A Disulfide in HBV Core Protein Dimer Allosterically Modifies Capsid Assembly and Stability

Abstract

During the Hepatitis B virus (HBV) life cycle, assembly and disassembly of the capsid need to be highly regulated. The HBV capsid is formed by 120 copies of the homodimeric core protein. Capsid assembly is allosterically regulated, implying that dimer transitions from assembly-inactive to assembly-active states. Indeed, the intra-dimer interface (which is distant from the site if inter-dimer contact) in free dimer is substantially different than in dimer from capsid (Packianathan et al (2010)). The intra-dimer interface contains a pair of completely conserved cysteines at position 61 that can form a disulfide bond.Within capsid, C61-C61 oxidized 7 times faster than in free dimer indicating dimer within capsid adopts a conformation that strongly promotes disulfide formation. However, compared to reduced dimer, oxidized protein assembled slowly and into lower yields of capsid. In addition, urea disassembly studies showed that capsids formed by oxidized dimer are less stable to urea treatment than reduced capsids.These results indicate that oxidized protein adopts a conformation unfavorable for capsid assembly. Sucrose gradient centrifugation and electron microscopy confirmed these findings and revealed that oxidized dimer forms a higher proportion of small, 90-dimer particles than reduced protein.Our results show that structural changes at the dimer interface can dramatically alter the assembly behavior and stability of the capsid. This distal effect is consistent with allosteric regulation of assembly. Our data also suggest an unsuspected biological role for the C61-C61 disulfide bond. We propose that newly expressed, reduced protein subunits assemble with high fidelity into capsids packaging the RNA genome. As the life cycle progresses oxidation of the capsid occurs resulting in metastable capsid particles that have a lower energy barrier to disassembly, facilitating the release of the genome.