Reconstitution of gene expression from a regulatory‐protein‐deficient hepatitis B virus genome by cell‐permeable HBx protein

Hafner, A.; Brandenburg, Boerries; Hildt, Eberhard

doi:10.1038/sj.embor.embor903

Cited by 28 publications

(26 citation statements)

References 33 publications

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“…In contrast to other enveloped viruses, the TLM-mediated escape from the endosome is not a fusogenic process. The difference from fusogenic mechanisms is that the TLM does not rest in the membrane at the end of the translocation process (14)(15)(16). Therefore, it is likely that the cleaved surface protein remains associated with the nucleocapsid during escape from the endocytotic pathway.…”

Section: Discussionmentioning

confidence: 99%

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Identification of a structural motif crucial for infectivity of hepatitis B viruses

Stoeckl

Funk²,

Kopitzki³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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Infectious entry of hepatitis B viruses (HBV) has nonconventional facets. Here we analyzed whether a cell-permeable peptide [translocation motif (TLM)] identified within the surface protein of human HBV is a general feature of all hepadnaviruses and plays a role in the viral life cycle. Surface proteins of all hepadnaviruses contain conserved functional TLMs. Genetic inactivation of the duck HBV TLMs does not interfere with viral morphogenesis; however, these mutants are noninfectious. TLM mutant viruses bind to cells and are taken up into the endosomal compartment, but they cannot escape from endosomes. Processing of surface protein by endosomal proteases induces their exposure on the virus surface. This unmasking of TLMs mediates translocation of viral particles across the endosomal membrane into the cytosol, a prerequisite for productive infection. The ability of unmasked TLMs to translocate processed HBV particles across cellular membranes was shown by confocal immunofluorescence microscopy and by infection of nonpermissive cell lines with HBV processed in vitro with endosomal lysate. Based on these data, we propose an infectious entry mechanism unique for hepadnaviruses that involves virus internalization by receptor-mediated endocytosis followed by processing of surface protein in endosomes. This processing activates the function of TLMs that are essential for viral particle translocation through the endosomal membrane into the cytosol and productive infection.cell permeability ͉ envelope protein ͉ virus entry I nfection with human hepatitis B virus (HBV) can cause acute or chronic inflammation of the liver (1, 2). HBV is the prototype member of the hepadnaviridae family, which encompasses members infecting woodchucks, ground squirrels, and avian viruses isolated from, e.g., pekin ducks, gray herons, and storks.Duck HBV (DHBV) is a well characterized model system of hepadnaviral infection (3). Cultures of primary duck hepatocytes (PDHs) can be readily established and efficiently infected (3, 4) and therefore provide a suitable tool for analyzing the early steps of hepadnaviral infection on the molecular level. As for HBV (5-7), it is known that DHBV infection is initiated by attachment of the virus particle to the hepatocyte surface via the pre-S domain of the viral surface protein L (8, 9). In DHBV there are two surface proteins embedded in the lipid envelope: The major S protein, a transmembrane protein that encompasses 167 aa, and the L protein, consisting of the S domain N-terminally extended by the 160-aa pre-S domain. Previous work suggested that DHBV enters the cell by receptor-mediated endocytosis (10-13). The mechanism that allows internalized viral particles to escape from the endocytic pathway remained elusive.Recently, a cell-permeable peptide [translocation motif (TLM)] was identified in the pre-S domain of HBV (14). The TLM is a 12-aa-encompassing domain that forms an amphipathic ␣-helix. It mediates an energy-and receptor-independent transfer of peptides, nucleic acids, and proteins when fus...

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Section: Discussionmentioning

confidence: 99%

“…Aliquots were polyethylene glycol-precipitated as previously described and resuspended in culture medium. Subcellular fractionation was performed as described (14,16).…”

Section: Methodsmentioning

confidence: 99%

Identification of a structural motif crucial for infectivity of hepatitis B viruses

Stoeckl

Funk²,

Kopitzki³

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…The peptide Cbz-VVRR-AMC that was derived from the HBV core protein (amino acids 141-151) was used as substrate (38) Stimulation with interferon ␣ or interferon ␥ was performed for 3 days using 10 2 and 10 3 units per ml. Recombinant purified HBx was isolated as described previously (34). All experiments were performed in triplicate.…”

Section: Methodsmentioning

confidence: 99%

“…Mutant HBV genomes lacking PreS2 activator function (pHBV⌬PreS2) or functional HBx (pHBV⌬HBx) or the activator-deficient double mutant (pHBV⌬PreS2/⌬HBx) were described recently (33,34). For silencing of HBV expression, the following siRNA was used, 5Ј-AAGACCUAGUCAGUUAUG-3Ј.…”

Section: Methodsmentioning

confidence: 99%

Hepatitis B Virus Induces Expression of Antioxidant Response Element-regulated Genes by Activation of Nrf2

Schaedler

Krause

Himmelsbach

et al. 2010

Journal of Biological Chemistry

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“…5 The TLM mediates receptor-and energy-independent transfer of peptides, proteins, and nucleic acids across membranes, when fused to them, without affecting cellular integrity or interfering with intracellular signal transduction cascades. 5,13,23,24 We describe the potential of the TLM to mediate the transport of fully assembled particles into the cytoplasm of target cells in a receptor-and endocytosis-independent manner. Based on this system, we established cell-permeable VLPs harboring marker genes enabling an efficient gene transfer into primary hepatocytes.…”

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confidence: 99%

A novel system for efficient gene transfer into primary human hepatocytes via cell-permeable hepatitis B virus-like particle

et al. 2005

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R ecently, a variety of protein transduction domains (PTDs) have been identified allowing the transfer of peptides, proteins, or nucleic acids across cellular membranes into cells. 1-9 PTDs have been used to successfully treat preclinical models of human disease such as cancer, psoriasis, and stroke. 10-13 Transfer of nano-particular structures across cellular membranes is of increasing importance for the development of novel diagnostic and therapeutic tools. The capacity of PTDs to mediate an endocytosis-independent transfer of particles across the plasma membrane into the cytoplasm is still unclear. To investigate the potential of PTD to mediate the transfer of nano-particles across the cell membrane, virus-like particles (VLPs) harboring a marker gene were instrumental.Hepatitis B virus (HBV) core particles represent a very well-characterized VLP model system. 14,15 The HBV core particle (capsid) is assembled by 180 or 240 core protein monomers (HBcAg), resulting in an icosahedral particle 30 or 34 nm in diameter, respectively. A characteristic of the core protein is a basic arginine-rich C-terminal region, which is responsible for the packaging of DNA 16 and for guidance of the capsid to the nucleus. 17 Based on extensive structural analysis, 18 the hepatitis B capsid was used as a carrier for foreign epitopes to develop new vaccines. 19,20 In the viral context, the core particle harbors the viral genome (nucleocapsid). Efficient in vivo packaging of nucleic acids into capsids requires HBV polymerase and its interaction with a defined secondary structure (termed epsilon) at the 5Ј end of the RNA to be packaged. 21 The ⑀ signal is the primary element of the hepadnaviral packag-

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Reconstitution of gene expression from a regulatory‐protein‐deficient hepatitis B virus genome by cell‐permeable HBx protein

Cited by 28 publications

References 33 publications

Identification of a structural motif crucial for infectivity of hepatitis B viruses

Identification of a structural motif crucial for infectivity of hepatitis B viruses

Hepatitis B Virus Induces Expression of Antioxidant Response Element-regulated Genes by Activation of Nrf2

A novel system for efficient gene transfer into primary human hepatocytes via cell-permeable hepatitis B virus-like particle

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