Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus.

Hijikata, Makoto; Mizushima, Hiroto; Tanji, Yasunori; Komoda, Yoshiyuki; Hirowatari, Yuji; Akagi, Tsuyoshi; Kato, Naoya; Kimura, Kôichi; Shimotohno, Kunitada

doi:10.1073/pnas.90.22.10773

Cited by 322 publications

(265 citation statements)

References 27 publications

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“…In the absence of a satisfactory cell culture system for propagating the virus, studies of its mode of gene expression have depended upon the use of heterologous systems for protein expression, including in vitro translation, transient expression in mammalian cells and production of recombinant vaccinia viruses. By these methods, it has been established that the NS3 region of the polyprotein contains a serine proteinase domain which is responsible for cleavage of the downstream polyprotein in at least four places (Bartenschlager et al, 1993(Bartenschlager et al, , 1994Eckart et al, 1993;Grakoui et al, 1993;Hijikata et al, 1993;Tomei et al, 1993 ;Manabe et al, 1994). By analogy with the yellow fever virus (Chambers et al, 1990) this proteinase is probably essential for the production of infectious virus, and hence represents a possible target for chemotherapeutic intervention.…”

Section: Introductionmentioning

confidence: 99%

Recombinant baculovirus-expressed NS3 proteinase of hepatitis C virus shows activity in cell-based and in vitro assays

Overton

McMillan

Gillespie

et al. 1995

Journal of General Virology

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Recombinant baculoviruses have been constructedwhich express the hepatitis C virus (HCV) NS3 proteinase and its substrates in insect cells. The expressed proteinase has been shown to carry out transcleavage at the NS3/4A, NS4A/4B, NS4B/5A and NS5A/5B junctions in a cell-based assay. When assayed in a cell-free system using in vitro translated substrates, the proteinase could perform trans-processing of the NS4A/4B and NS5A/5B junctions, but only when coexpressed with NS4A, either as an NS3-4A precursor or by co-infection of cells with NS3-and NS4A-expressing recombinant baculoviruses. Possible reasons for the absolute requirement of the NS3 proteinase for NS4A in vitro are discussed.

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Section: Introductionmentioning

confidence: 99%

Recombinant baculovirus-expressed NS3 proteinase of hepatitis C virus shows activity in cell-based and in vitro assays

Overton

McMillan

Gillespie

et al. 1995

Journal of General Virology

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“…The N-terminal third of the hepatitis C virus (HCV) 1 NS3 protein contains a trypsin-like serine protease that accomplishes four out of the five processing events that take place during maturation of the nonstructural portion of the HCV polyprotein, performing cleavages at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions (1)(2)(3)(4)(5)(6). It has been shown that cleavage between NS3 and NS4A is an intramolecular event, whereas all remaining junctions are processed in trans.…”

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confidence: 99%

Substrate Specificity of the Hepatitis C Virus Serine Protease NS3

Urbani¹,

Bianchi²,

Narjes³

et al. 1997

Journal of Biological Chemistry

110

122

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The substrate specificity of a purified protein encompassing the hepatitis C virus NS3 serine protease domain was investigated by introducing systematic modifications, including non-natural amino acids, into substrate peptides derived from the NS4A/NS4B cleavage site. Kinetic parameters were determined in the absence and presence of a peptide mimicking the protease co-factor NS4A (Pep4A). Based on this study we draw the following conclusions: (i) the NS3 protease domain has an absolute requirement for a small residue in the P1 position of substrates, thereby confirming previous modelling predictions. (ii) Optimization of the P1 binding site occupancy primarily influences transition state binding, whereas the occupancy of distal binding sites is a determinant for both ground state and transition state binding. (iii) Optimized contacts at distal binding sites may contribute synergistically to cleavage efficiency.The N-terminal third of the hepatitis C virus (HCV) 1 NS3 protein contains a trypsin-like serine protease that accomplishes four out of the five processing events that take place during maturation of the nonstructural portion of the HCV polyprotein, performing cleavages at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B junctions (1-6). It has been shown that cleavage between NS3 and NS4A is an intramolecular event, whereas all remaining junctions are processed in trans.In vivo, NS3 appears to form a heterodimer whereby the protease domain associates with the viral protein NS4A. The latter is a 54-residue protein that has been shown to bind to the N-terminal region of the protease via a central hydrophobic domain spanning residues 21-34 (7-13). NS4A acts as a cofactor of the protease enhancing cleavage at all sites and being an absolute requirement for processing of the NS4B/NS5A junction ex vivo (7). Several studies have shown that a peptide encompassing the central hydrophobic domain of NS4A is sufficient for eliciting activation of the protease (12, 14 -17).Serine proteases contact the P1 residue of their substrates through characteristic specificity pockets. The residues flanking the specificity pocket are important determinants of substrate recognition. Homology modelling of the S1 specificity pocket of the NS3 protease has predicted the presence of a phenylalanine as a prominent feature, thus rendering the pocket rather small and hydrophobic (18). These characteristics have led to the prediction of the preference for small, hydrophobic residues, ideally cysteine residues, in the P1 position of NS3 substrates. Radiosequencing of the single cleavage products has subsequently confirmed these predictions, yielding the consensus sequence (D/E)XXXXC(A/S) for all trans cleavage sites, with X being any amino acid and the scissile bond being located between Cys and Ala or Ser (2, 18). The homology model has been used to successfully redesign the enzyme's specificity, thereby increasing its validity. Very recentyl the three-dimensional structure of the protease has been solved by two different groups (20, 21...

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“…Previous studies have shown that NS4A, besides stabilizing NS3, is responsible for the localization of the NS3‐4A complex at the organelle's membranes 21, 30, 31. Tanji et al .…”

Section: Resultsmentioning

confidence: 99%

Hepatitis C virus NS3‐4A inhibits the peroxisomal MAVS‐dependent antiviral signalling response

Ferreira

Magalhães

Camões

et al. 2016

J Cellular Molecular Medi

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Hepatitis C virus (HCV) is the cause of one of the most prevalent viral infections worldwide. Upon infection, the HCV genome activates the RIG‐I‐MAVS signalling pathway leading to the production of direct antiviral effectors which prevent important steps in viral propagation. MAVS localizes at peroxisomes and mitochondria and coordinate the activation of an effective antiviral response: peroxisomal MAVS is responsible for a rapid but short‐termed antiviral response, while the mitochondrial MAVS is associated with the activation of a stable response with delayed kinetics.The HCV NS3‐4A protease was shown to specifically cleave the mitochondrial MAVS, inhibiting the downstream response.In this study, we have analysed whether HCV NS3‐4A is also able to cleave the peroxisomal MAVS and whether this would have any effect on the cellular antiviral response. We show that NS3‐4A is indeed able to specifically cleave this protein and release it into the cytosol, a mechanism that seems to occur at a similar kinetic rate as the cleavage of the mitochondrial MAVS. Under these conditions, RIG‐I‐like receptor (RLR) signalling from peroxisomes is blocked and antiviral gene expression is inhibited. Our results also show that NS3‐4A is able to localize at peroxisomes in the absence of MAVS. However, mutation studies have shown that this localization pattern is preferred in the presence of a fully cleavable MAVS. These findings present evidence of a viral evasion strategy that disrupts RLR signalling on peroxisomes and provide an excellent example of how a single viral evasion strategy can block innate immune signalling from different organelles.

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Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus.

Cited by 322 publications

References 27 publications

Recombinant baculovirus-expressed NS3 proteinase of hepatitis C virus shows activity in cell-based and in vitro assays

Recombinant baculovirus-expressed NS3 proteinase of hepatitis C virus shows activity in cell-based and in vitro assays

Substrate Specificity of the Hepatitis C Virus Serine Protease NS3

Hepatitis C virus NS3‐4A inhibits the peroxisomal MAVS‐dependent antiviral signalling response

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