CMV DISRUPTION OF CONSTITUTIVE MHC CLASS II

描述（摘自申请人摘要）人巨细胞病毒

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.