Hepatitis B virus (HBV) is an enveloped DNA virus with a spherical capsid (or core). The capsid is constructed from 120 copies of the homodimeric capsid protein arranged with T = 4 icosahedral symmetry. We examined in vitro assembly of purified E. coli expressed HBV capsid protein. After equilibration, concentrations of capsid and dimer were evaluated by size exclusion chromatography. The extent of assembly increased as temperature and ionic strength increased. The concentration dependence of capsid assembly conformed to the equilibrium expression: K(capsid) = [capsid]/[dimer](120). Given the known geometry for HBV capsids and dimers, the per capsid assembly energy was partitioned into energy per subunit-subunit contact. We were able to make three major conclusions. (i) Weak interactions (from -2.9 kcal/mol at 21 degrees C in low salt to -4.4 kcal/mol at 37 degrees C in high salt) at each intersubunit contact result in a globally stable capsid; weak intersubunit interactions may be the basis for the phenomenon of capsid breathing. (ii) HBV assembly is characterized by positive enthalpy and entropy. The reaction is entropy-driven, consistent with the largely hydrophobic contacts found in the crystal structure. (iii) Increasing NaCl concentration increases the magnitude of free energy, enthalpy, and entropy, as if ionic strength were increasing the amount of hydrophobic surface buried by assembly. This last point leads us to suggest that salt acts by inducing a conformational change in the dimer from an assembly-inactive form to an assembly-active form. This model of conformational change linked to assembly is consistent with immunological differences between dimer and capsid.

The capsids of most spherical viruses are icosahedral, an arrangement of multiples of 60 subunits. Though it is a salient point in the life cycle of any virus, the physical chemistry of virus capsid assembly is poorly understood. We have developed general models of capsid assembly that describe the process in terms of a cascade of low order association reactions. The models predict sigmoidal assembly kinetics, where intermediates approach a low steady state concentration for the greater part of the reaction. Features of the overall reaction can be identified on the basis of the concentration dependence of assembly. In simulations, and on the basis of our understanding of the models, we find that nucleus size and the order of subsequent "elongation" reactions are reflected in the concentration dependence of the extent of the reaction and the rate of the fast phase, respectively. The reaction kinetics deduced for our models of virus assembly can be related to the assembly of any "spherical" polymer. Using light scattering and size exclusion chromatography, we observed polymerization of assembly domain dimers of hepatitis B virus (HBV) capsid protein. Empty capsids assemble at a rate that is a function of protein concentration and ionic strength. The kinetics of capsid formation were sigmoidal, where the rate of the fast phase had second-power concentration dependence. The extent of assembly had third-power concentration dependence. Simulations based on the models recapitulated the concentration dependences observed for HBV capsid assembly. These results strongly suggest that in vitro HBV assembly is nucleated by a trimer of dimers and proceeds by the addition of individual dimeric subunits. On the basis of this mechanism, we suggest that HBV capsid assembly could be an important target for antiviral therapeutics.

Hepatitis B virus (HBV) is an enveloped virus with an icosahedral capsid. Its homodimeric capsid protein ("core antigen") assembles into particles of two sizes, one with T = 3 icosahedral symmetry (90 dimers) and the other with T = 4 symmetry (120 dimers). We have investigated this assembly process in vitro, using a variety of purified, bacterially expressed, capsid proteins. All of our constructs lacked the predominantly basic C-terminal 34 amino acids of the full-length capsid protein (183 amino acids) and were further truncated to terminate at specific points between residues 138 and 149. While the smallest construct (138 residues) did not assemble into capsids, those terminating at residue 140, and beyond, assembled into mixtures of T = 3 and T = 4 particles. The two kinds of capsids could be separated on sucrose gradients and did not interconvert upon protracted storage. The proportion of T = 3 capsids, assayed by sucrose gradient fractionation, analytical ultracentrifugation, and cryoelectron microscopy, was found to increase systematically with larger deletions from the C-terminus. The variant terminating at residue 149 formed approximately 5% of T = 3 capsids, while the 140-residue protein produced approximately 85% of this isomorph. For the 147-residue capsid protein, the structures of both capsids were determined to 17 A resolution by three-dimensional reconstruction of cryoelectron micrographs. In these density maps, the boundaries of the constituent dimers can be clearly seen and the quaternary structures of the two capsids compared. The arrangement of dimers around their icosahedral five-fold axes is almost identical, whereas the quasi-six-fold arrangements of dimers are distinctly different.

The assembly of virus capsids or other spherical polymers--empty, closed structures composed of hundreds of protein subunits--is poorly understood. Assembly of a closed spherical polymer is unlike polymerization of a filament or crystal, examples of open-ended polymers. This must be considered to develop physically meaningful analyses. We have developed a model of capsid assembly, based on a cascade of low-order reactions, that allows us to calculate kinetic simulations. The behavior of this model resembles assembly kinetics observed in solution (Zlotnick, A., J. M. Johnson, P. W. Wingfield, S. J. Stahl, and D. Endres. 1999. Biochemistry. 38:14644-14652). We exhibit two examples of this general model describing assembly of dodecahedral and icosahedral capsids. Using simulations based on these examples, we demonstrate how to extract robust estimates of assembly parameters from accessible experimental data. These parameters, nucleus size, average nucleation rate, and average free energy of association can be determined from measurement of subunit and capsid as time and concentration vary. Mathematical derivations of the analyses, carried out for a general model, are provided in an Appendix. The understanding of capsid assembly developed in this paper is general; the examples provided can be readily modified to reflect different biological systems. This enhanced understanding of virus assembly will allow a more quantitative analysis of virus stability and biological or antiviral factors that affect assembly.

Despite the development of vaccines, the hepatitis B virus remains a major cause of human liver disease. The virion consists of a lipoprotein envelope surrounding an icosahedral capsid composed of dimers of a 183-residue protein, 'core antigen' (HBcAg). Knowledge of its structure is important for the design of antiviral drugs, but it has yet to be determined. Residues 150-183 are known to form a protamine-like domain required for packaging RNA, and residues 1-149 form the 'assembly domain' that polymerizes into capsids and, unusually for a capsid protein, is highly alpha-helical. Density maps calculated from cryo-electron micrographs show that the assembly domain dimer is T-shaped: its stem constitutes the dimer interface and the tips of its arms make the polymerization contacts. By refining the procedures used to calculate the map, we have extended the resolution to 9 A, revealing major elements of secondary structure. In particular, the stem, which protrudes as a spike on the capsid's outer surface, is a 4-helix bundle, formed by the pairing of alpha-helical hairpins from both subunits.

Capsids of spherical viruses share a common architecture: an icosahedral arrangement of identical proteins. We suggest that there may be a limited number of common assembly mechanisms for such viruses. Previous assembly mechanisms were proposed on the basis of virion structure but were not rigorously tested. Here we apply a rigorous analysis of assembly to cowpea chlorotic mottle virus (CCMV), a typical, small, positive-strand RNA virus. The atomic resolution structure of CCMV revealed an interleaving of subunits around the quasi-sixfold vertices, which suggested that capsid assembly was initiated by a hexamer of dimers (Speir et al., 1995, Structure 3, 63-78). However, we find that the capsid protein readily forms pentamers of dimers in solution, based on polymerization kinetics observed by light scattering. Capsid assembly is nucleated by a pentamer, determined from analysis of the extent of assembly by size-exclusion chromatography. Subsequent assembly likely proceeds by the cooperative addition of dimers, leading to the T = 3 icosahedral capsid. At high protein concentrations, the concentration-dependent nucleation reaction causes an overabundance of five-dimer nuclei that can be identified by classical light scattering. In turn these associate to form incomplete capsids and pseudo-T = 2 capsids, assembled by oligomerization of 12 pentamers of dimers. The experimentally derived assembly mechanisms of T = 3 and pseudo-T = 2 CCMV capsids are directly relevant to interpreting the structure and assembly of other T = 3 viruses such as Norwalk virus and pseudo-T = 2 viruses such as the vp3 core of blue tongue virus.

Hepatitis B virus (HBV) is a leading cause of liver disease and hepatocellular carcinoma; over 400 million people are chronically infected with HBV. Specific anti-HBV treatments, like most antivirals, target enzymes that are similar to host proteins. Virus capsid protein has no human homolog, making its assembly a promising but undeveloped therapeutic target. HAP1 [methyl 4-(2-chloro-4-fluorophenyl)-6-methyl-2-(pyridin-2-yl)-1,4-dihydropyrimidine-5-carboxylate], a heteroaryldihydropyrimidine, is a potent HBV capsid assembly activator and misdirector. Knowledge of the structural basis for this activity would directly benefit the development of capsid-targeting therapeutic strategies. This report details the crystal structures of icosahedral HBV capsids with and without HAP1. We show that HAP1 leads to global structural changes by movements of subunits as connected rigid bodies. The observed movements cause the fivefold vertices to protrude from the liganded capsid, the threefold vertices to open, and the quasi-sixfold vertices to flatten, explaining the effects of HAP1 on assembled capsids and on the assembly process. We have identified a likely HAP1-binding site that bridges elements of secondary structure within a capsid-bound monomer, offering explanation for assembly activation. This site also interferes with interactions between capsid proteins, leading to quaternary changes and presumably assembly misdirection. These results demonstrate the plasticity of HBV capsids and the molecular basis for a tenable antiviral strategy. Perturbing HBV assembly, altering either the timing or the geometry of capsid formation, is expected to interfere with viral infection (42). In cultured cells, heteroaryldihydropyrimidine (HAP) molecules, such as BAY41-4109, lead to decreased production of virions and accelerated loss of capsid protein to proteasomal digestion (8). In vitro, excess BAY41-4109 led to misdirection of assembly (11). HAP1 [methyl 4-(2-chloro-4-fluorophenyl)-6-methyl-2-(pyridin-2-yl)-1,4-dihydropyrimidine-5-carboxylate], a related small molecule, was found to accelerate capsid assembly kinetics, while stoichiometric concentrations of HAP1 misdirect assembly, yielding nonicosahedral hexagonal arrays of capsid protein (26). HAP1 also bound preformed capsids and triggered disassembly, which was exacerbated by capsid-destabilizing conditions (26). Hepatitis B virus (HBV) isTo ascertain the structural basis for HAP1 activity, we have determined the 5.05-Å structure of a variant of the adyw strain HBV capsid (residues 1 to 149) complexed with HAP1 (referred to as ϩHAP1). To facilitate comparison, we have also determined the structure of this variant in the absence of HAP1 (ϪHAP1) to 3.95 Å. We find structural changes resulting from binding HAP1 that suggest a molecular basis for HAP1 activity. We also identify a putative HAP1-binding site, defining a target for assembly-directed anti-HBV molecules. MATERIALS AND METHODSSample preparation. Hepatitis B virus strain adyw, truncated at residue 149, had three native cyst...

HBV persistence and transmission require HBV replication, which depends on the assembly of a core particle composed of capsid protein (Cp), polymerase, and pregenomic RNA. Reverse transcription to produce infectious DNA-containing particles occurs solely within the core residing in the cytoplasm (9, 10). Thus, core assembly is likely to be a high value target for therapeutics (11).The capsid, the protein shell of the core, is built of 120 Cp dimers arranged with T ϭ 4 symmetry (12, 13). The dimer interfaces are evident as spikes (14-16) that are the major epitope of the capsid (17). Cp in low ionic strength solution is dimeric (18). We have studied ionic strength-dependent capsid assembly extensively in vitro by using the Cp assembly domain (Cp149) (residues 1-149) lacking the 34-residue C-terminal RNA-binding domain (19)(20)(21). Assembly is nucleated by a trimer of Cp dimers and proceeds without accumulating observable populations of intermediates (22). Interactions between dimers are weak but sum to give a globally stable capsid (23). These capsids persist, even under conditions where they are not thermodynamically favored, because of hysteresis to dissociation (24). Some Cp mutations lead to faster assembly and greater stability (25), indicating that wild-type Cp is suboptimal for assembly and suggesting that assembly is regulated in vivo, possibly by conformational change. In support of this assertion, we found that Zn 2ϩ alters the conformation of Cp dimers and enhances the rate of assembly, suggesting that capsid assembly is allosterically regulated (26).Recently, it was discovered that heteroaryldihydropyrimidines (HAPs) (Fig. 1) affect the accumulation of HBV capsids (27,28). HAP drugs decreased the yield of assembled core and HBV genomes from cells that constitutively produce HBV. Electron microscopy showed that Cp assembled in vitro in the presence of HAP drugs led to polymers that had abnormal morphology (29). Similarly, small molecules such as bis ANS {5,5-bis[8-(phenylamino)-1-naphthalenesulfonate]} alter Cp assembly in vitro (30). Recent reports suggest that other small molecules also inhibit normal HBV capsid assembly (31-33).Here, we describe the mechanism of a representative HAP compound, HAP-1 [methyl 4-(2-chloro-4-f luorophenyl)-6-methyl-2-(pyridin-2-yl)-1,4-dihydropyrimidine-5-carboxylate] (Fig. 1). In vitro, low concentrations of HAP-1 enhance both the rate and extent of assembly by favoring an assembly-active state; thus, HAP-1 acts like an allosteric effector. At higher concentrations, HAP-1 led to aberrant noncapsid polymers in vitro, even at the expense of preexisting capsids. We propose that both of these effects on assembly contribute to reducing HBV virion production. Materials and MethodsSynthesis of HAP-1. Preparation of racemic HAP-1 (Fig. 1) was adapted from the patent literature (27,28,34). Condensation of a pyridylamidine with a substituted ␣-carboxymethyl enone gave a 30% yield of Ͼ97% pure HAP-1 after chromatographic purification. HAP-1 was characterized by 1 H, 13 C, and 19 ...