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: 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
• 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

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===========================================

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个核苷酸内所含的蛋白质结合元件[3 '（+）42元件]相互作用。（间乌头酸酶），HSP 60和HSP 40，与MHV RNA，我们将采用荧光共振能量转移（FRET），免疫电子显微镜，和细胞渗透交叉-我们将进行一系列的生物化学研究，以确定这些蛋白质与MHV RNA的相互作用是否发生在完整的细胞中。在将这些蛋白输入线粒体之前，或可选地，如果这些MHV RNA相互作用蛋白从线粒体输出，则MHV RNA与mtHSP 70、m-乌头酸酶和HSP 60相互作用。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，我们将进行一系列实验以绘制m-乌头酸酶、mtHSP 70、HSP 60、和HSP 40，它们与MHV 3 ′（+）42蛋白质结合元件接触。我们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。第二线诱变实验将更精确地确定蛋白质结合所需的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失活突变体。我们已经确定mtHSP 70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与MHV 3'（+）的结合我们将进行一系列的药理学和遗传学操作，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。mtHSP 70，间乌头酸酶，HSP 60，将在一系列采用突变和过表达策略的实验中研究MHV RNA和HSP40对病毒复制的影响。==