Cytomegalovirus (CMV) infections are a major cause of morbidity and mortality in solid organ and bone marrow transplant recipients, and it is the long term goal of Dr. Sedmak's laboratory to understand the mechanisms of CMV disease in these patients. While it is known that these CMV infections frequently arise from dissemination of persistent virus acquired prior to transplantation, the underlying mechanisms of the persistence are poorly understood. The laboratory has discovered and reported the finding that CMV, in vitro, inhibits induced HLA class II protein and mRNA expression within infected endothelial cells and constitutive HLA class II protein within infected macrophages. This inhibition requires viable virus and is not secondary to the effect of alpha or beta interferons. More importantly, the investigators have recently demonstrated that this phenomenon occurs in vivo, as found in patients with pulmonary CMV infections. The molecular mechanisms by which CMV inhibits HLA class II antigen transcription are unknown. Based upon preliminary data, the investigators hypothesize that CMV immediate early proteins play a major role in the inhibition of class II transcription, which will be investigated in Specific Aim I. They also hypothesize that sequences of CMV genomic DNA homologous to conserved regions within the HLA class II promoter sequester critical HLA class II transcription factors, which will be investigated in Specific Aim II. These hypotheses will be tested using a number of techniques including transfection and co-transfection assays with CMV IE proteins expression plasmids and an HLA DRa reporter construct (pDRa-syn-LUC), electroporation of purified CMV IE proteins, immunoprecipitation, electrophoretic mobility shift assays (EMSA), and Western blot analysis.
