PICORNAVIRUS TRANSLATION/INHIBITION BY SMALL YEAST RNA

小核糖核酸病毒包括多种医学上重要的人类和哺乳动物病毒。 动物病毒包括那些诱发普通感冒的病毒（鼻病毒）， 脊髓灰质炎、传染性肝炎（甲型肝炎）、科萨基病毒和 口蹄疫病毒（FMDV）等。虽然加盖细胞 自然地，mRNA通过核糖体“扫描”机制翻译， 未加帽的小核糖核酸病毒mRNA通过一种独特的机制翻译 涉及核糖体进入5 '非翻译区的内部 病毒RNA，同时试图在酵母中表达感染性脊髓灰质炎病毒cDNA 酿酒酵母，偶然发现酵母细胞是 无法翻译脊髓灰质炎病毒RNA。这是由于一个小的交易 RNA（称为抑制剂RNA，I-RNA）存在于酵母细胞裂解物中， 能够抑制HeLa细胞提取物中PV RNA的翻译。I-RNA具有 纯化并制备了合成克隆。I-RNA合成 通过T7 RNA聚合酶的转录从克隆中， 核糖体的进入，从而抑制小核糖核酸病毒的翻译， 在体内和体外的细胞RNA翻译。初步研究 表明I-RNA与细胞蛋白（p52）相互作用， 翻译的内部启动。生物化学和遗传学 方法将用于确定I-RNA 选择性地抑制翻译的内部起始。我们将 研究除了翻译以外的病毒过程（如RNA） 合成或蛋白质加工）也受到I-RNA的影响。身份 和正常功能的I-RNA在酵母酿酒酵母将被解决 通过克隆和表征编码I-RNA序列的基因。I-RNA 基因“敲除”将被执行，以更好地了解I-RNA的作用， 在酵母细胞生长中。我们还将确定是否酵母蛋白， 与I-RNA结合在翻译中起作用。I-RNA是否有潜力 抑制不同的病毒（包括丙型肝炎），使用内部 将确定蛋白质合成的翻译起始。 最后，制备表达I-RNA的稳定HeLa细胞系， 确定I-RNA在动物细胞中长期表达的效果， 表达I-RNA的细胞是否能抵抗小RNA病毒 感染希望在此提出的研究不仅能提供一个 为抗病毒药物设计提供合理依据，也进一步推动我们的 对翻译内部启动机制的认识 真核细胞

Picornaviruses encompass a large variety of medically important human and animal viruses which include those inducing common cold (rhinovirus), poliomyelitis, infectious hepatitis (hepatitis A), Coxsackie virus, and foot-and-mouth disease virus (FMDV) among others. While capped cellular mRNAs are translated by a ribosomal 'scanning' mechanism, naturally uncapped picornaviral mRNAs are translated by a distinct mechanism involving internal entry of ribosomes within the 5'untranslated region of viral RNA, While trying to express infectious poliovirus cDNA in the yeast S.cerevisiae, it was discovered serendipitously that yeast cells were unable to translate poliovirus RNA. This was due to a transacting small RNA (called inhibitor RNA, I-RNA) present in yeast cell lysates that was able to inhibit translation of PV RNA in HeLa cell extracts. The I-RNA has been purified and a synthetic clone has been prepared. I-RNA synthesized from the clone by transcription with T7 RNA polymerase, blocks internal entry of ribosomes and thus inhibits picornaviral translation but not cellular RNA translation both in vivo and in vitro. Initial studies suggest that I-RNA interacts with a cellular protein (p52) involved in internal initiation of translation. Both biochemical and genetic approaches will be used to determine the mechanism by which I-RNA selectively inhibits internal initiation of translation. We will investigate whether viral processes other than translation (such as RNA synthesis or protein processing) is also affected by the I-RNA. Identity and normal function of I-RNA in the yeast S.cerevisiae will be addressed by cloning and characterizing the gene encoding the I-RNA sequence. I-RNA gene "knock out" will be performed to better understand the role of I-RNA in yeast cell growth. We will also determine whether yeast proteins which bind to I-RNA plays a role in translation. Whether I-RNA has potential to inhibit different viruses (including hepatitis C) that use internal initiation of translation for protein synthesis will be determined. Finally, a stable HeLa cell line expressing I-RNA will be prepared to determine the effect of long term expression of I-RNA in animal cells and whether cells expressing I-RNA will be resistant to picornavirus infection. It is hoped that studies proposed here will not only provide a rational basis for antiviral drug design but also further our understanding of the mechanism of internal initiation of translation in eukaryotic cells.