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Hepatitis B virus (HBV) is a leading cause of liver disease. Its success as a human pathogen is related to the immense production of subviral envelope particles (SVPs) contributing to viral persistence by interfering with immune f...
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Hepatitis B virus (HBV) is a leading cause of liver disease. Its success as a human pathogen is related to the immense production of subviral envelope particles (SVPs) contributing to viral persistence by interfering with immune functions. To explore cellular pathways involved in SVP formation and egress, we investigated host–pathogen interactions. Yeast‐based proteomics revealed Sec24A, a component of the coat protein complex II (COPII), as an interaction partner of the HBV envelope S domain. To understand how HBV co‐opts COPII as a proviral machinery, we studied roles of key Sec proteins in HBV‐expressing liver cells. Silencing of Sar1, Sec23, and Sec24, which promote COPII assembly concomitant with cargo loading, strongly diminished endoplasmic reticulum (ER) envelope export and SVP secretion. By analysing Sec paralog specificities, we unexpectedly found that the HBV envelope is a selective interaction partner of Sec24A and Sec23B whose functions could not be substituted by their related isoforms. In support, we found that HBV replication upregulated Sec24A and Sec23B transcription. Furthermore, HBV encountered the Sec24A/Sec23B complex via an interaction that involved the N‐terminal half of Sec24A and a di‐arginine motif of its S domain, mirroring a novel ER export code. Accordingly, an interference with the COPII/HBV cross‐talk might display a tool to effectively inhibit SVP release.
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Abstract Viruses are obligate intracellular pathogens that utilize cellular machinery for many aspects of their propagation and effective egress of virus particles from host cells is one important determinant of virus infectivity....
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Abstract Viruses are obligate intracellular pathogens that utilize cellular machinery for many aspects of their propagation and effective egress of virus particles from host cells is one important determinant of virus infectivity. Hijacking host cell processes applies in particular to the hepatitis B virus (HBV), as its DNA genome with about 3?kb in size is one of the smallest viral genomes known.?HBV is a leading cause of liver disease and still displays one of the most successful pathogens in human populations worldwide. The extremely successful spread of this virus is explained by its efficient transmission strategies and its versatile particle types, including virions, empty envelopes, naked capsids, and others. HBV exploits distinct host trafficking machineries to assemble and release its particle types including nucleocytoplasmic?shuttling transport, secretory, and exocytic pathways, the Endosomal Sorting Complexes Required for Transport pathway, and the autophagy pathway. Understanding how HBV uses and subverts host membrane trafficking systems offers the chance of obtaining new mechanistic insights into the regulation and function of this essential cellular processes. It can also help to identify potential targets for antiviral interventions. Here, I will provide an overview of HBV maturation, assembly, and budding, with a focus on recent advances, and will point out areas where questions remain that can benefit from future studies. Unless otherwise indicated, almost all presented knowledge was gained from cell culture‐based, HBV in vitro‐replication and in vitro‐infection systems.
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Hepatitis B virus (HBV) is an enveloped DNA virus that exploits the endosomal sorting complexes required for transport (ESCRT) pathway for budding. In addition to infectious particles, HBV-replicating cells release non-enveloped (...
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Hepatitis B virus (HBV) is an enveloped DNA virus that exploits the endosomal sorting complexes required for transport (ESCRT) pathway for budding. In addition to infectious particles, HBV-replicating cells release non-enveloped (nucleo)capsids, but their functional implication and pathways of release are unclear. Here, we focused on the molecular mechanisms and found that the sole expression of the HBV core protein is sufficient for capsid release. Unexpectedly, released capsids are devoid of a detectable membrane bilayer, implicating a non-vesicular exocytosis process. Unlike virions, naked capsid budding does not require the ESCRT machinery. Rather, we identified Alix, a multifunctional protein with key roles in membrane biology, as a regulator of capsid budding. Ectopic overexpression of Alix enhanced capsid egress, while its depletion inhibited capsid release. Notably, the loss of Alix did not impair HBV production, furthermore indicating that virions and capsids use diverse export routes. By mapping of Alix domains responsible for its capsid releasemediating activity, its Bro1 domain was found to be required and sufficient. Alix binds to core via its Bro1 domain and retained its activity even if its ESCRT-III binding site is disrupted. Together, the boomerang-shaped Bro1 domain of Alix appears to escort capsids without ESCRT.
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Hepatitis B virus (HBV) is a major cause of liver disease. Due to the tiny size of its genome, HBV depends on the critical interplay between viral and host factors for the generation of new viral particles from infected cells. Rec...
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Hepatitis B virus (HBV) is a major cause of liver disease. Due to the tiny size of its genome, HBV depends on the critical interplay between viral and host factors for the generation of new viral particles from infected cells. Recent work has illuminated a multiplicity of spatially and temporally coordinated virus-host interactions that accompany HBV particle genesis. These interactions include the requirement of cellular chaperones for the maturation of the three viral envelope proteins, the cellular factors involved in dynamic modification, maturation, and intracellular trafficking of the nucleocapsids, and the host components of the multivesicular body (MVB) pathway enabling virion budding at intracellular compartments. Beside infectious virions, HBV produces at least two other types of particles, subviral empty envelope particles and subviral naked capsid particles, likely as a result of the engagement of different host factors by the viral structural proteins. Accordingly, HBV exploits distinct cellular pathways to release its particle types. Here, I review recent progress in these areas of the cell biology of HBV genesis.
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The identity and functionality of biological membranes are determined by cooperative interaction between their lipid and protein constituents. Cholesterol is an important structural lipid that modulates fluidity of biological memb...
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The identity and functionality of biological membranes are determined by cooperative interaction between their lipid and protein constituents. Cholesterol is an important structural lipid that modulates fluidity of biological membranes favoring the formation of detergent-resistant microdomains. In the present study, we evaluated the functional role of cholesterol and lipid rafts for entry of hepatitis B viruses into hepatocytes. We show that the duck hepatitis B virus (DHBV) attaches predominantly to detergent-soluble domains on the plasma membrane. Cholesterol depletion from host membranes and thus disruption of rafts does not affect DHBV infection. In contrast, depletion of cholesterol from the envelope of both DHBV and human HBV strongly reduces virus infectivity. Cholesterol depletion increases the density of viral particles and leads to changes in the ultrastructural appearance of the virus envelope. However, the dual topology of the viral envelope protein L is not significantly impaired. Infectivity and density of viral particles are partially restored upon cholesterol replenishment. Binding and entry of cholesterol-deficient DHBV into hepatocytes are not significantly impaired, in contrast to their release from endosomes. We therefore conclude that viral but not host cholesterol is required for endosomal escape of DHBV.
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Hepatitis B virus (HBV) is an enveloped DNA virus that presumably buds at intracellular membranes of infected cells. HBV budding involves two endocytic host proteins, the ubiquitin-interacting adaptor γ2-adaptin and the Nedd4 ubi...
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Hepatitis B virus (HBV) is an enveloped DNA virus that presumably buds at intracellular membranes of infected cells. HBV budding involves two endocytic host proteins, the ubiquitin-interacting adaptor γ2-adaptin and the Nedd4 ubiquitin ligase. Here, we demonstrate that HBV release also requires the cellular machinery that generates internal vesicles of multivesicular bodies (MVBs). In order to perturb the MVB machinery in HBV-replicating liver cells, we used ectopic expression of dominant-negative mutants of different MVB components, like the ESCRT-III complex-forming CHMP proteins and the Vps4 ATPases. Upon coexpression of mutated CHMP3, CHMP4B, or CHMP4C forms, as well as of ATPase-defective Vps4A or Vps4B mutants, HBV assembly and egress were potently blocked. Each of the MVB inhibitors arrested virus particle maturation by entrapping the viral core and large and small envelope proteins in detergent-insoluble membrane structures that closely resembled aberrant endosomal class E compartments. In contrast, HBV subvirus particle release was not affected by MVB inhibitors, hinting at different export routes used by viral and subviral particles. To further define the role γ2-adaptin plays in HBV formation, we examined the effects of its overexpression in virus-replicating cells. Intriguingly, excess γ2-adaptin blocked HBV production in a manner similar to the actions of CHMP and Vps4 mutants. Moreover, overexpressed γ2-adaptin perturbed the endosomal morphology and diminished the budding of a retroviral Gag protein, implying that it may act as a principal inhibitor of the MVB sorting pathway. Together, these results demonstrate that HBV exploits the MVB machinery with the aid of γ2-adaptin.
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For the outcome of a hepatitis B virus (HBV) infection, the viral L envelope protein with its pre-S domain performs pivotal functions by mediating attachment of HBV to liver cells, envelopment of viral capsids, release of (sub)vir...
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For the outcome of a hepatitis B virus (HBV) infection, the viral L envelope protein with its pre-S domain performs pivotal functions by mediating attachment of HBV to liver cells, envelopment of viral capsids, release of (sub)viral particles, regulation of supercoiled DNA amplification, and transcriptional transactivation. To assess its multiple functions and host-protein assistance involved, we initiated a two-hybrid screen using the L-specific pre-S1 domain as bait. With this approach, we have identified γ2-adaptin, a putative member of the clathrin adaptor proteins responsible for protein sorting and trafficking, as a specific binding partner of L protein. Evidence for a physical interaction between L protein and γ2-adaptin was also demonstrated by affinity chromatography and coimmunoprecipitation, and the binding sites were mapped to the L-specific pre-S1 domain and the γ2-adaptin-specific ear domain. The specificity of the interaction was further sustained by the failure of γ1-adaptin, a closely related γ2-adaptin homologue, to associate with L protein. Analysis of an L mutant protein indicates that the L–γ2-adaptin interaction strictly depends on the pre-S1 domain of transmembrane L protein oriented to the cytosol and thus appears to occur in the cytosolic environment. Interestingly, coexpression of the two interacting partners in transfected cells resulted in recruitment of γ2-adaptin by L protein onto cis-Golgi-like structures, strongly indicating that the association is physiologically relevant. Together, the results suggest a role for γ2-adaptin in L-mediated processes of viral biogenesis and/or pathogenesis, such as facilitating and guiding HBV assembly.
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Liver damage caused by chronic hepatitis B virus (HBV) infection may be enhanced through the selection of deleted HBV preS mutants by intracellular accumulation of viral proteins and subsequent cell death. However, the prevalence ...
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Liver damage caused by chronic hepatitis B virus (HBV) infection may be enhanced through the selection of deleted HBV preS mutants by intracellular accumulation of viral proteins and subsequent cell death. However, the prevalence and impact of such mutants on the clinical course of infection have not yet been studied in children. Serum samples from 60 children (mean age 9.8 y) were investigated by means of PCR and direct sequencing of the entire preS region. Only one patient (1.5\%) was found with a mixed HBV population of a deletion spanning 183 nucleotides and wild-type sequences. This mutation alters the HBV large-surface protein and removes the small-surface promoter. To clarify the significance of this mutation, we studied 14 serial serum samples of the child within a follow-up of 10 y. After occurrence of the mutation, the liver enzymes increased, despite seroconversion to anti-HBe. Transfection of an HBV expression construct containing this deletion into human hepatoma cells by using an HBV in vitro replication system showed that the mutant lost the ability of nucleocapsid packaging as a result of alteration of the transmembrane topology of the large surface protein. This effect could not be restored by coexpression of wild-type large- or small-surface proteins in trans. In conclusion, the circulation of HBV preS deletion mutants is rare in childhood. However, our functional and clinical follow-up studies in one child suggest that such a mutant may have the potential to aggravate liver inflammation, especially if corroborated with larger numbers of children.Abbreviations: HBV, hepatitis B virus; Nt, nucleotide; ER, endoplasmic reticulum; EPR, endogenous polymerase reaction
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Unlike those of the S and the L envelope proteins, the functional role of the related M protein in the life cycle of the hepatitis B virus (HBV) is less understood. We now demonstrate that a single N glycan, specific for M, is req...
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Unlike those of the S and the L envelope proteins, the functional role of the related M protein in the life cycle of the hepatitis B virus (HBV) is less understood. We now demonstrate that a single N glycan, specific for M, is required for efficient secretion of M empty envelope particles. Moreover, this glycan mediates specific association of M with the chaperone calnexin. Conversely, the N glycan, common to all three envelope proteins, is involved neither in calnexin binding nor in subviral particle release. As proper folding and trafficking of M need the assistance of the chaperone, the glycan-dependent association of M with calnexin may thus play a crucial role in the assembly of HBV. Beyond being modified by N glycosylation, M is modified by O glycosylation occurring within its amino acid sequence at positions 27 to 47. The O glycans, however, were found to be dispensable for secretion of M but may rather support viral infectivity. Surprisingly, nonglycosylated M localizes exclusively to the cytosol, either for degradation or for a yet-unknown function.
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Many viruses take advantage of cellular trafficking machineries to assemble and release new infectious particles. Using RNA interference (RNAi), we demonstrate that the Golgi/autophagosome-associated Rab33B is required for hepatit...
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Many viruses take advantage of cellular trafficking machineries to assemble and release new infectious particles. Using RNA interference (RNAi), we demonstrate that the Golgi/autophagosome-associated Rab33B is required for hepatitis B virus (HBV) propagation in hepatoma cell lines. While Rab33B is dispensable for the secretion of HBV subviral envelope particles, its knockdown reduced the virus yield to 20% and inhibited nucleocapsid (NC) formation and/or NC trafficking. The overexpression of a GDP-restricted Rab33B mutant phenocopied the effect of deficit Rab33B, indicating that Rab33B-specific effector proteins may be involved. Moreover, we found that HBV replication enhanced Rab33B expression. By analyzing HBV infection cycle steps, we identified a hitherto unknown membrane targeting module in the highly basic C-terminal domain of the NC-forming core protein. Rab33B inactivation reduced core membrane association, suggesting that membrane platforms participate in HBV assembly reactions. Biochemical and immunofluorescence analyses provided further hints that the viral core, rather than the envelope, is the main target for Rab33B intervention. Rab33B-deficiency reduced core protein levels without affecting viral transcription and hampered core/NC sorting to envelope-positive, intracellular compartments. Together, these results indicate that Rab33B is an important player in intracellular HBV trafficking events, guiding core transport to NC assembly sites and/or NC transport to budding sites.
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