CMV DISRUPTION OF CONSTITUTIVE MHC CLASS II

CMV 破坏 MHC II 类结构

• 批准号: 6626385
• 负责人: William James Waldman
• 金额: $ 26.26万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6626385
• 关键词: MHC class II antigen; actins; confocal scanning microscopy; cytomegalovirus; flow cytometry; gene expression; genetic library; genetic mapping; immunofluorescence technique; immunoprecipitation; intracellular transport; latent virus infection; microtubules; molecular assembly /self assembly; plasmids; protein folding; transfection; vesicle /vacuole; virus cytopathogenic effect; virus infection mechanism; virus protein; western blottings

DESCRIPTION (Verbatim from the applicant's abstract) Human cytomegalovirus (CMV) is a significant viral cause of morbidity and mortality in transplant recipients. In these patients CMV disease often results from reactivation of latent and persistent virus. A central mechanism of persistence is the ability of CMV to escape detection by host T lymphocyte-mediated immuno-surveillance, which is mediated by both CD8+ T lymphocytes and CD4+ T lymphocytes. In an analogous fashion to CMV encoding multiple genes which disrupt MHC class I, it now appears that CMV has developed mechanisms to evade CD4+ T lymphocyte mediated immuno-surveillance through disruption of MHC class II molecule expression. Within infected cells, CMV inhibits IFN-g induced MHC class II expression by mechanisms that disrupt the IFN-g signaling pathway. Recently the effect of CMV on MHC class II expression has been expanded to include inhibition of constitutively expressed class II, by its gene US2. Using a constitutive HLA class II expressing cell line, we have found a major CMV-mediated decrease in surface class II expression that is independent of US2 or proteasomal degradation. Our Northern, Western and confocal microscopy studies suggest that the mechanism is a defect in trafficking of mature class II to the cell surface. Moreover, this mechanism specifically targets class II, since studies with mutant CMV lacking the genes that alter class I demonstrate normal to increased class I expression, but decreased class II surface expression. Based on our preliminary data, we will test the hypothesis that CMV specifically blocks class II trafficking by altering the intracellular vesicle trafficking machinery. In Specific Aim I, we will further characterize the effect of CMV infection on MHC class II expression. The transport and assembly of MHC class II molecules in infected cells will be examined using co-immunoprecipitation, Western blot analyses, and confocal microscopy. In Specific Aim II, the mechanism(s) of the CMV-mediated decrease in constitutive MHC class II expression will be investigated, specifically examining the role of CMV-mediated disruption of the actin and microtubule networks in this inhibition. We will quantify the effect of CMV on the polymerization, cleavage and integrity of these structures and determine the mechanism(s) CMV uses to inhibit their ability to traffic class II positive vesicles. In Specific Aim III, stable transfections of the U373-CIITA line with CMV Towne strain cosmid clones will be used, in conjunction with a CMV cDNA library, to identify and isolate the CMV genes responsible for the decrease in constitutive MHC class II surface expression.

描述（来自申请人摘要的逐字描述）人巨细胞病毒 (CMV)是移植中发病率和死亡率重要病毒原因 受惠人士在这些患者中，CMV疾病通常是由于 潜伏和持久病毒。坚持的核心机制是 CMV逃避宿主T淋巴细胞介导的免疫监视的检测， 其由CD 8 + T淋巴细胞和CD 4 + T淋巴细胞介导。中 与编码破坏MHC I类的多个基因的CMV类似， 现在看来，CMV已经开发出了逃避CD 4 + T淋巴细胞的机制， 通过破坏MHC II类分子介导的免疫监视 表情在感染的细胞内，CMV抑制IFN-γ诱导的MHC II类 通过破坏IFN-g信号通路的机制表达。最近 CMV对MHCII类表达的影响已经扩大到包括 通过其基因US 2抑制组成型表达的II类。使用 组成型HLA II类表达细胞系，我们已经发现了一个主要的 CMV介导的不依赖于US 2的表面II类蛋白表达降低 或蛋白酶体降解。我们的北方、西方和共聚焦显微镜 研究表明，成熟类的贩运机制存在缺陷 II至细胞表面。此外，这一机制专门针对第二类， 由于缺乏改变I类基因的突变CMV的研究表明， I类表达正常至增加，但II类表面减少 表情根据我们的初步数据，我们将测试CMV 通过改变胞内囊泡特异性阻断II类转运 贩卖机器在具体目标I中，我们将进一步描述 CMV感染对MHC II类分子表达影响。所述运输及组装 在感染细胞中的MHC II类分子将被检查， 免疫共沉淀、Western印迹分析和共聚焦显微镜。在 具体目标II，CMV介导的组成性降低的机制 将研究MHC II类表达，特别是研究其作用。 CMV介导的肌动蛋白和微管网络的破坏， 抑制作用我们将量化CMV对聚合、裂解和降解的影响， 这些结构的完整性，并确定CMV用于 抑制其运输II类阳性囊泡的能力。具体目标 III，用CMV Towne株粘粒稳定转染U373-CIITA系 克隆将与CMV cDNA文库结合使用，以鉴定和 分离导致组成型MHC II类减少的CMV基因， 表面表达