A dramatic shift in the transmembrane topology of a viral envelope glycoprotein accompanies hepatitis B viral morphogenesis.

Ostapchuk, Philomena; Hearing, Patrick; Ganem, Don

doi:10.1002/j.1460-2075.1994.tb06353.x

Cited by 141 publications

(141 citation statements)

References 24 publications

(20 reference statements)

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“…On the other hand, if this topology holds true for authentic NS4B, our findings would indicate that the COOH-terminal region in a minor fraction of NS4B molecules slips post-translationally through the membrane after cleavage by the viral protease. Comparable post-translational reorientations of membrane proteins have been described for other viral proteins, for example, for the M protein of the gastroenteritis corona virus (50,51), the hepatitis B virus large envelope protein (52)(53)(54)(55), and the NH 2 terminus of the HCV NS4B (26).…”

Section: Discussionmentioning

confidence: 83%

Subcellular Localization and Membrane Topology of the Dengue Virus Type 2 Non-structural Protein 4B

Miller

Sparacio

Bartenschlager

2006

Journal of Biological Chemistry

238

289

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Dengue virus (DV) is a member of the family Flaviviridae. These positive strand RNA viruses encode a polyprotein that is processed in case of DV into 10 proteins. Although for most of these proteins distinct functions have been defined, this is less clear for the highly hydrophobic non-structural protein (NS) 4B. Despite its possible role as an antagonist of the interferon-induced antiviral response, this protein may play an additional more direct role for viral replication. In this study we determined the subcellular localization, membrane association, and membrane topology of DV NS4B. We found that NS4B resides primarily in cytoplasmic foci originating from the endoplasmic reticulum. NS4B colocalizes with NS3 and double-stranded RNA, an intermediate of viral replication, arguing that NS4B is part of the membrane-bound viral replication complex. Biochemical analysis revealed that NS4B is an integral membrane protein, and that its preceding 2K signal sequence is not required for this integration. We identified three membrane-spanning segments in the COOH-terminal part of NS4B that are sufficient to target a cytosolic marker protein to intracellular membranes. Furthermore, we established a membrane topology model of NS4B in which the NH 2 -terminal part of the protein is localized in the endoplasmic reticulum lumen, whereas the COOH-terminal part is composed of three trans-membrane domains with the COOH-terminal tail localized in the cytoplasm. This topology model provides a good starting point for a detailed investigation of the function of NS4B in the DV life cycle. The mosquito-transmitted Dengue virus (DV)3 is the causative agent of dengue fever, the most prevalent arthropod-borne viral illness in humans with 50 -100 million individuals infected annually worldwide (1). Dengue fever is characterized by high fever, chills, body aches, and skin rash and ranges from mild, flu-like symptoms to severe forms, which are dengue hemorrhagic fever and the dengue shock syndrome. The four serotypes of DV identified so far (DV1-4) belong to the genus Flavivirus in the family Flaviviridae. Flaviviruses are small, enveloped viruses that have a single-stranded genomic RNA of positive polarity ϳ11 kb in length. This RNA serves as mRNA for translation of a large polyprotein (3,391 amino acids in case of DV2) at the rough endoplasmic reticulum. The viral polyprotein is co-and post-translationally processed into three structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The amino termini of prM, E, NS1, and NS4B are generated upon cleavage by the host signal peptidase in the lumen of the ER, whereas processing of most of the other NS proteins and the COOH terminus of the C protein is carried out by the viral two-component protease NS2B-3 in the cytoplasm of DV-infected cells (2-4). For cleavage of the COOH terminus of NS1 an unknown ER resident protease seems to be responsible (5). A Golgi-localized furin protease mediates cleavage of prM at a late state of infection to...

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Section: Discussionmentioning

confidence: 83%

Subcellular Localization and Membrane Topology of the Dengue Virus Type 2 Non-structural Protein 4B

Miller

Sparacio

Bartenschlager

2006

Journal of Biological Chemistry

238

289

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“…In addition to their role as structural proteins of the viral envelope, a transcriptional activator function has been ascribed to the PreS2 domain. Prerequisite for the activator function is the cytoplasmic orientation of the PreS2-domain, which can be observed in the case of LHBs [10][11][12] and of the Cterminally truncated MHBs proteins (MHBs t ). 13,14 The cytoplasmic PreS2 domain is a binding partner and activator of the protein kinase C (PKC).…”

Section: Introductionmentioning

confidence: 99%

Novel cell permeable motif derived from the PreS2-domain of hepatitis-B virus surface antigens

Oess

Hildt

2000

Gene Ther

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Efficient transfer of proteins or nucleic acids across cellular membranes is a major problem in cell biology. Recently the existence of a fusogenic sequence was predicted in the junction area of the PreS2-and S-domain of the hepatitis-B virus surface antigens. We have identified cell permeability as a novel property of the PreS2-domain. Cell permeability of PreS2 is not restricted to hepatocytes. PreS2 translocates in an energy-independent manner into cells and is evenly distributed over the cytosol. Detailed analysis revealed that cell-permeability is mediated by an amphipatic ␣-helix between amino acids 41 and 52 of PreS2. Destruction of this translocation motif (PreS2-TLM) abolishes cell permeability. PreS2-TLM per se can act as a shuttle for peptides and functional proteins (such as EGFP). This permits the highly

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“…In most of these cases, the topological diversity results in protein multifunctionality, suggesting regulatory mechanisms controlling translocational variations at the ER (3)(4)(5). One example of such a protein is the large L envelope protein of the hepatitis B virus (HBV), a polytopic membrane protein existing in a mixed topology (6)(7)(8).…”

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confidence: 99%

Chaperone action in the posttranslational topological reorientation of the hepatitis B virus large envelope protein: Implications for translocational regulation

Lambert

Prange

2003

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

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The large L envelope protein of the hepatitis B virus utilizes a new folding pathway to acquire a dual transmembrane topology in the endoplasmic reticulum (ER). The process involves cotranslational membrane integration and subsequent posttranslational translocation of its preS subdomain into the ER. Here, we demonstrate that the conformational and functional heterogeneity of L depends on the action of molecular chaperones. Using coimmunoprecipitation, we observed specific interactions between L and the cytosolic Hsc70, in conjunction with Hsp40, and between L and the ERresident BiP in mammalian cells. Complex formation between L and Hsc70 was abolished when preS translocation was artificially switched to a cotranslational mode, implicating Hsc70 to act as a preS holding and folding catalyst that controls partial preS posttranslocation. The functional role of Hsc70 in L topogenesis was confirmed through modulation of its in vivo activity by overexpressing its co-chaperones Hip and Bag-1. Overexpression of the Hsc70-stimulating molecule Hip led to increased entrapping of preS on the cytosolic ER face and hence to a decrease in preS posttranslocation, whereas the negative regulator Bag-1 had the opposite effects. Furthermore, Hip-mediated Hsc70 activation impaired virus production in hepatitis B virus-replicating hepatoma cells, likely due to the improper topological reorientation of L. Together, these results indicate that translocational regulation of protein topology by chaperones provides a means of generating structural and functional diversity. They also hint to the dynamic nature of the mammalian ER translocation machinery in handling co-and posttranslational substrates.I t is generally thought that all copies of a given membrane protein exist in a single orientation with respect to the membrane. However, certain proteins have been found to be expressed in two or more topological isoforms, with the heterogeneity apparently generated at the time of translocation at the endoplasmic reticulum (ER) membrane (1, 2). In most of these cases, the topological diversity results in protein multifunctionality, suggesting regulatory mechanisms controlling translocational variations at the ER (3-5). One example of such a protein is the large L envelope protein of the hepatitis B virus (HBV), a polytopic membrane protein existing in a mixed topology (6-8).On biogenesis, the HBV L protein, together with the structurally closely related middle M and small S envelope proteins, is expressed from a single ORF of the viral genome by differential translation initiation. As a consequence, the entire sequence of S is repeated at the C termini of M and L, which contain the additional preS2 domain or preS2 and preS1 domains, respectively (9). All three proteins are cotranslationally integrated into the ER membrane by the topogenic signals of the S region that also direct cotranslational translocation of the upstream preS2 region of M into the ER lumen (10, 11). In contrast, the preS2 plus preS1 (preS) domain of L fails to be transloc...

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A dramatic shift in the transmembrane topology of a viral envelope glycoprotein accompanies hepatitis B viral morphogenesis.

Cited by 141 publications

References 24 publications

Subcellular Localization and Membrane Topology of the Dengue Virus Type 2 Non-structural Protein 4B

Subcellular Localization and Membrane Topology of the Dengue Virus Type 2 Non-structural Protein 4B

Novel cell permeable motif derived from the PreS2-domain of hepatitis-B virus surface antigens

Chaperone action in the posttranslational topological reorientation of the hepatitis B virus large envelope protein: Implications for translocational regulation

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