| It has been estimated that approximately 3% of the world's population is infected with HCV.This represents nearly 170 million person worldwild. HCV infection often result in persistent infection and has been shown to be associated with high risk of liver cirrhosis and hepatocellular carcinoma. The progression of prevention and therapy of HCV infection was hampered by lacking reliable cell culcures in vitro and successful small animal model.Combination therapy with IFN- a and ribavirin -until recently, considered the treatment of choice for hepatitis C梚s relatively ineffective. Less than 40% of those given this treatment have a durable benefit, and most patients continue to have numerous and frequent side effects. Therefore, it's necessary to find novel therapeutic strategy to prevent and eliminate HCV infection. As one of the novel therapeutic strategy, gene therapy including antisense oligodeoxynucleotides, ribozymes,interfering peptide in vivo and inhibitor RNA etc, was identified andexploration on the strategy was important for the prevention and therapeutic procedure of HCV infection.The single-stranded, plus-polarity RNA genome of HCV, a member of the Flaviviridae, is approximately 9,500 nucleotides long. The 5' untranslated region of HCV RNA is approximately 340 nt long, is highly structured, and contains mutiple AUG codes. The 5' UTR is highly conserved among different strains of HCV. It is followed by a single large open reading frame that encodes a polyprotein which is proteolytically progressed to produce the mature structural and unstructural proteins of HCV. Nucleotides 40 to 370 of the 5' UTR of HCV have been shown to contain an internal ribosome entry site (IRES). HCV IRES contains similar structure of poliovirus and EMCV. IRES is crucial to the genome replication and translation of HCV. Therefore, it has been the therapeutic target of HCV infection.Coward and Dasgupta have shown that PV-IRES mediated translation is restricted in the yeast Saccharomyces cerevisidae, in part due to a trans-acting factor capable of inhibiting PV IRES-mediated translation in HeLa cell extracts. The inhibitor was purifed and subsequently shown to be a small (60-nt) RNA which specifically inhibited cap-indepent, IRES-mediated translation but had little or no effect on cap-dependent translation of cellular mRNAs. The yeast RNA (called IRNA) was found to bind strongly several cellular polypeptides which interact with the PV IRES element. Because HCV and PV IRES elements bind similar polypeptids. It was reasoned that IRNA might also interfere with HCV IRES-mediated translation.By using molecular cloning methods, we constructed protocaryotic and eukaryotic expression plasmid of IRNA and mlRNA which could produce thecorrect sequence and structure of IRNA and mIRNA by introducing the self-cleavage ribozyme sequences into both sides of the sequence of IRNA and mIRNA. Using HCV IRES-containing bicistronic construct pTTDCl-341 (a generous gift from A.Siddiqui), the inhibitor activity of IRNA and complement IRNA (cERNA) on HCV IRES-mediated translation in vitro were studied. Additionally, by using transient transfection of hepatoma cells and a hepatoma cell constitutively expressing IRNA, we demonstrated that IRNA could specificly inhibit HCV IRES- mediated translation in vivo. Additionally, hepatome cells constitutively expressing IRNA became refractory to infection by PV. Finally, the binding of the cellular factor to the HCV 5' UTR was specifically and efficiently competed by IRNA. RESULTSi. The protocaryotic and eukaryotic expression plasmids of IRNA and mIRNA were constructed which could produce the correct sequence and structure of IRNA and mIRNA by introducing self-cleavage ribozyme sequence into both sides of IRNA and mIRNA. ii. By using HCV IRES - containing bicistronic plasmid pTTDC 1-341 and HCV IRES - containing plasmid pCMVNCRluc, the inhibition activity of IRNA and cIRNA on HCV IRES - mediated translation in vitro were explorated.iii. By using transient transfection of he...
|
PDF Full Text Request