HCMV IN AIDS--DISRUPTION OF CELL CYCLE REGULATION

人巨细胞病毒（HCMV）感染是艾滋病患者最重要的机会性感染之一，并被认为是HIV-1疾病进展的辅助因素。流行病学证据已经确定HCMV是涉及异常细胞生长的疾病（如再狭窄和宫颈癌）的可能因子或辅因子。尽管最近在抗逆转录病毒疗法方面取得了进展，但与艾滋病毒相关的恶性肿瘤的发病率并未下降，如浸润性宫颈癌。由于许多体外研究表明HCMV的致癌潜力及其在人类恶性组织和艾滋病患者中的普遍存在，本研究将进一步探索和描述细胞和病毒因素之间的相互作用，可能导致宿主DNA复制机制的激活，并可能导致不受控制的细胞增殖，特别是在半许可或非生产性HCMV感染。特别注意将致力于阐明HCMV立即早期（IE）蛋白，IE 1 -72，如何与细胞p107和p53蛋白相互作用，以激活参与DNA复制的宿主基因，同时防止细胞凋亡反应的激活。我们的工作假设是：（a）感染后，IE 1 -72与P107及其相关的E2 F和细胞周期蛋白/cdk复合物相互作用，分别激活E2 F应答启动子和细胞周期蛋白/cdk激酶，诱导与细胞增殖相关的基因的表达;和（B）IE 1 -72还通过其与p53的相互作用防止p53激活和随后的p53介导的细胞凋亡。为了支持这些假设，我们提出了以下具体目标：（1）进一步表征IE 1 -72和P107之间的相互作用。将构建在IE 1 - 72中具有点突变或缺失突变的蛋白质和病毒的缺失突变体和点突变体，并用于访问相互作用所需的结构域。(2)描述IE 1 -72与p107-E2 F复合物结合导致p107介导的转录抑制功能减轻的机制。将检查IE 1 -72结合后p107-细胞周期蛋白/cdk复合物的解离和/或活化，以确定这是否导致p107介导的生长抑制的缓解。(3)了解p53和IE 1 -72之间相互作用的结果，并通过使用组成型表达IE 1 - 72的细胞系和缺乏p53的细胞系研究IE 1 - 72对TGF-β 1和p53信号转导通路的影响。将研究相互作用所必需的每种蛋白质上的结构域以及这种相互作用在防止细胞凋亡中所起的作用。将研究IE 1 -72和p53相互作用对p53应答基因如p21和mdm 2的功能意义，以确定IE 1 -72表达是否也具有转录抑制作用。这些研究应该为我们提供一个更清晰的HCMV在细胞周期畸变和潜在的肿瘤发生中的作用。

Human cytomegalovirus (HCMV) infection is one of the most important opportunistic infections in patients with AIDS, and has been implicated as a cofactor in the progression of HIV-1 disease. Epidemiological evidence has identified HCMV as a possible factor or cofactor in diseases involving abnormal cell growth like restenosis and cervical carcinoma. Despite the recent advance in antiretroviral therapy, the incidence of HIV-associated malignancies has not decreased, such as invasive cervical cancer. Because of the oncogenic potential of HCMV as demonstrated by many in vitro studies and its ubiquity in human malignant tissues and patients with AIDS, this study will explore further and delineate the interactions between cellular and viral factors that may lead to the activation of host DNA replication machinery and, potentially, uncontrolled cellular proliferation, particularly in semi-permissive or nonproductive HCMV infection. Specific attention will be devoted to elucidating how the HCMV immediate-early (IE) protein, IE1-72, interacts with the cellular p107 and p53 proteins to activate host genes involved in DNA replication, while preventing the activation of the cellular apoptotic response. Our working hypotheses are: (a) that upon infection IE1-72 interacts with P107 and its associated E2F and cyclin/cdk complexes to activate E2F responsive promoters and cyclin/cdk kinase, respectively, to induce expression of genes associated with cellular proliferation; and (b) that IE1-72 also acts to prevent p53 activation and subsequent p53-mediated apoptosis via its interaction with p53. To support these hypotheses, we propose the following specific aims: (l) To further characterize the interactions between IE1-72 and P107. Deletion and point mutants of both proteins and viruses with point or deletion mutation in IE1- 72 will be constructed and used to access the domains necessary for interaction. (2) To describe the mechanism by which IE1-72 binding to the p107-E2F complex results in the alleviation of p107-mediated transcriptional repression function. The dissociation and/or activation of the p107-cyclin/cdk complexes following IE1-72 binding will be examined to determine if this leads to the alleviation of the p107-mediated growth suppression. (3) To understand the consequences of the interaction between p53 and IE1-72, and to study the effect of IE1-72 on the TGF-beta1 and p53 signal transduction pathways through the use of cell lines constitutively expressing IE1-72 and cell lines lacking p53. The domains on each protein necessary for interaction and the role this interaction plays in the preventing cellular apoptosis will be studied. The functional significance of the IE1-72 and p53 interaction on p53-responsive genes like p21 and mdm2 will be investigated to determine whether IE1-72 expression also has a transcriptionally repressive effect. These studies should provide us with a clearer picture of HCMV's role in cell cycle aberrations and, potentially, oncogenesis.