| Summary: | 碩士 === 國立臺灣大學 === 生化學研究所 === 87 === Hepatitis C virus ( HCV ) is the major pathogen of post-transfusionally transmitted hepatitis. Some patients develop chronic infection and about 20 % of them progress into cirrhosis or hepatocellular carcinoma. HCV is postive-sense single-stranded RNA virus with a 9.5 kb RNA genome. The HCV genomic RNA contains a open reading frame and encodes a long polyprotein precursor. HCV polyprotein is translated via a mechanism of internal initiation controlled by its internal ribosome entry site (IRES). The HCV IRES occupies most of the 5’-untranslated region and extents to sequences downstream the AUG codon. Previously, we have defined a cellular factor p120 which can specifically binds to HCV IRES and is associated with IRES function. In this study, the p120 exhibits affinity when partially purified on an anion-exchange column. In addition, two-dimensional electrophoresis revealed that the pI value of p120 protein was around 5.0~6.0. Thus, p120 is a relatively acidic RNA binding protein. Recently, the subunit p116 of eukaryotic translation initiation factor 3 (eIF3) was demonstrated to interact with HCV IRES. In this study, a putative RNA recognition motif (RRM) was expressed in E. coli. Gel retardation experiments showed that the RRM domain specifically bound to the domain III of the HCV IRES. The recombinant RRM abolished the HCV IRES function in an in vitro translation system. Furthermore, the RRM competed away the binding of cellular factor p120 and p87 to the HCV IRES as demonstrated by UV-crosslinking experiments. Nevertheless, antibodies against RRM cannot recognize the p120/RNA complex. These results indicate that p120, p87 and p116 subunit of eIF3 may bind to the same site or in the proximity to the domain III of the HCV IRES. Further studies of interaction among translation initiation factors、cellular factors and HCV IRES will help us in understanding the control mechanisms of the internal initiation of HCV polyprotein synthesis.
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