Core Protein-Directed Antivirals and Importinβ Can Synergistically Disrupt HBV Capsids

Abstract

Viral structural proteins can have multiple activities. Antivirals that target structural proteins have potential to exhibit multiple antiviral mechanisms. Hepatitis B Virus (HBV) core protein (Cp) is involved in most stages of the viral lifecycle: it assembles into capsids, packages viral RNA, is a metabolic compartment for reverse transcription, interacts with nuclear trafficking machinery, and disassembles to release the viral genome into the nucleus. During nuclear localization, HBV capsids bind to host importins (e.g. Imp{beta}) via Cps C-terminal domain (CTD); the CTD is localized to the interior of the capsid and is transiently exposed on the exterior. We used HAP12 as a representative Cp Allosteric Modulators (CpAMs), a class of antivirals that inappropriately stimulates and misdirects HBV assembly and deforms capsids. CpAM impact on other aspects of the HBV lifecycle is poorly understood. We investigated how HAP12 influenced the interactions between empty or RNA-filled capsids with Imp{beta} and trypsin in vitro. We showed that HAP12 can modulate CTD accessibility and capsid stability, depending on the saturation of HAP12-binding sites. We demonstrated that Imp{beta} synergistically contributes to capsid disruption at high levels of HAP12 saturation, using electron microscopy to visualize disruption and rearrangement of Cp dimers into aberrant complexes. However, RNA-filled capsids resisted the destabilizing effects of HAP12 and Imp{beta}. In summary, we show host protein-induced catalysis of capsid disruption, an unexpected additional mechanism of action for CpAMs. Potentially, untimely capsid disassembly can hamper the HBV lifecycle and also cause the virus to become vulnerable to host innate immune responses. IMPORTANCEThe HBV core, an icosahedral complex of 120 copies of the homodimeric core (capsid) protein with or without packaged nucleic acid, is transported to the host nucleus by its interaction with host importin proteins. Importin-core interaction requires the core protein C-terminal domain, which is inside the capsid, to "flip" to the capsid exterior. Core-protein directed drugs that affect capsid assembly and stability have been developed recently. We show that these molecules can, synergistically with importins, disrupt capsids. This mechanism of action, synergism with host protein, has potential to disrupt the virus lifecycle and activate the innate immune system.