CMV ACCESSORY GENES AND RETINAL CELL INFECTION

CMV 辅助基因与视网膜细胞感染

• 批准号: 2888489
• 负责人: LENORE PALMA PEREIRA
• 金额: $ 22.93万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-09-30 至 2000-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2888489
• 关键词: MDCK cell; actins; cellular polarity; cytomegalovirus; cytomegalovirus retinitis; gene mutation; herpes simplex virus 1; immunofluorescence technique; immunoprecipitation; ion transport; laboratory mouse; molecular cloning; protein structure function; protein transport; recombinant proteins; retinal pigment epithelium; site directed mutagenesis; virus infection mechanism; virus protein

Retinal disease caused by the herpesviruses, cytomegalovirus (CMV), varicella zoster virus (VZV), and herpes simplex virus 1 (HSV-1), is the major cause of vision loss in patients with the acquired immunodeficiency syndrome (AIDS). CMV retinitis is a progressive disease which, if untreated, causes retinal detachment and blindness. It begins in the retinal capillaries and spreads directionally through the retina to the polarized retinal pigment epithelium (RPE). In cultures of polarized RPE cells, CMV preferentially infects the apical surface membrane and progeny virions spread from cell to cell across junctional complexes of the lateral membranes. Cell-cell spread is different from entry across the apical membrane, as in polarized cells these membranes contains different proteins. Preliminary experiments showed that CMV and HSV-1 mutants that lack certain glycoproteins, US8/US9 in CMV and gE/gI in HSV-1, are significantly impaired in cell-cell spread in polarized RPE cells, although they grow normally in unpolarized fibroblasts, and fail to alter tight junction proteins. This exciting finding revealed that CMV US9 and US8 are functional homologues of gE and gI in HSV-1 and VZV. HSV-1 and pseudorabies virus (PrV) gE function in pathogenesis of ocular infections by facilitating cell-cell spread of virus in the eye and in the central nervous system. CMV US8 and US9 are similar in amino acid sequence to alpha herpesvirus gE and gI and to Vibrio cholerae proteins in a virulence cassette, zonula occludens toxin (Zot) and accessory cholera entertoxin (Ace), which increase the permeability of intestinal epithelial cells. These herpesvirus and bacterial proteins are also similar to families of mammalian Ca2+ and C1- transporters, which predicts their possible functions. It is proposed to investigate the role of gE/gI homologues in pathogenesis and to elucidate their biological activities and the mechanisms by which they alter polarity of epithelial and neuronal cells. The aims are as follows. Aim 1. Determine the degree of functional similarity between CMV US9 and HSV-1 gE in altering cellular proteins that maintain polarity. Compare the properties of US9- and gE-expressing MDCK cells. Express US9 and gE in polarized RPE cells and assess changes in cell proteins that maintain polarity and integrity of the actin cytoskeleton. Evaluate effects of mutants in US9 and gE on other cell types affected in herpesvirus retinitis. Aim 2. Determine whether US9 and US8 function as a heterodimer similarly to HSV-1 gE/gI to promote cell-cell spread of virus. Study the synthesis, processing, and transport of CMV US8 and HSV- 1 gI in MDCK cells expressing these glycoproteins. Compare heterodimer formation between US8/US9 in parallel with HSV-1 gE/gI. Analyze the transport of US8/US9 and gE/gI heterodimers and their effect on patterns of cellular proteins. Evaluate the functional homology of CMV US8/US9 by constructing a HSV-1 recombinant. Aim 3. Investigate the mechanisms by which CMV US8 and US9, HSV-1 gE and gI, and their heterodimers alter the cellular proteins that maintain polarity. Determine whether US9 and gE, and US8 and gI, function as ion transporters. As an alternative, identify cellular ligands for these viral glycoproteins, as predicted experimentally by altered properties of the actin cytoskeleton. Examine the effect of site-directed mutations on the glycoprotein functions. Construct HSV-1 and CMV recombinants with mutated forms of US8/US9 and gE/gI. Findings of these studies will have far-reaching implications for understanding pathogenic mechanisms of herpesviruses and form a foundation for developing novel strategies to arrest cell-cell spread of retinal herpesvirus infections.

由疱疹病毒，巨细胞病毒（CMV），视网膜疾病， 水痘带状疱疹病毒（VZV）和单纯疱疹病毒1（HSV-1）是 获得性免疫缺陷患者的视力丧失原因 综合征（艾滋病）。 CMV视网膜炎是一种进行性疾病，如果 未经处理，会导致视网膜脱离和失明。它从 视网膜毛细血管并通过视网膜方向传播到 偏振视网膜色素上皮（RPE）。在极化RPE的培养物中 细胞，CMV优先感染顶部表面膜和后代 病毒体从细胞遍布细胞跨连接络合物 侧膜。细胞电池传播与进入 根尖膜，如偏光细胞中，这些膜包含不同的 蛋白质。初步实验表明，CMV和HSV-1突变体 缺乏某些糖蛋白，CMV中的US8/US9和HSV-1中的GE/GI是 在极化RPE细胞中的细胞细胞扩散中显着受损， 尽管它们正常生长在未成熟的成纤维细胞中，但无法改变 紧密连接蛋白。这一令人兴奋的发现表明CMV US9和 US8是HSV-1和VZV中GE和GI的功能同源物。 HSV-1和 伪标记病毒（PRV）GE在眼部感染的发病机理中的功能 通过促进病毒在眼睛和中央的细胞细胞扩散 神经系统。 CMV US8和US9在氨基酸序列中与 Alpha疱疹病毒GE和GI，以及在A中的弧菌霍乱蛋白 毒力盒，Zonula occludens毒素（ZOT）和辅助霍乱 肠毒素（ACE），增加了肠道的渗透性 上皮细胞。这些疱疹病毒和细菌蛋白也是 类似于哺乳动物Ca2+和C1-转运蛋白的家族 预测他们可能的功能。建议调查角色 发病机理中GE/GI同源物的生物学 活动及其改变上皮极性的机制 和神经元细胞。目的如下。目标1。确定学位 CMV US9和HSV-1 GE之间的功能相似性 维持极性的细胞蛋白。比较US9-的属性 和表达GE的MDCK细胞。在极化RPE细胞中表达US9和GE 并评估维持极性和完整性的细胞蛋白的变化 肌动蛋白细胞骨架。评估US9和GE中突变体对 其他细胞类型受到疱疹病毒性视网膜炎的影响。目标2。确定 US9和US8是否与HSV-1 GE/GI相似地充当异二聚体 促进病毒的细胞细胞扩散。研究合成，加工， 在MDCK细胞中CMV US8和HSV-1 GI的运输表达这些 糖蛋白。比较US8/US9并行的异二聚体形成 与HSV-1 GE/GI。分析US8/US9和GE/GI异二聚体的运输 及其对细胞蛋白模式的影响。评估 CMV US8/US9的功能同源性通过构建HSV-1重组。 目标3。调查CMV US8和US9，HSV-1 GE和 GI及其异二聚体改变了维持的细胞蛋白 极性。确定US9和GE以及US8和GI是否起离子的功能 运输商。作为替代方案，确定这些细胞配体 病毒糖蛋白，如通过改变性能的实验预测 肌动蛋白细胞骨架。检查位置定向突变的效果 在糖蛋白功能上。与使用HSV-1和CMV重组剂 US8/US9和GE/GI的突变形式。这些研究的结果将有 了解理解病原机制的深远影响 疱疹病毒并为制定新型策略的基础 视网膜疱疹病毒感染的停止细胞细胞扩散。