Assembly and disassembly of viral capsids are essential steps in the viral life cycle. Studies on their kinetics are mostly performed in vitro, allowing application of biochemical, biophysical and visualizing techniques. In vivo kinetics are poorly understood and the transferability of the in vitro models to the cellular environment remains speculative. We analyzed capsid disassembly of the hepatitis B virus in digitonin-permeabilized cells which support nuclear capsid entry and subsequent genome release. Using gradient centrifugation, size exclusion chromatography and immune fluorescence microscopy of digitonin-permeabilized cells, we showed that capsids open and close reversibly. In the absence of RNA, capsid re-assembly slows down; the capsids remain disintegrated and enter the nucleus as protein dimers or irregular polymers. Upon the presence of cellular RNA, capsids re-assemble in the nucleus. We conclude that reversible genome release from hepatitis B virus capsids is a unique strategy different from that of other viruses, which employs irreversible capsid destruction for genome release. The results allowed us to propose a model of HBV genome release in which the unique environment of the nuclear pore favors HBV capsid disassembly reaction, while both cytoplasm and nucleus favor capsid assembly. Viral capsids facilitate protection of the enclosed viral genome and participate in the intracellular transport of the genome. At the site of replication capsids have to release the genome, but after replication new capsids have to be assembled for encapsidation of the progeny genomes. Detailed data on stability of capsids and kinetics of their formation and dissociation are obtained for several viruses in vitro, allowing biophysical or electron microscopical techniques. These approaches, however, do not consider the impact of cellular interaction partners. Using digitonin-permeabilized cells which support hepadnaviral genome release actively, we analyzed the disassembly kinetic of the hepatitis B virus (HBV) capsid. Using different analytical methods we found that HBV capsids disintegrate to protein dimers which reassemble to capsids inside the nucleus. The study provides a link between in vitro and in vivo data showing that HBV uses a unique strategy. We propose a model in which the unique environment of the nuclear pore favors the disassembly reaction, while both cytoplasm and nucleus favor assembly.

A multivalent vaccine candidate against hepatitis B virus (HBV) and hepatitis C virus (HCV) infections was constructed on the basis of HBV core (HBc) virus-like particles (VLPs) as carriers. Chimeric VLPs that carried a virus-neutralizing HBV pre-S1 epitope corresponding to amino acids (aa) 20 to 47 in the major immunodominant region (MIR) and a highly conserved N-terminal HCV core epitope corresponding to aa 1 to 60 at the C terminus of the truncated HBcΔ protein (N-terminal aa 1 to 144 of full-length HBc) were produced in Escherichia coli cells and examined for their antigenicity and immunogenicity. The presence of two different foreign epitopes within the HBc molecule did not interfere with its VLP-forming ability, with the HBV pre-S1 epitope exposed on the surface and the HCV core epitope buried within the VLPs. After immunization of BALB/c mice, specific T-cell activation by both foreign epitopes and a high-titer antibody response against the pre-S1 epitope were found, whereas an antibody response against the HBc carrier was notably suppressed. Both inserted epitopes also induced a specific cytotoxic-T-lymphocyte (CTL) response, as shown by the gamma interferon (IFN-γ) production profile.

Hepatitis C virus (HCV) infection is accompanied by the induction of oxidative stress, mediated by several virus proteins, the most prominent being the nucleocapsid protein (HCV core). Here, using the truncated forms of HCV core, we have delineated several mechanisms by which it induces the oxidative stress. The N-terminal 36 amino acids of HCV core induced TGFβ1-dependent expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases 1 and 4, both of which independently contributed to the production of reactive oxygen species (ROS). The same fragment also induced the expression of cyclo-oxygenase 2, which, however, made no input into ROS production. Amino acids 37–191 of HCV core up-regulated the transcription of a ROS generating enzyme cytochrome P450 2E1. Furthermore, the same fragment induced the expression of endoplasmic reticulum oxidoreductin 1α. The latter triggered efflux of Ca2+ from ER to mitochondria via mitochondrial Ca2+ uniporter, leading to generation of superoxide anions, and possibly also H2O2. Suppression of any of these pathways in cells expressing the full-length core protein led to a partial inhibition of ROS production. Thus, HCV core causes oxidative stress via several independent pathways, each mediated by a distinct region of the protein.

The hepatitis B virus (HBV) core antigen (HBcAg) has a unique ability to bind a high frequency of naive human and murine B cells. The role of HBcAg-binding naive B cells in the immunogenicity of HBcAg is not clear. The HBcAg-binding properties of naive B cells were characterized using HBcAg particles with mutated spike region (residues 76–85) sequences. Deletion of residues 76–85 (HBcΔ76–85) destroyed naive B cell binding, whereas deletion of residues 79–85 did not. HBcAg particles with an Ile instead of the natural Ala at position 80 did not bind naive B cells, whereas reversion of Ile80→Ala restored B cell binding. Destroying the B cell-binding ability of HBcAg had a marginal effect on the overall B cell immunogenicity of the different particles, suggesting that they were equally efficient in priming T helper cells. Therefore, the importance of HBcAg-binding B cells is studied with relation to the priming of HBcAg-specific cytotoxic T cells (CTLs). The role of HBcAg-binding B cells in the priming of HBcAg-specific CTLs was evaluated by immunization with endogenous HBcAg (DNA immunization) and exogenous recombinant HBcAg particles. Endogenous HBcAg primed HBcAg-specific CTLs in wild-type and B cell-deficient mice, whereas exogenous HBcAg primed HBcAg-specific CTLs only in wild-type mice. Importantly, HBcΔ76–85 did not prime CTLs despite the presence of B cells. Thus, the ability of exogenous HBcAg particles to prime specific CTLs is B cell dependent, suggesting a possible role for HBcAg-binding B cells in HBV infections.