CrPV IRES function and animal virus replication in yeast

描述(申请人提供)：尽管世界卫生组织即将宣布消灭微小核糖核酸病毒家族中最知名的肠道病毒--脊髓灰质炎病毒，但仍有66种不同的人类肠道病毒仍在感染世界各地的人群。然而，目前还没有针对任何非脊髓灰质炎肠道病毒的疫苗或抗病毒治疗。开发有效的抗病毒药物的关键是了解支配病毒生命周期的分子过程。虽然在了解微小核糖核酸病毒蛋白的功能方面已经取得了相当大的进展，但对病毒基因扩增所需的宿主细胞因子知之甚少。由于缺乏易于接受的遗传系统，宿主细胞因子的鉴定一直受到阻碍。以前研究动物病毒在酵母中复制的尝试受阻，因为微小核糖核酸病毒通过帽不依赖的机制启动翻译，使用内部核糖体进入位点(IRES)，而IRES在酵母中不起作用。然而，最近的研究发现，来自昆虫病毒基因组的IRES在酵母中发挥作用，这为在酵母中开发一种系统来识别微小核糖核酸病毒复制所需的宿主细胞因子提供了可能性。板球麻痹病毒(CRPV)IRES的特性是非同寻常的，因为这种IRES可以在没有任何启动因子、tRNAs或GTP的情况下，由纯化的40S和60S亚基形成80S核糖体。在第一个具体目标中，现有的酵母突变将被用来深入了解CRPV IRES招募核糖体的机制。此外，还将确定酵母mRNAs可以通过与CRPV IRES类似的机制启动翻译。具体地说，酵母微阵列分析将用于鉴定Afun12突变酵母菌株中的多倍体mRNAs。该菌株缺乏编码elF5B的非必需FUN12基因，该基因参与核糖体亚单位的连接。因此，在突变株中，与mRNAs相关的核糖体较少。在第二个特定目标中，将构建嵌合的小核糖核酸RNA基因组，包含CRPV IRES来代替它们的正常IRES，并将检测它们在酵母中的复制。此外，还将构建复制子，包含可选择的酵母标记URA3来代替衣壳蛋白。将分离表达复制子的细胞，并分离复制子扩增有缺陷的突变细胞。然后，将在酵母和哺乳动物细胞中检查已识别基因的功能。总体而言，这些研究将有助于我们了解病毒IRESS的功能，并可能导致一种研究病毒繁殖所需宿主基因的新方法。

DESCRIPTION (provided by applicant): Although poliovirus, the most well known enterovirus in the picornavirus family, is soon to be declared eradicated by the World Health Organization, there are 66 other distinct human enteroviruses that are still infecting populations worldwide. However, there are no vaccines or antiviral treatments available for any of the non-polio enteroviruses. The key to developing an effective antiviral agent is to gain an understanding of the molecular processes that govern the viral life cycle. Although considerable progress has been made towards understanding the function of the picornaviral proteins, little is known about the host cell factors that are required for viral gene amplification. Identification of host cell factors has been hampered by the absence of an easily amenable genetic system. Previous attempts to study animal virus replication in yeast have been thwarted by the fact that the picornaviruses initiate translation by a cap-independent mechanism, using an internal ribosome entry site (IRES), which does not function in yeast. However, the recent finding that an IRES from an insect viral genome functions in yeast opened the possibility to develop a system in yeast to identify host cell factors required for picornavirus replication. The properties of the cricket paralysis virus (CrPV) IRES are extraordinary, because this IRES can form 80S ribosomes from purified 40S and 60S subunits in the absence of any initiation factors, tRNAs or GTP. In the first specific aim, existing yeast mutants will be used to gain insight into the mechanism by which the CrPV IRES recruits ribosomes. In addition, yeast mRNAs will be identified that can initiate translation by a mechanism similar to the CrPV IRES. Specifically, yeast microarray analysis will be used to identify polysomal mRNAs in a Afun12 mutant yeast strain. This strain lacks the non-essential FUN12 gene encoding elF5B, which is involved in ribosomal subunit joining. As a consequence, fewer ribosomes are associated with mRNAs in the mutant strain. In the second specific aim, chimeric picornaviral RNA genomes, containing the CrPV IRES in place of their normal IRES, will be constructed and their replication in yeast will be examined. In addition, replicons will be constructed, containing the selectable yeast marker URA3 in place of the capsid proteins. Replicon-expressing cells will be isolated and mutant cells that are defective replicon amplification will be isolated. The functions of identified genes will then be examined both in yeast and in mammalian cells. Overall, these studies will contribute to our understanding on the functioning of viral IRESs and potentially lead to a novel way of studying host genes that are required for virus propagation.