CMV DISRUPTION OF CONSTITUTIVE MHC CLASS II

CMV 破坏 MHC II 类结构

基本信息

批准号：
6288030
负责人：
Daniel D Sedmak
金额：
$ 25.73万
依托单位：
OHIO STATE UNIVERSITY
依托单位国家：
美国
项目类别：
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

项目摘要

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（逐字化申请人摘要）人类巨细胞病毒（CMV）是移植中发病率和死亡率的重要病毒原因收件人。在这些患者中，CMV疾病通常是由潜在病毒。持久性的中心机制是能力 CMV的宿主T淋巴细胞介导的免疫监视的逃避检测，这是由CD8+ T淋巴细胞和CD4+ T淋巴细胞介导的。在类似于CMV编码多个破坏MHC I类的基因现在看来CMV已经开发了逃避CD4+ T淋巴细胞的机制通过中断MHC II类分子的介导的免疫护理表达。在受感染的细胞中，CMV抑制IFN-G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MHC II类表达的影响已扩展到包括通过其基因US2抑制组成型表达的II类。使用组成型HLA II类表达细胞系，我们发现了一个主要 CMV介导的表面II类表达的降低与US2无关或蛋白酶体降解。我们的北部，西部和共聚焦显微镜研究表明，该机制是成熟阶级运输的缺陷 II到细胞表面。此外，该机制专门针对II类由于使用突变CMV缺乏改变类别的基因证明的基因正常表达I类表达，但II类表面降低表达。根据我们的初步数据，我们将测试CMV的假设特异性通过改变细胞内囊泡来阻止II类贩运贩运机械。在特定目的I中，我们将进一步描述 CMV感染对MHC II类表达的影响。运输和集会将使用感染细胞中的MHC II类分子的共免疫沉淀，蛋白质印迹分析和共聚焦显微镜。在特定的目标II，CMV介导的本构降低的机制将研究MHC II类表达式，专门研究该角色 CMV介导的肌动蛋白和微管网络的破坏抑制。我们将量化CMV对聚合，裂解的影响这些结构的完整性，并确定CMV使用的机制抑制其交通II类阳性囊泡的能力。在特定目标中 iii，使用CMV Towne菌株Cosmid的U373-CIITA线的稳定转染克隆将与CMV cDNA库一起使用，以识别和分离构成构型MHC II类减少的CMV基因表面表达。