Human cytomegalovirus (CMV) is a major pathogen affecting the developing fetus and immunocompromised individuals. This virus is an important opportunistic pathogen and possible cofactor in AIDS. This grant will dissect the regulatory role of two murine CMV genetic elements, the enhancer and an alpha gene (ie2), in latency using biochemical and genetic approaches. Work will focus on these two genetic elements because they are already known to influence latency and we will test the hypothesis that this effect may be at the level of transcriptional regulation. Preliminary work with murine CMV mutants in latently infected BALB/c mice has established that ie2 is important for efficient reactivation of latent murine CMV and that enhancer function may override the need for ie2 for latency reactivation. Further, ie2 has significant sequence similarity to the Epstein-Barr virus Z transactivator, which is thought to regulate reactivation, as well as other transcription factors. The cellular localization of ie2 will be evaluated with specific antibody and the functional domains of ie2 will be identified through construction of mutants, particularly in putative leucine zipper and charged regions of ie2. The function of ie2 as a transactivator of the enhancer will be investigated using transient transfection assays and the importance of the enhancer and enhancer elements as targets for ie2-mediated transactivation will be determined using viral mutants and transient assays. The human CMV enhancer elements that confer efficient ie2-independent reactivation will also be mapped. Biochemical assays will be used to identify and study the interaction of ie2 or other proteins with enhancer target sequences. Furthermore, ie2:protein interactions will be identified by immunoprecipitation and mobility shift analyses. Viral plaque assays as well as sensitive in situ assays for the detection, quantitation and localization of recombinant virus and viral gene expression in infected tissues will be used to help determine the phenotype of different mutants. We will use the expression of beta-galactosidase from wild type and mutant viruses to localize virus and to study viral gene expression in cells that harbor acute, persistent and latent virus, paying particular attention to differences between wild type and mutant viruses. This analysis will identify the cell type(s) in which murine CMV remains latent. This project will provide direct information on specific mechanisms of murine CMV latency, tissue tropism and pathogenesis. Based on the presumed or established existence of genetic elements similar to the enhancer and ie2 in human CMV, an attempt will be made to test function of human CMV homologues both in transient transfection assays and in the murine CMV genome. Should these approaches be possible, specific information on the role of human CMV genetic elements in latency may result from this grant as well.
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