CrPV IRES function and animal virus replication in yeast

CrPV IRES 功能和酵母中动物病毒的复制

基本信息

批准号：
6705331
负责人：
Sunnie R Thompson
金额：
$ 16.05万
依托单位：
UNIVERSITY OF ALABAMA AT BIRMINGHAM
依托单位国家：
美国
项目类别：
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

项目摘要

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（由申请人提供）：虽然脊髓灰质炎病毒是小核糖核酸病毒家族中最著名的肠道病毒，很快就会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世界人群。然而，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。开发有效抗病毒药物的关键是了解控制病毒生命周期的分子过程。尽管在了解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，但对于病毒基因扩增所需的宿主细胞因子却知之甚少。由于缺乏易于使用的遗传系统，宿主细胞因子的鉴定受到阻碍。之前研究动物病毒在酵母中复制的尝试因小核糖核酸病毒利用内部核糖体进入位点（IRES）通过帽独立机制启动翻译这一事实而受阻，而该位点在酵母中不起作用。然而，最近发现昆虫病毒基因组的 IRES 在酵母中发挥作用，这为在酵母中开发系统来识别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 的特性非同寻常，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 40S 和 60S 亚基形成 80S 核糖体。在第一个具体目标中，现有的酵母突变体将用于深入了解 CrPV IRES 招募核糖体的机制。此外，还将鉴定酵母 mRNA 可以通过类似于 CrPV IRES 的机制启动翻译。具体来说，酵母微阵列分析将用于鉴定 Afun12 突变酵母菌株中的多聚体 mRNA。该菌株缺乏编码 elF5B 的非必需 FUN12 基因，该基因参与核糖体亚基连接。因此，突变株中与 mRNA 相关的核糖体较少。在第二个具体目标中，将构建嵌合小核糖核酸病毒RNA基因组，其中包含CrPV IRES代替其正常IRES，并将检查它们在酵母中的复制。此外，将构建复制子，其中包含选择性酵母标记 URA3 代替衣壳蛋白。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 IRES 的功能，并可能带来一种研究病毒传播所需宿主基因的新方法。