Human cytomegalovirus (HCMV), a member of the herpesvirus group, is capable of causing serious medical problems that affect all age groups. This virus is one of the principal pathogens in patients with AIDS and has been implicated as a potential cofactor for the development or progression of the disease. The multiple pathogenic effects of HCMV are likely manifested through a complex interplay of viral gene products and induced and repressed cellular gene functions. HCMV immediate early and early gene expression clearly play a central role in this scheme. The research in my laboratory has been directed towards molecular analysis of HCMV genome organization and gene expression. Specific focus has been on the regulation of expression of three representative early transcription units including: the 2.7 kb and 1.2 kb RNA transcripts encoded by the repeat bonding the long unique segment of the genome; and the 2.2 kb family of RNA transcripts containing the cell-related sequences encoded by HCMV strain AD 169 EcoRI fragments R and d. In this renewal application, we propose to continue our studies on the cis-acting sequences and the trans-acting factors (both viral and cellular) necessary for control of these three early genes. For these studies, we will couple site-directed mutagenesis and functional in vivo transient expression assays with DNA-protein binding assays. To elucidate the means by which specific HCMV immediate early gene products (particularly those expressed by the IE1 and IE2 regions) activate transcription of the early genes, we propose to overexpress the proteins, purify them to homogeneity, and analyze their biochemical interaction with the early promoter elements as well as with the basic RNA polymerase II transcription machinery. With the use of in vitro transcription assays, we will analyze the effect of the IE proteins on: the overall transcription reaction: transcription initiation: commitment of the transcription complex to the DNA template; and the assembly and transcription of nucleosome templates. 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 the basis for understanding the process by which this virus causes disease. The knowledge is essential for the development of effective strategies for prevention and treatment.
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