Hepatitis C virus-encoded nonstructural protein NS4A has versatile functions in viral protein processing

Tanji, Yasunori; Hijikata, Makoto; Satoh, Shinya; Kaneko, Toshinobu; Shimotohno, Kunitada

doi:10.1128/jvi.69.3.1575-1581.1995

Cited by 225 publications

(133 citation statements)

References 23 publications

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“…4B). These results show concordance with those of previous studies (14,16,50). The sodium carbonate wash data indicated that NS4B integrated less efficiently in the microsomal membranes than our control protein, Lep (a known integral protein) (17), despite the clear ER localization of NS4B shown above by microscopy.…”

Section: Resultssupporting

confidence: 93%

Topology of the Membrane-Associated Hepatitis C Virus Protein NS4B

Lundin

Monné

Widell

et al. 2003

J Virol

182

220

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Hepatitis C virus (HCV) belongs to the Hepacivirus genus in the Flaviviridae family. Among the least known viral proteins in this family is the nonstructural protein NS4B, which has been suggested to be a part of the replication complex. Hydrophobicity plots indicate a common profile among the NS4B proteins from different members of the Flaviviridae family, suggesting a common function. In order to gain a deeper understanding of the nature of HCV NS4B, we have determined localization and topology of this protein by using recombinant HCV NS4B constructs. The protein localized to the endoplasmic reticulum (ER), but also induced a pattern of cytoplasmic foci positive for markers of the ER. Computer predictions of the membrane topology of NS4B suggested that it has four transmembrane segments. The N and C termini were anticipated to be localized in the cytoplasm, because they are processed by the cytoplasmic NS3 protein. By introducing glycosylation sites at various positions in HCV NS4B, we show that the C terminus is cytoplasmic and the loop around residue 161 is lumenal as predicted. Surprisingly, the N-terminal tail was translocated into the lumen in a considerable fraction of the NS4B molecules, most likely by a posttranslational process. Interestingly, NS4B proteins of the yellow fever and dengue viruses also have their N termini located in the ER lumen due to an N-terminal signal peptide not found in NS4B of HCV. A shared topology achieved in two different ways supports the notion of a common function for NS4B in Flaviviridae.

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Section: Resultssupporting

confidence: 93%

Topology of the Membrane-Associated Hepatitis C Virus Protein NS4B

Lundin

Monné

Widell

et al. 2003

J Virol

182

220

View full text Add to dashboard Cite

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“…However, the soluble protease/helicase NS3 protein associates with the membrane by interaction with NS4A, a cofactor of the protease domain of NS3. NS4A is a 54-amino acid cofactor for both serine protease and helicase activities of NS3, and its cofactor activity requires stable complex formation between NS3 and NS4A, an interaction that also stabilizes NS3 (Bartenschlager et al, 1995;Pang et al, 2002;Tanji et al, 1995). NS3 is found in association with ER or ER-like membranes when coexpressed with NS4A, but it is distributed diVusely throughout the cytoplasm and nucleus when expressed in the absence of NS4A (Wolk et al, 2000).…”

Section: A Viral Replication Associated With Membranes Derived From mentioning

confidence: 99%

Interactions Between Virus Proteins and Host Cell Membranes During the Viral Life Cycle

Villanueva

Rouillé

Dubuisson

2005

International Review of Cytology

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The structure and function of cells are critically dependent on membranes, which not only separate the interior of the cell from its environment but also define the internal compartments. It is therefore not surprising that the major steps of the life cycle of viruses of animals and plants also depend on cellular membranes. Indeed, interactions of viral proteins with host cell membranes are important for viruses to enter into host cells, replicate their genome, and produce progeny particles. To replicate its genome, a virus first needs to cross the plasma membrane. Some viruses can also modify intracellular membranes of host cells to create a compartment in which genome replication will take place. Finally, some viruses acquire an envelope, which is derived either from the plasma membrane or an internal membrane of the host cell. This paper reviews recent findings on the interactions of viral proteins with host cell membranes during the viral life cycle.

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“…For instance, this domain can unwind double-stranded RNA and DNA in vitro [36], but direct evidence is lacking that it binds to or unwinds viral RNA during the replication cycle in vivo. The small NS4A protein binds to NS3, acts as a cofactor for both NS3 activities [28,37], and anchors the NS3-4A heterodimer in the membrane [38]. NS4B is a polytopic membrane protein that can oligomerize and may serve as a scaffold for replicase assembly [39,40].…”

Section: Introductionmentioning

confidence: 99%

Hepatitis C Virus RNA Replication Depends on Specific Cis- and Trans-Acting Activities of Viral Nonstructural Proteins

et al. 2015

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Many positive-strand RNA viruses encode genes that can function in trans, whereas other genes are required in cis for genome replication. The mechanisms underlying trans- and cis-preferences are not fully understood. Here, we evaluate this concept for hepatitis C virus (HCV), an important cause of chronic liver disease and member of the Flaviviridae family. HCV encodes five nonstructural (NS) genes that are required for RNA replication. To date, only two of these genes, NS4B and NS5A, have been trans-complemented, leading to suggestions that other replicase genes work only in cis. We describe a new quantitative system to measure the cis- and trans-requirements for HCV NS gene function in RNA replication and identify several lethal mutations in the NS3, NS4A, NS4B, NS5A, and NS5B genes that can be complemented in trans, alone or in combination, by expressing the NS3–5B polyprotein from a synthetic mRNA. Although NS5B RNA binding and polymerase activities can be supplied in trans, NS5B protein expression was required in cis, indicating that NS5B has a cis-acting role in replicase assembly distinct from its known enzymatic activity. Furthermore, the RNA binding and NTPase activities of the NS3 helicase domain were required in cis, suggesting that these activities play an essential role in RNA template selection. A comprehensive complementation group analysis revealed functional linkages between NS3-4A and NS4B and between NS5B and the upstream NS3–5A genes. Finally, NS5B polymerase activity segregated with a daclatasvir-sensitive NS5A activity, which could explain the synergy of this antiviral compound with nucleoside analogs in patients. Together, these studies define several new aspects of HCV replicase structure-function, help to explain the potency of HCV-specific combination therapies, and provide an experimental framework for the study of cis- and trans-acting activities in positive-strand RNA virus replication more generally.

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Hepatitis C virus-encoded nonstructural protein NS4A has versatile functions in viral protein processing

Cited by 225 publications

References 23 publications

Topology of the Membrane-Associated Hepatitis C Virus Protein NS4B

Topology of the Membrane-Associated Hepatitis C Virus Protein NS4B

Interactions Between Virus Proteins and Host Cell Membranes During the Viral Life Cycle

Hepatitis C Virus RNA Replication Depends on Specific Cis- and Trans-Acting Activities of Viral Nonstructural Proteins

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