HUMAN CYTOMEGALOVIRUS

这项研究的目的是阐明 导致人类疾病进展的细胞和病毒因素 巨细胞病毒(HCMV)感染和激活细胞周期。 将特别注意了解人类巨细胞病毒是如何启动的 其基因表达及HCMV主要即刻早期(IE)蛋白 影响细胞因子调节下游病毒基因表达和 启动大分子合成。我们的工作假设是：(A) 人巨细胞病毒糖蛋白gB和/或Gh与其相互作用 细胞受体触发信号转导通路导致IKB 核转录因子-kB的磷酸化、核转位和反式激活 HCMV Majore IE启动子(以及其他)，以及(B)所得到的IE2 产品(S)单独作用，或与IE1合作，反转激活 表达第二层病毒和细胞转录因子， 包括核因子-kB。目标启动子包括病毒和 调控参与DNA合成的酶和细胞的表达 扩散。为了检验这些假设，我们提出了以下建议 方法：(1)高效表达和纯化昆虫来源的人巨细胞病毒gB和Gh 哺乳动物细胞系统，并检测它们对活化的影响。 核转录因子，包括核因子-kB，在未感染的细胞中。 抗这些蛋白的抗体和相关的激酶抑制物 信号转导将被用来试图阻止这些影响 并阐明它们的激活机制。(2)识别 能够磷酸化IkB的病毒粒子相关蛋白激酶(S)，以及 研究(I)IkB/NF-kB胞浆池的调节和(Ii) 人巨细胞病毒感染后核转录因子-kB活性的变化。我们假设第二个 核因子-kB的激活(假设(B))是由于表达增加所致 新合成的病毒IE对编码核因子-kB蛋白基因的比较 蛋白质。因此，我们将研究核因子-kB诱导的动力学， 确定核因子-kB在IE时间和 探讨核因子-KB第二阶段诱导的机制 发生。核因子-kB的p65和p50亚基的启动子，以及 将使用SP-1共识序列来检查它们对 IE蛋白反式激活；(3)检测和鉴定重要病毒 和细胞蛋白质直接或间接与IE2相互作用 负责激活电迁移率的蛋白质 移位分析(EMSA)、免疫共沉淀、DNA足迹、Western 分析和体外转录将被用来研究 互动。(4)确定IE2和IE2之间的相互作用域 细胞因子，如TFIID、SP-1等。 研究IE2、Rb、Cyclins与E2F之间、IE2与E2F之间的相互作用 TFIID，以及IE2和SP-1之间。IE2的DNA序列特异性结合 通过SP-1、E2F或NF-kB将通过EMSA、DNA足迹和 电子显微镜分析。

The objective of this study is to elucidate the interactions between cellular and viral factors which results in the progression of human cytomegalovirus (HCMV) infection and activation of the cell cycle. Specific attention will be devoted to understanding how HCMV initiates its gene expression and how HCMV major immediate-early (IE) proteins affect cellular factors to regulate downstream viral gene expression and activate macromolecular synthesis. Our working hypotheses are (a) upon infection, interactions of HCMV glycoprotein gB and/or gH with their cellular receptors trigger signal transduction pathways resulting in IkB phosphorylation, the nuclear translocation of NF-kB and transactivation of the HCMV majore IE promoter (among others), and (b) the resultant IE2 product(s) acting alone, or in cooperation with IE1, transactivate the expression of a second tier of viral and cellular transcription factors, including NF-kB. Target promoters include viral as well as those regulating the expression of enzymes involved in DNA synthesis and cell proliferation. To test these hypotheses, we propose the following approaches: (1) To overexpress and purify HCMV gB and gH from insect or mammalian cell system, and examine their effects on the activation of nuclear transcription factors, including NF-kB, in uninfected cells. Antibodies against these proteins and inhibitors of kinases associated with signal transduction will be used in attempts to block these effects and elucidate their mechanisms of activation. (2) To identify the virion-associated protein kinase(s) capable of phosphorylating IkB, and investigate the regulation of (i) cytosolic pools of IkB/NF-kB and (ii) nuclear NF-kB activity upon HCMV infection. We postulate that the second tier of NF-kB activation (Hypothesis (b)) is due to increased expression of genes encoding NF-kB proteins by the newly synthesized viral IE proteins. Therefore, we shall study the kinetics of NF-kB induction, determine the means by which NF-kB is released at IE times and investigate the mechanism by which the second phase of NF-Kb induction occurs. Promoters of p65 and p50 subunits of NF-kB, and promoter with SP-1 consensus sequence will be used to examine their responsiveness to IE proteins transactivation; (3) To detect and identify important viral and cellular proteins directly or indirectly interacting with IE2 proteins which are responsible for the activation of electromobility shift assay (EMSA), immuno-coprecipitation, DNA footprinting, Western analyses and in vitro transcription will be used to study the interactions. (4) To identify the interacting domains between IE2 and cellular factors, such as TFIID, SP-1, etc. Attention will be devoted to study interactions among IE2, RB, cyclins and E2F, between IE2 and TFIID, and between IE2 and SP-1. DNA sequence-specific binding of IE2 via SP-1, E2F, or NF-kB will be studied by EMSA, DNA footprinting and electron microscopic analysis.