Substrate Specificity of the NS3 Serine Proteinase of Hepatitis C Virus as Determined by Mutagenesis at the NS3/NS4A Junction

Leinbach, Susan S.; Bhat, Ramesh A.; Xia, Shi-Mao; Hum, Wah-Tung; Stauffer, Barbara; Davis, Alan R.; Hung, Paul P.; Mizutani, Satoshi

doi:10.1006/viro.1994.1520

Cited by 30 publications

(26 citation statements)

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“…The NS3 serine proteinase has characteristics both similar to and different from the trypsin family as follows: it was proved through mutational analyses that a reactive Ser residue and two additional amino acids (Asp and His) are essential for cleavage at NS3/4A, NS4A/4B, NS4B/NS5A and NS5A/NS5B (Bartenschlager et al, 1993;Eckart et al, 1993;Grakoui et al, 1993 ;Hijikata et al, 1993;Tomei et al, 1993); the optimum pH of NS3 proteinase is neutral to slightly basic as presented here, which is similar to that of wellcharacterized serine proteases such as trypsin, chymotrypsin or thrombin; as for the substrate specificity, it was proposed that amino acid requirements for the P1 position are restrictive and that Cys and Ser residues are ideal at that position for the substrate (Grakoui et al, 1993;Leinbach et al, 1994;Pizzi et al, 1994); cellular serine proteinases, however, cleave mainly after basic residues and these amino acids are usually found at the P1 and P2 positions for the substrates of flaviviral serine proteinases (Chambers et al, 1990;Rice & Strauss, 1990).…”

Section: Discussionmentioning

confidence: 64%

“…The ideal residues at the P1 position of the cleavage site were proposed from the configuration of the pocket (Pizzi et al, 1994). The P1 position of the polyprotein substrate was suggested to be more important in defining the specificity than the P6 or P1 position whose amino acid sequences are well-conserved among different HCV strains reported (Leinbach et al, 1994).…”

Section: Introductionmentioning

confidence: 99%

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In vivo and in vitro trans-cleavage activity of hepatitis C virus serine proteinase expressed by recombinant baculoviruses

Suzuki¹,

Sato²,

Chieda³

et al. 1995

Journal of General Virology

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By the use of recombinant baculoviruses, the transcleavage of hepatitis C virus (HCV) non-structural polyprotein was studied. The viral serine proteinase encoded by the NS3 gene was expressed efficiently in insect cells infected with a baculovirus recombined with HCV cDNA corresponding to amino acids 1046-1243 and the signal sequence of the rabies virus G protein.Coinfection studies showed the in vivo trans-cleavage activity of the expressed protein by the use of a recombinant producing NS5 as a substrate. We also fou ad that the partially purified NS3 serine proteinase prepared from the recombinant-infected cells could cleave NS5A/5B substrate. Characterization of the proteinase obtained will provide basic knowledge on processing of the HCV polyprotein.

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Section: Discussionmentioning

confidence: 64%

Section: Introductionmentioning

confidence: 99%

In vivo and in vitro trans-cleavage activity of hepatitis C virus serine proteinase expressed by recombinant baculoviruses

Suzuki¹,

Sato²,

Chieda³

et al. 1995

Journal of General Virology

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“…Substrate specificity of the NS3 protease has been investigated by several groups in a qualitative way using transient transfection (22,23), in vitro translation (24), or intracellular processing of fusion proteins in Escherichia coli (25). Availability of quantitative data using peptidic substrates has been so far hampered by difficulties in expressing and purifying sufficient amounts of enzymatically active recombinant NS3 protease.…”

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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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“…We tried to found the most recent and accurate samples to build the prediction model. We used the last accurate datasets used [10][11][12][13][14][15][16][17][18] in previous work. The collected datasets are divided into two parts:…”

Section: Data Collectionmentioning

confidence: 99%

A Data Mining Approach for the Prediction of Hepatitis C Virus protease Cleavage Sites

eldin¹

2011

IJACSA

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Abstract-Summary: Several papers have been published about the prediction of hepatitis C virus (HCV) polyprotein cleavage sites, using symbolic and non-symbolic machine learning techniques. The published papers achieved different Levels of prediction accuracy. the achieved results depends on the used technique and the availability of adequate and accurate HCV polyprotein sequences with known cleavage sites. We tried here to achieve more accurate prediction results, and more Informative knowledge about the HCV protein cleavage sites using Decision tree algorithm. There are several factors that can affect the overall prediction accuracy. One of the most important factors is the availably of acceptable and accurate HCV polyproteins sequences with known cleavage sites. We collected latest accurate data sets to build the prediction model. Also we collected another dataset for the model testing. Results: The ease to use and to understand of the decision tree enabled us to create simple prediction model. We used here the latest accurate viral datasets. Decision tree achieved here acceptable prediction accuracy results. Also it generated informative knowledge about the cleavage process itself. These results can help the researchers in the development of effective viral inhibitors. Using decision tree to predict HCV protein cleavage sites achieved high prediction accuracy.

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Substrate Specificity of the NS3 Serine Proteinase of Hepatitis C Virus as Determined by Mutagenesis at the NS3/NS4A Junction

Cited by 30 publications

References 0 publications

In vivo and in vitro trans-cleavage activity of hepatitis C virus serine proteinase expressed by recombinant baculoviruses

In vivo and in vitro trans-cleavage activity of hepatitis C virus serine proteinase expressed by recombinant baculoviruses

Substrate Specificity of the Hepatitis C Virus Serine Protease NS3

A Data Mining Approach for the Prediction of Hepatitis C Virus protease Cleavage Sites

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