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: 7159356
• 负责人: JULIAN L LEIBOWITZ
• 金额: $ 24.14万
• 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 2008-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7159356
• 关键词: Aconitate Hydratase; Binding; Binding Proteins; Biochemical; Cells; Complex; Dominant-Negative Mutation; Elements; Fluorescence; Genome; Genomics; Heat-Shock Proteins 70; Immunoelectron Microscopy; Infection; Maps; Mitochondria; Mitochondrial Proteins; Murine hepatitis virus; Mutagenesis; Nucleotides; Phosphorylation; Play; Principal Investigator; Protein Binding; Protein Export Pathway; Proteins; RNA; RNA Binding; RNA-Binding Proteins; Role; Series; Signal Pathway; Tyrosine; Tyrosine Phosphorylation; Viral; Virus Replication; base; cis acting element; crosslink; genetic manipulation; programs; research study

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 trar_sfer (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.

我们最近鉴定了四种与蛋白质结合元件[3‘(+)42元件]相互作用的蛋白质 包含在小鼠肝炎病毒基因组的最后42个核苷酸中。在本申请中，我们建议 为了研究线粒体HSP70(MtHSP70)、线粒体乌头酸酶这四种蛋白质之间的相互作用 (M-乌头酸酶)、HSP60和HSP40与MHV RNA。我们将使用荧光共振能量 TRAR_SFER(FRET)，免疫电子显微镜和细胞杂交研究，以验证 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是否与mtHSP70，m-乌头酸酶和HSP60在 将这些蛋白质输入线粒体，或者如果这些MHV RNA相互作用的蛋白质 是从线粒体输出的。我们还将确定线粒体功能在MHV期间是否发生改变 感染。为了更好地描述这些相互作用的结构基础，我们将执行一系列 间乌头酸酶、线粒体热休克蛋白70、热休克蛋白60和热休克蛋白40残基的作图实验 MHV 3‘(+)42蛋白结合元件。我们随后将进行一系列的诱变 绘制RNA结合所需的这些蛋白质残基的实验。第二条诱变路线 实验将更准确地定义蛋白质结合所需的RNA序列。我们有 确定了这些蛋白质在结合过程中相互作用，因此显性负突变将特别 被追寻。我们已经确定mtHSP70是酪氨酸磷酸化的，并且mtHSP70的磷酸化状态 MHV 3‘(+)42结合蛋白调节其与MHV 3’(+)42元件的结合。我们将表演一系列节目 用药理学和遗传操作研究酪氨酸磷酸化在调控中的作用 这些蛋白与MHV RNA的相互作用。线粒体HSP70相互作用的功能意义， M-乌头酸酶、HSP60和HSP40与MHV RNA对病毒复制的影响将在一系列 采用突变和过度表达策略的实验。

项目成果

Mouse hepatitis virus stem-loop 4 functions as a spacer element required to drive subgenomic RNA synthesis.

小鼠肝炎病毒茎环 4 充当驱动亚基因组 RNA 合成所需的间隔元件。

• DOI: 10.1128/jvi.05092-11
• 发表时间: 2011
• 期刊: Journal of virology
• 影响因子: 5.4
• 作者: Yang,Dong;Liu,Pinghua;Giedroc,DavidP;Leibowitz,Julian
• 通讯作者: Leibowitz,Julian

In vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication.

• DOI: 10.1016/j.virol.2004.10.045
• 发表时间: 2005-02-05
• 期刊: Virology
• 影响因子: 3.7
• 作者: Youn S;Leibowitz JL;Collisson EW
• 通讯作者: Collisson EW

SHAPE analysis of the RNA secondary structure of the Mouse Hepatitis Virus 5' untranslated region and N-terminal nsp1 coding sequences.

• DOI: 10.1016/j.virol.2014.11.001
• 发表时间: 2015-01-15
• 期刊: VIROLOGY
• 影响因子: 3.7
• 作者: Yang, Dong;Liu, Pinghua;Wudeck, Elyse V.;Giedroc, David P.;Leibowitz, Julian L.
• 通讯作者: Leibowitz, Julian L.

The virion N protein of infectious bronchitis virus is more phosphorylated than the N protein from infected cell lysates.

• DOI: 10.1016/j.virol.2005.04.029
• 发表时间: 2005-08-15
• 期刊: Virology
• 影响因子: 3.7
• 作者: Jayaram J;Youn S;Collisson EW
• 通讯作者: Collisson EW