CrPV IRES function and animal virus replication in yeast

CrPV IRES 功能和酵母中动物病毒的复制

• 批准号: 7101037
• 负责人: Sunnie R Thompson
• 金额: $ 10.8万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-08-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7101037
• 关键词: Cardiovirus; Picornaviridae; Saccharomyces; capsid; fungal genetics; genetic strain; genetic translation; host organism interaction; messenger RNA; microarray technology; molecular assembly /self assembly; polysomes; recombinant virus; replicon; reporter genes; ribosomes; tissue /cell culture; virus genetics; virus infection mechanism; virus protein; virus replication

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.

描述（由申请方提供）：尽管脊髓灰质炎病毒（小核糖核酸病毒家族中最知名的肠道病毒）即将被世界卫生组织宣布根除，但仍有66种其他不同的人类肠道病毒仍在感染全球人群。然而，没有任何疫苗或抗病毒治疗可用于任何非脊髓灰质炎肠道病毒。开发有效抗病毒剂的关键是了解控制病毒生命周期的分子过程。虽然已经取得了相当大的进展，对理解的小核糖核酸病毒蛋白质的功能，很少有人知道的宿主细胞因子所需的病毒基因扩增。由于缺乏一个易于操作的遗传系统，宿主细胞因子的鉴定受到阻碍。先前研究动物病毒在酵母中复制的尝试受到以下事实的阻碍：小核糖核酸病毒通过帽独立机制启动翻译，使用内部核糖体进入位点（IRES），其在酵母中不起作用。然而，最近发现，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以鉴定小核糖核酸病毒复制所需的宿主细胞因子提供了可能性。蟋蟀麻痹病毒（CrPV）IRES的特性是非凡的，因为这种IRES可以在没有任何起始因子、tRNA或GTP的情况下从纯化的40 S和60 S亚基形成80 S核糖体。在第一个具体目标中，现有的酵母突变体将用于深入了解CrPV IRES招募核糖体的机制。此外，将鉴定可通过类似于CrPV IRES的机制启动翻译的酵母mRNA。具体而言，酵母微阵列分析将用于鉴定Afun12突变酵母菌株中的多核糖体mRNA。该菌株缺乏编码eIF5B的非必需FUN12基因，其参与核糖体亚基连接。因此，在突变株中与mRNA相关的核糖体较少。在第二个具体目标中，将构建含有CrPV IRES代替其正常IRES的嵌合小核糖核酸病毒RNA基因组，并检查其在酵母中的复制。此外，将构建复制子，其含有替代衣壳蛋白的选择性酵母标记URA3。将分离复制子表达细胞，并分离复制子扩增缺陷的突变细胞。然后将在酵母和哺乳动物细胞中检查所鉴定的基因的功能。总的来说，这些研究将有助于我们了解病毒IRES的功能，并可能导致一种新的方式来研究病毒繁殖所需的宿主基因。