Interaction of MHV RNA with mtHSP70 and m-aconitase

MHV RNA 与 mtHSP70 和 m-乌头酸酶的相互作用

基本信息

批准号：
6834617
负责人：
JULIAN L LEIBOWITZ
金额：
$ 25.46万
依托单位：
TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
依托单位国家：
美国
项目类别：
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/6834617
关键词：
Interaction MHV RNA mtHSP70 aconitase

项目摘要

EXCEED THE SPACE PROVIDED -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 (mtHSPT0), 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 PERFORMANCE SITE ========================================Section End===========================================

超出所提供的空间 - 我们最近鉴定了四种蛋白质，它们与小鼠肝炎病毒基因组最后 42 个核苷酸中包含的蛋白质结合元件 [3'(+)42 元件] 相互作用。在本申请中，我们建议检查这些蛋白质的相互作用四种蛋白质，线粒体 HSP70 (mtHSPT0)、线粒体乌头酸酶 (m-乌头酸酶)、HSP60 和 HSP40，以及 MHV RNA 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 的相互作用发生在完整细胞中。我们将进行一系列生化研究，以确定 MHV RNA 是否与 mtHSP70 相互作用， m-乌头酸酶和 HSP60 在这些蛋白质输入线粒体之前，或者，如果这些 MHV RNA 相互作用蛋白质从线粒体输出，我们还将确定线粒体功能是否在MHV 感染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来绘制与 MHV 3'(+)42 蛋白结合元件接触的 m-乌头酸酶、mtHSP70、HSP60 和 HSP40 的残基图谱。随后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图第二线诱变实验将更精确地定义蛋白质结合的 RNA 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，因此将特别寻找显性失活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它们与 MHV 3'(+) 的结合42 元件我们将进行一系列的药理学和遗传操作来研究酪氨酸磷酸化在调节这些蛋白质与 MHV RNA 相互作用中的作用相互作用的功能意义mtHSP70、m-乌头酸酶、HSP60 和 HSP40 以及 MHV RNA 对病毒复制的影响将在一系列采用突变和过表达策略的实验中进行检查性能站点 ================= =======================章节结束========================= =================