BAY 41-4109 has multiple effects on Hepatitis B virus capsid assembly

Here we report the effect of a heteroaryldihydropyrimidine (HAP) antiviral compound, BAY 41-4109, on Hepatitis B virus (HBV) capsid assembly and on preformed HBV capsids. The HBV capsid is an icosahedral complex of 120 capsid protein dimers. BAY41-4109 inhibits virus production in vivo by a mechanism that targets the viral capsid. We found that BAY 41-4109 was able to both accelerate and misdirect capsid assembly in vitro. As little as one HAP molecule for every five HBV dimers was sufficient to induce formation of non-capsid polymers. Unlike the related molecule HAP-1 (Stray et al., Proc. Natl. Acad. Sci. USA 102:8138-43, 2005), no stable assembly intermediates were observed in assembly reactions with BAY 41-4109, indicating that accelerated assembly by BAY 41-4109 was still kinetically regulated by the nucleation rate. Preformed capsids were stabilized by BAY 41-4109, up to a ratio of one inhibitor molecule per two dimers. However, at BAY 41-4109:dimer ratios of 1:1 and greater, capsids were destabilized to yield very large non-capsid polymers. These data suggest the existence of two functionally distinguishable classes of drug-binding sites on HBV capsids. Occupation of the first class of site stabilizes capsid, while binding at the second class requires or induces structural changes that cannot be tolerated without destabilizing the capsid. Our data suggest that HAP compounds may inhibit virus replication by inducing assembly inappropriately and, when in excess, by misdirecting assembly decreasing the stability of normal capsids.