Interaction of MHV RNA with mtHSP70 and m-aconitase

描述（由申请人提供）：我们最近鉴定了四种蛋白质，它们与包含在小鼠肝炎病毒基因组的最后42个核苷酸内的蛋白质结合元件[3 ′（+）42元件]相互作用。在这个应用程序中，我们建议检查这四种蛋白质，线粒体HSP 70（mtHSP 70），线粒体乌头酸酶（m-aconitase），HSP 60和HSP 40，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来验证这些蛋白质与MHV RNA的相互作用发生在完整细胞中。我们将进行一系列的生物化学研究，以确定MHV RNA是否与线粒体HSP 70，间乌头酸酶和HSP 60相互作用之前，这些蛋白质输入到线粒体，或者，如果这些MHV RNA相互作用的蛋白质从线粒体输出。我们还将确定线粒体功能是否在MHV感染期间改变。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与MHV 3 '（+）42蛋白结合元件接触的m-乌头酸酶、mtHSP 70、HSP 60和HSP 40的残基。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负突变体。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，m-aconitase，HSP 60和HSP 40与MHV RNA的相互作用对病毒复制的功能意义将在一系列采用突变和过表达策略的实验中进行研究。

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.