TRANSLATION-INHIBITION OF HEPATITIS C AND POLIO VIRUSES

微微核糖核酸病毒和黄病毒包括多种医学上重要的人类病毒，包括那些引起脊髓灰质炎(脊髓灰质炎病毒)、传染性(HEP.A)和慢性肝炎(丙型肝炎)、感冒(鼻病毒)、心肌炎和脑炎(柯萨奇)等。这些病毒的一个共同特征是它们用来合成病毒蛋白质的策略。细胞内的mRNAs是通过帽依赖的“扫描”机制翻译的，而病毒RNA是通过一种独特的机制翻译的，该机制涉及核糖体进入病毒RNA的5‘非翻译区(5’UTR)(IRES介导的翻译)。两种抑制剂(一种小RNA，iRNA和一种小肽，LAP)将在体内和体外有效地阻断丙型肝炎和脊髓灰质炎病毒IRES介导的翻译(但不是细胞翻译)。将使用生化和遗传方法来确定iRNA和LAP优先抑制丙型肝炎病毒和丙型肝炎病毒RNA的翻译而不是细胞mRNA的机制。参与IRES介导的翻译的细胞蛋白将被纯化，并将确定它们在丙型肝炎病毒RNA翻译中的作用。将进行iRNA的结构-功能分析。LAP进入肝细胞的机制将被确定。我们将通过克隆和鉴定酿酒酵母中的iRNA基因来解决其在酵母中的特性和正常功能。为了更好地了解iRNA在酵母中的作用，将进行iRNA基因敲除。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，我们将建立表达iRNA和LAP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有效性。希望这些研究不仅能加深我们对真核细胞内启动翻译机制的理解，还能为开发有效的抗丙型肝炎药物提供新的策略。

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.