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：
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
项目类别：
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

项目摘要

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.

我们最近确定了与蛋白质结合元件相互作用的四种蛋白质[3'（+）42个元素] 包含在小鼠肝炎病毒基因组的最后42个核苷酸中。在此应用程序中，我们建议要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，带有MHV RNA。我们将采用荧光共振能量 trar_sfer（FRET），免疫电子显微镜和细胞Pereant交联研究，以验证是否验证这些蛋白质与MHV RNA的相互作用发生在完整的细胞中。我们将进行一系列生化研究以确定MHV RNA是否与MTHSP70，M- aconitase和Hsp60相互作用。如果这些MHV RNA相互作用蛋白从线粒体出口。我们还将确定MHV期间线粒体功能是否改变感染。为了更好地表征这些相互作用的结构基础，我们将执行一系列绘制与M-Aconitase，MTHSP70，HSP60和HSP40残基的实验 MHV 3'（+）42蛋白结合元件。随后我们将进行一系列诱变实验绘制RNA结合所需的这些蛋白质残基的实验。第二行诱变实验将更精确地定义RNA对蛋白质结合的序列要求。我们有确定这些蛋白质在结合过程中相互作用，因此，主要的负突变体将特别是寻求。我们已经确定MTHSP70是酪氨酸磷酸化的，并且是磷酸化状态 MHV 3'（+）42结合蛋白调节其结合MHV 3'（+）42个元素。我们将执行系列研究酪氨酸磷酸化在调节中的作用的药理和遗传操作这些蛋白与MHV RNA的相互作用。 MTHSP70相互作用的功能意义，在病毒复制中，将检查具有MHV RNA的M- aconitase，HSP60和HSP40，将在一系列中检查采用突变和过表达策略的实验。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

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

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.

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