Cell-Free Hepatitis B Virus Capsid Assembly Dependent on the Core Protein C-Terminal Domain and Regulated by Phosphorylation

Abstract

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, rabbit reticulocyte lysate (RRL), was able to assemble into capsids when (low-nanomolar) HBc concentrations 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, the NTD was insufficient for capsid assembly and the CTD was also required. The CTD likely facilitated assembly under these conditions via RNA binding and protein-protein interactions. Moreover, the CTD underwent phosphorylation and dephosphorylation events in RRL similar to those seen in vivo which regulated capsid assembly. Importantly, the NTD alone also failed to accumulate in mammalian cells, likely resulting from its failure to assemble efficiently. Coexpression 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 the 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. IMPORTANCE Hepatitis B virus (HBV) is an important global human pathogen and the main cause of liver cancerworldwide. 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 assembles into capsids under near-physiological conditions. In this cell-free system, as in mammalian cells, capsid assembly depends on the C-terminal domain (CTD) of HBc, in contrast to other assembly systems in which HBc assembles into capsids independently of the CTD under conditions of nonphysiological protein and salt concentrations. Furthermore, the phosphorylation state of the CTD regulates capsid assembly and RNA encapsidation in the cell-free system in a manner similar to that seen 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.

Discovery and Mechanistic Study of Benzamide Derivatives That Modulate Hepatitis B Virus Capsid Assembly

“…8B). Consistent with a previous report, although Cp144 was able to assemble into capsids, Cp149 failed to accumulate in AML12 cells, presumably due to assembly of unstable capsids (46). Interestingly, like wild-type core protein, most of the truncated core proteins assembled into a major species of slower-migrating and minor species of faster-migrating capsids.…”

“…Information on the sequence and construction of the wild-type or NUC-resistant HBV genotype B and C plasmids, including pHY536207, pHY634, pHY6923, and pHY6945, has been published previously (76). The plasmids pCI-HBc-WT, pCI-HBc-3A, pCI-HBc-3E, pCI-HBc-7A, and pCI-HBc-7E were gifts of Jianming Hu at Pennsylvania State University (46). pHBV1.3-derived plasmids expressing mutant polymerase Y63F or mutant core proteins with F97L, V124A, V124F, V124L, or V124W mutation were generated by an overlapping PCR strategy.…”

“…The amino acid residues 150 to 183 constitute the C-terminal domain (CTD) of HBV core protein. This arginine-rich domain contains 3 major and 4 minor phosphorylation sites and plays an important role in the regulation of pgRNA encapsidation, DNA replication, nucleocapsid uncoating, and nuclear transport of rcDNA (46)(47)(48)(49)(50). Because a recent report showed that phosphorylation of the three major serines in the CTD stabilized capsids (51), we tested whether the phosphorylation status of CTD modified the effects of capsid assembly modulators.…”

Capsid Phosphorylation State and Hepadnavirus Virion Secretion

“…To support empty virion formation, abundant levels of empty HBV capsids are assembled in human cells, in addition to the aforementioned NCs. In contrast to capsid assembly in bacteria, which leads to packaging of nonspecific RNAs such as the cellular tRNAs in the absence of the RT protein or pgRNA, HBV capsid assembly in human cells does not lead to nonspecific RNA packaging, likely as a result of HBc CTD phosphorylation, which on one hand helps to facilitate specific packaging of the RT-pgRNA complex into NCs and on the other blocks nonspecific RNA packaging when the RT-pgRNA complex is absent or limiting (13,25). To reconcile the envelopment of mature, dsDNA-containing NCs as well as empty capsids, but not immature pgRNA-or ssDNA-containing NCs, we proposed a single-strand blocking model to explain selective HBV virion morphogenesis, whereby a negative signal is triggered by the presence of the (single-stranded) pgRNA or ssDNA in immature NCs to inhibit their envelopment (25).…”

“…The N-terminal two-thirds (the N-terminal domain [NTD], the assembly domain) of the relatively small core protein, approximately 21 kDa (for HBV) or 32 kDa (for DHBV), forms the capsid shell (5)(6)(7)(8), while its highly basic C-terminal domain (CTD) plays essential roles in viral pgRNA packaging and DNA synthesis (9)(10)(11)(12). While traditionally thought to be dispensable for capsid assembly, the CTD has also been shown recently to be required to facilitate capsid assembly under physiological conditions (13).…”