KINETIC AND STRUCTURAL-ANALYSES OF HEPATITIS-C VIRUS POLYPROTEIN PROCESSING

Recombinant vaccinia viruses were used to study the processing of hepa titis C virus (HCV) nonstructural polyprotein precursor. HCV-specific proteins and cleavage products were identified by size and by immuno-p recipitation with region-specific antisera. A polyprotein beginning wi th 20 amino acids derived from the carboxy terminus of NS2 and ending with the NS5B stop codon (amino acids 1007 to 3011) was cleaved at the NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B sites, whereas a polyprotein in which the putative active site serine residue was replaced by an alan ine remained unprocessed, demonstrating that the NS3-encoded serine-ty pe proteinase is essential for cleavage at these sites. Processing of the NS3'-5B polyprotein was complex and occurred rapidly. Discrete pol ypeptide species corresponding to various processing intermediates wer e detected, With the exception of NS4AB-5A/NS5A, no clear precursor-pr oduct relationships were detected. Using double infection of cells wit h vaccinia virus recombinants expressing either a proteolytically inac tive NS3'-5B polyprotein or an active NS3 proteinase, we found that cl eavage at the NS4A/4B, NS4B/5A, and NS5A/5B sites could be mediated in trans. Absence of trans cleavage at the NS3/4A junction together with the finding that processing at this site was insensitive to dilution of the enzyme suggested that cleavage at this site is an intramolecula r reaction. The trans-cleavage assay was also used to show that (i) th e first 211 amino acids of NS3 were sufficient for processing at all t rans sites and (ii) small deletions from the amino terminus of NS3 sel ectively affected cleavage at the NS4B/5A site, whereas more extensive deletions also decreased processing efficiencies at the other sites. Using a series of amino-terminally truncated substrate polyproteins in the trans-cleavage assay, we found that NS4A is essential for cleavag e at the NS4B/5A site and that processing at this site could be restor ed by NS4A provided in cis (i.e., together with the substrate) or in t rans (i.e., together with the proteinase). These results suggest that in addition to the NS3 proteinase, NS4A sequences play an important ro le in HCV polyprotein processing.