An Assembly-Activating Site in the Hepatitis B Virus Capsid Protein Can Also Trigger Disassembly

Abstract

The Hepatitis B Virus (HBV) core protein homodimers self-assemble to form an icosahedral capsid that packages the viral genome. Disassembly occurs in the nuclear basket to release the mature genome to the nucleus. Small molecules have been developed that bind to a pocket at the interdimer interface to accelerate assembly and strengthen interactions between subunits; these are under development as antiviral agents. Here, we explore the role of the dimer-dimer interface by mutating sites in the drug-binding pocket to cysteine and examining the effect of covalently linking small molecules to them. We find that ligands bound to the pocket may trigger capsid disassembly in a dose-dependent manner. This result indicates that, at least transiently, the pocket adopts a destabilizing conformation. We speculate that this pocket also plays a role in virus disassembly and genome release by binding ligands that are incompatible with virus stability, "unwanted guests." Investigating protein-protein interactions, especially large protein polymers, offers new and unique challenges. By using an engineered addressable thiol, we provide a means to examine the effects of modifying an interface without requiring drug-like properties for the ligand.

Figure 1.. Locations of engineered cysteines at the dimer-dimer interface of the HBV core protein.

(a) 120 copies of homodimeric core protein (Cp) form T=4 icosahedral capsids. One icosahedral asymmetric unit is highlighted to show the four quasi-equivalent environments: A (light grey), B (dark grey), C (light blue) and D (medium blue). (b) The CpAM pocket, viewed from the capsid interior, highlighting the location of V124 (in red) at 5-fold and 6-fold vertices. (c) CpAM pocket mutants. Four single cysteines mutants were created at the pocket, L30C (purple), I105C (green), S106C (orange), and V124C (red). Residue V124 is in the final helix of the assembly domain; the loop just C-terminal of V124 fits into a groove in the neighboring subunit to support protein-protein interaction. The models shown here are based on PDB: 1QGT.

Figure 2.. Biophysical characterization of pocket mutants.

(a-e) Pseudo-critical concentrations of assembly were determined for Cp mutants 300 mM NaCl by size exclusion chromatography. (f-i) Negative stain TEM images of assembled particles show morphologically normal capsids. The ionic strength was chosen to maximize the amount of assembly while minimizing the possibility of kinetically trapped incomplete capsids.

Figure 3.. Accessibility of cysteines for labeling.

Absorbance spectra of purified dimer (a) and (b) capsid from reactions of Cp149–3CA cysteine mutants with BoDIPYm. Spectra show the absorbance of protein, 280 nm, and BoDIPYm, predominantly at 504nm. SDS-PAGE of dimer and capsid labeled with Coomassie blue (inset top panel) and imaged with a fluorescence detector (inset bottom) recapitulate the evaluation of labeling in the chromatographs. UV-vis spectra indicate that I105C, S106C, and V124C were all accessible in free dimer. Only V124C was accessible for labeling in assembled capsids.

Figure 4:. Capsid disassembles when treated with BoDIPYm and is dose dependent.

(a) Size exclusion chromatography of Cp149–3CAV124C: capsid (solid line) and after treatment with BoDIPYm (dashed line). (b) V124C capsids incubated with other ligands show dissociation varies. BoDIPYm (blue) has the greatest impact on dissociation followed by Fluoresceinmaleimide (red). Some dissociation was observed with iodoacetamide (cyan). Dissociation was not observed with FITC (green) or N-ethyl maleimide (magenta). Disassembly is considered to be site-specific, since no disassembly was observed with FITC.

Figure 5:. V124C capsid can be protected from disassembly by ligands.

V124C capsids were incubated overnight with (a) iodoacetamide, (b) N-ethyl maleimide (NEM), or (c) HAP 12. The following day, samples were treated with a 20-fold molar excess of BoDIPYm for 2h, excess dye was removed, and samples were resolved by size exclusion chromatography to determine the percent dissociation (closed circles) and the percent of maximal BoDIPY labeling of the capsid peak (open circles). A molar excess of iodoacetamide is required to protect the Cp149–3CA-V124C from BoDIPYm labelling, indicating that it is not a preferred ligand. NEM and HAP12 protect V124C linearly with concentration. For iodoacetamide and NEM there is good agreement between the amount of protection from dissociation and the amount of BoDIPYm labelling. For HAP12, there is a gap between the amount of labeling and the amount of protected capsid. At stoichiometric ratios of HAP, 2 HAPs/dimer, about 25% of the subunits are labeled, even though the capsids remain intact. Measurements were in triplicate except the first three points in panel a, where the low signal for capsid precluded calculation of percent dissociation, and the value was set to 100%.