title: Hepatitis C NS3 protease: restoration of NS4A cofactor activity by N-biotinylation of mutated NS4A using synthetic peptides.
abstract: The NS3 serine protase of Hepatitis C virus (HCV) requires NS4A protein as a cofactor for efficient cleavage at four sites in the nonstructural region. The cofactor activity has been mapped to the central hydrophobic region (aa 22-34) of this 54-amino-acid NS4A protein, and site-directed mutagenesis has identified alternating hydrophobic amino acids, particularly Ile25 and Ile29, as critically important. A double mutant of NS4A cofactor peptide, I25A/I29A, completely abolished the cofactor activity. We now report that the cofactor peptide activity in the I25A/I29A double mutant can be restored specifically by introducing a biotin-aminohexanoic acid fusion at the N-terminus. In addition, a similar N-terminal fusion of biotin-aminohexanoic acid with the wild-type 4A peptide significantly enhanced cofactor activity. Our data corroborate the crystal structure-based hypothesis of hydrophobic interaction between the N-terminus of NS4A and the N-terminal alpha(0) helix of NS3 protease.
authors.affiliation: Department of Antiviral Therapy, Schering-Plough Research Institute, Kenilworth, New Jersey, 07033, USA.
authors.forename: N J
authors.initials: NJ
authors.lastname: Butkiewicz
authors.forename: N
authors.initials: N
authors.lastname: Yao
authors.forename: J
authors.initials: J
authors.lastname: Wright-Minogue
authors.forename: R
authors.initials: R
authors.lastname: Zhang
authors.forename: L
authors.initials: L
authors.lastname: Ramanathan
authors.forename: J Y
authors.initials: JY
authors.lastname: Lau
authors.forename: Z
authors.initials: Z
authors.lastname: Hong
authors.forename: B
authors.initials: B
authors.lastname: Dasmahapatra
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D019921:Biotinylation; D003001:Cloning, Molecular; D016174:Hepacivirus / Q000201:enzymology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases