Mutations that affect dimer formation and helicase activity of the hepatitis C virus helicase

Interaction between viral proteins is necessary for viral replication and v iral particle assembly. We used the yeast two-hybrid assay to identify inte ractions among all the mature proteins of the hepatitis C virus. The intera ction between NS3 and NS3 was one of the strongest viral protein-protein in teractions detected. The minimal region required for this interaction was m apped to a specific subdomain of 174 amino acids in the N terminus of the h elicase region. Random mutations in the minimal region were generated by PC R, and mutants that failed to interact with a wild-type minimal fragment we re isolated using the yeast two-hybrid assay as a screen. Three of these mu tations resulted in a reduction or a loss of interaction between helicases. Analytical gel filtration showed that in the presence of an oligonucleotid e, wild-type helicases form dimers whereas the mutants re main mostly monom eric. 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 indicat e 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.