The overall objective is to elucidate the nature and mechanism of action of those host and viral-encoded factors which regulate the transcription of both viral and host genes in cells productively or abortively infected by, or transformed by, adenovirus 2. Using purified DNA templates we have demonstrated accurate in vitro transcription of host and adenoviral (early, intermediate, land late) class II genes and host and viral (VA RNA) class III genes in systems reconstituted with crude extracts or purified RNA polymerases and partially purified host factors. We now propose to (1) complete the resolution and purification to homogeneity of the various host transcription factors, using a battery of conventional and more sophisticated techniques, (2) to determine the nature, specificity, and site and mechanism of action of each, as well as quantitative or qualitative variations in each during infection/transformation, (3) to identify, by complementation of purified systems with known viral proteins or with partially purified components from infected cells, viral regulatory factors and to similarly characterize these, (4) as part of the above, to analyze the structure and function of various natural and reconstituted nucleoprotein templates/transcription complexes and their role in transcriptional regulation (particularly by viral proteins), (5) as part of the above, to use site-directed mutagenesis to determine specific DNA sequences important for expression and regulation in vitro, and (6) to complement (and verify) the in vitro analyses with corresponding determinations of those cis-acting DNA sequences and transacting viral genes (products) required for expression and regulation in vivo (using various viral infection and viral gene transfection/transformation protocols). While adenovrirus genes will be emphasized initially, a long-range goal is to include similar studies of virus-modulated host genes.
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