title: Mutations that affect dimer formation and helicase activity of the hepatitis C virus helicase.
abstract: Interaction between viral proteins is necessary for viral replication and viral particle assembly. We used the yeast two-hybrid assay to identify interactions among all the mature proteins of the hepatitis C virus. The interaction between NS3 and NS3 was one of the strongest viral protein-protein interactions detected. The minimal region required for this interaction was mapped to a specific subdomain of 174 amino acids in the N terminus of the helicase region. Random mutations in the minimal region were generated by PCR, and mutants that failed to interact with a wild-type minimal fragment were isolated using the yeast two-hybrid assay as a screen. Three of these mutations resulted in a reduction or a loss of interaction between helicases. Analytical gel filtration showed that in the presence of an oligonucleotide, wild-type helicases form dimers whereas the mutants remain mostly monomeric. All three mutants were partially or almost inactive when assayed for helicase activity in vitro. Mixing a mutant helicase (Y267S) with wild-type helicase did not dramatically affect helicase activity. These data indicate that dimerization of the helicase is important for helicase activity. The mutations that reduce self-association of the helicase may define the key residues involved in NS3-NS3 dimerization.
authors: Collaborative Anti-Viral Research Group, Institute of Molecular and Cell Biology, Singapore 117609.
authors: Y L
authors: YL
authors: Khu
authors: E
authors: E
authors: Koh
authors: S P
authors: SP
authors: Lim
authors: Y H
authors: YH
authors: Tan
authors: S
authors: S
authors: Brenner
authors: S G
authors: SG
authors: Lim
authors: W J
authors: WJ
authors: Hong
authors: P Y
authors: PY
authors: Goh
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D019281:Dimerization; D008969:Molecular Sequence Data; D009154:Mutation; D020365:RNA Helicases / Q000737:chemistry* / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases
chemical_list: D020365:RNA Helicases; D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases