Analysis of the interaaction between HCV IRES and the p116 subunit of eIF3

• Main Authors: Pei-Yu Chu, 朱珮瑜
• Other Authors: 張鑫
• Format: Others
• Language: zh-TW
• Published: 2006
• Online Access: http://ndltd.ncl.edu.tw/handle/60284113070772111601

碩士 === 國立臺灣大學 === 微生物學研究所 === 94 === Hepatitis C virus (HCV), the major infectious agent of non-A, non-B hepatitis, often causes chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV is a positive, single-stranded RNA virus with genomic size of approximately 9.6 kb. The viral genome consists of a 5’ noncoding region (NCR), a large open reading frame encoding a polyprotein of approximately 3010 amino acids, and a 3’ NCR. Sequences in the 5’ NCR are highly conserved among HCV isolates. Translation initiation of HCV is mediated by an internal ribosome entry site (IRES) element which encompasses almost the entire 5'' NCR and about 30 nt of the core protein coding region immediately downstream the AUG codon. In addition, the IRES folds into a stable secondary and tertiary structure and has been demonstrated to functionally replaces several initiation factors by directly recruiting the 40S ribosomal subunit, eukaryotic initiation factor 3 (eIF3), and other cellular factors. Nevertheless, the mechanism of HCV internal initiation is poorly understood. Eukaryotic translation initiation factor 3 (eIF3) is a large multisubunit complex that plays a central role in the initiation of translation. eIF3 has an aggregate molecular mass of ~800 kDa and comprises at least 13 subunits. Four subunits of eIF3 complex, p170, p116, p66 and p47 have been demonstrated to specifically bind to the HCV IRES. According to secondary structure prediction, p116 contains a putative RNA recognition motif (RRM) near the N terminal region (a.a. 185-268). Independent studies have demonstrated interactions between p116-RRM and the domain Ⅲ (nt134/314) of HCV IRES, and between the p116 subdomain from amino acid 227 to 320 and the IRES domain Ⅱ (nt 44-118 apical part). In our laboratory, p116-RRM was previously found to interact with both the HCV IRES domain Ⅱ (nt 65-102) and the domain Ⅲabcd (nt 131-278). In this study, the interaction between HCV IRES and the p116 subunit of eIF3 was further examined. His•RRM fusion protein was purified and used to examine its RNA binding ability by filter binding assay, gel shift assay, and UV-crosslinking experiments. The results indicate that IRES domain Ⅲab (nt 123-232) has a stronger RRM-binding activity then the domain Ⅲabcd (nt 131-278) does. Interesting, HCV413-581 that represents a part of the core protein sequence was also found to interact with p116-RRM. By performing in vitro translation competition assay, RNA subdomains important for the HCV-IRES-mediated translation of core protein were examined. RNA subdomains that had stronger ability to interact with p116-RRM shown stronger effect on translation-inhibition when used as a RNA competitor. So the interaction between HCV IRES and p116-RRM has essential effect on the IRES-mediated translation.