TRANSLATION-INHIBITION OF HEPATITIS C AND POLIO VIRUSES

小核糖核酸病毒和黄病毒包括多种医学上重要的人类病毒，其包括诱导脊髓灰质炎（脊髓灰质炎病毒）、传染性（Hep. A）和慢性肝炎（丙型肝炎）、普通感冒（鼻病毒）、心肌炎和脑炎（科萨基）等。 这些病毒的一个共同特征是它们用于合成病毒蛋白的策略。 虽然细胞mRNA通过帽依赖性“扫描”机制翻译，但病毒RNA通过涉及核糖体内部进入病毒RNA的5'非翻译区（5' UTR）内的独特机制翻译（IRES介导的翻译）。 将研究两种抑制剂（一种小RNA，IRNA和一种小肽，peptide）在体内和体外有效阻断丙型肝炎病毒和脊髓灰质炎病毒IRES介导的翻译（但不是细胞翻译）。 生物化学和遗传学方法将用于确定IRNA和IRNA优先抑制HCV和PV RNA翻译而不是细胞mRNA的机制。 将纯化参与IRES介导的翻译的细胞蛋白，并确定它们在HCV RNA翻译中的作用。将进行IRNA的结构-功能分析。 将确定β-内酰胺酶进入肝细胞的机制。酵母S.将通过克隆和表征IRNA基因来解决酿酒酵母的问题。将进行IRNA基因敲除以更好地了解IRNA在酵母中的作用。 我们将研究酵母中的IRNA结合蛋白是否在IRES介导的翻译（在酵母中）中发挥任何作用。 最后，我们将开发表达IRNA和IRNA的转基因小鼠，以确定IRNA在动物中的长期表达以及IRNA在转基因动物中阻断病毒感染的功效。 希望本文提出的研究不仅能加深我们对真核细胞内翻译起始机制的理解，而且能为开发有效抗丙型肝炎的抗病毒药物提供新的策略。

Picorna and Flaviviruses encompass a large variety of medically important human viruses which include those inducing poliomyelitis (poliovirus), infectious (Hep. A) and chronic hepatitis (hepatitis C), common cold (rhinovirus), and myocarditis and encephalitis (coxsackie) among others. A common feature of these viruses is the strategy they employ for synthesis of viral proteins. While cellular mRNAs are translated by cap-dependent "scanning" mechanism, the viral RNAs are translated by a distinct mechanism involving internal entry of ribosomes within the 5' untranslated region (5' UTR) of viral RNA (IRES-mediated translation). Two inhibitors (a small RNA, IRNA and a small peptide, LAP) which efficiently block hepatitis C and poliovirus IRES-mediated translation (but not cellular translation) both in vivo and in vitro, will be studied. Both biochemical and genetic approaches will be used to determine the mechanism by which IRNA and LAP preferentially inhibit translation of HCV and PV RNAs over cellular mRNAs. Cellular proteins involved in IRES-mediated translation will be purified and their roles in HCV RNA translation will be determined. Structure-function analysis of IRNA will be performed. The mechanism of entry of LAP into hepatocytes will be determined. Identity and normal function of IRNA in the yeast S. cerevisiae will be addressed by cloning and characterizing the IRNA gene. IRNA gene knockout will be performed to better understand the role of IRNA in yeast. We will examine whether IRNA-binding proteins in yeast play any role in IRES-mediated translation (in yeast). Finally, we will develop transgenic mice expressing IRNA and LAP to determine the long term expression of IRNA in animals as well as efficacy of IRNA in blocking virus infections in transgenic animals. It is hoped that the studies proposed here will not only further our understanding of the mechanism of internal initiation of translation in eukaryotic cells, but also provide novel strategies to develop antivirals effective against hepatitis C.