J Virol. 2018 Aug 8. pii: JVI.01082-18. doi: 10.1128/JVI.01082-18. [Epub ahead of print]
Assembly properties of Hepatitis B Virus core protein mutants correlate with their resistance to assembly-directed antivirals.
Ruan L1, Hadden JA2, Zlotnick A3.
Author information
1
Molecular and Cellular Biochemistry, Indiana University, Bloomington IN 47405.
2
Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716 [email protected].
3
Molecular and Cellular Biochemistry, Indiana University, Bloomington IN 47405 [email protected].
Abstract
Hepatitis B Virus (HBV) capsid or core protein (Cp) can self-assemble to form an icosahedral capsid. It is now pursued as a target for small molecule antivirals that enhance the rate and extent of its assembly to yield empty and/or aberrant capsids. These small molecules are thus called Core protein Allosteric Modulators (CpAMs). We sought to understand the physical basis of CpAM-resistant mutants and how CpAMs might overcome them. We examined the effects of two closely related CpAMs, HAP12 and HAP13, which differ by a single atom but have drastically different antiviral activity, on the assembly of wildtype Cp and three T109 mutants (T109M, I, S) that display a range of resistance. The T109 side chain forms part of the mouth of the CpAM-binding pocket. A T109 mutant that has substantial resistance even to a highly active CpAM strongly promotes normal assembly. Conversely, a mutant that weakens assembly is more susceptible to CpAMs. In crystal and cryo-electron microscopy (cryo-EM) structures of T=4 capsids with bound CpAMs, the CpAMs preferentially fit into two of four quasi-equivalent sites. In these static representations of capsid structures, T109 does not interact with the neighboring subunit. However, all-atom molecular dynamics simulations of an intact capsid show that T109 of one of the four classes of CpAM site has a hydrophobic contact with the neighboring subunit at least 40% of the time, providing a physical explanation for the mutation's ability to affect capsid stability, assembly, and sensitivity to CpAMs.ImportanceThe HBV core protein and its assembly into capsids have become important targets for development of core protein allosteric modulators (CpAMs) as antivirals. Naturally occurring T109 mutants have been shown to be resistant to some of these CpAMs. We found that mutation of T109 led to changes in capsid stability and recapitulated resistance to a weak CpAM but much less so to a strong CpAM. Examination of HBV capsid structures, determined by cryo-EM and crystallography, could not explain how T109 mutations change capsid stability and resistance. However, by mining data from a long-duration all-atom molecular dynamics simulation we found that the capsid was extraordinarily flexible and that T109 can impede entry to the CpAM binding site. In short, HBV capsids are incredibly dynamic and that molecular mobility must be considered when discussing antiviral mechanism.