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.

Current therapies rarely cure chronic hepatitis B virus (HBV) infection due to the persistence of the viral episome, the covalently closed circular DNA (cccDNA), in hepatocytes. The hepatitis B core-related antigen (HBcrAg), a mixture of the viral precore/core gene products, has emerged as one potential marker to monitor the levels and activities of intrahepatic cccDNA. Here, a comprehensive characterization of precore/core gene products revealed that HBcrAg components included the classical hepatitis B core antigen (HBc) and e antigen (HBeAg), and additionally, the precore-related antigen, PreC, retaining the N-terminal signal peptide. Both HBeAg and PreC antigens displayed heterogeneous proteolytic processing at their C-terminus resulting in multiple species, which varied with viral genotypes. HBeAg was the predominant form of HBcrAg in HBeAg-positive patients. Positive correlations were found between HBcrAg and PreC, between HBcrAg and HBeAg, and between PreC and HBeAg, but not between HBcrAg and HBc. Serum HBeAg and PreC shared similar buoyant density and size distributions, and both displayed density and size heterogeneity. HBc, but not HBeAg or PreC antigens, was found as the main component of capsids in DNA-containing or empty virions. Neither HBeAg nor PreC proteins were able to form capsids in cells or in vitro under physiological conditions. In conclusion, our study provides important new quantitative information on levels of each component of precore/core gene products as well as their biochemical and biophysical characteristics, implying that each component may have distinct functions and applications in reflecting intrahepatic viral activities. IMPORTANCE Chronic hepatitis B virus (HBV) infection afflicts approximately 257 million people, who are at high risk of progressing to chronic liver diseases, including cirrhosis, fibrosis, and hepatocellular carcinoma. Current therapies rarely achieve cure of HBV due to the persistence of HBV episome, covalently closed circular DNA (cccDNA), in the nucleus of infected hepatocytes. Peripheral markers of cccDNA levels and transcriptional activities are urgently required to guide antiviral therapy and drug development. Serum hepatitis B core-related antigen (HBcrAg) is one such emerging peripheral marker. We have herein characterized the components of HBcrAg in HBV infected patients as well as in cell cultures. Our results provide important new quantitative information on levels of each HBcrAg component, as well as their biochemical and biophysical characteristics. Our findings further suggest that each HBcrAg component may have distinct functions and applications in reflecting intrahepatic viral activities.

SUMMARY Complete virions of hepatitis B virus (HBV) contain a DNA genome that is enclosed in a capsid composed of the HBV core antigen (HBcAg), which is in turn surrounded by a lipid envelope studded with viral surface antigens (HBsAg). In addition, HBV-infected cells release subviral particles composed of HBsAg only (HBsAg ‘spheres’ and ‘filaments’) or HBsAg enveloping HBcAg but devoid of viral DNA (‘empty virions’). The hepatitis B e antigen (HBeAg), a soluble antigen related to HBcAg, is also secreted in some HBV-infected patients. The goals of this study were to explore the levels of empty virions in HBV-infected patients before and during therapy with the nucleotide analog tenofovir disoproxil fumarate (TDF) that inhibits HBV DNA synthesis and the relationships of empty virions to complete virions, HBsAg and HBeAg. HBV DNA, HBcAg and HBsAg levels were determined in serum samples from 21 patients chronically infected with HBV and enrolled in clinical TDF studies. Serum levels of empty virions were found to exceed levels of DNA-containing virions, often by ≥100-fold. Levels of both empty and complete virions varied and were related to the HBeAg status. When HBV DNA replication was suppressed by TDF, empty virion levels remained unchanged in most but were decreased (to the limit of detection) in some patients who also experienced significant decrease or loss of serum HBsAg. In conclusion, empty virions are present in the serum of chronic hepatitis B patients at high levels and may be useful in monitoring response to antiviral therapy.