Human cytomegalovirus (HCMV), a member of the herpesvirus family, is an important pathogen capable of causing a variety of diseases. HCMV is the leading viral cause of birth defects and poses a serious health threat to immunocompromised individuals, particularly those with AIDS. The multiple pathogenic effects of HCMV are likely manifested through a complex interplay of viral gene products and induced and repressed cellular functions. The research in Dr. Spector's laboratory has been directed towards characterizing the sequence requirements and trans- acting factors (both viral and cellular) necessary for the control of three representative early transcription units including: the 2.7 kb and 1.2 kb RNA transcripts encoded by the repeat bounding the long unique segment of the genome; and the 2.2 kb family of RNA transcripts encoding 4 related nuclear phosphoproteins. In this renewal application, the investigator proposes to continue their studies on the regulation of HCMV early gene expression and the functional properties of the major viral transactivator, an immediate early protein designated IE2 86. The approach is to couple functional in vivo genetic analyses with in vitro biochemical and molecular assays to achieve the following aims: (1) determination of the cis-acting regulatory elements on HCMV early promoters when the promoters are part of the infectious viral genome; (2) analysis of the function of the IE2 86 protein in the context of mutant recombinant viruses; (3) identification and characterization of the trans-acting cellular factors interacting with the HCMV 1.2 kb and 2.2 kb early RNA promoters; (4) use of in vitro RNA pol I transcription reactions to assess the functional basis of interaction of the IE2 86 protein with the upstream binding factor UBF; and (5) utilization of in vitro RNA pol II transcription reactions to determine the molecular mechanisms by which the IE2 86 protein stimulates HCMV early promoters. The long range goals of this research are to define at the molecular level the mechanisms which operate to control HCMV gene expression, thus providing an important foundation for understanding how the complex interactions of viral and host functions relate to the pathogenesis of the virus.
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