Substrate Specificity of the Hepatitis C Virus Serine Protease NS3

Abstract

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...

Evasive Maneuvers by Hepatitits C Virus

“…NS3-4A is therefore thought to bind its substrate via an extensive network of weak interactions, spanning over 10 substrate residues. 20,21 These observations posed a challenge to the development of traditional competitive substrate inhibitors. A breakthrough in this arena was the unusual observation that N-terminal cleavage products (i.e., the nonprime side) bind NS3-4A and competitively block substrate recognition.…”

In Vitro Selection and Characterization of Hepatitis C Virus Serine Protease Variants Resistant to an Active-Site Peptide Inhibitor

“…Both domains can be expressed in isolation as fully active and stably folded proteins. In line with functional studies, the X-ray structure of the full-length NS3 protein showed that the protease and helicase domains are segregated and connected by a single strand (52).NS3-4Ap specificity has been defined by identification (17, 44) and mutagenesis (5,23,49,53) of the natural cleavage sites and selection of optimized cleavage sites using peptide libraries (21, 41). The NS3/NS4A junction is cleaved in cis and tolerates substitutions at all positions except P1, where a threonine residue is found in all isolates.…”

High Affinity Peptide Inhibitors of the Hepatitis C Virus NS3-4A Protease Refractory to Common Resistant Mutants

“…S4C) resulting in a more remote positioning of the scissile bond. Similarly, the adjacent Leu-10, which binds to the subsite S1Ј, moves the peptide backbone away from the active site, although the S1Ј pocket was shown to bind larger side chains than the natural P1Ј serine (29). Additionally, Leu-10 causes dislocation of a nearby NS3-4A loop (Thr-38 -Gln-41) (Fig.…”