virus Gn tail.
NMR Structure DeterminationCCHF virus Gn729 805
showed a well dispersed two-dimensional 1H-15N HSQC (Fig.
3A). Complete backbone assignments were obtained from
three-dimensional HNCA, CBCA(CO)NH, HNCACB, and
15
N-edited NOESY-HSQC. The C, H, and C secondary
chemical shifts (Fig. 3B) showed the presence of three short
81
FIGURE 2. CCHF virus Gn zinc finger domain (residues 729 805) relies on
Zn2 for proper folding. Addition of 4 mM EDTA to a sample of 15N-labeled
Gn729 805 effectively narrows the HSQC spectrum into a characteristic of an
unfolded protein (A). Likewise, addition of a metal chelator causes a downward shift at 208 nm in the CD spectra toward random coil (Y axis: molar
ellipticity per residue, degcm2 dmol1residue1 104) (B). Titration of zinc
sulfate back into the sample recovers the original CD trace (B).
FIGURE 3. The CCHF virus Gn zinc finger yielded a well dispersed two-dimensional 1H-15N HSQC spectrum (A). The smaller peak in the tryptophan (W801)
side-chain suggested a minor conformation of the tryptophan ring possibly
due to ring flip-flop. Secondary chemical shifts for 13C, 1H, and 13C suggest the presence of three short helices interspersed with two short hairpins (B).
776 were involved in Zn2 coordination. Notably, His-752 H1

shares an NOE with Cys736 Hs. Likewise, His-776 H1 and
H2 share NOEs with Cys-761 and Cys-780 Hs. A two-dimensional 15N HMQC (23) spectrum showed that His-752 and
His-776 coordinated Zn2 through the N1 and N2 atoms
(supplemental Fig. S1), respectively. Manual analysis of threedimensional 15N- and 13C-edited NOESY spectra identified
1193 unambiguous interproton NOE distance restraints. The
NOE restraints together with 26 and 26 dihedral angle
restraints and zinc coordination restrains (Table 1) were used
in structure calculation and refinement in CYANA and
-helices with an intervening random coil region between the

second and third helix. Two more regions in random coil orientations flanked the central sequence of the domain as indicated by the heteronuclear {1H}-15N NOE (Fig. 4C). Side chain
assignments were completed using two-dimensional 1H-13C
HMQC, three-dimensional HBHA(CO)NH, and three-dimensional 13C edited HMQC-NOESY. There were six invariant cysteine and two histidine residues (His-752 and His-776) in
Gn729 805 (Fig. 1), all of which were involved in Zn2 coordination. Long distance NOEs confirmed that His-752 and His82

nated to residues Cys-736, Cys-739, His-752, and Cys-756 and
forms the classical zinc finger fold. Cys-736 and Cys-739
form part of a short -hairpin. Thr-737 and Ile-738 form a loop
with Cys-736 and Cys-739 on either side of the hairpin. The
structure contains helix 1 formed by Ile-747 to Ser-757 that
folds back toward the -hairpin, forming the zinc finger
fold. Cys-756 forms the fourth Zn2-coordinating residue and
is located on the same surface of helix 3 with His-752.
Likewise, the second CCHC-zinc finger array (ZF2) consists
of a second Zn2 ion coordinated to residues Cys-761, Cys-764,
His-776, and Cys-780 into a classical zinc finger fold. Cys761 and Cys-764 are positioned on either side of a short -hairpin. Pro-762 and Tyr-763 form a loop between Cys-761 and
Cys-764. The structure is followed by a short helix 2 formed by
Leu773-Cys-780 and folded back toward the -hairpin, forming the zinc finger fold. His-776 is located toward the middle of helix 3, and the final coordinating cysteine (Cys-780) is
located at the end of helix 3. Although ZF2 also resembles the
classical fold, a minor difference exists when compared
with ZF1. The helix 2 of ZF2 is shorter than helix 1 by three
residues. This is due to the presence of helix breakers Gly-772
and Pro-781 located at either end of helix 2.
Unlike many classical zinc fingers which form independently folded domains like beads-on-a-string, the two
CCHF virus zinc fingers were tightly stuck together, with over
65 NOEs observed between ZF1 and ZF2. These NOEs fix the
relative orientation of ZF1 with respect to ZF2. Among these
NOEs, the strongest were observed between Met-751 (ZF1) and
Tyr-763 (ZF2), Cys-739 (ZF1) and Ala-774 (ZF2), His-752
(ZF1) and Val-777 (ZF2), and Thr-741 (ZF1) and Val-777 (ZF2).
In addition to the two classical fold zinc fingers, the
structure contains an additional helix, helix 3, formed by Lys782 to Glu-791 that packs against the dual zinc finger fold. A
hydrophobic interaction between Val-744 of ZF1 and Val-787
keeps helix 3 pinned to the core structure. The orientation of
helix 3 to ZF2 is partially determined by the helix breaker
Pro-781, the residue immediately following ZF2. Pro-781 is
100% identical among Nairoviruses (Fig. 1) and serves as a kink
between helix 2 and 3. Strong Cys C to Pro-718 C NOEs
indicated a trans proline isomer. The C-terminal 13 residues
(Leu-792 to Lys-805) are primarily unstructured. NOEs
between Ile-799 C2 and Met-751 C indicate the unstructured tail is pinned to the rest of the structure.
The 15N backbone relaxation rates (R1 and R2) as well as the
heteronuclear {1H}-15N NOE (Fig. 4) showed that the ZF1,
linker (residue 757760), and ZF2 regions behave with nearly
similar amide backbone dynamics. The average R1 values for
ZF1, ZF2 and linker regions were well within each other, with
values of 1.60 ( 0.15), 1.81 ( 0.40), and 1.65 ( 0.04) s1,
respectively (Fig. 4A). ZF1 and the linker had essentially similar
R1 values, however, within ZF2, the R1 values increased for residues 770 and 771 of the loop connecting 4 and 2, indicating
increased mobility of this region. Likewise, the average R2 values for ZF1, ZF2, and linker regions were similar to each other,
with values of 13.8 ( 1.8), 13.8 ( 1.8), and 15.6 ( 3.1) s1,
respectively. Interestingly, the first linker residue, Ser-756,
showed increased R2 without a corresponding increase in R1,
which suggested chemical exchange on the s-ms timescale for
FIGURE 4. Amide backbone relaxation rates R1 (A), R2 (B), and heteronuclear {1H}-15N NOE (C) of the CCHF virus Gn zinc finger.
TABLE 1
NMR restraints and structural statistics for the 20 refined NMR
structures
Total distance restraints

Intraresidue (i, i)
Sequential (i, i 1)
Long Range (i-j) 4)
1193
246
407
307
Total dihedral angle restraints

Phi
Psi
52
26
26
RMS deviation from mean structure

Backbone atoms (N, C, C) ()
All heavy atoms (C, N, O) ()
0.25
0.76
NOE violations
Max distance violation ()
Max dihedral angle violation ()
0.47
5.3
Energies (kcal/mol)
Mean GBa-AMBER energy
Mean restraint energy
3359
79
Ramachandran plot
Most favorable region (%)
Additionally allowed regions (%)
Generously allowed regions (%)
Disallowed regions (%)
79.2
20.1
0.6
0.2
Generalized Born potential.
AMBER. The 20 low energy NMR structures of Gn729 805 converged into a family of structures (Fig. 5) with low restraint
violations and good Ramachandran plot statistics (Table 1).
Structure of CCHF Virus Zinc FingerThe NMR structure of
Gn729 805 reveals a rigid, compact three-helix structure with
four short -strands (Fig. 5). The structure contains a pair of
tightly associated, back to back zinc fingers connected by a
short four residue linker (Ser757-Ile760) (Fig. 5). The first
CCHC-zinc finger array (ZF1) consists of a Zn2 ion coordi83
FIGURE 5. NMR structure of CCHF virus Gn tail zinc finger. Stereoview of the superposition of 20 lowest energy NMR structures of CCHF virus Gn zinc finger
(A). CCHF virus Gn zinc finger domain folds into a compact three-helix structure consisting of two back-to-back zinc fingers with helix 3 pinned
underneath the core zinc finger structure (B).
Ser-756. The average heteronuclear {1H}-15N NOE for the ZF1,

linker and ZF2 regions was 0.8 (Fig. 4C), indicating reduced
flexibility for the dual zinc finger domain including the linker
region. In brief, the NMR amide backbone relaxation parameters (Fig. 4) confirmed that the two zinc fingers essentially tumble as one entity, that the linker between the two zinc fingers
was rigid and tumble at the same rate as the zinc fingers, and
that the loops and tails were flexible.
CCHF Virus Zinc Finger Contains Conserved Electrostatic
SurfacesAnalysis of the surface electrostatics of the CCHF
virus Gn729 805 reveals clustering of positive and negatively
charged surfaces on opposite faces of the structure (Fig. 6). Surface residues Glu-740, Glu-750, and Asp-753 of ZF1 converge
with surface residues Glu-789 and Glu-791 of helix 3 and Glu797 of the C-terminal unstructured region to form a large,
nearly contiguous negatively charged surface. Similarly, Arg767 and Arg-775 of ZF2 converge with Lys-782, Lys-784, and
Lys-786 of helix 3 and Arg-798 of the C-terminal unstructured
region to form a large contiguous positively charged surface. Of
the charged surface residues, only Glu-750, Glu-797, Lys-784,
and Arg-798 are not conserved in Nairoviruses. Most of the
surface electrostatics, therefore, is a conserved feature of the
Nairoviruses zinc finger domain.
CCHF Virus Gn Tail Binds RNARNA electrophoretic

mobility shift binding assays (EMSA) were carried out using
two different proteins: the zinc finger domain of CCHF virus
consisting of Gn729 805, and the zinc finger domain G1534 599
of the related Andes hantavirus. The RNA sequences used in
the EMSA were a 58-mer RNA of the Andes hantavirus and a
51-mer RNA of the CCHF virus (Fig. 7A). Both RNA sequences
contain 2326 nucleotides at the 5 and 3 termini of the M
genomic segments of the Andes and CCHF viruses, and these 5
and 3 strands are complementary to each other and are
expected to form into hairpin-like panhandle structures (Fig.
7A). On a 12% native acrylamide gel, the Andes hantavirus RNA
traveled as a single band consistent with a 58-mer hairpin (Fig.
7B), however, the CCHF virus 51-mer RNA migrated as two
bands, a higher molecular weight form and a lower band
migrating at a size consistent with a 51-mer hairpin (Fig. 7B).
Incubation of the Andes hantavirus protein with the 58-mer
RNA failed to affect the migration of RNA (Fig. 7B). The RNA
bands in the presence of the Andes hantavirus protein were
similar to the free RNA band (Fig. 7B). However, incubation of
the CCHF virus zinc finger protein notably affected the migration of the CCHF virus RNA, as demonstrated by the appearance of an additional band (marked with asterisk, Fig. 7B)
84

migrating between the hairpin and the higher MW form of the
CCHF virus RNA. The protein-RNA complex band was most
likely formed between the zinc finger protein and the RNA
hairpin. Because the 51-mer CCHFV RNA existed in two forms
(the hairpin and the higher MW form), the relative amounts of
each form must be in equilibrium and binding of the CCHFV
zinc finger protein to the hairpin RNA will also affect the
amount of the RNA B form as seen in Fig. 7B. We are designing
new RNA sequences that will only form hairpins for future
studies of the protein-RNA interaction of the CCHF virus zinc
finger. Nevertheless, the main conclusion from the EMSA
results demonstrated that the CCHF Nairovirus Gn tail (residues 729 805) interacted with RNA whereas the Andes hantavirus G1 tail (residues 534 599) did not.
To test our hypothesis that conserved basic residues in the
CCHF virus zinc finger protein may be involved in RNA binding, point mutations were introduced in conserved lysine and
arginine residues into aspartic acid and the mutant proteins
were used in EMSA. The point mutants K783D, K786D, and
R767D retained the ability to bind RNA suggesting these basic
residues may not be critical in RNA binding (supplemental Fig.
S2A). However, the K782D mutation disrupted the proteinRNA interaction, as its shift pattern resembles that of RNA
alone. Coincidentally, Lys-782 is located at the kink between
helix 2 and 3, and is pointed away from the zinc finger
domain (supplemental Fig. S2B), suggesting that the kink
between helix 2 and 3 may be important for RNA binding
interaction of the CCHF virus Gn zinc finger.
FIGURE 6. Comparison of the surface electrostatics of the CCHF virus (A)

and Andes hantavirus (B) zinc fingers. Analysis of the surface electrostatics
of the CCHF virus Gn zinc finger reveals a contiguous cluster of basic charges
(colored blue) that cover the entire half of the structure (A). Rotating the structure 180 reveals an equally large cluster of acidic charges (colored red) on the
opposite face (A). Surface electrostatics of the Andes hantavirus zinc fingers
(PDB 2K9H) in the same orientation as the CCHF virus structure (B). Notably,
the clustering of conserved basic surface in Nairovirus (A) is absent in Hantavirus (B).
DISCUSSION
We report here that an envelope glycoprotein of a Nairovirus
contains a dual CCHC-type zinc finger (Figs. 25) domain that
FIGURE 7. RNA electrophoretic mobility shift assay comparing the Andes Hantavirus and the CCHF virus zinc fingers. RNA sequences used in the
EMSA: Andes Hantavirus RNA (58 nt) and CCHF virus RNA (51 nt) (A). CCHF virus RNA traveled in two forms (marked with ), lower band consistent with
a 51-nt hairpin form, and a higher molecular weight band above 100 bp (B). While the Andes hantavirus zinc finger fails to alter the mobility of Andes
hantavirus RNA, increasing the amount of CCHF virus zinc finger causes the appearance of an additional band (marked with *) for the CCHF virus RNA,
thus suggesting a protein-RNA complex (B). Each lane contained 0.24 mol RNA, proteins came from 0.4 mM stock diluted accordingly (B). Surface
electrostatics combined with EMSA results of the Gn tail provide mechanistic insight into RNP packaging (C). We propose a model in which packaging
consists of a zinc finger-RNA complex. Studies in related viruses suggest a nucleocapsid, Gn tail interaction, presented here as a possible additional
packaging site (C).
85

Proposed Role in Viral AssemblyGiven the data available
regarding the Bunyaviridae Gn role in viral assembly (8 10), it
is likely that the surface electrostatics play an important role in
assembly of the CCHF virus, presumably via direct interaction
with some component of the ribonucleoprotein. The large positively charged surface of the Gn tail would suggest RNA binding, as is the traditional role for zinc fingers in retroviruses (33,
35). Our EMSA results (Fig. 7) suggest that this may in fact be
the case. Using increasing amounts of Gn729 805 revealed the
migration of an additional RNA band (Fig. 7B), which likely
represents a protein-RNA complex consisting of Gn729 805 and
the hairpin-like M segment panhandle. While the observed
complex is weakly bound and therefore likely to be nonspecific,
these results suggest RNA interaction mediated by some of the
conserved residues in the Gn tail. Additionally, these results
suggest the possibility of an interaction between the Gn tail and
the RNA component of the ribonucleoproteins (Fig. 7C). A
reverse genetics system for studying the CCHF virus has only
recently been developed (36), thus allowing testing of this
model in the future.
In summary, we present the NMR structure of a zinc finger
domain in the Gn tail of the CCHF virus. Currently, this is the
only available atomic structure for a protein component of the
Nairovirus genus. The global fold of this zinc finger is similar to
that of the Hantavirus zinc finger, which represents a unique
fold of two classical-type -zinc fingers that are stuck
together (in contrast, individual classical -zinc fingers
behave as independent domains, like beads-on-a-string). We
also demonstrated that the CCHF virus Gn tail binds RNA in
vitro, thus suggesting the possibility of an interaction between
the Gn tail with the viral RNA. Taken together, our results
contribute novel mechanistic insight toward understanding the
CCHF virus life cycle.
binds RNA. Although zinc fingers have been known in viruses,

in particular, the HIV-1 nucleocapsid protein zinc fingers (33)
are critical in RNA packaging and viral assembly, zinc fingers
are rarely found in viral envelope glycoproteins. Including the
zinc finger in this report, there are currently only three known
structures of viral envelope glycoprotein zinc fingers. First is
the zinc finger of the Hantavirus G1 envelope glycoprotein (11),
and second is the zinc finger of the Junin virus envelope glycoprotein (34) (also an RNA virus, of family Arenaviridae). In all
three cases, the cytoplasmic tails of the envelope glycoproteins
contain the zinc finger domains. The Junin virus zinc fingers
(34) form a unique fold that do not show any structural nor
sequence similarity with the Nairovirus and Hantavirus zinc
fingers. Although the Nairovirus and the Hantavirus zinc fingers (11), which both belong to family Bunyaviridae (Fig. 1),
show an overall similar global fold (supplemental Fig. S3), they
also have major structural differences (Fig. 6) and properties
(Fig. 7).
Key Differences between Nairovirus and Hantavirus Gn Zinc
FingersExamination of the surface electrostatics of the CCHF
virus Gn tail reveals key differences when compared with the
Andes hantavirus structure (Fig. 6). Whereas the CCHF Gn tail
displays sharp clustering of conserved charges that form a large
contiguous swath on the protein surface, the Andes hantavirus
zinc fingers display charges that are predominately negative
and apparently randomly dispersed (Fig. 6). The variation in
charge conservation is also evident in the sequence analysis
(supplemental Fig. S3). Whereas the spacing of CCHC motif
is mostly conserved, the spacing between conserved charges is
highly variable. The CCHF virus displays conserved negative
charges on ZF1 and conserved positive charges on ZF2. The
Andes hantavirus sequence, however, displays clustering of
conserved positive charges on the sequences flanking the negatively charged core zinc finger structure.
Moreover, the CCHF virus Gn tail contains a structural motif
that is absent in the Andes hantavirus structure. Helices 2 and
3 (Fig. 3B), residues Leu-773 to Leu-792, form a helix-kinkhelix motif due the positioning of the conserved helix breaker
Pro-781. While not an uncommon motif, this structural aspect
in the CCHF Gn tail forms the core scaffold for a large positively
charged surface partly composed of the conserved charges at
Arg-775, Lys-782, and Lys-786 (Fig. 6A). By contrast, the Andes
hantavirus structure contains neither the corresponding proline nor the charges to support a similar motif (Fig. 1). Instead,
it contains the non-conserved helix breaker Gly-598 followed
by conserved charges exclusively on the C-terminal end of ZF2
(Fig. 1). In this respect, the surface electrostatics of the CCHF
virus Gn tail may more closely resemble that of Orthobunyaviruses, with conserved helix breakers flanked by conserved basic
charges (Fig. 1). Perhaps not coincidentally, Nairoviruses and
Orthobunyaviruses are both arthropod-borne, whereas Hantaviruses are rodent-borne (3). Overall, the general preservation
of the fold indicates that the dual zinc finger motif plays a general but important role in the life cycle of both Nairoviruses and
Hantaviruses. However, the major differences in the surface
electrostatics of the CCHF virus and Hantavirus cytoplasmic
tail structures (Fig. 6) also suggests that while general, the function of the tail may be species specific.
AcknowledgmentsWe thank Lou Altimura (United States Army

Medical Research Institute of Infectious Diseases), Gaya Amarasinghe (Iowa State University), Sunhwan Jo (University of Kansas),
Gerard Kroon (Scripps Research Institute), Dan McElheny (University of Illinois at Chicago), and Asokan Anbanandam (University of
Kansas) for helpful discussions.
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5HIHUHQFH=KDQJ4HWDO  'HVLJQLQJIDFLDODPSKLSKLOHVIRUWKH

The Journal of Biological Chemistry

2011 COMPENDIA COLLECTION: Recent Advances in Pathogenic

3 Recent Advances in Pathogenic Human Viruses. H. Smith

53 Identification of Interactions in the E1E2 Heterodimer of

and Charles E. Samuel

Hepatitis C Virus Important for Cell Entry. Guillemette Maurin,

7 Inhibitors of Histone Deacetylases. CORRELATION BETWEEN

S ISOFORM SPECIFICITY AND REACTIVATION OF HIV TYPE 1 (HIV-1) FROM

65 Identification of Cis-Acting Elements in the 3-Untranslated

15 Host Protein Ku70 Binds and Protects HIV-1 Integrase from

S Region of the Dengue Virus Type 2 RNA That Modulate

Proteasomal Degradation and Is Required for HIV Replication.

S Heat Shock Protein 90AB1. Pheroze Joshi and Cheryl A. Stoddart

41 A Chimeric HIV-1 Envelope Glycoprotein Trimer with an

Embedded Granulocyte-Macrophage Colony-stimulating

S Hemorrhagic Fever Virus Gn Tail Provides Insight into Virus

S Online version of this article contains supplemental material.

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This paper is available online at www.jbc.org

Printed in the U.S.A.

Recent Advances in Pathogenic Human Viruses*

Edited and curated by Charles E. Samuel1

Written histories of virology research frequently start with

Its All Very Retro

* Translated into Chinese by Xing Guo, University of California, San Diego.

The Nobel Prize in Physiology or Medicine 1975 was awarded jointly to

PROLOGUE: Recent Advances in Pathogenic Human Viruses

retroviral infection have also entered center stage in the effort

Flaviviridae: Hepatitis C Virus and Dengue Virus

PROLOGUE: Recent Advances in Pathogenic Human Viruses

The Gn Protein of the Crimean Congo Hemorrhagic Fever

PROLOGUE: Recent Advances in Pathogenic Human Viruses

clinical opportunities, whereas others shed light not only on

Inhibitors of Histone Deacetylases

Combination antiretroviral therapy (cART)3 can effectively

The on-line version of this article (available at http://www.jbc.org) contains

Inhibition of HDAC3 Required for Reactivation of Latent HIV-1

The results from this study suggest that potent inhibition of

Inhibition of HDAC3 Required for Reactivation of Latent HIV-1

apicidin, and trichostatin A) were able to stimulate HIV-1

RESULTS AND DISCUSSION

Inhibition of HDAC3 Required for Reactivation of Latent HIV-1

FIGURE 1. Chemical structures of HDACI used in this study.

IC50 against HDAC1

To gain insight into the mechanisms by which oxamflatin

Inhibition of HDAC3 Required for Reactivation of Latent HIV-1

isoforms, although scriptaid exhibited excellent activity against

Data represent the mean S.D. from three replicate experiments.

Inhibition of HDAC3 Required for Reactivation of Latent HIV-1

Data represent the mean S.D. from three replicate experiments.

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