Chapter 6

Polyomaviruses: SV40

Chapter Outline
6.1 Discovery of Polyomaviruses 85 6.4 Effects on Host 91
6.2 The Genome Structure of SV40 87 6.5 Perspectives 94
6.3 The Life Cycle of SV40 88 6.6 Summary 94
6.3.1 Early Phase 89 Study Questions 94
6.3.2 Late Phase 89

Simian virus 40 (SV40) had been extensively studied during the 1970s
1980s. A reason for that is, perhaps, because it
was easier to grow SV40 than other animal viruses in tissue culture, and it was relatively easier to handle SV40 genome
experimentally due to its small size. Further, SV40 has been employed as a model to study cell transformation and viral
oncogenesis and became a prototype DNA tumor virus. As a consequence, SV40 contributed greatly to our current
knowledge of cancer biology.

6.1 DISCOVERY OF POLYOMAVIRUSES

Discovery: Polyomaviruses1 were first discovered in the 1950s in the rodent tissues as a tumor-associated virus
(Table 6.1). As its name implies, it causes diverse kinds of tumors in rodents. On the other hand, SV40 was discovered
in a monkey cell line that was utilized in the 1960s for poliovirus vaccine production. In fact, it is believed that a
considerable number of people had been unknowingly infected by SV40 that was inadvertently contained in the vaccine
preparation. The observation that SV40 causes tumors in hamsters led to a realization that SV40 is related to the rodent
polyomavirus. Moreover, the discovery of a primate polyomavirus (ie, SV40) strongly supported a notion that a related
virus could be found in humans as well.
Human polyomaviruses: Two human polyomaviruses—JCV2 and BKV3—were discovered in 1965 and 1971, respec-
tively (see Table 6.1). Although their relatedness to SV40 is nearly 70%, evidence for their association with human can-
cers has not been reported. They are transmitted via respiration during infancy, and nearly all adults (40
90%) are
persistently infected without any apparent clinical symptoms. Since these viruses coexist with humans, it is believed
that these polyomaviruses somehow establish a symbiotic relationship with the human body. Nonetheless, JCV can
rarely cause progressive multifocal leukoencephalopathy (PML4) in immune compromised individuals. Recently, a
novel human polyomavirus was discovered in Merkel cell carcinoma (MCC), a rare kind of skin cancer. MCV is of
interest, since it is the first polyomavirus that has been found to cause cancer in humans (Box 6.1).
Since the discovery of human polyomaviruses, and in particular since the discovery of MCV, more attention is
being paid to human polyomaviruses. However, this chapter mainly focuses on SV40, since our current knowledge on
polyomaviruses has been largely acquired from earlier studies on SV40 (Table 6.2).

1. Polyomaviruses The term polyoma is derived from Greek word for “many”-poly and “tumor” -oma.
2. JCV JCV was discovered in 1965 from a patient with progressive multifocal leukoencephalopathy (PML). It was named using two initials of a
patient.
3. BKV BKV was discovered in 1971 from the urine of a renal transplant patient, initials B.K.
4. PML PML is a rare and usually fatal viral disease characterized by progressive damage (-pathy) or inflammation of the white matter (leuko-) of the
brain (-encephalo-) at multiple locations (multifocal). It occurs almost exclusively in people with severe immune deficiency, such as transplant patients
on immunosuppressive medications.
Molecular Virology of Human Pathogenic Viruses. DOI: http://dx.doi.org/10.1016/B978-0-12-800838-6.00006-0
© 2017 Elsevier Inc. All rights reserved. 85
86 PART | II DNA Viruses

TABLE 6.1 The Classification of Human Polyomaviruses

Virus Host Sero-Prevalence Disease

Simian virus 40 (SV40) Monkey
Tumor in rodents
Polyomavirus (PyV) Mouse
Tumor
JC virus (JCV) Human B81% Progressive multifocal leukoencephalopathy
BK virus (BKV) Human 82
99% Cystitis, nephropathy
Merkel cell polyomavirus (MCV) Human 60
81% Merkel cell carcinoma

BOX 6.1 Merkel Cell Polyomavirus

A novel human polyomavirus—Merkel cell polyomavirus (MCV)—was discovered in 2008 from Merkel cell carcinoma, a rare but
aggressive form of skin cancer. Merkel cells are oval receptor cells found in the dermis of skin that are associated with the sense of
light touch discrimination. MCV is suspected to cause the majority of cases of Merkel cell carcinoma. Approximately 80%
of Merkel cell carcinoma (MCC) tumors have been found to be infected with MCV. MCV appears to be a common—if not
universal—infection of older children and adults (60
81% sero-prevalence). Most MCV viruses found in MCC tumors have
at least two mutations that render the virus nontransmissible: (1) The virus is integrated into the host genome in a monoclonal fash-
ion and (2) The viral T-antigen has truncation mutations that make T-antigen defective in initiating DNA replication. Importantly,
the integration into the host chromosome precedes the viral oncogenesis, a feature that is not obligatory in the virus life cycle.

Merkel cell carcinoma (MCC) lesion and MCV genome. (left) Clinical appearance of Merkel cell carcinoma (MCC) lesion. Red, raised,
nodular MCC lesion on the arm of a patient. (right) Genomic organization of Merkel cell polyomavirus. NCRR (noncoding regulatory region)
contains a bidirectional promoter and the origin of replication. LT (large T-antigen), sT (small T-antigen), and 57-kT antigen constitute the early
gene products.

TABLE 6.2 The Defining Features of Polyomaviruses

Genome Virion Structure Genome Replication

Circular dsDNA (5 kb) Nonenveloped Host DNA polymerase
Minichromosome Icosahedral symmetry T-antigen

Life Cycle Interaction with Host Diseases

Early-to-late switch T-antigen-p53 interaction Tumor
Cell cycle deregulation T-antigen-Rb interaction Merkel cell carcinoma
 Polyomaviruses: SV40 Chapter | 6 87

6.2 THE GENOME STRUCTURE OF SV40

Virion structure: SV40 virions are small, nonenveloped capsid particles with T 5 7 icosahedral symmetry (Fig. 6.1).
VP1 pentamers constitute the vertices of icosahedron, in which one molecule of VP2/VP3 underlies. In particular, VP2
is modified by myristate, a saturated fatty acid with 14 carbons. The fatty acid moiety facilitates an interaction with cell
membrane. Inside the capsid, the viral DNA genome is found in association with histones, forming a nucleosome-like
structure called “minichromosome.” Unlike cellular nucleosome, notably, H1 linker histone is absent in the minichro-
mosome. The nucleosome-like structure of the SV40 genome is one of the features that makes SV40 suitable for a
model for eukaryotic DNA replication.
Genome structure: The viral genome is a small double-stranded circular DNA of approximately 5.2 kb in length
(Fig. 6.2). All regulatory elements, including the replication origin5 (Ori), promoter of early genes, and promoter of
late genes, are clustered within a segment called the regulatory region. The viral mRNAs are transcribed from both
strands of the DNA genome, as two promoters lie in the opposite direction (ie, bidirectional promoter). Both early and
late RNAs are alternatively spliced and processed into multiple mRNAs for the synthesis of viral proteins.

FIGURE 6.1 Virion structure of SV40. (A) Diagram depicting the cross section of the SV40 particle. It is an icosahedral capsid made of
72 capsomers, each composed of VP1 pentamers, with T 5 7 symmetry. Each VP1 pentamer is underlined by one molecule of VP2/VP3. The viral
genomic DNA is associated with histones. (B) Electron micrograph of polyomavirus particles.

FIGURE 6.2 Genome structure of SV40. Early and late genes are transcribed from respective promoters embedded into “regulatory region” in
opposite direction. The origin of replication (Ori) is also positioned in the regulatory region. The ORFs for early genes (ie, small and large T-antigens)
and late genes (ie, VP1, VP2, and VP3) are depicted by arrowed boxes. Polyadenylation sites for early and late mRNAs are denoted by A with an
arrow, respectively.

5. Replication origin (Ori) a cis-acting element where the DNA synthesis begins.
88 PART | II DNA Viruses

Protein coding: Early mRNAs are translated into two early proteins: small T-antigen and large T-antigen,6 while
late mRNAs are translated into three capsids proteins, VP1, VP2, and VP3.

6.3 THE LIFE CYCLE OF SV40

Cell culture: SV40 lytically infects monkey cells, such as CV-1 cell, a cell line derived from the kidney of an African
green monkey.
Entry: Diverse glycolipid molecules are utilized as a receptor for the entry of polyomaviruses. In particular, SV40
utilizes GM1 ganglioside7 as a receptor for the entry (Fig. 6.3). The VP1 of SV40 capsid binds to GM1 ganglioside
via the galactose and sialic acid residues. The capsid enters cells via a receptor-mediated endocytosis. SV40 particles
are subsequently internalized inside on endosome which is called “caveolae.”8 In other words, SV40 enters the cell via
caveolin/lipid raft-mediated endocytosis, as opposed to clathrin-mediated endocytosis (see Fig. 3.5). Virions are then
transported inside these vesicles through the cytosol to the lumen of the endoplasmic reticulum, near the nuclear
membrane. The lower pH inside the endosome induces conformational change of VP2 such that the now exposed
N-terminus of VP2 attaches to the membrane via the modified myristate, thereby triggering membrane fusion. Being
small capsids, the viral capsids enter the nucleus via a nuclear pore (see Fig. 3.7). The nuclear localization signal
(ie, NLS) on VP1 drives the nuclear import of the capsid. Once in the nucleus, VP1 dissociates from the viral
minichromosome, which then serves as a template for viral transcription.
The life cycle of SV40 can be divided into early and late phase by the onset of the viral genome replication, a feature
that is shared by all DNA viruses.

FIGURE 6.3 The life cycle of SV40. SV40 enters the cell via endocytosis. The viral DNA is uncoated in the nucleus following nuclear entry of the
viral capsid via nuclear pore. The viral genome replication occurs in the nucleus largely by host DNA replication machinery. Early and late phase are
divided by the onset of viral genome replication.

6. T-antigen It was discovered as a tumor-specific antigen (ie, tumor antigen) in SV40-induced tumors. thus named as “T-antigen.”
7. GM1 ganglioside a kind of glycolipid (ie, glycosphingolipid) that was further modified with sialic acid.
8. Caveolae Caveolae are a special type of lipid raft, which are small (50
100 nm) invaginations of the plasma membrane found in many cell types.
 Polyomaviruses: SV40 Chapter | 6 89

6.3.1 Early Phase

Immediately following the nuclear entry of the viral DNA, the viral transcription starts.
Transcription: Early RNAs are transcribed by the cellular RNA polymerase II from early promoter (Fig. 6.4). Early
promoter represents a typical eukaryotic promoter having a “TATA box” and upstream elements, such as SP1 binding
sites. Early transcripts are alternatively spliced into two mRNAs, which are translated to large T-antigen and small
T-antigen (see Fig. 6.2). Early RNAs encode two proteins from one transcript via alternative splicing, resulting in an
extension of the coding capacity.
Switching to late phase: T-antigen begins to bind to the T-antigen binding sites, starting from the T-antigen binding
site I to III, as it accumulates in the nucleus (see Fig. 6.4). T-antigen binding to the viral genome triggers the switch
from early to late phase. Specifically, the engagement of T-antigen to the binding site I leads to transcription suppres-
sion from the early promoter. The suppression is also called “autoregulation,” because T-antigen regulates its own
expression (Fig. 6.5). The engagement of T-antigen to the binding site II triggers the initiation of viral genome replica-
tion. Lastly, the engagement of T-antigen to the binding site III activates transcription from the late promoter, a process
also called “transactivation.”

6.3.2 Late Phase

The late phase begins with the initiation of viral DNA replication.

FIGURE 6.4 Regulatory region of SV40. Almost all cis-acting elements essential for the viral genome replication are embedded into the 0.5 kb
so-called “regulatory region”: (1) early promoter, (i2) late promoter, and (3) origin of replication. Early and late transcripts are transcribed in the oppo-
site direction, as denoted by arrows. Early promoter constitutes TATA box, SP1 binding sites, and 72-bp repeat enhancer region. Late promoter does
not have TATA box, but have numerous 50 initiation site. Three T-antigen binding sites (ie, I, II, and III) are denoted. The origin of replication (Ori)
overlaps with T-antigen binding site II.

FIGURE 6.5 Switch from early to late phase. T-antigen plays a pivotal role in the switch from early phase to late phase. Autoregulation of early
gene transcription and transactivation of late gene transcription by T-antigen is schematically depicted for the purpose of explanation.
90 PART | II DNA Viruses

Genome replication: SV40 genome replication heavily relies on the cellular replication machinery, including not
only DNA polymerase but also other accessory factors such as topoisomerase and PCNA.9 In fact, T-antigen
is sufficient to orchestrate the viral genome replication in infected cells. As a matter of fact, the diverse biochemical
activities of T-antigen contribute to DNA synthesis: (1) DNA helicase, (2) ATPase, and (3) DNA polymerase
binding activity (Fig. 6.6).
SV40 has served as a model in studying eukaryotic DNA replication, as it heavily relies on cellular DNA replication
machinery. The DNA synthesis had been demonstrated in vitro using a template DNA containing SV40 origin (Ori),
when cellular extracts (ie, DNA replication machinery) was complemented by the purified T-antigen. Note that
T-antigen is the only viral protein required to execute the DNA synthesis. Establishment of the SV40 DNA replication
in vitro was instrumental for subsequent identification of the cellular factors contributing to eukaryotic DNA replica-
tion. The DNA synthesis is initiated by binding of two hexamers of T-antigen to the origin (Ori) on the SV40 genome,
thereby melting the duplex DNA (Fig. 6.7). Subsequently, cellular DNA replication machineries are recruited to
constitute the so-called SV40 replisome10 that can execute DNA synthesis (Fig. 6.8). Importantly, all cellular factors

FIGURE 6.6 The functional domains of large T-antigen. T-antigen is divided into six domains, each of which has a biological activity: from
N-terminus, (1) J domain, (2) LXCXE motif, (3) a nuclear localization sequence, (4) DNA-binding domain (DBD), (5) a helicase domain, and (6) a
host range domain (HR). Four T-antigen binding proteins are drawn to their respective binding domains: retinoblastoma-associated protein (RB) binds
to LXCXE motif; DNA polymerase α subunit (POLα) binds to DBD domain; p53 and topoisomerase 1 (TOP1) bind to the helicase domain.

FIGURE 6.7 Initiation of SV40 DNA replication by T-antigen. SV40 DNA replication begins when the large T-antigen binds to the origin of
replication (Ori). Two hexamers of T-antigen form a head-to-head orientation at the origin, unwinding the viral DNA followed by bidirectional
replication.

FIGURE 6.8 SV40 replisome versus host replisome. Cellular factors contributing to SV40 replisome are almost identical to those in host
replisome, except that the leading strand DNA polymerase ε is replaced by DNA polymerase δ, and Mcm2-7 DNA helicase is replaced by T-antigen.
RPA (replication protein A), PCNA (proliferating cell nuclear antigen).

9. PCNA (proliferating cell nuclear antigen) PCNA was discovered as a nuclear antigen in a dividing cell, as its name implies. It acts as a sliding
clamp at the replication fork, leading to increase of the “processivity” of DNA polymerase.
10. Replisome a complex molecular machine that carries out replication of DNA.
 Polyomaviruses: SV40 Chapter | 6 91

involved in the SV40 replisome are essentially identical with those involved in the “host replisome,” except that
Mcm2-7/Cdc45 DNA helicase is replaced by T-antigen (ie, viral DNA helicase).
Deregulation of cell cycle: SV40 deregulates cell cycle control to achieve multiple round of the viral DNA replica-
tion. SV40 overrides cell cycle control in two distinct mechanisms. First, T-antigen impedes the progress of S phase in
the cell cycle, thereby prolonging the S phase (ie, S phase arrest). How does T-antigen hold the progress of S phase?
T-antigen induces the ATR11 /ATM12-mediated DNA damage response (DDR13) pathway (Box 6.2). Such activated
ATR directly activates the p53 isoform Δp53, leading to upregulation of the Cdk inhibitor p21, which forces the host
cell to stay in the replicative S phase. This pathway is called ATR-Δp53-p21 pathway14 (see Journal Club).
Intriguingly, ultraviolet (UV) irradiation of cells at G1/S transit also induces cell cycle arrest at S phase via ATR-
Δp53-p21 pathway. Similar to UV irradiation, SV40 infection induces the DDR pathway to prolong the S phase. Then,
what is the benefit for SV40 of inducing the DDR pathway? Since SV40 replication depends on the host factors, such
as DNA polymerase α plus δ, topoisomerases, and other factors that are functional only in S phase, the prolonged S
phase facilitates the progeny virus production.
Second, T-antigen overrides the “re-replication block,” which is the central regulatory mechanism to maintain the
integrity of the host chromosome. The re-replication block is to ensure that origins are utilized only once per cell cycle
so that all chromosome DNA are equally duplicated. Intriguingly, although SV40 heavily relies on host DNA machin-
ery, SV40 overrides the re-replication block so that it induces multiple rounds of cellular DNA synthesis, giving rise to
polyploid cells. How does the T-antigen override the re-replication block? A recent report showed that T-antigen binds
to Nbs115 and blocks the function of Nbs1, which is involved in the re-replication block. The deregulation of the re-
replication block by SV40 T-antigen represents the viral strategy to coopt the host’s cell cycle control for its own
benefit.
Late Gene Transcription: The onset of the viral genome replication cues the late gene transcription from the late
promoter (see Fig. 6.5). The late RNAs are alternatively spliced into multiple mRNAs, which are translated into VP1 to
VP3 proteins. These capsid proteins are translocated to the nucleus, where the viral capsid assembly occurs.
Assembly and Release: The viral capsids are assembled in the nucleus, and then extracellularly released via cell lysis
(see Fig. 6.3). It remained uncertain as to how cell lysis is triggered. Recently, a novel viral capsid protein, called VP4,
was identified. It is translated from the third AUG codon of VP2 ORF, sharing the C-terminus with VP2 and VP3.
Importantly, VP4 was shown to exert “viroporin” function, which induces pore formation in the membrane. It is VP4
that triggers cell lysis by disrupting the cell membrane.

6.4 EFFECTS ON HOST

For a permissive host, such as primates (ie, monkeys and human), the viral life cycle is fully executed in that viral
genome replication occurs, and the progeny virus are produced (Fig. 6.9). SV40 induces cell lysis and forms plaques on
monolayer cells of the permissive cells. In contrast, for nonpermissive hosts,16 such as rodents (mouse and hamster),
only the early phase of viral life cycle is executed, whereas the late phase (ie, the viral genome replication) is blocked.
Instead, however, cell transformation17 is observed (see Fig. 6.9). Thus, the consequence of SV40 infection is different
depending upon whether the host is permissive or nonpermissive for SV40 infection.
Tumor formation: SV40 infection leads to tumor formation in rodents, a nonpermissive host. Upon infection,
T-antigen is continually expressed, although the late gene expression is not permitted. Which function of T-antigen con-
tributes to cell transformation? As described above, T-antigen has multiple biochemical activities such as DNA binding

11. ATR (ataxia telangiectasia and RAD3-related) ATM-related gene. It is a serine/threonine kinase that is involved in sensing DNA damage and
activating the DNA damage checkpoint, leading to cell cycle arrest.
12. ATM (ataxia telangiectasia-mutated) a tumor suppressor gene identified as a mutated gene in ataxia telangiectasia. Ataxia telangiectasia is a
rare, neurodegenerative, inherited disease causing severe disability. ATM, a serine/threonine protein kinase, is activated by double-strand break
(DSB), acting as a sensor of the DNA damage.
13. DNA damage response (DDR) it refers to the signaling pathway that is induced by DNA damage (see Box 6.2).
14. ATR-Δp53-p21 pathway it refers to a signaling pathway that is triggered by ATR. The activated ATR activates an isoform of p53 (ie, Δp53) via
phosphorylation, which then leads to induction of p21 expression. Such induced p21 induces cell cycle arrest. Δp53, an isoform of p53, resulted from
alternative splicing. Unlike p53, it does not bind to T-antigen.
15. Nbs1 a component of MRN complex (Mre11, Rad50, and Nbs1) that is involved in initial processing of double-strand DNA break repair.
16. Nonpermissive host a host that permits only the early phase of the viral life cycle.
17. Cell transformation It refers to a process by which cells acquires the properties of cancer cells.
BOX 6.2 DNA Damage Response and DNA Viruses
Genome stability is an essential feature for cell survival. Eukaryotic cells have evolved a complex set of pathways to repair DNA
and ensure the faithful duplication of the genome. Diverse kinds of genotoxic stresses, such as UV irradiation and reactive
oxygen species, cause DNA damage. In addition to genotoxic stresses, double-strand break (DSB), a fatal DNA damage, takes
place inevitably during DNA replication. To overcome such DNA damages, cells are equipped with diverse DNA repair
mechanisms. In this process, the signaling pathway that senses the DNA damage and activates the DNA repair mechanisms is
collectively called DNA damage response (DDR).
To what extent is DDR related to DNA viruses? Being a parasite, a virus co-opts diverse cellular functions. In fact, DDR is the
one that is most extensively exploited by DNA viruses. Intriguingly, some viruses trigger DDR to induce the viral DNA synthesis
in resting cells they infect, while other viruses suppress DDR. For instance, SV40 and HPV induce ATM/ATR-mediated DDR, in
that the activation of S phase checkpoint results in S phase delay or arrest, which prolongs the ensuing viral genome replication
(Table 6.B1). In contrast, adenovirus blocks DDR for efficient viral genome replication. In fact, DDR is induced by adenovirus
infection, since the adenoviral DNA itself is recognized as double-strand break damage (see Box 8.1). Consequently, the conca-
temers of the viral genome are formed, unless the DSB repair is blocked. Thus, the inhibition of DSB repair by the virally coded
proteins is critical for the efficient adenoviral genome replication. In the case of herpesviruses, the replication intermediates of
linear DNA genomes are recognized by ATM/ATR without invoking the DDR signaling. In cells infected by herpes simplex virus
(HSV-1), ATM/ATR-mediated DDR is also blocked by the virally coded proteins. It is apparent that DNA viruses have evolved
to acquire functions that block DDR signaling in order to avoid unwanted DNA products, as the linear DNA genomes are
inevitably recognized by ATM/ATR as DNA damages.

(A) DNA damage response signaling pathway in DNA

viruses. (A) ATM signaling and ATR signaling. ATM
kinase is activated when the double-strand break DNA dam-
age is sensed via a mechanism involving MRN (Mre11/
Rad50/Nbs1). The activated ATM then phosphorylates the
histone variant H2AX. The phosphorylated H2AX (γH2AX)
binds to the mediator of DNA damage checkpoint protein-1
(MDC1), leading to recruitment of additional ATM-MRN
complexes and further phosphorylates H2AX. The ATM
also phosphorylates downstream targets, CHK2 kinase, lead-
ing to cell cycle arrest and DSB repair. ATR kinase is acti-
vated by sensing DNA damage via a mechanism involving
Replication protein A (RPA). ATR/ATRIP and Rad9-Rad1-
Hus1 (also known as 9-1-1) are independently recruited to
the damaged sites. Then, ATR activator topoisomerase-
binding protein-1 (TOPBP1) is recruited via interaction with
Rad9 of 9-1-1. TOPBP1 binds and activates ATR, leading to
phosphorylation of CHK1. In response to DNA damage or
replication stress, cell cycle arrest is induced. (B) The roles
of DNA damage response (DDR) signaling pathway in DNA
viruses. The activation of DDR signaling is essential for
cells to enter S phase that ensure the viral DNA synthesis.
For some viruses such as SV40 and HPV, virus-encoded
proteins, T-antigen and E7, induce the DDR, while linear
DNA genome itself could induce DDR in adenovirus and
C HSV-1 infected cells. On the other hand, DNA viruses hav-
ing the linear DNA genome, the activated DDR needs to be
(B) blocked for efficient viral DNA replication.

(Continued )
 Polyomaviruses: SV40 Chapter | 6 93

BOX 6.2 (Continued)

TABLE BOX 6.2 DNA Damage Response and DNA Viruses

Family Virus DNA Damage Response Host Factors Viral Function

Signaling Targeted by Proteins
Polyomavirus SV40 ATM signaling activated
T-antigen S phase delay
Papillomavirus HPV-31 ATM signaling activated
E7 S phase arrest
Adenovirus Ad5 ATM signaling inhibited MRN complex E1B-55/E4 orf6 Blockade of DSB
repair
Herpesvirus HSV-1 ATM signaling inhibited DNA-PK ICP0 Blockade of DSB
repair
ATR signaling inhibited ATR/ATRIP/RPA ICP8/UL8/UL5/UL52 Blockade of DSB
complex complex repair

FIGURE 6.9 Permissive host versus nonpermissive host. The viral life cycle is fully executed in a monkey cell, a permissive host, and the progeny
virions are released by cell lysis. In other word, the cells are killed. In nonpermissive cells, ie, rodent cells, the early phase of the virus life cycle is
executed, while the late phase is not permitted. As a result, the cells are transformed.

activity and DNA helicase activity. In fact, these biochemical functions are not directly related to cell transformation.
Instead, the T-antigen’s ability to interact with host proteins is directly related to its cell transformation function.
Specifically, T-antigen binds to two gene products of tumor suppressor genes: p53 and Rb18 protein (ie, pRB105)
(see Fig. 6.6). Indeed, co-immunoprecipitation of cell lysate with anti-T-antigen antibody brought down a protein hav-
ing a size of 53 kDa, revealing the protein—protein interaction between T-antigen and p53 (Fig. 6.10). Mechanistically,
T-antigen blocks the tumor suppressor function of p53 via the protein—protein interaction, thereby contributing to cell
transformation (see Fig. 24.4). Likewise, co-immunoprecipitation of cell lysate with anti-Rb antibody brought down
T-antigen as well as pRB105 or vice versa, revealing the protein—protein interaction between T-antigen and pRB105
(Fig. 6.10). After all, T-antigen blocks the tumor suppressor function of pRB105 via protein—protein interaction,
thereby contributing to cell transformation (see Fig. 24.3).

18. Rb (retinoblastoma) a tumor suppressor gene that was first identified as a defective gene in retinoblastoma.
94 PART | II DNA Viruses

FIGURE 6.10 Evidence for the interactions between T-antigen and p53 and between T-antigen and Rb protein. (A) A diagram illustrating the
strategy of co-immunoprecipitation to examine the protein—protein interaction. Antibody specific for the protein A can bring down the protein B, if
the protein B binds to the protein A in cells. The protein G, which has the ability to bind to the Fc fragment of IgG, linked to Sepharose bead is uti-
lized to precipitate the antigen—antibody complex. The protein A-interacting proteins can be revealed by SDS-PAGE. (B) Cell lysates were precipi-
tated with either anti-T-antigen antibody or anti-Rb antibody, and the precipitates were resolved by SDS-PAGE.

Altogether, SV40 induces tumor formation via dysregulation of two important tumor suppressor proteins—p53 and
Rb—by T-antigen (see Fig. 24.8).

6.5 PERSPECTIVES
Polyomaviruses have served as a model for molecular oncology as well as eukaryotic molecular biology. A payoff
was the discovery of p53 and Rb protein as tumor suppressor gene products, in which their tumor suppressor functions
are suppressed by T-antigen via a protein—protein interaction. Until recent discoveries of human polyomaviruses, such
as BK virus and JC virus, the research on polyomaviruses served as a model, and seemed to be unrelated to human dis-
eases. More recently, quite a few human polyomaviruses, including MCV, were discovered by the advent of new
sequencing technology termed next generation sequencing. In particular, MCV has drawn a lot of attention, as it is clearly
found to be associated with MCC, an aggressive form of skin cancer. Knowledge accumulated from studies on animal
polyomaviruses during the 1970s and 1980s now serves as a keystone for the investigation of human polyomaviruses.

6.6 SUMMARY
G Polyomavirus: Polyomavirus possess a small circular DNA genome (B5.3 kb in size).
G SV40 genome: SV40, the prototype of polyomavirus, infects lytically permissive monkey cells but transforms
nonpermissive rodent cells.
G Human polyomavirus: MCV is found associated with MCC, an aggressive form of skin cancer.
G Virion structure: Nonenveloped capsid structure, inside which the viral DNA genome is found in association with
histone molecules.
G Genome replication: The viral replication heavily relies on host DNA replication machinery. T-antigen is the only
viral protein essential for viral genome replication.
G Host effect: SV40 infection leads to tumor formation in rodent cells. The binding ability of T-antigen to p53 and Rb
protein is critical for tumorigenesis.

STUDY QUESTIONS
6.1 The life cycle of SV40 can be divided into early and late phase by the onset of the viral genome replication, where
a switch from early to late phase is facilitated by T-antigen. Explain the molecular mechanisms underlying the
early to late switch.
 Polyomaviruses: SV40 Chapter | 6 95

6.2 Describe to what extent the viral life cycle proceeds in the following SV40 mutants, when these mutants DNA
were transfected into CV-1 cell (monkey cell) or COS cell (T-antigen expressing monkey cell). (1) mutant B
(T-antigen helicase mutant), and (2) mutant B (Origin-defective mutant)
6.3 SV40 mutants (ie, Ori-defective) are frequently used in establishing human cell lines from a primary cell. Explain
why the Ori-defective mutants are used, instead of wild-type SV40.

SUGGESTED READING
Chang, Y., Moore, P.S., 2012. Merkel cell carcinoma: a virus-induced human cancer. Annu. Rev. Pathol. 7, 123
144.
DeCaprio, J.A., Garcea, R.L., 2013. A cornucopia of human polyomaviruses. Nat. Rev. Microbiol. 11 (4), 264
276.
Jiang, M., Zhao, L., Gamez, M., Imperiale, M.J., 2012. Roles of ATM and ATR-mediated DNA damage responses during lytic BK polyomavirus
infection. PLoS Pathog. 8 (8), e1002898.
Lilley, C.E., Schwartz, R.A., Weitzman, M.D., 2007. Using or abusing: viruses and the cellular DNA damage response. Trends Microbiol. 15 (3),
119
126.
Sowd, G.A., Fanning, E., 2012. A wolf in sheep’s clothing: SV40 co-opts host genome maintenance proteins to replicate viral DNA. PLoS Pathog.
8 (11), e1002994.

JOURNAL CLUB
G Rohaly, G., Korf, K., Dehde, S., Dornreiter, I., 2010. Simian virus 40 activates ATR-Δp53 signaling to override cell cycle and DNA replication
control. J. Virol. 84 (20), 10727
10747.
Highlight: SV40 overrides cell cycle control, thereby prolonging the S phase. This paper demonstrated that SV40 T-antigen induces S phase
arrest by activating ATR-Δp53-p21 pathway.