Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids

Abstract

Defining mechanisms of direct-acting antivirals facilitates drug development and our understanding of virus function. Heteroaryldihydropyrimidines (HAPs) inappropriately activate assembly of hepatitis B virus (HBV) core protein (Cp), suppressing formation of virions. We examined a fluorophore-labeled HAP, HAP-TAMRA. HAP-TAMRA induced Cp assembly and also bound pre-assembled capsids. Kinetic and spectroscopic studies imply that HAP-binding sites are usually not available but are bound cooperatively. Using cryo-EM, we observed that HAP-TAMRA asymmetrically deformed capsids, creating a heterogeneous array of sharp angles, flat regions, and outright breaks. To achieve high resolution reconstruction (<4 Å), we introduced a disulfide crosslink that rescued particle symmetry. We deduced that HAP-TAMRA caused quasi-sixfold vertices to become flatter and fivefold more angular. This transition led to asymmetric faceting. That a disordered crosslink could rescue symmetry implies that capsids have tensegrity properties. Capsid distortion and disruption is a new mechanism by which molecules like the HAPs can block HBV infection.

Local stabilization of subunit-subunit contacts causes global destabilization of Hepatitis B virus capsids

“…The crystal structures of both apo capsid (1qgt) [74] and HAP-TAMRA bound capsid (6bvf) [66] were used as starting points for flexible model refinement using the PHENIX, COOT, and eLBOW software programs. [75][76][77] The model validation statistics we report were obtained from the final output of the Phenix real space refinement tool.…”

Expression of quasi-equivalence and capsid dimorphism in the Hepadnaviridae

“…Apart from gaining an improved understanding of viral capsid structure, assembly, and disassembly-for instance as a target for assembly-disrupting drugs [5,[38][39][40][41] and inhibitory antibodies [42]-there is also the prospect of engineering capsids for epitope display and as delivery vehicles [43][44][45][46]. These considerations have motivated the determination of a substantial number of structures for HBV capsids.…”

“…Currently, the Electron Microscopy Data Bank (EMDB) and the Protein Data Bank (PDB) include 51 non-redundant HBV capsid-related structures (33 in the EMDB, 18 in the PDB) (S1 Table). Of these, all but four are T = 4 structures, and of the T = 3 structures, two are from Nackednaviruses reconstructed to low (8.0-9.0 Å) resolution [37], one to intermediate resolution (5.6 Å) [47], and one (at 4.0 Å) is ligand-bound [41]. The highest resolution reported for T = 4 capsids by X-ray crystallography is 3.4 Å [4] and more recently by cryo-EM (3.5 Å) [8].…”

“…The highest resolution reported for T = 4 capsids by X-ray crystallography is 3.4 Å [4] and more recently by cryo-EM (3.5 Å) [8]. The highest resolution attained for the T = 3 capsid (4.0 Å) is for the liganded structure [41]. No high-resolution structure for the T = 3 apo form has been reported.…”

Pregenomic RNA: How to assist the management of chronic hepatitis B?

“…Note that the kinetics of the serum HBV RNA and HBV DNA declines during CAMs treatment were very similar, but there are also some differences . The inhibition of the establishment of HBV infection could be due to an accelerated breakdown of capsid in cytoplasm before rcDNA could be delivered to nucleus and converted into cccDNA or to a stabilization of the capsid structure, which would prevent uncoating at the nuclear pore . In addition, CAMs could interfere with the nuclear functions of HBV viral core, including regulation of cccDNA transcription and host‐gene expression .…”