This project is designed to replace ZO1 AI000570 since Dr. Jens Bukh, the Principal Investigator of that project, is no longer a full-time employee of the NIH. Hepatitis C virus (HCV) is an important human pathogen that previously could be studied only in the chimpanzee model because the virus could not be grown in cultured cells. Growth of the virus in cultured hepatoma cells was recently achieved by Wakita et al. but even now growth is strain-specific and inefficient and infectious titers rarely reach even 100,000 viruses per ml of culture medium. Prior to discovery of this culture system, we relied heavily on a retrovirus pseudotyped particle system to study neutralization of HCV and to perform mutational analysis of the two glycoproteins of HCV. Our recent studies using pseudoparticles constructed from different genotypes of HCV suggest there may be more than one serotype of virus. Mutagenesis studies utilizing pseudoparticles have identified amino acid residues that are important for incorporation of the glycoproteins into the pseudoparticle or that affect glycosylation in trans. We are now also developing the cell culture system since it should more closely model the natural replication system of this virus. We have successfully transfected cell cultures with the JFH1 strain, the only strain identified to date that replicates relatively efficiently in cultured cells. We have constructed a number of recombinant viruses that encode the structural proteins of virus strains other than JFH1 but that rely on the JFH1 nonstructural proteins for genome replication. We are serially passaging chimeric viruses and the JFH1 virus in order to select mutants which will replicate more efficiently in these cells and thus far we have isolated one mutant which grows to a 100-fold higher titer than does its parent. Through a combination of the pseudeoparticle system and the infection system, we have made significant progress in identifying host-cell proteins that facilitate entry of HCV into cells.
