BAY 41-4109-mediated aggregation of assembled and misassembled HBV capsids in cells revealed by electron microscopy

Abstract

HBc is a small protein essential for the formation of the icosahedral HBV capsid. Its multiple roles in the replication cycle make this protein a promising target for the development of antiviral molecules. Based on the structure of HBc, a series of HBV assembly inhibitors, also known as capsid assembly modulators, were identified. We investigated the effect of BAY 41–4109, a heteroaryldihydropyrimidine derivative that promotes the assembly of a non-capsid polymer. We showed, by confocal microscopy, that BAY 41–4109 mediated HBc aggregation, mostly in the cytoplasm of Huh7 cells. Image analysis revealed that aggregate size depended on BAY 41–4109 concentration and treatment duration. Large aggregates in the vicinity of the nucleus were enclosed by invaginations of the nuclear envelope. This deformation of the nuclear envelope was confirmed by transmission electron microscopy (TEM) and immuno-TEM. These two techniques also revealed that the HBc aggregates were accumulations of capsid-like shells with an electron-dense material consisting of HBV core fragments. These findings, shedding light on the ultrastructural organization of HBc aggregates, provide insight into the mechanisms of action of BAY 41–4109 against HBV and will serve as a basis for comparison with other HBV capsid assembly inhibitors.