Reconstitution of hepatitis C virus protease activities in yeast

The hepatitis C virus (HCV) protease genes (NS2/3 and NS3) were expressed i n yeast with their natural substrates fused to a ligand-dependent transcrip tional activator, the retinoic acid receptor (RAR beta). RAR beta can activ ate transcription in yeast cells in response to retinoic acids. We hypothes ized that cis-cleavage at the NS2-3 or NS3-4A junctions by the appropriate HCV proteases would release RAR beta, thereby activating transcription of a reporter gene. Our results from Western blot analyses and reporter gene ac tivation indicate that the wild-type NS2/3 and NS3 enzymes are catalyticall y active in yeast cells, whereas mutations in the catalytic domain of NS2(C 993V) and NS3(S 1165A) lead to inactive enzymes. We conclude that HCV NS2/3 and NS3 protease activities can be reconstituted in yeast. (C) 2001 Federa tion of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.