Research on Hepatitis C (HC) has followed several lines. Following cloning and sequencing the genome of the H strain of HCV, we have constructed a putative full length cDNA clone of the genome. We have evaluated that clone in cell culture systems as well as by direct transfection of RNA transcripts of this clone into chimpanzee liver. To date this clone has not proved to be infectious and we are in the process of modifying it with the hopes of having an infectious clone. We have continued the analysis of HCV polyprotein processing in collaboration with C. Rice. We previously identified the cleavage events specified by the NS3 protease and showed that the NS2 also had protease activity and is responsible for the cleavage between NS2 and NS3. We have extended these studies to finer detail of the processing of the core protein. Using monoclonal antibodies of defined specificities we have shown that the core protein expressed by both recombinant baculovirus and vacciniavirus is further processed from the theoretical 191 amino acid protein called C22 to a 14kDa protein representing the C terminus of the C22. In addition, it appears that the hydrophobic C terminus from about amino acid 178 is also cleaved off presumably by signal peptidase. The core processing event may be autocatalytic as has been seen in pestiviruses. We have a recombinant vaccinia virus containing the entire open reading ?rame of HCV genome which we will evaluate in the chimpanzee model as a potential vaccine.In vitro cell culture of HCV is being actively pursued. Using a liver cell line, THLE5b, we have demonstrated the low level replication of HCV by quantitative PCR, in situ PCR and immunofluorescence. We have made over 5 passages of the virus in these cells but have not been able to detect any increased level of replication and in fact have lost the virus at that passage level. Blind passages and repeat passages are now being attempted.
