title: NS3-4A of hepatitis C virus is a chymotrypsin-like protease.
abstract: The polyprotein encoded by a single open reading frame of hepatitis C virus (HCV) is processed by host- and virus-encoded proteases. The viral protease NS3 is responsible for the cleavage of at least four sites (NS3/4A, NS4A/4B, NS4B/5A, and NS5A/5B junctions) in the nonstructural protein region. To characterize the protease function of NS3 and NS4 on various target sites, efficient cis- and trans-cleavage assay systems were developed by using in vitro transcription and translation. Deletion of the C-terminal two-thirds from NS3 in an NS3-NS4A-4B polypeptide (NS3 delta C-4A-4B) hampered cleavage of the NS3/4A junction but not that of the NS4A/4B junction. As a consequence, expression of NS3 delta C-4A-4B containing an internal deletion of NS3 results in an NS3 delta C-4A fusion protein. NS3 delta C-4A shows very efficient and specific trans-cleavage activity at NS4A/4B, NS4B/5A, and NS5A/5B junctions. In addition, the biochemical properties of HCV NS3 delta C-4A were further elucidated by adding known protease inhibitors in trans-cleavage reactions. The HCV protease NS3-4A is inhibited by chymotrypsin-specific inhibitors N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), chymostatin, and Pefabloc SC but not by trypsin-like protease inhibitors antipain, leupeptin, and N-alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK) or by the protease inhibitors E-64, bestatin, pepstatin, and phosphoramidon. This finding strongly suggests that HCV protease NS3-4A is a chymotrypsin-like serine protease.
authors.affiliation: Department of Life Science, Pohang University of Science and Technology, Kyungbuk.
authors.forename: B
authors.initials: B
authors.lastname: Hahm
authors.forename: D S
authors.initials: DS
authors.lastname: Han
authors.forename: S H
authors.initials: SH
authors.lastname: Back
authors.forename: O K
authors.initials: OK
authors.lastname: Song
authors.forename: M J
authors.initials: MJ
authors.lastname: Cho
authors.forename: C J
authors.initials: CJ
authors.lastname: Kim
authors.forename: K
authors.initials: K
authors.lastname: Shimotohno
authors.forename: S K
authors.initials: SK
authors.lastname: Jang
mesh_terms: D001483:Base Sequence; D002918:Chymotrypsin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004265:DNA Helicases / Q000235:genetics; D006526:Hepatitis C / Q000201:enzymology*; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D017384:Sequence Deletion; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D002918:Chymotrypsin; D004265:DNA Helicases; D009838:Oligodeoxyribonucleotides; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases