The long range goals of this research are to define at the molecular level the expression of the human cytomegalovirus (HCMV) genome. Specific focus has been on the regulation of expression of three representative early transcription units including the major 2.7 kb and 1.2 kb RNAs and the 2.2kb family of transcripts. In this application, the PI proposes to continue studies on the cis- acting sequences and trans-acting factors (both viral and cellular) necessary for the control of these three early genes. For these studies, site-directed mutagenesis and in vivo transient expression assays will be coupled with DNA-protein binding and in vitro transcription 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 early genes. The proteins will be overexpressed, 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. The proposed studies are important not only to advance the knowledge of the regulation of the viral life cycle, but also to help elucidate the general mechanisms that operate to control eukaryotic gene expression.
