Global Structural Changes in Hepatitis B Virus Capsids Induced by the Assembly Effector HAP1

Abstract

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...

Thermodynamic origins of protein folding, allostery, and capsid formation in the human hepatitis B virus core protein

“…Others have shown that inserting short linkers at the tip of the four-helix bundle mediates a 40°rotation of the contact domain relative to the bundle (within capsids) (23). Further, small molecules have been reported that bind to HBc and induce global structural rearrangements that impair HBV capsid formation (11)(12)(13)50).…”

“…capsid assembly have inferred the existence of assembly-active (HBc Ass ) and assembly-incompetent (HBc Inc ) HBc conformations (12,13,21,24,25). However, there are few detailed insights on the thermodynamic origins of structure, allostery, and dynamics for the dimeric HBc 1-149 protomer, where structural plasticity must originate.…”

“…Although excellent vaccines exist, there are no effective cures for extant chronic infections (5,6). In addition to capsid formation, HBc plays many essential roles in HBV replication (7)(8)(9), making it an attractive drug target (10)(11)(12)(13)(14)(15).…”

Isothiafludine, a novel non-nucleoside compound, inhibits hepatitis B virus replication through blocking pregenomic RNA encapsidation

“…HAPs, including Bay 41-4109, Bay 38-7690, and Bay 39-5493, were first identified to prevent the formation of the HBV capsid [20,21] . These analogues inhibited HBV replication through binding within the assembly domain of HBcAg at a site that bridged elements of the secondary …”

“…HAPs bind at a sequence of the assembly domain that is involved in forming inter-dimer contacts, whereas DHBV-derived recombinant capsids show no binding [7,20] ; therefore, HAPs did not inhibit DHBV replication in cultured cells or in a DHBV-infected duck model.…”