| Translation of the Hepatitis C virus polyprotein is an essential step of the viral lifecycle and is mediated by an internal ribosome entry site (IRES) located in the 5' non-coding region of the viral genome. The HCV IRES forms a binary complex with the 40S ribosomal subunit and its translation requires fewer translation initiation factors than canonical cap-dependent translation initiation. Initial studies were undertaken to characterize HCV IRES function. Mutational analysis of the IRES revealed that its ability to bind 40S subunits does not always correlate with translation activity. This result suggests that the mechanism of HCV IRES translation initiation requires more than just 40S binding, implying that other initiation factors have a role in HCV IRES function. It was also determined that the HCV IRES was active in a number of different translation systems, indicating that the mechanism of HCV IRES activity is evolutionarily conserved. HCV IRES-mediated translation was also examined in an in vitro reconstituted translation system. It was determined that under certain conditions, the HCV IRES can initiate protein synthesis in the absence of translation initiation factors. This demonstrated an alternate mechanism of HCV IRES-mediated translation initiation, which may benefit HCV during infection, perhaps in evading the innate immune response. Another aspect of this research was to examine how the unique character of the HCV IRES could be exploited as a drug target. In an attempt to identify inhibitors of the HCV IRES, we constructed a cell line that expressed a selectable marker under the translational control of the HCV IRES. We transduced this reporter cell line with a retroviral conformational constrained peptide (CCP) library. While no specific inhibitory CCPs were identified, we isolated two mutant cell lines in which the HCV IRES was inhibited. At this time, the mechanism of HCV IRES inhibition in these cell lines is unclear. Future characterization of mutant cell lines CL22 and CL52 may reveal important trans-factors required for HCV IRES function and possible targets for anti-viral therapies. In summary, several approaches were undertaken, which together provide new insight into the mechanism of HCV IRES-mediated translation initiation. |
