CYTOMEGALOVIRUS GENE REGULATION IN IMMUNODEFICIENCY

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Human cytomegalovirus (CMV) is a major opportunistic pathogen affecting AIDS patients. This virus has been suggested to be one of several possible contributors to progression in this disease. Viral pathogenesis and determinants of replication, persistence and latency are key to reactivation and dissemination of CMV in AIDS patients but mechanisms remain poorly defined because of a lack of appropriate model systems for this species specific virus. Bone marrow derived hematopoietic cells have been thought to be an important natural reservoir of latent virus. This application will identify bone marrow cell types capable of harboring the viral genome, focussing on the granulocyte-macrophage (GM) lineage, and will investigate the extent of viral gene expression and the form that the viral genome takes in these cells. First, we will investigate the quiescent infection of the GM lineage cells in culture as a model of viral latency, studying the expression of the ie1 gene that has been detected in these cells, identifying other viral transcripts and analyzing the form of the viral genome. This information will be used to guide investigations in experimentally infected SCID-hu (bone/bone marrow) mice and naturally infected bone marrow cells from seropositive individuals. Second, we will investigate the signal(s) and mechanism(s) of viral reactivation when infected GM precursors are subjected to long-term coculture with permissive human fibroblast cells. Factors that stimulate differentiation and growth of GM precursors will be evaluated as a means to increase efficiency of reactivation. Parameters for reactivation that efficiently reactivate virus in the cultured GM cell model and in SCID-hu mice will be applied to peripheral blood or bone marrow cells of naturally infected individuals. The long term aim is to establish in biological assay for a process that has never yielded to experimental manipulation; virus has only been shown to reactivate when an individual is severely immunosuppressed, or when latently infected blood or tissues are transfused or transplanted into another human being. Third, we will investigate the viral genetic basis for latency (establishment and reactivation) using the GM cell culture and SCID-hu mouse models. Individual regulatory genes (ie1, ie2) and newly identified latent infection associated genes will be deleted from the viral genome and the resultant mutant viruses will be evaluated for their biological phenotype in establishment of latency and ability to reactivate. Taken together, the proposed research will define the biological spectrum of infection in bone marrow and will establish the contribution of viral functions that control latency and reactivation. This information will provide information on viral latency that should directly impact on the approaches to control reactivation following immunosuppression and transplantation.