Human cytomegalovirus is a nearly ubiquitous pathogen that can be associated with high morbidity and mortality in the immunocompromised including organ transplant and AIDS patients and can cause serious CNS sequelae in the developing fetus or newborn infants. Despite encoding more than 170 distinct proteins, HCMV, like other DNA viruses, utilizes just a small number of transacting regulatory factors and specialized cis-acting promoter-enhancer regions to initiate and control the productive lytic cycle infection process. These immediate-early promoter-enhancers are in essence designed to sense the differentiation state and environment of a cell into which the viral genome enters and to subsequently either operate at high efficiency, shut-down or respond to appropriate induction events depending on the particular combination of transcription factors present. The corresponding regulatory transactivator proteins in turn either stimulate appropriate cellular genes and downstream promoters in the viral gene expression cascade or trigger the switch into or out of a """"""""quiescent"""""""" latent state. The particular combination of cis and trans-acting components found in HCMV and SCMV are unique to the beta-class of herpes viruses and have presumably evolved to become tailored to the specialized biological """"""""niche"""""""" occupied by cytomegaloviruses in the human host. Knowledge of the detailed mechanisms of the trans-acting virus-cell understanding of the factors that govern permissive, persistent and latent infections and could provide a basis for eventual successful therapeutic intervention. During the current budget period we propose to continue our extensive structure/function analysis of the role of the 82-kD IE2 transactivator protein of HCMV in initiating productive infection, as well as to use molecular genetic approaches to further evaluate the mechanism of positive and negative regulation by IE2. The experimental approaches used include site-directed or PCR mutagenesis and domain swapping plus transient DNA- cotransfection assays to map functional domains together with in vitro transcription and translation to detect DNA:protein and protein:protein interactions.
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