HCMV, CELL GROWTH STATE, AND TRANSCRIPTION FACTOR E2F

HCMV、细胞生长状态和转录因子 E2F

• 批准号: 6362610
• 负责人: Jane Azizkhan-Clifford
• 金额: $ 21.91万
• 依托单位: MCP HAHNEMANN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 2003-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6362610
• 关键词: cell cycle; cell free system; cytomegalovirus; enzyme activity; gene mutation; phosphorylation; polymerase chain reaction; protein kinase; tissue /cell culture; transcription factor; transfection; virus infection mechanism

DESCRIPTION: The interaction of viral and host cell factors determines the fate of viral infection. Dr. Azizkhan has been studying the effect of human cytomegalovirus (HCMV) infection and expression of HCMV-immediate early (IE) genes on the cell cycle/growth regulated dihydrofolate reductase (DHFR) gene. The E2F family of transcription factors are involved in basal transcription and cell cycle regulation of the DHFR gene. The E2F binding site is required for trans activation of DHFR by the 72 kDa IE1 gene product (IE72), and E2F-1 and -2 interact with IE72 in cells and in cell-free assay. Further functional and molecular analyses of HCMV IE72 and E2F are proposed to identify HCMV-host cell interactions that regulate viral replication. To this end, they will: (1) characterize the role of IE72 and its E2F-mediated effects in HCMV infection; (2) characterize the effects of IE72 on cell proliferation, cell cycle kinetics and cellular gene expression; and (3) determine the mechanism of activation of E2F-dependent transcription by HCMV IE72. In order to determine the precise role of IE72 in HCMV infection and the associated cellular response, they will determine the effect of viral infection on E2F synthesis and its association with other cellular proteins. To determine whether the interaction of IE72 with E2Fs and the kinase activity of IE72 are essential for viral replication, dominant negative mutants of IE72 that preclude these activities will be used. Experiments to address the second aim utilize a stable cell line with inducible IE72 expression. The effect of IE72 on host cell proliferation and gene expression and the role of host cell growth state in modulating its response to IE72 will be ascertained. Experiments to address the third aim involve establishing an in vitro transcription system to determine whether the effects of IE72 and E2F-dependent transcription. The role of the kinase activity of IE72 and its dissociation of E2F from specific pocket proteins in transcriptional activation will be addressed. In summary, their identification of E2F as a target of HCMV-IE72 directed them to explore the role of E2F as an essential mediator of virus effects. The goal of these experiments is to establish how the effects of HCMV on cellular transcription factors and other factors involved in cell cycle control relate to host cell infectivity and to activation of both cellular and viral DNA synthesis and gene expression.

描述：病毒和宿主细胞因子的相互作用决定了 病毒感染的命运 阿兹汗博士一直在研究 巨细胞病毒（HCMV）感染和HCMV立即早期（IE）表达 细胞周期/生长调节二氢叶酸还原酶（DHFR）基因 基因 转录因子E2 F家族参与了基底膜的形成。 DHFR基因的转录和细胞周期调控。 E2 F结合 72 kDa IE 1基因产物反式激活DHFR需要一个位点 （IE72），并且E2 F-1和-2在细胞中和在无细胞测定中与IE72相互作用。 本文对HCMV IE72和E2 F的功能和分子生物学特性进行了进一步的研究 以鉴定调节病毒复制的HCMV-宿主细胞相互作用。 到 为此，他们将：（1）表征IE72及其E2 F介导的作用， （2）研究IE72对HCMV感染细胞的影响， 增殖、细胞周期动力学和细胞基因表达;和（3） 确定HCMV激活E2 F依赖性转录的机制 IE72。 为了确定IE72在HCMV感染中的确切作用， 相关的细胞反应，他们将决定病毒的影响， 感染对E2 F合成及其与其他细胞蛋白质的关联。 为了确定IE72与E2 Fs和激酶的相互作用是否 IE72活性对病毒复制至关重要，显性阴性 将使用排除这些活性的IE72突变体。 实验来 利用具有诱导型IE72的稳定细胞系解决第二个目标 表情 IE72对宿主细胞增殖和基因表达的影响 表达和宿主细胞生长状态在调节其反应中的作用 第72章一定会被发现 实现第三个目标的实验包括 建立体外转录系统，以确定是否 IE72和E2 F依赖性转录的影响。 激酶的作用 IE72的活性及其将E2 F从特异性口袋蛋白解离 转录激活将得到解决。 总之，他们将E2 F鉴定为HCMV-IE72的靶点， 他们探索E2 F作为病毒效应的重要介质的作用。 这些实验的目的是确定HCMV如何影响细胞的增殖。 细胞转录因子和其他参与细胞周期的因子 控制涉及宿主细胞感染性和细胞 以及病毒DNA合成和基因表达。