title: Use of the fused NS4A peptide-NS3 protease domain to study the importance of the helicase domain for protease inhibitor binding to hepatitis C virus NS3-NS4A.
abstract: The NS3 protein of hepatitis C virus is unusual because it encodes two unrelated enzymatic activities in linked protease and helicase domains. It has also been intensively studied because inhibitors targeting its protease domain have potential to significantly improve treatment options for those infected with this virus. Many enzymological studies and inhibitor discovery programs have been carried out using the isolated protease domain in complex with a peptide derived from NS4A which stimulates activity. However, some recent publications have suggested that the NS3 helicase domain may influence inhibitor binding and thus suggest work should focus on the full-length NS3-NS4A protein. Here we present the characterization of a single-chain protease in which the NS4A peptide activator is linked to the N-terminus of the NS3 protease domain. This protein behaves well in solution, and its protease activity is very similar to that of full-length NS3-NS4A. We find that this fusion protein, as well as the noncovalent complex of the NS4A peptide with NS3, gives similar Ki values, spanning 3 orders of magnitude, for a set of 25 structurally diverse inhibitors. We also show that simultaneous mutation of three residues on the surface of the helicase domain which has been hypothesized to interact with the protease does not significantly affect enzymatic activity or inhibitor binding. Thus, the protease domain with the NS4A peptide, in a covalent or noncovalent complex, is a good model for the protease activity of native NS3-NS4A.
authors.affiliation: Boehringer Ingelheim (Canada) Ltd., 2100 Cunard Street, Laval, QC, Canada H7S 2G5.
authors.forename: Diane
authors.initials: D
authors.lastname: Thibeault
authors.forename: Marie-Josée
authors.initials: MJ
authors.lastname: Massariol
authors.forename: Songping
authors.initials: S
authors.lastname: Zhao
authors.forename: Ewald
authors.initials: E
authors.lastname: Welchner
authors.forename: Nathalie
authors.initials: N
authors.lastname: Goudreau
authors.forename: Rock
authors.initials: R
authors.lastname: Gingras
authors.forename: Montse
authors.initials: M
authors.lastname: Llinàs-Brunet
authors.forename: Peter W
authors.initials: PW
authors.lastname: White
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018360:Crystallography, X-Ray; D019281:Dimerization; D004789:Enzyme Activation / Q000235:genetics; D016174:Hepacivirus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D047908:Intracellular Signaling Peptides and Proteins; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D020365:RNA Helicases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D011480:Protease Inhibitors; D020365:RNA Helicases; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D017361:Viral Nonstructural Proteins; D014764:Viral Proteins; C084422:NS3 protein, hepatitis C virus; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C455041:NS4A cofactor peptide, Hepatitis C virus