HCMV DYSREGULATES ENDOTHELIAL CELL FUNCTIONS

本研究的长期目标是确定人类巨细胞病毒（HCMV）感染如何扰乱内皮细胞功能并阐明所涉及的分子机制。血管内皮细胞是HCMV的体内宿主细胞之一，是一组重要的血管生成细胞，构成完整的血管系统，分泌血管化和细胞外基质形成的因子。HCMV感染与几种心血管疾病有关，如动脉粥样硬化和冠状动脉再狭窄。在过去的资助期内，我们发现HCMV使用表皮生长因子受体（EGFR）作为病毒进入和信号传导的主要细胞受体。因此，在本研究中，我们假设HCMV感染人脐静脉内皮细胞（HUVE）依赖于EGFR，并且HCMV糖蛋白和立即早期蛋白分别通过与其表面受体和细胞周期调节蛋白相互作用在启动病毒发病机制中起主要作用。为了验证我们的假设，我们提出：(1)研究HCMV感染HUVE细胞是否依赖于EGFR。研究EGFR在HUVE、HEL和乳腺癌细胞表面的状态，以及HCMV感染的后果，以确定EGFR在细胞对HCMV感染易感性中的作用。(2)确定HCMV gB与EGFR结合的相互作用域。截断的、定点突变的和胰蛋白酶消化的gB片段将被表达和纯化，用于在转染erbB1和erbB3的CHO细胞中进行相互作用研究。我们还将使用噬菌体展示方法来识别结合egfr的gB结构域。利用细菌人工染色体（BAG）技术制备gB突变病毒，研究病毒背景下受体-配体相互作用；(3)确定EGFR上作为gB结合受体结构域的相互作用结构域。erbB1分子的截断和定点突变体将在EBFR(-) CHO或MB453细胞上表达，噬菌体展示方法将用于识别EGFR上的活性相互作用域(s)。(4)寻找靶向病毒结合和进入的抗hcmv药物。靶向EGFR激酶活性、EGFR降解或模拟细胞受体活性结构域的化合物将被研究和开发。本项目提出的研究将提供一个全面的

The long-term objectives of this study are to define how human cytomegalovirus(HCMV) infection upsets endothelial cell functions and to elucidate the molecular mechanisms involved. Vascular endothelial cells, one of in vivo host cells for HCMV, are a group of important angiogenic cells that form an integral vascular system, and secrete factors for vascularization and formation of the extracellular matrix. HCMV infection is associated with several cardiovascular diseases, such as atherosclerosis and coronary restenosis. In the past funding period we identified that HCMV uses the epidermal growth factor receptor (EGFR) as a primary cellular receptor for viral entry and signaling. Therefore, in this study we hypothesize that HCMV infection of human umbilical vein endothelial (HUVE) cells is EGFR dependent and that HCMV glycoproteins and immediate-early proteins play major roles in initiating viral pathogenesis by interacting with its surface receptors and cell cycle regulating proteins, respectively. To assess our hypothesis, we propose: (1) To investigate whether HCMV infection of HUVE cells is EGFR dependent. The status of EGFR on cell surfaces of HUVE, HEL, and breast cancer cells, and the consequences of HCMV infection will be studied to determine the role of EGFR in cell susceptibility to HCMV infection, (2) To define the interacting domains of HCMV gB that bind to EGFR. The truncated, site-directed point mutant, and tryptic-digested gB fragments will be expressed and purified for interaction studies in CHO cells transfected with erbB1 and erbB3. We will use also the phage display approach to identify the domains of gB that bind EGFRs. The gB mutant virus will be developed by using the bacterial artificial chromosome (BAG) technique to study the receptor-ligand interaction in the context of viruses, (3) To determine the interaction domains on EGFR that serve as receptor domains for gB binding. Truncated and site-directed mutants of erbB1 molecules will be expressed on EBFR(-) CHO or MB453 cells and the phage display approach will be employed to identify the active interaction domain(s) on EGFR, and (4) To search for anti-HCMV agents targeting viral binding and entry. Compounds targeting EGFR kinase activity, EGFR degradation, or mimicking the active domain(s) of cellular receptor will be studied and developed. Studies proposed in this project would provide a comprehensive understanding of how HCMV infects cell types crucial for angiogenesis. Thus, potential anti-HCMV agents that target viral entry and signaling can be developed.