TRANSLATION-INHIBITION OF HEPATITIS C AND POLIO VIRUSES

丙型肝炎和脊髓灰质炎病毒的翻译抑制

• 批准号: 6534167
• 负责人: ASIM DASGUPTA
• 金额: $ 28.09万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6534167
• 关键词: RNA; RNA binding protein; Saccharomyces cerevisiae; autoantigens; chemical structure function; fungal genetics; gene targeting; genetic library; genetic translation; genetically modified animals; hepatitis C virus; inhibitor /antagonist; laboratory mouse; molecular cloning; nucleic acid sequence; nucleic acid structure; poliovirus; protein purification; ribosomes; translation factor; virus RNA; virus genetics

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.

小RNA病毒和黄病毒包括多种医学上重要的人类病毒，其中包括诱发脊髓灰质炎（脊髓灰质炎病毒）、传染性肝炎（甲型肝炎）和慢性肝炎（丙型肝炎）、普通感冒（鼻病毒）、心肌炎和脑炎（柯萨奇病毒）等的病毒。 这些病毒的一个共同特征是它们合成病毒蛋白所采用的策略。 虽然细胞 mRNA 通过帽子依赖性“扫描”机制进行翻译，但病毒 RNA 通过一种独特的机制进行翻译，该机制涉及核糖体内部进入病毒 RNA 的 5' 非翻译区 (5' UTR)（IRES 介导的翻译）。 将研究两种抑制剂（一种小RNA，IRNA和一种小肽，LAP），它们可以在体内和体外有效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）。 生化和遗传学方法将用于确定 IRNA 和 LAP 相对于细胞 mRNA 优先抑制 HCV 和 PV RNA 翻译的机制。 参与 IRES 介导翻译的细胞蛋白将被纯化，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LAP进入肝细胞的机制。酿酒酵母中 IRNA 的身份和正常功能将通过 IRNA 基因的克隆和表征来解决。将进行 IRNA 基因敲除，以更好地了解 IRNA 在酵母中的作用。 我们将检查酵母中的 IRNA 结合蛋白是否在 IRES 介导的翻译（酵母中）中发挥作用。 最后，我们将开发表达IRNA和LAP的转基因小鼠，以确定IRNA在动物体内的长期表达以及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进一步加深我们对真核细胞内部翻译起始机制的理解，而且能为开发有效对抗丙型肝炎的抗病毒药物提供新策略。