Molecular model of the specificity pocket of the hepatitis C virus protease: implications for substrate recognition.

Abstract

We have built a model of the specificity pocket of the protease of hepatitis C vfrus on the basis of the known structures of trypsin-like serine proteases and of the conservation pattern of the protease sequences among various hepatitis C strains. The model allowed us to predict that the substrate of this proteas should have a cysteine residue in position P1. This hypothesis was subsequently proved by N-terminal sequencing of two products of the protease. The success of this "blind" test increases our confidence in the overall correctness of our proposed alment of the enzyme sequence with those of other proteases of known ure and constitutes a first step in the construction of a complete model of the viral protease domain.Hepatitis C virus (HCV) is the major cause of parentally transmitted non-A non-B hepatitis and is also implicated as the cause in many sporadic cases ofnon-A non-B hepatitis (1, 2). Chronic infection with HCV has also been linked to the development of hepatocellular carcinoma (3-5).HCV is an enveloped virus with a positive-strand RNA genome of -9.5 kb (6-13). This virus is probably related to flaviviruses and pestiviruses in view of the similarity of the deduced amino acid sequences and hydropathy profile of the viral proteins (14). As for pestiviruses and flaviviruses, the HCV genomic RNA includes a single open reading frame encoding a precursor polyprotein that is cleaved co-or posttranslationally into mature viral polypeptides. The putative viral structural proteins are encoded by the 5' quarter of the genome, whereas the remaining part encodes the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) that are believed to be components of the viral replication apparatus (15).The gene order in the HCV genome has been shown to be the following: 5'-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-3' (16,17 (14,25). Consistent with this prediction, when the proposed catalytic Ser-1165 of the polyprotein was mutated into an Ala, processing at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B sites was abolished (16, 21). Interestingly, cleavage at three of the four sites of the mutated polyprotein could be restored by supplying a functional NS3 in trans, while processing at the NS3-NS4A only occurred in cis (16). This finding indicates that the cleavage of the latter site is an intramolecular event.The comparative analysis of the three-dimensional structure of serine proteases obtained so far, with or without bound inhibitors, has shed light on the way diverse cleavage specificities are generated by the substrate binding sites. Structural information on the protease domain of HCV NS3, and in particular on the architecture of its substrate binding pocket, would be extremely helpful in making predictions on the specificity of the enzyme, as well as in designing structure-based inhibitors that could be used as therapeutic agents for treatment of HCV infections. To this end, we decided to undertake the modeling of the threedimensional structure of the enzyme by taking advantage of the wealth of st...

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

“…Cells coinfected with AcRV3440 and Ac6690 were separated on a 7'5 % SDSpolyacrylamide gel and transferred to a PVDF membrane, then the Coomassie blue-stained band corresponding to the processed NS5BAC protein was subjected to protein sequencing. The N-terminal sequence obtained was in good agreement with those previously reported (Grakoui et al, 1993;Pizzi et al, 1994) i.e., the N terminus of the processed NS5BAC protein was mapped at residue 2421 (Fig. 3 b, in parentheses).…”

“…All the H C V strains heretofore molecularly cloned have this structure in common in their NS3 region (Grakoui et al, 1993;Hijikata et al, 1993;Miller & Purcell, 1990;Pizzi et al, 1994). Many studies of cell-free transcription/translation of HCV cDNAs and their expression in cultured cells support this (Bartenschlager et al, 1993;Eckart et al, 1993;Grakoui et al, 1993;Hijikata et al, 1993;Tomei et al, 1993).…”

“…A molecular model of substrate binding pocket of the NS3 serine proteinase was built on the basis of the comparative analysis of the structures of other known serine proteinases and the NS3 of different HCV strains. 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).…”

Substrate Specificity of the Hepatitis C Virus Serine Protease NS3

“…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.…”

“…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.…”