PICORNAVIRUS TRANSLATION/INHIBITION BY SMALL YEAST RNA

小核糖核酸病毒包括多种具有重要医学意义的人类和 动物病毒，包括那些引起普通感冒的病毒(鼻病毒)， 小儿麻痹症、传染性肝炎(甲型肝炎)、柯萨奇病毒和 口蹄疫病毒(FMDV)等。而被封顶的手机 自然地，mRNAs是由核糖体“扫描”机制翻译的 无帽小核糖核酸通过一种不同的机制进行翻译 涉及核糖体在5‘非翻译区的内部进入 病毒RNA在酵母中表达传染性脊髓灰质炎病毒的研究 在酿酒酵母中，人们偶然发现酵母细胞 无法翻译脊髓灰质炎病毒RNA。这是由于一笔小额的交易 存在于酵母细胞裂解物中的RNA(称为抑制物RNA，i-RNA) 能够抑制HeLa细胞提取物中PV RNA的翻译。I-RNA已经 已经被提纯，并制备了一个合成克隆。I-RNA合成 通过T7 RNA聚合酶转录从克隆中，阻断内部 核糖体进入，从而抑制微小核糖核酸病毒的翻译，但不 体内和体外的细胞RNA翻译。初步研究 提示i-RNA与一种细胞蛋白(P52)相互作用 翻译的内部启蒙。包括生化和遗传 将使用各种方法来确定i-RNA的机制 选择性地抑制翻译的内部启动。我们会 调查除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 以及i-RNA在酿酒酵母中的正常功能 通过克隆和鉴定编码i-RNA序列的基因。I-RNA 将进行基因“敲除”以更好地理解i-RNA的作用 在酵母细胞的生长过程中。我们还将确定酵母蛋白是否 与i-RNA的结合在翻译中起着作用。I-RNA是否具有潜在的 抑制不同病毒(包括丙型肝炎)使用内部 蛋白质合成的翻译启动将被确定。 最后，将制备稳定表达i-RNA的HeLa细胞系，以 确定i-RNA在动物细胞中长期表达的影响 表达i-RNA的细胞对小核糖核酸病毒是否具有抵抗力 感染。希望在这里提出的研究不仅将提供一个 抗病毒药物设计的合理基础，也进一步推动了我们的 对翻译的内部启动机制的理解 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.