CONSTRUCTION OF HEPATITIS C-SIN VIRUS RECOMBINANTS WITH REPLICATIVE DEPENDENCY ON HEPATITIS-C VIRUS SERINE-PROTEASE ACTIVITY

An in vivo assay system was developed for the serine protease of hepat itis C virus (HCV) using the sindbis (SIN) viral replication system in which HCV serine protease activity is essential for the replication o f the HCV-SIN chimeric virus. Two chimeric viral cDNA clones were cons tructed by inserting the NS3/4A region and NS3/4A region with the puta tive helicase deleted, into the N-terminal region of SIN core protein. The constructs were named Tpro CT and Tpro T, respectively. BHK-21 ce lls transfected with the in vitro transcribed RNAs from Tpro CT and Tp ro T showed specific cytopathic morphology and produced chimeric virus es, Vpro CT and Vpro T. In contrast, in vitro transcribed RNAs from Tp ro CTI and Tpro TI, in which serine of catalytic triad of HCV protease was changed to alanine, were not infectious. When the chimeric viruse s were passaged in BHK-21 cells at about 0.1 multiplicity of infection (MOI), Vpro T, but not Vpro CT, stably expressed HCV protease for up to five passages. Surprisingly, the cell culture media of BHK-21 cells infected with Vpro T, compared to wild-type sindbis virus, showed rap id pH changes by more than 0.8 pH degree at 72 h post-infection. HCV-S IN hybrid viruses could be used in screening the HCV protease-inhibito r in cell culture systems. (C) 1997 Elsevier Science B.V.