Multiple subunits of the hepatitis B virus (HBV) core protein (HBc) assemble into an icosahedral capsid that packages the viral pregenomic RNA (pgRNA). The N-terminal domain (NTD) of HBc is sufficient for capsid assembly, in the absence of pgRNA or any other viral or host factors, under conditions of high HBc and/or salt concentrations. The C-terminal domain (CTD) is deemed dispensable for capsid assembly although it is essential for pgRNA packaging. We report here that HBc expressed in a mammalian cell lysate, the rabbit reticulocyte lysate (RRL), was able to assemble into capsids when HBc concentrations (in low nM) mimicked those achieved under conditions of viral replication in vivo and were far below those used previously for capsid assembly in vitro. Furthermore, at physiologically low HBc concentrations in RRL, NTD was insufficient for capsid assembly and CTD was also required. CTD likely facilitated assembly under these conditions via RNA binding and protein-protein interactions. Moreover, CTD underwent phosphorylation and dephosphorylation events in RRL as in vivo, which regulated capsid assembly. Importantly, NTD alone also failed to accumulate in mammalian cells, likely resulting from its failure to assemble efficiently. Co-expression of the full-length HBc rescued NTD assembly in RRL as well as NTD expression and assembly in mammalian cells, resulting in the formation of mosaic capsids containing both full-length HBc and NTD. These results have important implications for HBV assembly during replication and provide a facile cell-free system to study capsid assembly under physiologically relevant conditions including its modulation by host factors.
Hepatitis B virus (HBV) is an important global human pathogen and the main cause of liver cancer worldwide. An essential component of HBV is the spherical capsid composed of multiple copies of a single protein, the core protein (HBc). We have developed a mammalian cell-free system in which HBc is expressed at physiological (low) concentrations and assemble into capsids under near physiological conditions. In this cell-free system, as in mammalian cells, capsid assembly depends on the C-terminal domains (CTD) of HBc, in contrast to other assembly systems in which HBc assembles into capsids independent of CTD under non-physiological protein and salt concentrations. Furthermore, the phosphorylation state of CTD regulates capsid assembly and RNA encapsidation, in the cell-free system as in mammalian cells. This system will facilitate detailed studies on capsid assembly and RNA encapsidation under physiological conditions and identification of antiviral agents that target HBc.
Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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