The cytomegaloviruses (CMV) are large species-specific DNA viruses in the herpesvirus group, which are capable of producing persistent and latent infections. The serious problems human CMV (HCMV) transmission poses in blood transfusions and organ transplantation and the increased morbidity and mortality associated with HCMV infection in immunocompromised individuals make it imperative to understand the pathogenesis of this virus and to develop strategies for treatment and prevention. The major objective of our research is to utilize murine CMV (MCMV) infections in mice as an animal model system for uniting the studies on the molecular biology and in vivo pathology of the virus. We have already cloned and characterized subgenomic fragments representative of the entire genome of MCMTV and have used these reagents to study the molecular organization of the MCMV genome, the transcriptional patterns during the permissive infection, the mechanisms involved in the replication of MCMV DNA, and the pathogenesis of the virus in vivo. Our studies have shown that during the acute infection, the primary target in the spleen is the factor VIII-related antigen positive sinusoidal lining cell and that latent MCMV is associated with the stromal cells in the spleen. We also have recently identified the putative MCMV DNA polymerase and glycoprotein B genes and have initiated studies to create viral mutants in essential viral functions. In this renewal application, we plan to continue to study the molecular biology of the in vivo infections. In particular, we propose to use cell fractionation, in vitro reactivation, PCR analysis, in situ cytohybridization, and immunohistochemical staining to precisely identify the latently infected cell(s). We also plan to use viral mutants in the MCMV early el gene and the DNA polymerase gene to examine the viral functions governing the establishment and maintenance of latency. In addition, we propose to develop a vaccine consisting of three vaccine virus recombinants individually expressing the MCMV immediate early IE1, the early el, and the glycoprotein B gene products and to test its ability to prevent infection and establishment of latency by MCMV. The long range goals of this research are to provide the essential foundation for defining the molecular events involved in CMV pathogenesis in humans and for developing approaches for interception of the virus.
