Molecular Biology Of Hepatitis C Virus

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Hepatitis C virus (HCV) is a major cause of community-acquired viral hepatitis. Prototype strains of the various genotypes of HCV, including some of those discovered in this laboratory, have been biologically amplified in chimpanzees, packaged and distributed for use as challenge inocula in studies of passive and active immunoprophylaxis, etc. Full-length cDNA clones of HCV (genotypes 1a, 1b and 2a) have been constructed and transcribed RNA used to transmit hepatitis C to chimpanzees by in vivo hepatic transfection. Chimpanzees, transfected with infectious cDNA clones of HCV, are being followed to determine the natural history of infection. Infectivity pools have been prepared from chimpanzees infected with monoclonal HCV (derived by in vivo transfection with RNA transcripts of infectious cDNA); these have been titered for infectivity in other chimpanzees. With the reagents we have developed (infectious cDNA clones that are infectious by in vivo transfection; titered pools of polyclonal and monoclonal HCV representing different strains, subgenotypes and genotypes) we are pursuing a collaborative study of the immunopathogenesis of HCV infections. We have demonstrated that resolution versus progression to chronicity is a function of the host, not the virus, since infections with monoclonal viruses have yielded both results. We have shown that humoral immunity appears not to be important in control of the virus, at least in the control of infection or in preventing reinfection. We have demonstrated that sterilizing immunity can be achieved by repeated infection of chimpanzees, but that this sterilizing immunity is strain-specific. We are currently examining the role of CD4 versus CD8 cells in the control of HCV infection. When taken together, these studies will provide an in-depth analysis of humoral versus cellular immune responses to HCV infection in chimpanzees, a surrogate of man. In addition, the availability of infectious cDNA clones of HCV has permitted for the first time a mutational analysis of genomic regions. For example, individual portions of the 3' NCR have been deleted from the full-length clone and the resultant deletion mutant clones inoculated into chimpanzees by intrahapatic transfection. Certain regions of the NCR have been identified as critical for in vivo replication of HCV. In other studies we have deleted the hypervariable region 1 (HVR1) of the E2 protein of HCV, the region that contains a neutralization epitope. Surprisingly, the deletion mutant virus was viable but attenuated when transfected into chimpanzees. We are also performing a mutational analysis of the gene that encodes a small protein designated "p7". An analogous protein is found in related pestiviruses. We have demonstrated that p7 in HCV is critical for replication and we are now mapping the critical region of the gene. We have constructed an infectious cDNA clone of GB virus-B (GBV-B), a monkey virus that is the closest relative to HCV. In addition, we have prepared challenge pools of GBV-B and have determined the infectivity titer of these in tamarins. We are currently using the GBV-B tamarin system to study characteristics of the virus that it shares with HCV, a virus which must be studied in chimpanzees. In other studies, we have constructed chimeric genomes from infectious cDNA clones of HCV and bovine viral diarrhea virus. These genomes can replicate in transfected cells but the resultant viral products cannot assemble into infectious virus in the absence of helper virus. However, the transfected genome expresses large quantities of structural proteins in susceptible cells; such cells are a useful substrate for immunofluorescence studies. Similar studies are being carried out with dengue 4 virus and HCV. We have determined the genetic heterogeneity of HCV isolates that were recovered from patients who were infected following transfusion. The sequence of the hypervariable region and adjacent portions of envelope proteins 1 and 2 were determined for multiple clones obtained from patients who had fulminant hepatitis, from patients who convalesced following acute hepatitis and from patients who progressed to chronic hepatitis C. Distinctive patterns of dynamic change in the sequence of clones during the first several weeks of infection were observed. Patients with fulminant or resolving hepatitis had few changes in the sequences of clones, whereas there were many changes in the sequences of clones from patients who progressed to chronic hepatitis. Thus, the outcome of an HCV infection could be predicted in the first few weeks of the infection. Similar studies have been carried out in patients chronically infected with HCV and who were undergoing therapy with interferon. Patients could be separated into four groups, based upon their response to interferon therapy: a) long-term responders, b) those who relapsed following cessation of treatment, c) those who responded but had a break-through of viral replication while still on therapy and d) those who failed to respond (nonresponders). As with acutely infected HCV patients, distinctive patterns of dynamic change in the sequence and heterogeneity of HCV clones obtained early in therapy were predictive of outcome. Long-term responders demonstrated a marked decrease in heterogeneity of HCV, resulting in eradication of the virus. Relapsing patients also demonstrated a change in heterogeneity and a decrease in viral titer but a new dominant strain usually emerged following cessation of therapy. Nonresponders maintained the same dominant strain throughout therapy, suggesting that an interferon-resistant strain already existed before therapy. Patients who experienced a breakthrough during therapy had patterns that were similar to those of nonresponders, suggesting that this was a mixed group of patients. These findings may be useful in predicting the outcome of therapy with interferon early in the course of treatment.