The replication cycle of hepatitis B virus (original) (raw)
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Control of cccDNA function in hepatitis B virus infection
Journal of Hepatology, 2009
The template of hepatitis B virus (HBV) transcription, the covalently closed circular DNA (cccDNA), plays a key role in the life cycle of the virus and permits the persistence of infection. Novel molecular techniques have opened new possibilities to investigate the organization and the activity of the cccDNA minichromosome in vivo, and recent advances have started to shed light on the complexity of the mechanisms controlling cccDNA function. Nuclear cccDNA accumulates in hepatocyte nuclei as a stable minichromosome organized by histone and non-histone viral and cellular proteins. Identification of the molecular mechanisms regulating cccDNA stability and its transcriptional activity at the RNA, DNA and epigenetic levels in the course of chronic hepatitis B (CH-B) infection may reveal new potential therapeutic targets for anti-HBV drugs and hence assist in the design of strategies aimed at silencing and eventually depleting the cccDNA reservoir. Ó
Reverse transcription-associated dephosphorylation of hepadnavirus nucleocapsids
Proceedings of the National Academy of Sciences, 2005
Hepatitis B viruses are pararetroviruses that contain a partially dsDNA genome and replicate this DNA through an RNA intermediate (the pregenomic RNA, pgRNA) by reverse transcription. Viral assembly begins with the packaging of the pgRNA into nucleocapsids (NCs), with subsequent reverse transcription within NCs converting the pgRNA into the characteristic dsDNA genome. Only NCs containing this dsDNA (the so-called "mature" NCs) are enveloped by the viral envelope proteins and secreted as virions; "immature" NCs, i.e., those containing pgRNA or immature reverse transcription intermediates, are excluded from virion formation. This phenomenon is thought to be caused by the emergence of an intrinsic maturation signal only on the mature NCs. To define the maturation signal, we have devised a method to separate mature from immature duck hepatitis B virus NCs and have compared them to NCs derived from secreted virions. Detailed mass spectrometric analyses revealed that the core protein from immature NCs was phosphorylated on at least six sites, whereas the core protein from mature NCs and that from secreted virions was entirely dephosphorylated. These results, together with the known requirement of core phosphorylation for pgRNA packaging and DNA synthesis, suggest that the NC undergoes a dynamic change in phosphorylation state to fulfill its multiple roles at different stages of viral replication. Although phosphorylation of the NCs is required for efficient RNA packaging and DNA synthesis by the immature NCs, dephosphorylation of the mature NCs may trigger envelopment and secretion.
2006
of pEGFP-Core Fusion Protein and its Redundants 3.5. Effect of Staurosporine on the Localization of EGFP-Core 3C and 3 SV40 NLS 3.6. Effect of FCS on Intracellular Localization of EGFP-Core Fusion Proteins 3.6.1. Effect of FCS on Cell Division 3.6.2. Effect of Serum on the Localization of EGFP-Core 3 SV40 NLS in HuH-7 Cells 3.7. Nuclear Localization of EGFP-Core Fusion Proteins in HepG2 Cells 3.8. Dimerization of EGFP-Core 1C 3.9. Effect of the Assembly Inhibitor Bayer 41-4109 on the Localization of EGFP Core Fusion Proteins 4. Discussion 4.1. Nuclear Localization of HBV Capsids After Transfection of the Entire HBV genome into HuH-7 Cells 4.2. Structure of the Fusion Protein 4.3. Localization in HuH-7 cells 4.3.1. Transport Competence of EGFP-Core Fusion Proteins 4.3.2. Effect of Cell Cycle and Phosphorylation 4.4. Localization in HepG2 Cells 4.5. Molecular Implication for the Nuclear Import and the Viral Life Cycle 5. Summary 6. Zusammenfassung 7. References 8. Acknowlegements 9. Appendixes 9.1. List of Figures 9.2. List of Tables 10. Curriculum Vitae rc DNA relaxed circular DNA RT reverse transcriptase SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis SV40 Tag simian virus 40 large T antigen SHBs small hepatitis B surface protein TAE tris acetate EDTA TE tris-EDTA °C degree celsius Prange, 2003). The MHBs contains only the preS2 and S domain, and the SHBs consists of only of the S domain. In infected hepatocytes, only a minority of the surface proteins is part of the virion but the majority is secreted as subviral particles (SVPs). Dependent upon the composition of the 3 surface proteins SVPs either form filaments or spheres of 20 nm. A schematic presentation of virus and SVPs is given in Figure 1. The surface proteins of the virus and SVP are assembled at the endoplasmic reticulum (ER) and bud into the lumen of postER intermediate compartment. Thus the lipids in the outer protein shell or the HBs particles are derived from an intracellular compartment and not from the plasma membrane. Within virions the surface proteins enclose the capsid. The envelopment is mediated by an interaction of the core particle with the internally localized preS1 domain of LHBs (Bruss, 1997; Ponsel and Bruss, 2003). The envelopment requires DNA synthesis within the capsid (Gerelsaikhan et al., 1996) implying that the genome maturation induces a structural change of the capsid surface. After synthesis in the cytoplasm the core molecules form dimers (Zhou and Standring, 1992) that trimerize (Zlotnick et al., 1999). The resulting hexamers assemble to capsids. This assembly occurs spontaneously and is independent upon other viral proteins. In vivo a complex of pregenome, polymerase and the heat shock proteins Hsp40
Signals for Bidirectional Nucleocytoplasmic Transport in the Duck Hepatitis B Virus Capsid Protein
Journal of Virology, 2001
Hepadnavirus genome replication involves cytoplasmic and nuclear stages, requiring balanced targeting of cytoplasmic nucleocapsids to the nuclear compartment. In this study, we analyze the signals determining capsid compartmentalization in the duck hepatitis B virus (DHBV) animal model, as this system also allows us to study hepadnavirus infection of cultured primary hepatocytes. Using fusions to the green fluorescent protein as a functional assay, we have identified a nuclear localization signal (NLS) that mediates nuclear pore association of the DHBV nucleocapsid and nuclear import of DHBV core protein (DHBc)-derived polypeptides. The DHBc NLS mapped is unique. It bears homology to repetitive NLS elements previously identified near the carboxy terminus of the capsid protein of hepatitis B virus, the human prototype of the hepadnavirus family, but it maps to a more internal position. In further contrast to the hepatitis B virus core protein NLS, the DHBc NLS is not positioned near phosphorylation target sites that are generally assumed to modulate nucleocytoplasmic transport. In functional assays with a knockout mutant, the DHBc NLS was found to be essential for nuclear pore association of the nucleocapsid. The NLS was found to be also essential for virus production from the full-length DHBV genome in transfected cells and from hepatocytes infected with transcomplemented mutant virus. Finally, the DHBc additionally displayed activity indicative of a nuclear export signal, presumably counterbalancing NLS function in the productive state of the infected cell and thereby preventing nucleoplasmic accumulation of nucleocapsids.
Hepatitis B Virus Core Gene Mutations Which Block Nucleocapsid Envelopment
Journal of Virology, 2000
Recently we generated a panel of hepatitis B virus core gene mutants carrying single insertions or deletions which allowed efficient expression of the core protein in bacteria and self-assembly of capsids. Eleven of these mutations were introduced into a eukaryotic core gene expression vector and characterized by trans complementation of a core-negative HBV genome in cotransfected human hepatoma HuH7 cells. Surprisingly, four mutants (two insertions [EFGA downstream of A11 and LDTASALYR downstream of R39] and two deletions [Y38-R39-E40 and L42]) produced no detectable capsids. The other seven mutants supported capsid formation and pregenome packaging/viral minus-and plus-strand-DNA synthesis but to different levels.
Journal of Biological Chemistry, 2003
Viral nucleocapsids compartmentalize and protect viral genomes during assembly while they mediate targeted genome release during viral infection. This dual role of the capsid in the viral life cycle must be tightly regulated to ensure efficient virus spread. Here, we used the duck hepatitis B virus (DHBV) infection model to analyze the effects of capsid phosphorylation and hydrogen bond formation. The potential key phosphorylation site at serine 245 within the core protein, the building block of DHBV capsids, was substituted by alanine (S245A), aspartic acid (S245D) and asparagine (S245N), respectively. Mutant capsids were analyzed for replication competence, stability, nuclear transport, and infectivity. All mutants formed DHBV DNA-containing nucleocapsids. Wild-type and S245N but not S245A and S245D fully protected capsid-associated mature viral DNA from nuclease action. A negative ionic charge as contributed by phosphorylated serine or aspartic acidsupported nuclear localization of the viral capsid and generation of nuclear superhelical DNA. Finally, wildtype and S245D but not S245N virions were infectious in primary duck hepatocytes. These results suggest that hydrogen bonds formed by non-phosphorylated serine 245 stabilize the quarterny structure of DHBV nucleocapsids during viral assembly, while serine phosphorylation plays an important role in nuclear targeting and DNA release from capsids during viral infection.
Virology, 2006
Synthesis of hepadnaviral DNA is dependent upon both the viral DNA polymerase and the viral core protein, the subunit of the nucleocapsids in which viral DNA synthesis takes place. In a study of natural isolates of duck hepatitis B virus (DHBV), we cloned full-length viral genomes from a puna teal. One of the clones failed to direct viral DNA replication in transfected cells, apparently as a result of a 3 nt inframe deletion of histidine 107 in the core protein. Histidine 107 is located in the center of a predicted helical region of the "insertion domain", a stretch of 45 amino acids which appears to be at the tip of a spike on the surface of the nucleocapsid. The mutation was introduced into a well-characterized strain of DHBV for further analysis. Core protein accumulated in cells transfected with the mutant DHBV but was partially degraded, suggesting that it was unstable. Assembled nucleocapsids were not detected by capsid gel electrophoresis. Interestingly, the mutant protein appeared to form chimeric nucleocapsids with wild-type core protein. The chimeric nucleocapsids supported viral DNA replication. These results suggest that the insertion domain of the spike may play a role either in assembly of stable nucleocapsids, possibly in formation of the dimer subunits, or in triggering nucleocapsid disintegration, required during initiation of new rounds of infection.
Journal of virology, 1997
The coronavirus mouse hepatitis virus (MHV) contains a large open reading frame embedded entirely within the 5' half of its nucleocapsid (N) gene. This internal gene (designated I) is in the +1 reading frame with respect to the N gene, and it encodes a mostly hydrophobic 23-kDa polypeptide. We have found that this protein is expressed in MHV-infected cells and that it is a previously unrecognized structural protein of the virion. To analyze the potential biological importance of the I gene, we disrupted its expression by site-directed mutagenesis using targeted RNA recombination. The start codon for I was replaced by a threonine codon, and a stop codon was introduced at a short interval downstream. Both alterations created silent changes in the N reading frame. In vitro translation studies showed that these mutations completely abolished synthesis of I protein, and immunological analysis of infected cell lysates confirmed this conclusion. The MHV I mutant was viable and grew to ...