Hepatitis C virus NS3 serine proteinase: trans-cleavage requirements and processing kinetics

Abstract

The hepatitis C virus H strain (HCV-H) polyprotein is cleaved to produce at least 10 distinct products, in the order of NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NSSA-NS5B-COOH. An HCV-encoded serine proteinase activity in NS3 is required for cleavage at four sites in the nonstructural region (3/4A, 4A/4B, 4B/5A, and 5A/SB). In this report, the HCV-H serine proteinase domain (the N-terminal 181 residues of NS3) was tested for its ability to mediate trans-processing at these four sites. By using an NS3-SB substrate with an inactivated serine proteinase domain, trans-cleavage was observed at all sites except for the 3/4A site. Deletion of the inactive proteinase domain led to efficient trans-processing at the 3/4A site. Smaller NS4A-4B and NS5A-5B substrates were processed efficiently in trans; however, cleavage of an NS4B-5A substrate occurred only when the serine proteinase domain was coexpressed with NS4A. Only the N-terminal 35 amino acids of NS4A were required for this activity. Thus, while NS4A appears to be absolutely required for trans-cleavage at the 4B1/5A site, it is not an essential cofactor for serine proteinase activity. To begin to examine the conservation (or divergence) of serine proteinase-substrate interactions during HCV evolution, we demonstrated that similar trans-processing occurred when the proteinase domains and substrates were derived from two different HCV subtypes. These results are encouraging for the development of broadly effective HCV serine proteinase inhibitors as antiviral agents. Finally, the kinetics of processing in the nonstructural region was examined by pulse-chase analysis. NS3-containing precursors were absent, indicating that the 2/3 and 3/4A cleavages occur rapidly. In contrast, processing of the NS4A-5B region appeared to involve multiple pathways, and significant quantities of various polyprotein intermediates were observed. NS5B, the putative RNA polymerase, was found to be significantly less stable than the other mature cleavage products. This instability appeared to be an inherent property of NS5B and did not depend on expression of other viral polypeptides, including the

Continuous human cell lines inducibly expressing hepatitis C virus structural and nonstructural proteins

“…Whereas NS5B mRNA was easily detected by Northern blot analyses with use of a NS5B-specific probe (data not shown), we encountered difficulties in demonstrating the presence of NS5B protein in UHCV cells. This may be due to the instability of NS5B encoded by the pBRTM/HCV1-3011 prototype clone, 39 which differs from the recently determined HCV H consensus sequence by 7 amino acid substitutions. A clone reflecting this consensus sequence encodes a more stable NS5B cleavage product (Read CM, Rice CM, unpublished data, January 1997) and is also functional for initiating In the presence of tetracycline, 10 4 UHCV-11 cells per well were seeded into 6-well tissue culture plates.…”

Human and murine antibody recognition is focused on the ATPase/Helicase, but not the protease domain of the hepatitis C virus nonstructural 3 protein

“…8,10 The enzymatic activity of the NS3 protease is partly dependent on formation of a complex with NS4a. 9,[11][12][13][14][15] Chraracterization of the functional properties of the carboxy-terminal portion of NS3 of flaviviruses and HCV have revealed adenosine phosphatase (ATPase) and helicase activities that are involved in viral replication. 16,17 A portion of the NS3 sequence expressed as a fusion protein with superoxide dismutase (C33) appears to be serologically reactive during the early phase of HCV infections, and for this reason, C33 is routinely incorporated in clinical diagnostic HCV antibody immunoassays.…”

Hepatitis C virus NS4A inhibits cap-dependent and the viral IRES-mediated translation through interacting with eukaryotic elongation factor 1A

“…Further deletion analysis indicated that the central domain of NS4A from amino acid residues 21-34 [GST-NS4A(21-34)] is sufficient for the binding of eEF1A. In addition, NS4A (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) represents a second domain that, independent of NS4A (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34), is capable of interacting with eEF1A.…”

“…Nonstructural protein 4A (NS4A) is a multifunctional protein with 54 amino acid residues. It acts as a cofactor of NS3 serine protease and plays an essential role in the NS4A-dependent cleavage at the NS3-NS4A and NS4B-NS5A junctions [13,14]. Both NS4A and NS4B proteins were previously demonstrated to suppress translation in culture cells [15,16].…”