TRANSLATIONAL CONTROL IN INFLUENZA VIRUS INFECTED CELLS

流感病毒感染细胞中的翻译控制

• 批准号: 6631717
• 负责人: MICHAEL G KATZE
• 金额: $ 35.56万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-01-01 至 2005-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6631717
• 关键词: HeLa cells; Orthomyxoviridae; RNA binding protein; alkaline phosphatase; gel mobility shift assay; gene expression; genetic mapping; genetic translation; genetically modified animals; host organism interaction; interferons; laboratory mouse; messenger RNA; nucleic acid sequence; phosphorylation; polymerase chain reaction; protein biosynthesis; protein kinase; site directed mutagenesis; stress proteins; tissue /cell culture; virus RNA; virus genetics

Influenza virus is a major public health problem in the United States and worldwide. To better understand the cellular events that occur during influenza virus infection, we have been studying the shut-off of host cell protein synthesis and the selective translation of viral mRNAs in influenza virus-infected cells. We have accumulated evidence that the molecular strategies employed by influenza virus to accomplish these goals are intimately intermeshed with the host cell defense and stress-response pathways. In Specific Aim 1, we will further define the mechanisms of selective translation. We have identified a cellular RNA-binding protein, GRSF-1, that binds to the 5' untranslated region (UTR) of influenza virus mRNAs. This is significant, since we earlier demonstrated that the 5' UTR was both necessary and sufficient to redirect the host cell protein synthesizing machinery to translate only viral mRNAs. We hypothesize that GRSF-1 interacts with the 5' UTR of influenza virus mRNAs to upregulate viral protein synthesis. To test this hypothesis, we will perform separate in vitro functional assays and in vivo experiments with wild-type and transdominant mutant GRSF-1. Specific Aim 2 focuses on the stress-response pathway activated by influenza virus to ensure efficient viral mRNA translation. Influenza virus recruits the cellular TPR protein, P58IPK, PK, to down-regulate the interferon-induced PKR protein kinase, thereby keeping protein synthetic activity high in a virus-infected cell. In the absence of this regulation, activation of PKR by viral RNAs results in the phosphorylation of the eukaryotic initiation factor, eIF-2alpha and inhibition of protein synthesis initiation. In this aim, we will dissect the P58IPK/PKR pathway and examine the roles played by the P58IPK regulators, hsp40, P52rIPK, and the molecular chaperone hsp70, which we now hypothesize plays a key role in the downregulation of PKR. Finally, in Aim 3, we propose to construct a knockout mouse with a deletion of the P58IPK gene. We will examine the effects of deleting P58IPK on viral and cellular mRNA translation and gene expression, both in the null mice and in fibroblasts prepared from these mice. Together, the studies outlined will contribute to a better understanding of eukaryotic protein synthesis regulation, which may ultimately provide insights into novel antiviral therapeutics.

流感病毒是美国和全球的主要公共卫生问题。 为了更好地了解流感病毒感染过程中发生的细胞事件，我们一直在研究宿主细胞蛋白合成的关闭以及在感染流感病毒感染细胞中病毒mRNA的选择性翻译。 我们积累了证据表明，流感病毒采用的分子策略实现这些目标是与宿主细胞防御和应激反应途径密切相关的。 在特定目标1中，我们将进一步定义选择性翻译的机制。 我们已经确定了一种细胞RNA结合蛋白GRSF-1，该蛋白与流感病毒mRNA的5'未翻译区（UTR）结合。 这很重要，因为我们早些时候证明了5'UTR是必要且足以重定向宿主细胞蛋白合成机制以仅转化病毒mRNA的必要和足够的。 我们假设GRSF-1与流感病毒mRNA的5'UTR相互作用，以上调病毒蛋白的合成。 为了检验这一假设，我们将使用野生型和跨型突变体GRSF-1进行单独的体外功能测定和体内实验。 具体目标2的重点是流感病毒激活的应力反应途径，以确保有效的病毒mRNA翻译。 流感病毒募集细胞TPR蛋白P58IPK，PK，以下调干扰素诱导的PKR蛋白激酶，从而在病毒感染的细胞中保持较高的蛋白质合成活性。 在没有这种调节的情况下，通过病毒RNA激活PKR会导致真核起始因子，EIF-2alpha和蛋白质合成起始的抑制作用。 在此目标中，我们将剖析P58IPK/PKR途径，并检查P58IPK调节剂HSP40，P52RIPK和Molecular Chaperone HSP70的作用，我们现在假设这些作用在PKR下调中起关键作用。 最后，在AIM 3中，我们建议用p58IPK基因缺失构建敲除小鼠。 我们将在零小鼠和由这些小鼠制备的成纤维细胞中检查删除P58IPK对病毒和细胞mRNA翻译和基因表达的影响。 总之，概述的研究将有助于更好地理解真核蛋白质合成调节，这最终可能会提供对新型抗病毒疗法的见解。