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 ...

The relationship between the physical chemistry and biology of self-assembly is poorly understood, but it will be critical to quantitatively understand infection and for the design of antivirals that target virus genesis. Here we take advantage of heteroaryldihydropyrimidines (HAPs), which affect hepatitis B virus (HBV) assembly, to gain insight and correlate in vitro assembly with HBV replication in culture. Based on a low-resolution crystal structure of a capsid-HAP complex, a closely related series of HAPs were designed and synthesized. These differentially strengthen the association between neighboring capsid proteins, alter the kinetics of assembly, and give rise to aberrant structures incompatible with a functional capsid. The chemical nature of the HAP variants correlated well with the structure of the HAP binding pocket. The thermodynamics and kinetics of in vitro assembly had strong and predictable effects on product morphology. However, only the kinetics of in vitro assembly had a strong correlation with inhibition of HBV replication in HepG2.2.15 cells; there was at best a weak correlation between assembly thermodynamics and replication. The correlation between assembly kinetics and virus suppression implies a competition between successful assembly and misassembly, small molecule induced or otherwise. This is a predictive and testable model for the mechanism of action of assembly effectors.Hepatitis B virus (HBV), which chronically infects approximately 400 million people and contributes to 1 million deaths per year, is an enveloped virus with an icosahedral core. The core is assembled in the cytoplasm from core (capsid) protein, viral pregenomic RNA, viral reverse transcriptase, and a few host proteins. The core plays indispensable roles in viral DNA synthesis from the pregenome and intracellular trafficking (10). The predominant antiviral strategy against HBV is to attack the reverse transcriptase. Unsurprisingly, HBV reverse transcriptase inhibitors lead to drug-resistant mutants in HBV (7,24,30) and also human immunodeficiency virus (14). Resistance can have broader consequences because of the extensive gene overlap in HBV, since some reverse transcriptase mutations lead to surface protein that is insensitive to antibodies generated by the HBV vaccine (25). An alternative therapeutic strategy would be targeting capsid assembly (15, 33). Heteraryldihydropyrimidines (HAPs), first identified by scientists at Bayer AG as having anti-HBV activity in a cell culture-based screen, act in a capsid protein-specific manner (8,11,(19)(20)(21)(22).The mechanism of HAP activity has been studied in vitro using the 149-residue assembly domain of the HBV capsid protein, Cp149, which is always found as a homodimer (11,21,22). Assembly of empty capsids is nucleated by slow formation of a trimer of dimers followed by rapid addition of subsequent dimers (34). We previously reported that HAP 1 (Fig. 1), a variant of the original HAP structure identified by the Bayer laboratory (8,20), increases the rate of assembly by...

Understanding self-assembly of icosahedral virus capsids is critical to developing assembly-directed antiviral approaches and will also contribute to the development of self-assembling nanostructures. One approach to controlling assembly would be through the use of assembly inhibitors. Here we use Cp149, the assembly domain of the hepatitis B virus capsid protein, together with an assemblydefective mutant, Cp149-Y132A, to examine the limits of the efficacy assembly inhibitors. By itself, Cp149-Y132A will not form capsids. However, Cp-Y132A will co-assemble with wildtype protein based on light scattering and size exclusion chromatography. The resulting capsids appear indistinguishable from normal capsids. However, co-assembled capsids are more fragile, with disassembly observed by chromatography under mildly destabilizing conditions. The relative persistence of capsids assembled under conditions where association energy is weak compared to the fragility of those where association is strong suggests a mechanism of "thermodynamic editing" that allows replacement of defective proteins in a weakly associated complex. There is fine line between weak assembly, where assembly-defective protein is edited from a growing capsid, and relatively strong assembly, where assembly defective subunits may dramatically compromise virus stability. Thus, attempts to control virus self-assembly (with small molecules or defective proteins) must take into account the competing process of thermodynamic editing.Viruses have a long history as pathogens (see (1) and references therein) and a much shorter history as tools for the development of nanostructures (2-8). Biologically, virus assembly requires high fidelity and rapid kinetics. Virus assembly is a paradigm for nanostructure selfassembly. Controlling virus assembly therefore has value for development of new classes of antiviral agents and for developing viruses as platforms for nanotechnology.The object of this study, hepatitis B virus (HBV) is an unusually serious public health problem as a major contributing factor to cirrhosis and hepatocellular carcinoma that chronically infects approximately 400 million individuals (9). HBV is an enveloped DNA virus with an icosahedral core. The protein shell of HBV's core, the capsid, is a self-assembling complex of 120 core protein homodimers (10,11). In vivo, the nucleoprotein core is critical to virus lifecycle (9,(12)(13)(14)(15). In vitro, empty capsids can be recapitulated by the 149 residue core protein assembly domain (Cp149) (16). The interdimer contacts that form a capsid are compact, though not tightly packed, and dominated by hydrophobic residues (17,18). Small molecules that occupy gaps at this interface are able to strengthen the interdimer association energy and divert (19,20). HBV assembly has been characterized in detail with the aid of model-based analyses specific for icosahedral assembly (21,22). There is a welldefined nucleation step, formation of a trimer of homodimers, which is critical for avoiding kinetic traps...