We propose to test the effect of pokeweed antiviral protein (PAP) on translation mediated by the internal ribosome entry sites (IRES) of hepatitis C virus (HCV). HCV is the major cause of chronic hepatitis which can result in life threatening cirrhosis and hepatocellular carcinoma. A global survey by WHO in 1999 estimated that at least 170 million people in the world are chronically infected with HCV. Presently interferon is the best treatment for chronic hepatitis. However, it is associated with relatively poor efficacy and unfavorable side effects. PAP is a 29-kDa antiviral/antifungal protein isolated from Phytolacca americana. It belongs to a group of proteins called the ribosome inactivating proteins (RIPs). RIPs are known to catalytically remove a specific adenine residue from the highly conserved alpha-sarcin/ricin loop (SRL) in the large rRNA of eukaryotic and prokaryotic ribosomes, a process called depurination. Our study has indicated that in addition to the previously identified adenine (A4324), PAP removes another adenine (A4321) and a guanine (G4323) from the eukaryotic large rRNA resulting in inhibition of translation. HCV RNA translation is mediated by a complex RNA element called the internal ribosome entry site (IRES), located within the 5' untranslated region of the viral RNA. The current structural model of HCV IRES indicates the presence of four major stem-loops I-IV and a pseudoknot. Except for stem-loop I, all are required for translation initiation. Two SRL motifs have been found on stem-loops II and Illd. Our preliminary studies have indicated that PAP can affect the HCV IRES and reduce the reporter firefly luciferase activity significantly. In addition, a non- toxic PAP mutant can inhibit the translation mediated by HCV IRES as efficiently as wild type PAP. PAP may exert its detrimental effect through depurination, possibly on the SRL motifs found on the HCV IRES element. Therefore, specific aims of this application are to: (1) determine if nontoxic PAP mutants inhibit HCV IRES directed translation and whether they destabilize the reporter mRNA, (2) determine if PAP inhibits HCV IRES-mediated translation by binding to the HCV IRES and depurinating the SRL motifs, (3) identify the PAP binding and depurination sites on the HCV IRES and determine if these sequences arp critical for inhibition on IRES-mediated translation. This study will lay the groundwork for designing novel drugs against HCV for the treatment of chronic hepatitis. ? ?
