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: 6760101
• 负责人: JULIAN L LEIBOWITZ
• 金额: $ 32.74万
• 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/6760101
• 关键词: Interaction; MHV; RNA; mtHSP70; aconitase

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）：我们最近确定了四种与蛋白结合元件相互作用的蛋白质[3'（+）42个元素]，该蛋白包含在小鼠肝炎病毒基因组的最后42个核苷酸中。在此应用中，我们建议检查这四种蛋白质的相互作用，即线粒体HSP70（MTHSP70），线粒体刺激酶（M- aconitase），HSP60和HSP40与MHV RNA的相互作用。我们将采用荧光共振能量转移（FRET），免疫电子显微镜和细胞渗透交联研究，以验证这些蛋白质与MHV RNA的相互作用是否在完整的细胞中发生。我们将进行一系列的生化研究，以确定MHV RNA是否与MTHSP70，M- aconitase和Hsp60相互作用，然后再将这些蛋白质导入线粒体，或者如果这些MHV RNA相互作用的蛋白相互作用，则从线粒体中导出。我们还将确定在MHV感染期间线粒体功能是否改变。为了更好地表征这些相互作用的结构基础，我们将执行一系列实验，以绘制与MHV 3'（+）42蛋白结合元件接触的M- aconitase，MTHSP70，HSP60和HSP40的残基。随后，我们将进行一系列诱变实验，以绘制RNA结合所需的这些蛋白质的残基。第二行诱变实验将更精确地定义RNA在蛋白结合中的序列要求。我们已经确定这些蛋白质在结合过程中相互作用，因此将特别寻求主导的阴性突变体。我们已经确定MTHSP70是酪氨酸磷酸化的，并且MHV 3'（+）42结合蛋白的磷酸化状态调节其结合其MHV 3'（+）42个元素。我们将执行一系列药理和遗传操作，以研究酪氨酸磷酸化在调节这些蛋白与MHV RNA相互作用中的作用。在采用突变和过表达策略的一系列实验中，将检查MTHSP70，M- aconitase，Hsp60和Hsp40与MHV RNA与MHV RNA相互作用的功能意义。