CMV DISRUPTION OF CONSTITUTIVE MHC CLASS II

人类巨细胞病毒描述(逐字摘自申请者摘要) 巨细胞病毒(CMV)是导致移植患者发病和死亡的重要病毒原因。 收件人。在这些患者中，巨细胞病毒病通常是由于重新激活 潜伏的和持久的病毒。持之以恒的一个核心机制是 通过宿主T淋巴细胞介导的免疫监视逃脱CMV的检测， CD8T淋巴细胞和CD4T淋巴细胞均可介导。在一个 类似于CMV编码多个基因扰乱MHC I类的方式，它 现在看来，CMV已经开发出逃避CD4T淋巴细胞的机制 通过破坏MHC-II类分子介导的免疫监测 表情。在感染细胞内，CMV抑制干扰素-g诱导的MHC II类 通过干扰干扰素-g信号通路的机制来表达。最近一段时间 巨细胞病毒对MHC-II类分子表达的影响已扩大到包括 通过其基因US2抑制组成性表达的第II类。使用 构建表达人类白细胞抗原II类的细胞系，我们发现了一个主要的 巨细胞病毒介导的不依赖US2的表面II类表达的降低 或蛋白酶体降解。我们的北方显微镜、西方显微镜和共聚焦显微镜 研究表明，这种机制是成熟阶级贩运中的一个缺陷。 II到细胞表面。此外，这一机制专门针对第二类， 由于对缺乏改变I类基因的突变CMV的研究表明 I类表达正常至增加，但II类表面表达减少 表情。根据我们的初步数据，我们将检验CMV 通过改变细胞内的小泡来特异性地阻断第二类转运 贩卖机器。在具体目标一中，我们将进一步描述 巨细胞病毒感染对MHC-II类分子表达的影响运输和组装 感染细胞中MHC II类分子的数量将通过 免疫共沉淀、蛋白质印迹分析和共聚焦显微镜。在……里面 特异靶II，巨细胞病毒介导的本构降低的机制(S) 将调查MHC II类的表达，特别是检查其作用 巨细胞病毒介导的肌动蛋白和微管网络的破坏 抑制力。我们将量化CMV对聚合、裂解的影响 和这些结构的完整性，并确定了(S)CMV用来 抑制他们运输II类阳性囊泡的能力。以特定的目标 CMV Towne株COSMID对U373-CIITA系的稳定转化 克隆将与CMV cDNA文库结合使用，以鉴定和 分离导致结构性MHC II类分子减少的CMV基因 表面表达。

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.