Hepatitis B Virus Replication and Release Are Independent of Core Lysine Ubiquitination

Abstract

Ubiquitin conjugation to lysine residues regulates a variety of protein functions, including endosomal trafficking and degradation. While ubiquitin plays an important role in the release of many viruses, the requirement for direct ubiquitin conjugation to viral structural proteins is less well understood. Some viral structural proteins require ubiquitin ligase activity, but not ubiquitin conjugation, for efficient release. Recent evidence has shown that, like other viruses, hepatitis B virus (HBV) requires a ubiquitin ligase for release from the infected cell. The HBV core protein contains two lysine residues (K7 and K96), and K96 has been suggested to function as a potential ubiquitin acceptor site based on the fact that previous studies have shown that mutation of this amino acid to alanine blocks HBV release. We therefore reexamined the potential connection between core lysine ubiquitination and HBV replication, protein trafficking, and virion release. In contrast to alanine substitution, we found that mutation of K96 to arginine, which compared to alanine is more conserved but also cannot mediate ubiquitin conjugation, does not affect either virus replication or virion release. We also found that the core lysine mutants display wild-type sensitivity to the antiviral activity of interferon, which demonstrates that ubiquitination of core lysines does not mediate the interferon-induced disruption of HBV capsids. However, mutation of K96 to arginine alters the nuclear-cytoplasmic distribution of core, leading to an accumulation in the nucleolus. In summary, these studies demonstrate that although ubiquitin may regulate the HBV replication cycle, these mechanisms function independently of direct lysine ubiquitination of core protein.The hepatitis B virus (HBV) particle consists of an enveloped nucleocapsid that contains the viral polymerase (Pol) and an incomplete 3.2-kb double-stranded DNA genome (9). In the cytoplasm, the viral core structural proteins interact to form homodimers, which further self-assemble into capsid particles that package Pol and the viral pregenomic RNA. Encapsidated Pol subsequently reverse transcribes pregenomic RNA to give rise to mature double-stranded relaxed circular DNAcontaining capsids. HBV DNA-containing capsids are released from the cell as mature virions after acquiring an envelope consisting of cellular membrane lipids and the viral small, middle, and large envelope proteins (4, 9, 41). Due to the directed insertion of the envelope proteins in the endoplasmic reticulum and Golgi membrane, and the requirement of the large envelope protein for virion release, nucleocapsids are hypothesized to bud at intracellular membranes for release through the constitutive secretory pathway (5). Although the mechanism and site of HBV nucleocapsid envelopment and release remain poorly understood, emerging evidence indicates that the cellular ubiquitin pathway may play a role in this process.Structural proteins of some enveloped RNA viruses contain highly conserved sequences [PPXY, P(T/S)AP, an...

Hepatitis B Virus Covalently Closed Circular DNA Formation in Immortalized Mouse Hepatocytes Associated with Nucleocapsid Destabilization

“…As shown in Fig. 2, both differentiated and undifferentiated (transiently transfected) HBV-Met.4 cells could produce high levels of core DNA as reported previously (23,27). However, HBV-Met.4 cells produced much lower (less than 2%) levels of PF-RC DNA than did AML12HBV10 cells, as judged by the ratio of the PF-RC DNA to core RC DNA (Fig.…”

“…HBV-Met.4 cells can be induced to express the viral genome upon differentiation by treatment with DMSO (23). Alternatively, undifferentiated HBV-Met.4 cells can be transiently transfected with HBV DNA to initiate viral gene expression and replication (27). As shown in Fig.…”

Inhibition of Cellular Proteasome Activities Mediates HBX-Independent Hepatitis B Virus Replication In Vivo

“…Using recombinant adenoviruses or baculoviruses expressing replicating HBV or WHV genomes with or without a functional X gene, we determined the effects of proteasome inhibitors on the functions of the X protein in hepadnaviral replication and demonstrated that proteasome inhibitors restored the replication defect of X-negative HBV and WHV (30). On the other hand, Garcia et al recently showed that proteasome inhibition blocks HBV release in cell culture, presumably by depletion of free cellular ubiquitin (10).…”

Bortezomib Inhibits Hepatitis B Virus Replication in Transgenic Mice

“…Furthermore, this block occurred at a step downstream of HBV mRNA and protein expression. The inhibition of HBV replication by bortezomib in the transgenic mice was surprising, since we have previously found that although proteasome inhibition blocks HBV release in cell culture, it does not affect the levels of intracellular DNA replication intermediates (7,31). Although the reason for the difference between cell culture and mice is unclear, it is possible that the proteasome-sensitive aspect of the HBV replication cycle is more accurately reflected in the liver in vivo.…”

“…The interaction of HBx with the proteasome may be functionally important for virus replication, since the replication defect of an HBx-deficient HBV mutant is suppressed by proteasome inhibition in cell culture (46). We have also recently found that the IFN-regulated proteasome catalytic subunits can influence the specificity of the HBV-specific CD8 T-cell response (30) and that depletion of free cellular ubiquitin through proteasome inhibition blocks HBV release in cell culture (7).…”