Hepatitis C virus (HCV) is a positive-strand RNA virus which causes important health problems worldwide. New antiviral therapies and vaccines are urgently needed. The long-term goal of this work is to develop better methods of prevention and treatment based on an understanding of how HCV-RNA is translated within infected cells. HCV-RNA translation is know to be controlled by an internal ribosome entry site (IRES), an RNA sequence which overlaps the 5' untranslated region of the viral genomic RNA.
The specific aims of this proposal are to identify liver-derived HCV IRES-binding proteins, to clone the genes that encode these proteins, and to characterize the proteins in terms of their binding and functional properties. HCV IRES-binding proteins will be purified from extracts of mouse and human liver tissue, using ion-exchange and affinity chromatography techniques. The genes will be cloned from human liver cDNA libraries using hybridization and yeast three-hybrid screening techniques. Characterization of the proteins will involve use of additional techniques, including site-directed mutagenesis, heterologous expression, RNA-protein crosslinking, and in vitro translation. The proposed work will be important in developing a better understanding of HCV pathogenesis. In addition, since HCV-RNA is translated by a cap-independent (i.e. IRES-dependent) mechanism while cellular mRNAs are translated by a cap-dependent mechanism, HCV-RNA translation is a promising target for antiviral therapies. The identification and characterization of liver-derived HCV IRES-binding proteins will facilitate the development of such agents. Finally, the proposed work should provide optimal training for the candidate. The sponsoring investigators, laboratories and departments offer a broad range of scientific and technical resources which will support the candidate in his development as an independent investigator.
