Hepatitis C virus is a leading cause of human liver disease worldwide. Recent discovery of the JFH-1 isolate, capable of infecting cell culture, opens new avenues for studying HCV replication. We describe the development of a high-throughput, quantitative, genome-scale, mutational analysis system to study the HCV cis-elements and protein domains that are essential for virus replication. An HCV library with 15-nucleotide random insertions was passaged in cell culture to examine the effect of insertions at each genome location by insertion-specific fluorescent-PCR profiling. Of 2399 insertions identified in 9517 nucleotides of the genome, 374, 111, and 1914 were tolerated, attenuating, and lethal, respectively, for virus replication. Besides identifying novel functional domains, this approach confirmed other functional domains consistent with previous studies. The results were validated by testing several individual mutant viruses. Furthermore, analysis of the 3′ non-translated variable region revealed a spacer role in virus replication, demonstrating the utility of this approach for functional discovery. The high-resolution functional profiling of HCV domains lays the foundation for further mechanistic studies and presents new therapeutic targets as well as topological information for designing vaccine candidates.
Hepatitis C virus (HCV) is a major human health concern that causes fatal liver diseases. Currently no vaccine is available to prevent HCV infection. Though the HCV was identified two decades ago, the virus has only recently been successfully grown in cell culture conditions. The role of HCV protein and regulatory element sub-domains during virus growth is poorly understood. We have developed a mutational analysis method to identify the function of HCV sub-domains at a high resolution. A collection of HCV mutants containing 15-nucleotide random insertions was tested for growth in cell culture. The precise location of the insertions and their effects on virus growth were analyzed by capillary genotyping technology and bioinformatics. Out of the total 2399 HCV mutants identified, 374 mutants grew normally, 111 mutants demonstrated reduced growth, and 1914 mutants failed to grow in cell culture. This mutational analysis method was validated by testing many individual mutant viruses. The present study identified several HCV functional sub-domains required for virus growth, presenting novel therapeutic targets. The HCV mutant viruses identified with the property of reduced growth can be used for designing vaccine candidates.