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Hepatitis B Virus Capsids Have Diverse Structural Responses to

Small-Molecule Ligands Bound to the Heteroaryldihydropyrimidine
Pocket
Balasubramanian Venkatakrishnan,a Sarah P. Katen,b Samson Francis,a,b Srinivas Chirapu,c,d M. G. Finn,c,d Adam Zlotnicka
Molecular and Cellular Biochemistry Department, Indiana University, Bloomington, Indiana, USAa; Assembly Biosciences, Bloomington, Indiana, USA, and San Francisco,
California, USAb; Department of Chemistry and the Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USAc; Georgia Institute of
Technology, School of Chemistry and Biochemistry, Atlanta, Georgia, USAd

ABSTRACT
Though the hepatitis B virus (HBV) core protein is an important participant in many aspects of the viral life cycle, its best-char-
acterized activity is self-assembly into 240-monomer capsids. Small molecules that target core protein (core protein allosteric
modulators [CpAMs]) represent a promising antiviral strategy. To better understand the structural basis of the CpAM mecha-
nism, we determined the crystal structure of the HBV capsid in complex with HAP18. HAP18 accelerates assembly, increases
protein-protein association more than 100-fold, and induces assembly of nonicosahedral macrostructures. In a preformed cap-
sid, HAP18 is found at quasiequivalent subunit-subunit interfaces. In a detailed comparison to the two other extant CpAM
structures, we find that the HAP18-capsid structure presents a paradox. Whereas the two other structures expanded the capsid
diameter by up to 10 Å, HAP18 caused only minor changes in quaternary structure and actually decreased the capsid diameter by
⬃3 Å. These results indicate that CpAMs do not have a single allosteric effect on capsid structure. We suggest that HBV capsids
present an ensemble of states that can be trapped by CpAMs, indicating a more complex basis for antiviral drug design.

IMPORTANCE
Hepatitis B virus core protein has multiple roles in the viral life cycle—assembly, compartment for reverse transcription, intra-
cellular trafficking, and nuclear functions—making it an attractive antiviral target. Core protein allosteric modulators (CpAMs)
are an experimental class of antivirals that bind core protein. The most recognized CpAM activity is that they accelerate core
protein assembly and strengthen interactions between subunits. In this study, we observe that the CpAM-binding pocket has
multiple conformations. We compare structures of capsids cocrystallized with different CpAMs and find that they also affect
quaternary structure in different ways. These results suggest that the capsid “breathes” and is trapped in different states by the
drug and crystallization. Understanding that the capsid is a moving target will aid drug design and improve our understanding
of HBV interaction with its environment.

H epatitis B virus (HBV) causes degenerative liver disease and is

the leading cause of liver cancer, with 240 million chronically
infected individuals (1, 2). Current antiviral therapies control the
progression of the disease but fail to eliminate the virus (3, 4).
There is a need for improved therapies to combat chronic infec-
tions. One approach is to target virus assembly (5–7).
HBV is an enveloped, double-stranded DNA virus with an ico-
sahedral nucleoprotein core. The HBV capsid is the protein shell
of the core. Beyond genome protection, the capsid is involved in
intracellular trafficking, interaction with nuclear import machin-
ery, regulation of reverse transcription, signaling, completion of
reverse transcription, RNA chaperoning, and envelope acquisi-
tion (8). Capsid assembly is central to HBV replication. In vivo
HBV capsids assemble around an RNA copy of the viral genome
bound to the viral reverse transcriptase to form immature HBV
cores (9). These cores mature in the host cytoplasm through the
reverse transcription of the pregenomic RNA to a relaxed circular
double-stranded DNA (10).
A homodimer is the basic functional unit of the HBV core
protein (Cp) (Fig. 1d) (11). Cp has no human homolog, which
contributes to its value as an antiviral target. Cp dimers self-as-
semble to form capsids in a tightly regulated and dynamic process
(12–14). Wild-type Cp is 183 residues long and consists of two
domains: the N-terminal assembly domain (residues 1 to 149),
which forms the contiguous shell, and the C-terminal RNA-bind-
ing domain (residues 150 to 183) (15). The assembly domain
(Cp149) is capable of self-assembly in vitro and in vivo, forming
capsids that are indistinguishable from wild-type capsids (15–17).
Comparisons of free and capsid-constrained Cp149 reveal a stable
central chassis connected by glycine or proline hinges to sub-
domains: the spikes (residues 63 to 94), interdimer contact region
(residues 111 to the C terminus), and a fulcrum that mechanically
connects the other two subdomains (residues 10 to 25) (18).
The capsid has a T⫽4 icosahedral symmetry, whereby the 60
asymmetric units are each comprised of four Cp monomers (des-
ignated A, B, C, and D or AB dimers and CD dimers) in
Received 4 December 2015 Accepted 27 January 2016
Accepted manuscript posted online 3 February 2016
Citation Venkatakrishnan B, Katen SP, Francis S, Chirapu S, Finn MG, Zlotnick A.
2016. Hepatitis B virus capsids have diverse structural responses to small-molecule
ligands bound to the heteroaryldihydropyrimidine pocket. J Virol 90:3994 – 4004.
doi:10.1128/JVI.03058-15.
Editor: W. I. Sundquist
Address correspondence to Adam Zlotnick, azlotnic@indiana.edu.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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 HBV Capsid with Bound Drug

symmetry) contain two each of the B, C, and D subunits. Crystal

FIG 1 Core protein allosteric modulators and the Cp dimer. (a) HAP18; (b)
HAP1; (c) AT-130; (d) a Cp dimer with secondary structural elements labeled.
quasiequivalent environments that differ slightly from each other
(Fig. 2a) (19, 20). The A subunit interfaces with other A subunits
to form the 12 5-fold vertices of the capsid. The 20 3-fold vertices
contain three CD dimers each. The 30 quasi-6-fold vertices (2-fold
structures, showing multiple conformations of Cp in capsid and
dimer forms, have been determined (11, 18, 21–23).
In vitro HBV capsid assembly depends on a nucleus, believed to
be a trimer of dimers, followed by rapid elongation (14). Assembly
is entropically driven by burial of the hydrophobic surface (12,
24). Dimers associate with weak contact energy, which allows
“thermodynamic editing” to remove incorrectly and weakly asso-
ciated dimers (25). Aggressive assembly conditions (increased
temperature, Cp concentration, and ionic strength) that result in
stronger association energies overnucleate the assembly reaction
and lead to the formation of kinetically trapped intermediates (12,
25, 26).
Small-molecule core protein allosteric modulators (CpAMs)
(Fig. 1) can activate Cp assembly and lead to the formation of a
variety of nonuniform kinetically trapped structures (27–30).
They act by favoring an assembly-active form of Cp and by stabi-
lizing protein-protein interactions (21, 27, 30–32), implying a
competition between functional assembly and misassembly (29,
33). Besides being noninfective, misassembled structures also se-
quester and deplete functional Cp dimers that would otherwise be
involved in virion assembly. These molecules are predominantly
hydrophobic and were originally discovered as potent nonnucleo-
side inhibitors of HBV replication (31). Heteroaryldihydropy-
rimidines (HAPs) and phenylpropenamides are two families of
CpAMs that lead to suppression of HBV infection (34, 35). In vitro
studies have established a strong correlation between inhibition of
HBV replication and HAP-induced assembly (29). At saturating
concentrations, some CpAMs (e.g., HAPs but not phenylpropen-
amides) induce the formation of aberrant structures (36). Crystal

FIG 2 Crystal structure of HAP18-bound capsid and its comparison to the apo-capsid. (a) A cartoon representation of the quaternary structure of an HBV capsid
complexed with HAP18. A capsid asymmetric unit of two dimers is highlighted by surface representation, with the A subunit in blue, B subunit in red, C subunit
in green, and D subunit in yellow. The positions of the HAP18 molecules in the asymmetric unit are marked with numbered black circles: 1, B-associated HAP18;
2, C-associated HAP18. (b) Close-up of a CD dimer showing HAP18 (sphere representation) in the C subunit binding pocket, which in the context of a capsid
will be capped by the neighboring D subunit. The bottom of this image is on the interior of the capsid; the partially unraveled four-helix bundle forms the spike
that punctuates the capsid outer surface. (c) A stereoimage of the CD dimer (red) with the 2Fo-Fc map (blue) at 1.5 ␴ contouring shows the fit of the model to
the electron density.

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Venkatakrishnan et al.

structures have been determined for preassembled HBV capsids TABLE 1 Crystallographic data collection and refinement statistics for
bound to a representative HAP molecule, HAP1, and a represen- HAP-18-bound capsida
tative phenylpropenamide, AT-130 (Fig. 1) (21, 23). These mole- Characteristic (unit) Value
cules bind to the same hydrophobic pocket at a dimer-dimer in- Data collection
terface. Both affect the quaternary structure of the capsid, altering Space group C2
the spatial relationship between dimers. These changes were large Unit cell parameters
enough to cause these capsids to crystallize with different unit cell a, b, c (Å) 529.37, 367.41, 542.00
parameters from capsids without bound CpAM (apocapsids). Angles (°) ␣ ⫽ ␥ ⫽ 90, ␤ ⫽ 104.79
However, while the quaternary changes observed for these struc- Resolution (Å) 35.68–3.89 Å
tures were in the same mode, i.e., expansion around the 5-fold Rsym(%) 13.3 (44.7)
Average I/␴ 12.7 (4.3)
axes, the extent of these changes were distinctly different; HAP1
Completeness (%) 90.6 (77.8)
induced a much more dramatic quaternary rearrangement than Redundancy 3.1 (2.8)
AT-130. Also, there was not a consensus on a preferred binding No. of unique reflections 677,647
site; HAP1 bound preferentially to C subunits, while AT-130
bound preferentially to B subunits. AT-130 binding also resulted Refinement
in significant tertiary structural changes, while HAP1 binding did Noncrystallographic symmetry 60-fold
not (23). These tertiary changes were suggested to be compensa- R factor (%) 27.8
tory, to allow the formation of morphologically normal capsids, in Correlation coefficient (%) 91.66
No. of atoms 4,610
contrast to HAP1, where the quaternary changes alone appeared
RMSD bond length (Å) 0.004
to result in assembly misdirection. RMSD bond angle (°) 0.964
Based on the structure of the HAP1-bound capsid, we devel- Ramachandran outliers (%) 3
oped a series of HAP derivatives that differentially affected assem- Rotamer outliers (%) 11.2
bly (29). One of these compounds, HAP18 (Fig. 1C), increased the Ramachandran favored (%) 88.1
stability of protein-protein interactions (considerably more than Mean B factor size (Å2)
HAP1), increased the rate of Cp assembly (less than HAP1), and Protein 180.9
induced the formation of large, regular complexes— believed to HAP18 267.1
a
be tubes—that were hundreds of micrometers long (29). As Values in parentheses are for the highest-resolution shell of data (3.98 to 3.89 Å). Rfree
is not reported because it is highly correlated to Rwork due to noncrystallographic
HAP18 is larger than other HAPs, its orientation in the HAP site
symmetry averaging during refinement and is not statistically independent.
was likely to be readily discerned, even at a modest resolution.
Thus, to confirm the HAP binding site, identify the orientation of
HAP and its derivatives in the binding site, and understand the
Diffraction data collection. Selected crystals for data collection were
effect of CpAM size on HBV capsid structure, we determined the cryoprotected as described previously (21). Cryosoaked crystals were fro-
crystal structure the HBV capsid in complex with HAP18 at a zen in a stream of gaseous nitrogen at ⫺170°C before they were trans-
3.9-Å resolution. Comparison to other CpAMs shows a diversity ported in liquid nitrogen to the Advanced Photon Source (APS) synchro-
of conformational responses, suggesting that these molecules al- tron. Data were collected on the 14BMC beamline with an exposure time
low us to observe individuals from an ensemble of structures. of 30 s, 0.2° oscillations, and a detector distance of 500 mm.
Structure determination and refinement. Diffraction data were pro-
cessed using the HKL2000 program (39). Molecular replacement and sub-
MATERIALS AND METHODS
sequent refinement were carried out with programs in the Phenix suite
Sample preparation. Hepatitis B subtype adyw 3CA Cp150 (a Cp149 (40). A previously determined HBV capsid structure (PDB ID 1QGT) was
variant with the three native cysteines mutated to alanine and a C-termi- used as the initial phasing model (11). Initial phases from the 1QGT
nal cysteine appended) capsid protein dimers were expressed and purified model were calculated to a resolution of 6 Å. The phases were then ex-
as described in detail previously (37, 38). Briefly, Escherichia coli cells tended in eight steps in subsequent refinement cycles to a final resolution
expressing the capsid protein were lysed by sonication and the assembled of 3.89 Å. This is the same approach as that used with other HBV-CpAM
HBV capsid in the cell lysate was purified by size exclusion chromatogra- structures to minimize model bias (21, 23).
phy. T⫽4 and T⫽3 3CA Cp150 capsids from pooled fractions were iso- For refinement of the final molecular model, global noncrystallo-
lated by sucrose gradient centrifugation and immediately dialyzed into 10 graphic symmetry restraints were used in the refinement with isotropic
mM HEPES buffer at pH 7.5 with 150 mM NaCl. group B-factor refinement. This allowed averaging of the 60 copies of the
Crystallization. 3CA Cp150 T⫽4 capsids at a 17.5-mg/ml concentra- icosahedral asymmetric unit comprised of one AB dimer and one CD
tion were mixed with a 1:1 molar concentration (based on the molar dimer. Using Ramachandran restraints in the initial cycles of refinement
concentration of a Cp149 protein dimer) of a racemic mixture (R and S improved the R factors, model fitting, and the overall bonds and angles.
conformers) of HAP18 in dimethyl sulfoxide (DMSO) at a 2:1 molar ratio Manual model building with refinement in the density was performed
of HAP18 mixture to 3CA Cp150 dimer, prior to crystallization. The with Coot (41). Though a 5% test set of structure factors was reserved for
crystallization trials were carried out at room temperature with 4 ␮l sitting cross-validation, the values of Rwork and Rfree were highly correlated due
drops. The final crystallization well solutions were composed of 5 to 10% to the extensive noncrystallographic averaging and did not provide a valid
polyethylene glycol 5000 monomethyl ether, 0 to 5% polyethylene glycol comparison. The models of the HAP18 R and S stereoisomers were gen-
8000 monomethyl ether, 10 to 16% 2,3-butanediol, 100 mM Tris (pH erated using the Prodrg program (42). Geometrical restraints were gener-
9.0), 150 mM NaCl, and 300 mM KCl. The crystals took about a week to ated for the molecule using the eLBOW program (43) in Phenix. The
grow, although diffraction quality crystals were relatively rare. The crystal HAP18 was modeled into the binding site density after phase extension
used for this data set came from a well with 5% polyethylene glycol 5000 and visual inspection of the density. The model developed with Phenix.re-
monomethyl ether, 5% polyethylene glycol 8000, 14% 2,3-butanediol, fine resulted in a final Rwork of 27.8% and a correlation coefficient of
100 mM Tris (pH 9.0), 150 mM NaCl, and 300 mM KCl. 91.66% (Table 1). PyMOL (The PyMOL Molecular Graphics System, ver-

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 HBV Capsid with Bound Drug

FIG 3 HAP18, HAP1, and AT-130 each lead to distinctly different changes in tertiary and quaternary structures at the level of the capsid. Overlays of
CpAM-bound capsids (magenta) on apo-capsid (cyan) when viewed down the 5-fold reveal systematic differences in capsid structure in the exterior (upper
panels) and interior (lower panels). (a) There is a negligible size difference between the HAP18-bound capsid and the apo-capsid. Variegation of the coloring on
interior and exterior views indicates the close level of agreement. (b) The HAP1-bound capsid has a significantly larger diameter, shown by the excess of magenta
on the exterior and cyan on the interior. This difference is most evident at the 5-fold vertices, formed by A subunits. On the exterior view, the cyan patch ringing
the 5-fold is the side of the CD spike. (c) The AT-130-bound capsid is also expanded compared to the apo-capsid to an even greater degree than the HAP1
structure. Most of the exterior cyan-colored surface is due to differences in the tertiary structure of the C subunit.

sion 1.8; Schrödinger, LLC, New York, NY) was used to generate figures.
The Maestro program (Maestro, version 10.1; Schrödinger, LLC, New
York, NY) was used to identify interacting residues and generate two-
dimensional (2-D) projections. Buried surface areas and binding energies
were calculated using the PDBe PISA tool (44). Superpositions and root
mean square deviation (RMSD) calculations were done using Phenix
tools.
Protein structure accession number. Coordinates and structure fac-
tors for HAP18-capsid have been deposited in the Protein Data Bank
under accession code 5D7Y.
RESULTS
HAP18 leads to minimal quaternary and tertiary structural
changes compared to the apocapsid. For crystallography with
CpAMs, we used a Cp149 variant that crystallizes more readily,
3CA Cp150, in which the three native cysteines were mutated to
alanine and a C-terminal cysteine was appended (21). The C-ter-
minal cysteine cross-links and stabilizes capsids. This mutant has
been used previously for crystal structure determination in apo,
HAP1-bound, and AT-130-bound forms (21, 23). 3CA Cp150
capsids and HAP18 were cocrystallized at a 2:1 HAP18-to-dimer
ratio. Because HAP18 is a racemic mixture with only one active
enantiomer, this meant that there was one active HAP per dimer
(35). Like other CpAM-capsid crystallization experiments, initial
crystals formed within 1 week of mixing protein with precipitant;
by comparison, capsids without CpAMs take at least 1 month to
crystallize (21). The crystal packing and unit cell dimensions for
the HAP18-HBV capsids were very similar to those of previously
investigated CpAM-HBV crystals and distinct from crystals of
capsid without bound CpAM. Indeed, we hypothesized that
CpAMs influence packing by distorting the capsid quaternary
structure to favor a HAP-capsid unit cell and expected to see the
same quaternary changes in the HAP18-bound capsid (21, 23).
When we examined the structure of the capsid, determined by
molecular replacement, we were surprised to find that the quater-
nary structure of the HAP18-bound capsid was unlike that of the
HAP1-capsid complex and instead was very similar to apo forms
(PDB ID 1QGT and 2G33) (11, 21) (Fig. 2a). Previous HBV-
CpAM complexes showed clear and significant deformation of the
tertiary and/or quaternary structure, resulting in expansion of the
average capsid diameter by as much as 10 Å, particularly around
the 5-fold axes, while still retaining icosahedral symmetry (21, 23).
The structure of the HBV-HAP18 capsid was a T⫽4 arrange-
ment of 120 tetravalent dimers (Fig. 2a). Each dimer is roughly
shaped like an upside-down T with the intradimer contact, com-
prised of a four-helix bundle, forming a protruding spike (Fig. 2b
and c). Interdimer contacts were made at the extremities of the
dimer base. The surface of the capsid is fenestrated by two classes
of pores: those between trimers of dimers at 3-fold and quasi-3-
fold groups of trimers of dimers and those at quasi-6-fold vertices.
A superposition of the HAP18-bound capsid structure on the
apo structure revealed that the capsid diameter was very similar
(Fig. 3a). The HAP18-bound capsid, in fact, seemed to be radially

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Venkatakrishnan et al.

FIG 4 Tertiary structure changes in the HBV capsid are associated with HAP18 binding. (a) The two-dimer asymmetric unit from the HAP18-bound capsid is
overlaid on the asymmetric unit (A, blue; B, red; C, yellow; D, blue) of the apo structure (cyan), based on superposition of complete capsids. (b) The structures
are quantitatively compared by plotting displacement of C␣s against residue number. As seen in the molecular comparisons in panels a, c, and d, the major
changes occur near the spike tips, notably helix 4a, of the C and D subunits and the C termini of all the subunits. (c) Overlay of the AB dimer, extracted from the
all-capsid overlay in panel a, shows little structural difference between these dimers. (d) Overlay of the CD dimer, extracted from the all-capsid overlay in panel
a, shows major changes at the spikes, indicated by the brace bracket.

compressed at the 5-folds by about 3 Å. Given the lack of striking
global structural changes, in contrast to previous HBV-CpAM
structures (Fig. 3b and c), detailed comparisons of the HAP18-
capsid structure to the apo structure were then performed. We
observed an absence of quaternary structure change at the level of
the whole capsid, the two dimers in the icosahedral asymmetric
unit, and the individual dimeric asymmetric units. The two-dimer
icosahedral asymmetric unit from a whole-capsid-based superpo-
sition of the HAP18-bound structure on the asymmetric unit of
the apocapsid had an RMSD of only 0.98 Å for ␣-carbons; we note
that the C-terminal 20 amino acids demonstrate a systematic shift
(Fig. 4). The relative positions of the Cp monomers were all but
indistinguishable from the apocapsid.
Despite the lack of quaternary structural changes, tertiary
structure differences were observed on the exterior surface near
the 5-fold vertices, the exterior surface near the quasi-6-fold ver-
tices, the walls of 3-fold pores, and the helical domains that form
the quasi-6-fold spikes (Fig. 3a and 4a). There were changes in the
C␣ positions near the C-terminal subdomains of all four subunits
in the capsid asymmetric unit, with most changes mapping to
positions after Gly123 in the middle of the C-terminal helix (Fig.
3a). Differences in the exterior and interior surfaces near the
5-fold vertices were a result of C-terminal tertiary changes in the A
subunit, leading to a decrease in internal diameter, as measured
5-fold to 5-fold. Structural differences near the quasi-6-fold ver-
tices were a result of corresponding C-terminal conformational
changes in the quasiequivalent B and C subunits. Differences in
the inner walls of the 3-fold pores resulted from C-terminal struc-
tural changes in the D subunit (Fig. 3a). Contacts between sub-
units are modulated by the C-terminal helix-loop-extended struc-
ture; we find that these motifs can move slightly to accommodate
a CpAM without altering quaternary structure.
Superposition of the asymmetric units of HAP18-bound and
apocapsid structures allowed visualization and analysis of tertiary
structural changes (Fig. 4a). A plot of C␣ displacement versus
residue number, comparing the monomers in the HAP18-capsid
to the corresponding subunits in the 1QGT structure, showed
specific structural changes particularly in helix 4a (residues 80 to
92) and in the latter half of helix 5 and the following turn (residues
123 to 135) (Fig. 4b). The electron density and corresponding
model at the spike tips (residues 75 to 79) were weaker than the
rest of the structure, and the model does not provide a reliable
indication of movement. The AB dimer from the HAP18-bound
structure superposed with the AB dimer from the apo structure
had a small mean displacement (RMSD, 0.50 Å), but with system-
atic shifts in the C-terminal residues after amino acid 120, in helix
5, and in helix 2, which crosses helix 5 (Fig. 4c and d and 1d).
Superposition of the CD dimers had a larger mean displacement
(RMSD, 1.41 Å), but most of the difference was localized to large
conformational shifts at the spikes, specifically shifts in helices 3

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FIG 5 HAP density and the HAP-binding site viewed from the capsid interior. The HAP-binding site is delineated by a C-shaped four-helix motif (orange
polygon), with one of the helices donated by the capping subunit. (a) The B-associated HAP site, viewed from the capsid interior, shows the R enantiomer of
HAP18 binding at the interface of the B (red) and C (dark green) subunits. The HAP pocket is viewed from the interior of the capsid here and in panel b. (b) The
C-associated HAP18, also an R enantiomer, is capped by the D subunit (yellow). (c) Viewed from the capsid exterior, electron density (black mesh) is shown for
the B-associated HAP18 (magenta) and the C-associated HAP18 (green) at a contour of 1.5 ␴. The R enantiomer fits density well. Shape complementarity of the
CpAMs for the pockets establishes the orientation of the CpAMs in their binding sites. Each molecular model is shown with its chlorophenyl group (Fig. 1)
pointing to the top of the figure.

(near residue 70) and the junction of helix 4a and 4b (residues 82
to 95), similar to those observed in the AT-130-bound capsid (Fig.
4d) (23). The systematic movement of the spikes, distal to the
HAP site, may reflect an allosteric response. A comparison to a
second apo structure (PDB ID 2G33) (21) showed similar mini-
mal quaternary structure differences between it and the HAP18-
bound capsid.
Electron density establishes the orientation of the HAP mol-
ecule in its hydrophobic pocket. The CpAM-binding pocket is a
hydrophobic site buried at the interface of neighboring dimers,
previously identified to bind HAP1 and AT-130. As in previous
studies, CpAM density was observed in B and C subunits but not
A and D (21, 23). The site is comprised of a pocket in one subunit
that is capped by residues from the neighboring subunit. For A
and D subunits, the pocket is occluded by a neighboring A subunit
or a neighboring B subunit, respectively. In contrast, the B pocket
is capped by the C subunit within the asymmetric unit (Fig. 5a),
and the C subunit is capped by the neighboring D subunit (Fig.
5b). Most of the residues forming the site are from the C-terminal
subdomains of the two contributing subunits. The site is bounded
by a “C-shaped” 4-helix motif, which includes helices 2, 4b, and 5
from the subunit with the pocket and helix 5 from the capping
subunit (Fig. 5a and b). This site is not open to the exterior surface
but is open to the capsid interior, particularly in the case of the
C-associated pocket (Fig. 5b). The curvature of the pocket im-
poses a preferred orientation of the HAP18 molecule, and helix 5
of the capping subunit clamps down on the molecule. In the ab-
sence of a HAP molecule, there is a naturally occurring gap be-
tween the hydrophobic surfaces of the two adjacent subunits.
HAP molecules fill this gap to effectively increase buried hydro-
phobic surface at the protein-protein interaction site, promoting
aggressive, error-prone assembly (25).
Previously, we observed that HAP1 bound preferentially to the
C subunit while AT-130 bound preferentially to the B subunit,
with very weak density in the other available site, though both had
weaker densities than observed for HAP18 (21, 23). The HAP18
density was equally strong in B and C subunits (Fig. 5c) and sim-
ilar in strength to the nearby protein. The asymmetric shape of

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Venkatakrishnan et al.
HAP18 and the relatively high-quality electron density con-
strained the placement and orientation of HAP18 in its binding
sites despite the modest resolution of the structure and the rela-
tively high B factor required for the molecule (Fig. 5c).
Though the HAP18 used in crystallization experiments was a
racemic mixture, only the R enantiomer has assembly activity and
suppresses HBV replication (30, 35). The R enantiomer was ob-
served in the crystal structure (Fig. 5c). In the ⬃5-Å HAP1-bound
capsid structure, the enantiomer could not be identified (21); in
fact, we were cautious of assigning the HAP to the weak density
observed. To examine the basis of selectivity with HAP18, we per-
formed parallel refinements with both the R and S enantiomers.
Refinement with the S enantiomer in both binding sites did not
yield improvements in the R factor (data not shown). It was ob-
served that the halogenated phenyl moiety in the S enantiomer of
HAP18 sterically clashed with the Phe23 side chain of both the B
and C subunits. The S enantiomer in the B-subunit-binding site
(which is sterically more restrained) also clashed with Trp102 and
Tyr132 from the C chain. The R enantiomer improved refinement
and showed complementarity with electron density in both sites
(Fig. 5c).
HAP18 binds different structural states in quasiequivalent
sites. Quasiequivalence is reflected in the HAP18 sites. The dialkyl
moiety of the HAP18 in the C-associated pocket was more ex-
posed to the capsid interior. The buried surface area of the B-as-
sociated HAP18 in its binding site was 299.6 Å2. The surface area
for the C-associated HAP18 was only slightly smaller, at 274.2 Å2,
even though the dialkyl moiety was solvent exposed. In contrast,
the substantial change in quaternary structure in the HAP1-
bound capsid (21) increased the solvent exposure at the C-associ-
ated site (the interfacial area between HAP1 and the site was only
233 Å2).
HAP18 had strong shape complementarity to both B and C
sites (Fig. 5). Most of the interactions in the binding sites were
based on the association of hydrophobic surfaces between HAP18
and amino acid side chains. However, specific interactions varied
between the two sites. In the B-associated site, the B subunit
Trp102 indole nitrogen hydrogen bonded to the HAP18 pyrimi-
dine ring. In the C-associated site, the guanidine group of Arg133
of the capping D subunit interacted with both the dihydropyrimi-
dine and pyridine ring of the C-associated HAP18. The C-associ-
ated HAP18 also appeared to have a ␲-stacking interaction with
Phe23 from the C subunit. Hydrophobic interactions in the B-as-
sociated site were observed to result from residues from both the B
and C subunits, while the interactions observed in the C-associ-
ated site were primarily from the C subunit.
The two HAP18 molecules in the icosahedral asymmetric unit
flank the C subunit C-terminal subdomain (residues following
G123). Conformational shifts in the C subunit, compared to the
apo structure, created room for the molecules in both sites where
HAP18-binding traps a specific capsid conformation (Fig. 6).
There were minimal conformational changes in the C terminus of
the B subunit, suggestive of lock-and-key binding, to accompany
the observed shift in the capping C subunit. For the C-associated
HAP18, the C-terminal tail of the C subunit was moved away from
the bound HAP18 compared to the apo structure, suggesting in-
duced fit, creating room to accommodate the ligand in the pocket.
A modest conformational change was also observed in the main
chain of the capping D subunit; the loop at the end of helix 5 bent
toward the binding site. In the apo structure, the loop faced away
4000 jvi.asm.org Journal of Virology
FIG 6 The C-terminal helix-turn-extended structure of the C subunit medi-
ates binding of both B- and C-associated HAP-18 molecules. An overlay of the
HAP18 structure on the apo structure (gray) reveals structural changes near
the HAP binding sites. To bind the B-associated HAP18 (magenta), minimal
changes are seen in the residues of the B subunit (red), but there is a shift in the
C subunit (dark green) C-terminal loop, after helix 5, to create room in the
B-associated HAP pocket. To bind the C-associated HAP18, the loop between
helices 4 and 5 of the neighboring D subunit (yellow) shifts by up to 3 Å
compared to the apo structure to form contacts with the C-associated HAP18
while the C terminus of the C subunit shifts away to create a binding pocket for
the CpAM.
from the pocket. The observed shift allowed Arg133 from the D
subunit loop to interact with HAP18. The movement of the C-ter-
minal subdomain of the C subunit correlates with distal structural
changes in the helices of the dimer interface, a correlation previ-
ously noted in a free-core dimer structure (18).
Comparing CpAM-bound capsids. The same site binds
HAP1, AT-130, and HAP18, but each CpAM led to distinctly dif-
ferent crystal structures (Fig. 3). HAP1 and AT-130 both ex-
panded capsids, while HAP18 led to a contraction. In overlays, the
differences are most readily visible when looking at the capsid
interiors, where the HAP18 structure is a clear outlier (Fig. 7).
However, the mechanisms of structural change for each CpAM
had striking differences. HAP1 minimally affected the tertiary
structure of the subunits but introduced large global quaternary
structure changes; the icosahedral asymmetric unit pivoted on a
point near the center of the quasi-6-fold so that the A subunit was
elevated by about 5 Å and the CD subunit was tilted to offset the
dimer interface spike, with respect to the apo structure (Fig. 7, top
panels, and 3b). We do note that the low resolution of the HAP1-
capsid structure (⬃5 Å) may have obscured tertiary structural
changes (21). AT-130 introduced both tertiary and quaternary
changes in the capsid, including a 4-Å upward rigid body shift in
the A and B subunits, similar to, but to a lesser extent than, what
was observed in HAP1, and substantial refolding of the CD inter-
face at the spike tip (Fig. 7, bottom panels, and 3c). These obser-
vations initially suggested that CpAM bound in the interfacial
hydrophobic pocket to induce quaternary changes in the capsid
structure and in turn led to the alternate unit cell.
While the HAP18-bound capsids crystallized with the same
unit cell dimensions as other CpAM-bound capsids, the quater-
nary structure of the capsid was very similar to that of an apocap-
sid. HAP18 interacts with capsid protein almost entirely through
atoms that it has in common with HAP1. While HAP1 led to a
large shift in quaternary structure compared to the apocapsid, the
almost identical binding interactions resulted in subunit move-
ment limited to modest tertiary changes in the C-terminal sub-
domain of the A, C, and D subunits and the intradimer contacts of
the CD spikes. The shift of the C-terminal subdomain is thus the
basis of the smaller internal diameter in HAP18 capsids.
April 2016 Volume 90 Number 8
 HBV Capsid with Bound Drug

FIG 7 HAP18, HAP1, and AT-130 each lead to distinctly different changes in tertiary and quaternary structures at the level of the dimer. Though the same
HAP-binding sites are filled, the effect on local structure is distinct in each case. (Top panels) Overlay of AB and CD dimers from the HAP1-bound capsid
structure (2G34, red) on corresponding subunits from the HAP18-bound capsid structure (yellow), based on the superposition of capsid asymmetric units.
There are minimal differences in structure, as HAP1 induces primarily quaternary structural changes affecting dimer-dimer position rather than structure.
(Bottom panels) Overlay of AB and CD dimers from the AT-130 phenylpropenamide-bound capsid structure (4G93, magenta) on corresponding subunits from
the HAP18-bound capsid structure (yellow). There are large differences in tertiary structure, especially in the CD dimer. The upper half of helix 4 in both the C
and D subunits is displaced by about a half turn.

A commonality seen in all the CpAM-bound capsid structures
is that minimal structure changes were observed in the AB dimer.
HAP1 preferentially bound the C pocket and caused quaternary
changes. AT-130 preferentially bound the B pocket and yet
changed the tertiary structure of the CD dimer as well as the capsid
quaternary structure. HAP18 bound both B and C subunits, caus-
ing no detectable quaternary changes, but affected the tertiary
structure of the CD dimer.
DISCUSSION
We have compared the structures of HBV capsids with and with-
out bound CpAMs. We had hypothesized that CpAMs would
expand capsid structure by distorting interdimer interfaces,
but this was incorrect. Unlike previous HBV-CpAM structures,
the HAP18-capsid has essentially the same quaternary structure as
the apocapsid. This is all the more surprising as the protein-CpAM
interactions with HAP18 and HAP1, the latter of which led to
gross changes in quaternary structure, are the same. The absence
of quaternary change in the HAP18-capsid structure is not due to
lack of CpAM binding in the HAP site, and based on the strength
of the density the sites are filled with high occupancy.
Thus, the expansion of the capsid seen with HAP1 and AT-130
(Fig. 3b and c) was not specifically the effect of filling the CpAM-
binding site. The range of HBV capsid protein structures, liganded
and apo, icosahedral and nonicosahedral, led us to suggest that in
solution Cp can be found in a diverse ensemble of quaternary
structures that can be biased by CpAM binding, mutations, and
crystal contacts. An analogous observation was made with exper-
imental antiviral ligands that bind to picornavirus capsids, which
make small tertiary structure changes to the capsid and stabilize
capsids to inhibit nucleic acid release; these ligands appear to act
by modifying picornavirus capsid dynamics, entropically stabiliz-
ing them (45–49).
CpAM binding, allostery, Cp assembly, and structure. The
CpAM-binding site is believed to act as a regulatory site for Cp
dimer conformation that affects subsequent assembly by triangu-
lating subdomains (18). The CpAMs studied thus far bind to the
pockets in B and C subunits around the quasi-6-fold vertices.
Unlike the HAP1 and AT-130 structures, where either B or C
pockets were preferentially filled (21, 23), HAP18 has equal occu-
pancy in both quasiequivalent sites, leading to 120 HAP18 mole-
cules per capsid. Filling the HAP site allows CpAMs to move one
end of helix 4b to alter the spikes, which thus allosterically interact
with the other half of the dimer. This effect is seen most clearly in
the C-associated pocket.
Though HAP18 is the largest CpAM yet examined, it showed
the smallest structural shifts. Nonetheless, HAP18 accelerated as-
sembly and stabilized protein-protein interactions, each more
than 100-fold (29). Indeed, small differences in the CpAM led to
large, unpredictable differences in products of assembly reactions
(21, 23, 27, 29, 30, 50). We expected that the piperidinyl dialkyl
moiety of HAP18 would result in additional interactions with core
protein (compared to HAP1 [Fig. 1a and b]). We unexpectedly
found that all HAP18 interactions with Cp were via the core HAP1
structure, with no contributions from the additional moiety.
Though HAP18 led to aberrant structures, it had very modest
effects on a preformed capsid structure, unlike HAP1; this is con-
sistent with an effect on structural dynamics rather than a static
effect.
When present in molar excess during assembly reactions,
CpAMs can alter assembly geometry. HAP1 led to large baglike
complexes with large patches of hexameric sheet (27). BAY41-

April 2016 Volume 90 Number 8 Journal of Virology jvi.asm.org 4001

Venkatakrishnan et al.
4109 led to similar, but usually incomplete, particles (30, 32). AT-
130, which has a completely different chemistry, yielded near-
normal capsids (33). HAP18 led to sheets of uniform width and
irregular length; we suggest that these are flattened tubes of regular
diameter (29). Similarly, single mutations that alter the HAP site
led to altered assembly and altered solution properties (18, 24, 50).
CpAMs preferentially bind at quasi-6-fold vertices; this forms a
basis for hexamer-rich macrostructures generated by stoichio-
metric excess of HAPs (27, 29). Without the curvature provided
by 5-fold vertices, the hexameric HAP-bound Cps form sheetlike
structures (27, 29). The differences in macrostructure features
likely are based on CpAM-modified quasiequivalence. The pro-
pensity for HAP18 to form tubes presumably correlates with its
ability to evenly fill four sites in the quasi-6-fold vertex (two B sites
and two C sites in the context of a capsid). We suggest that HAP18
introduces a 2-fold polarity in hexamers. During assembly in the
presence of excess HAP18, this would be the basis for polymeriz-
ing a uniform hexagonal lattice.
In capsids, AB dimers show little change in tertiary structure
with bound CpAM but can shift their position with reference to
capsid quaternary structure (23). This provides a rationale for
assembly of spherical capsids in the presence of substoichiometric
concentrations of CpAM (30). In the absence of excess CpAM,
5-fold vertices form at the correct locations for icosahedral geom-
etry. For AT-130, which led exclusively to “spherical” capsids (33,
51), it appeared that tertiary changes in the CD dimer compen-
sated for the CpAM and led to AB quaternary shifts that preserved
the icosahedral symmetry (23). We suggest that the choice be-
tween aberrant and regular capsid assembly is determined by the
structural state of the quasi-6-fold.
Capsid dynamics. The HBV capsid is plastic. Hydrogen-deu-
terium (H-D) exchange experiments showed that although Cp149
has a stable fold, the contained peptide amides are very labile (52).
Furthermore, binding antibodies to capsids changed H-D ex-
change rates distal to the epitope site. When Cp149 capsids were
treated with trypsin, the first cut was at Arg127 in helix 5 for both
free dimer and capsid (53). For Arg127 to fit the protease active
site, this helix must transiently partially unfold. Similarly, re-
straining subunit mobility through a highly conserved intradimer
disulfide interfered with capsid assembly (54). Paradoxically, the
component cysteines readily oxidize in capsids (54, 55). These
results are consistent with a fluctuating ensemble of icosahedral
states, which can be modified by bound CpAM.
The allosteric changes in HBV capsid and dimer and the asso-
ciated ensemble of structural states play pivotal roles in the HBV
life cycle. For example, capsids appear to be responsive to changes
in nucleic acid conformation, as reverse transcription generates
double-stranded DNA from an RNA template (56). Nucleic acid-
induced changes to virus structure also modulate interactions
with surface protein. Conversely, changes in interdimer contacts
affected reverse transcription (24). Mature capsids are less stable
than RNA-filled capsids, presumably due to both the tension from
the packaged double-stranded DNA and the oxidation of the C61
disulfide (54, 57, 58). The C-terminal domains of full-length Cp
become at least transiently exposed as a function of genome mat-
uration and phosphorylation (59–62). Therefore, limiting Cp
conformation by blocking allostery could be devastating to the
virus life cycle.
Crystallization also suggests that CpAMs affect capsid dynam-
ics. Crystallization of structurally heterogeneous proteins can be
4002 jvi.asm.org Journal of Virology
facilitated by solutes that favor a more homogeneous form (63).
One commonality between the CpAM-bound capsid crystals is
that they grow much faster (on the order of days) than apocapsid
crystals (on the order of months), which could be explained by a
decrease in capsid dynamics with CpAM binding allowing for
more-rapid crystal elongation (11, 21, 23, 37). Capsid-CpAM
crystals and apo crystals also have differences in the way capsids
are packed, i.e., they are not isomorphic, despite the fact that the
HAP18-capsid structure is very similar to the native structure.
Strikingly, the crystals of CpAM-bound capsids have fewer pack-
ing interactions than capsids of the same protein without bound
CpAM (21, 23). We now observe that the difference in crystalliza-
tion is not a function of capsid quaternary structure as previously
hypothesized. Therefore, we suggest that the difference is a func-
tion of capsid stability and protein dynamics.
Antiviral mechanism and final comments. HAP18 has a
marked antiviral effect but modest quaternary structural effects.
In cell culture, HAP18 suppressed HBV replication, though not as
effectively as HAP1 and other related HAPs (29). A comparison of
various HAPs and phenylpropenamides showed that a reduction
in viral load correlates with the CpAM’s effects on the kinetics of
assembly (29, 33). While HAP18 stabilized the interdimer inter-
actions to a greater extent than HAP1, its effects on the rate of
assembly were weaker. HAP18 had a 50% effective concentration
(EC50) much closer to that of AT-130, which has a comparable
kinetic effect while having a negligible effect on intersubunit asso-
ciation energy (23, 29). The differential kinetic effect could arise
from the interactions between CpAM-bound subunits and/or
from the ability of the bound CpAM to alter subunit conforma-
tions. The differences between binding energy, kinetic effect, and
antiviral activity indicate a complex structure-activity relation-
ship. We suggest that HAP18’s ability to redirect the structural
states of the capsid protein (36) may have a role in its antiviral
effect. These results have led to ongoing studies on HBV structure
and dynamics.
Recently, Klumpp et al. published a description of the activities
of a HAP molecule (NVR-010-001-E2, here HAP-NVR) including
a high-resolution crystal structure (1.95 Å). HAP-NVR is a close
relative of HAP12 (29). In their study, HAP-NVR had been soaked
into crystals of Cp149-Y132A, an assembly-deficient core protein
mutant (64). Their structural results are largely consistent with
those reported here. In Cp149-Y132A crystals, the protein forms
an approximately trigonal sheet with a repeat of three, nearly
2-fold symmetric dimers forming a closed triangle. As with the
HAP18-capsid structure, they observed that the HAP core of
HAP-NVR interacted with residues of the HAP site but there was
little interaction with the HAP-NVR morpholino group, equiva-
lent in position to the piperidinyl moiety in HAP18. The biophys-
ical effects of HAP18 and HAP-NVR also had similarities. HAP18
was found to stabilize dimer-dimer interactions by ⫺1.8 kcal/mol
per contact, HAP12 increased stability by ⫺1.9 kcal/mol (29), and
HAP-NVR was found to raise the unfolding temperature of Cp149
capsids by about 10°C to 89°C. A critical difference between the
two structures arises from the quasiequivalence of a T⫽4 capsid:
there are substantial differences in HAP-protein interactions at
quasiequivalent B and C sites, whereas HAP-NVR in Cp149-
Y132A appears to be limited to a single environment that is dis-
tinct from those in a capsid. The molecular detail provided by the
high-resolution structure, therefore, does not explain or predict
April 2016 Volume 90 Number 8

the multiple binding modes for the same HAP molecule seen in a
biological context.
In this study, we have established the structural basis for
HAP18 binding and activity and that not all CpAMs share a struc-
tural effect. The exposure of the acetylene tails of HAP18 toward
the capsid interior would make it an attractive motif for additional
modifications. Our findings show that very modest structural
changes, possibly transient changes, can still disrupt capsid assem-
bly to significant antiviral effect. We therefore suggest a dynamic
as well as a structural basis for HAP antiviral activity. A dynamic
capsid is not unique to HBV, as demonstrated in studies with
picornaviruses (49, 65) and HIV (66). We therefore propose that
small molecules that limit dynamic ensembles can serve as an ef-
fective approach to antiviral treatment.
ACKNOWLEDGMENTS
We thank the Macromolecular Crystallography Facility at Indiana Uni-
versity, Bloomington, for facilitating preliminary X-ray screens and freez-
ing of crystals and the BioCARS staff at beamline 14BM-C at the Advanced
Photon Source, Argonne National Laboratory, for assistance with data
collection.
Competing financial interests: A.Z. has an interest in a biotechnology
company (Assembly Biosciences) and acknowledges a conflict of interest.
S.P.K. and S.F. are employed by Assembly Biosciences.
FUNDING INFORMATION
HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)
provided funding to Adam Zlotnick under grant number R01 AI067417.
Some elements of this work were supported by Assembly Biosciences,
including a supported research project to Adam Zlotnick. Beam time at
the Advanced Photon Source, Argonne National Laboratory, was sup-
ported by the U.S. Department of Energy, Basic Energy Sciences, Office of
Science, under contract W31-109-Eng-38. This work was supported by
grant R01 AI067417 from the NIH to A.Z.
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April 2016 Volume 90 Number 8