Interaction of MHV RNA with mtHSP70 and m-aconitase

MHV RNA 与 mtHSP70 和 m-乌头酸酶的相互作用

• 批准号: 6680484
• 负责人: JULIAN L LEIBOWITZ
• 金额: $ 12.73万
• 依托单位: TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-07-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6680484
• 关键词: RNA binding protein; affinity chromatography; biological signal transduction; fluorescence resonance energy transfer; genetic regulatory element; immunoelectron microscopy; mitochondria; mitochondrial membrane; murine hepatitis virus; mutant; phosphorylation; protein localization; protein protein interaction; protein purification; protein quantitation /detection; protein sequence; site directed mutagenesis; virus infection mechanism; virus protein; virus replication

DESCRIPTION (provided by applicant): We have recently identified four proteins which interact with a protein binding element [3'(+)42 element] contained within the last 42 nucleotides of the mouse hepatitis virus genome. In this application we propose to examine the interaction of these four proteins, mitochondrial HSP70 (mtHSP70), mitochondrial aconitase (m-aconitase), HSP60, and HSP40, with MHV RNA. We will employ fluorescence resonance energy transfer (FRET), immuno-electron microscopy, and cell permeant cross-linking studies to verify that the interaction of these proteins with MHV RNA takes place in intact cells. We will undertake a series of biochemical studies to determine if the MHV RNA interacts with mtHSP70, m-aconitase, and HSP60 prior to the importation of these proteins into mitochondria, or alternatively, if these MHV RNA interacting proteins are exported from mitochondria. We will also determine if mitochondrial function is altered during MHV infection. To better characterize the structural basis for these interactions we will perform a series of experiments to map the residues of m-aconitase, mtHSP70, HSP60, and HSP40 which are in contact with the MHV 3'(+)42 protein binding element. We will subsequently carry out a series of mutagenesis experiments to map residues of these proteins required for RNA binding. A second line of mutagenesis experiments will more precisely define the sequence requirements of the RNA for protein binding. We have determined that these proteins interact during binding, thus dominant negative mutants will be particularly sought. We have determined that mtHSP70 is tyrosine phosphorylated, and that the phosphorylation state of the MHV 3'(+)42 binding proteins regulates their binding the MHV 3'(+)42 element. We will perform a series of pharmacologic and genetic manipulations to investigate the role of tyrosine phosphorylation in regulating the interaction of these proteins with MHV RNA. The functional significance of the interaction of mtHSP70, m-aconitase, HSP60, and HSP40 with MHV RNA on virus replication will be examined in a series of experiments employing mutational and over-expression strategies.

描述(由申请人提供)：我们最近鉴定了四种与小鼠肝炎病毒基因组最后42个核苷酸中包含的蛋白结合元件[3‘(+)42元件]相互作用的蛋白质。在这个应用中，我们建议研究这四种蛋白质，线粒体HSP70(MtHSP70)，线粒体乌头酸酶(m-乌头酸酶)，HSP60和HSP40，与MHV RNA的相互作用。我们将使用荧光共振能量转移(FRET)、免疫电子显微镜和细胞交联研究来验证这些蛋白质与MHV RNA的相互作用发生在完整的细胞中。我们将进行一系列的生化研究，以确定MHV RNA在将mtHSP70、m-乌头酸酶和HSP60输入线粒体之前是否与这些蛋白质相互作用，或者是否这些MHV RNA相互作用蛋白是从线粒体输出的。我们还将确定MHV感染期间线粒体功能是否发生变化。为了更好地表征这些相互作用的结构基础，我们将进行一系列实验，以定位与MHV 3‘(+)42蛋白结合元件接触的m-乌头酸酶、mtHSP70、HSP60和Hsp40的残基。随后，我们将进行一系列突变实验，以定位这些蛋白质与RNA结合所需的残基。第二行的突变实验将更准确地定义蛋白质结合所需的RNA序列。我们已经确定这些蛋白质在结合过程中相互作用，因此将特别寻找显性负突变。我们已经确定mtHSP70是酪氨酸磷酸化的，MHV 3‘(+)42结合蛋白的磷酸化状态调节它们与MHV 3’(+)42元件的结合。我们将进行一系列的药理学和遗传操作，以研究酪氨酸磷酸化在调节这些蛋白质与MHV RNA相互作用中的作用。MtHSP70、m-乌头酸酶、HSP60和HSP40与MHV RNA的相互作用对病毒复制的功能意义将通过一系列突变和过度表达策略进行检测。