Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions

Bartenschlager, Ralf; Ahlborn-Laake, L; Mous, Jan; Jacobsen, Helmut

doi:10.1128/jvi.67.7.3835-3844.1993

Cited by 418 publications

(185 citation statements)

References 34 publications

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“…Given that the NS3-4A-4B complementation grouping was reciprocal, it was surprising that 3m6-and 4Bm1-mutant replicons replicated well on cells expressing the 4Am1-mutant form of NS3-5B. Furthermore, it was surprising that NS3 and NS4A activities segregated independently given that proteolytic processing of the NS3/NS4A junction is autocatalytic [121] and is predicted to lead to cis-preferential interaction between NS3 and NS4A [122]. One possible explanation is that NS3-4A dimerizes, such that one monomer provides NS3 activity while the other provides NS4A activity ( Fig 8H).…”

Section: The Cis and Trans Activities Of Ns5bmentioning

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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Section: The Cis and Trans Activities Of Ns5bmentioning

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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“…It was based on the sequence of NS4B genotype 1a (pCV/H77C), which was analyzed with the programs TMHMM (24), HMMTOP (51), and MEMSAT (18). Since the NS3 protease, which is responsible for processing the polyprotein in the nonstructural region, is located on the cytoplasmic side of the ER membrane (1,12,14,53,54), we further assumed that both the N terminal and the C terminal of the protein were cytoplasmic.…”

Section: Prediction Of the Topology Of Ns4b In The Membranementioning

confidence: 99%

Topology of the Membrane-Associated Hepatitis C Virus Protein NS4B

Lundin

Monné

Widell

et al. 2003

J Virol

183

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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“…Core to NS2 is primarily involved in the formation of infectious virus (see chapter "Virion Assembly and Release", this volume), whereas the nonstructural proteins NS3 to NS5B are primarily involved in viral RNA replication, forming the viral replication complex ), which will be the focus of this chapter. NS3 is a multifunctional protein, consisting of an aminoterminal protease domain required for processing of the NS3 to NS5B region (Bartenschlager et al 1993) and a carboxyterminal helicase/nucleoside triphosphatase domain (Suzich et al 1993;Kim et al 1995). NS4A is a cofactor that activates the NS3 protease function by forming a heterodimer (Bartenschlager et al 1995).…”

Section: Introductionmentioning

confidence: 99%

Hepatitis C Virus RNA Replication

Lohmann

2013

Current Topics in Microbiology and Immunology

113

133

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Genome replication is a crucial step in the life cycle of any virus. HCV is a positive strand RNA virus and requires a set of nonstructural proteins (NS3, 4A, 4B, 5A, and 5B) as well as cis-acting replication elements at the genome termini for amplification of the viral RNA. All nonstructural proteins are tightly associated with membranes derived from the endoplasmic reticulum and induce vesicular membrane alterations designated the membranous web, harboring the viral replication sites. The viral RNA-dependent RNA polymerase NS5B is the key enzyme of RNA synthesis. Structural, biochemical, and reverse genetic studies have revealed important insights into the mode of action of NS5B and the mechanism governing RNA replication. Although a comprehensive understanding of the regulation of RNA synthesis is still missing, a number of important viral and host determinants have been defined. This chapter summarizes our current knowledge on the role of viral and host cell proteins as well as cis-acting replication elements involved in the biogenesis of the membranous web and in viral RNA synthesis.

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Nonstructural protein 3 of the hepatitis C virus encodes a serine-type proteinase required for cleavage at the NS3/4 and NS4/5 junctions

Cited by 418 publications

References 34 publications

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

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

Topology of the Membrane-Associated Hepatitis C Virus Protein NS4B

Hepatitis C Virus RNA Replication

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