HUMAN CYTOMEGALOVIRUS

本研究的目的是阐明 细胞和病毒因素，导致人类疾病的进展， 巨细胞病毒（HCMV）感染和细胞周期的激活。 特别注意将致力于了解HCMV如何启动 它的基因表达和HCMV如何主要立即早期（IE）蛋白 影响细胞因子以调节下游病毒基因表达， 激活大分子合成。 我们的工作假设是（a） 感染、HCMV糖蛋白gB和/或gH与它们的相互作用 细胞受体触发信号转导途径，导致IkB 磷酸化、NF-κ B核转位和反式激活 HCMV主要IE启动子（其中），和（B）所得IE 2 单独或与IE 1协同作用的产物，反式激活 第二层病毒和细胞转录因子的表达， 包括NF-κ B。 靶启动子包括病毒启动子以及那些 调节参与DNA合成和细胞增殖的酶的表达， 增殖 为了验证这些假设，我们提出以下建议： 方法：（1）从昆虫或哺乳动物中高效表达和纯化HCMV gB和gH， 哺乳动物细胞系统，并检查它们对激活 核转录因子，包括NF-κ B，在未感染的细胞。 针对这些蛋白质的抗体和相关激酶的抑制剂 与信号转导将被用于试图阻止这些影响 并阐明其激活机制。 (2)识别 能够磷酸化IkB的病毒体相关蛋白激酶，和 研究（i）IkB/NF-kB的胞质池和（ii） HCMV感染后核NF-kB活性。 我们假设第二个 NF-κ B激活的一层（假设（B））是由于表达增加 通过新合成的病毒IE， proteins. 因此，我们将研究NF-kB诱导的动力学， 确定在IE时间释放NF-kB的方式，以及 研究NF-Kb诱导的第二阶段的机制， 发生。 NF-kB的p65和p50亚基的启动子，以及具有 SP-1共有序列将用于检查它们对以下的反应性： IE蛋白的反式激活;（3）检测和鉴定重要的病毒 和直接或间接与IE 2相互作用的细胞蛋白质 负责激活电迁移率的蛋白质 迁移试验（EMSA），免疫共沉淀，DNA足迹，Western印迹 分析和体外转录将用于研究 交互. (4)为了识别IE 2和 细胞因子，如TFIID、SP-1等。 研究IE 2、RB、cyclins和E2 F之间的相互作用，IE 2和 TFIID，以及IE 2和SP-1之间。 IE 2的DNA序列特异性结合 通过SP-1，E2 F或NF-kB的表达将通过EMSA，DNA足迹和 电镜分析

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.