During the replicative cycle of DNA-containing viruses there is a dramatic switch from early to late gene expression, concurrent with the onset of viral DNA replication. The mechanisms by which this switch occurs however are poorly understood. The overall goal of this proposal is to investigate the positive and negative regulatory mechanisms that operate in the late phase of adenovirus infection, an effort previously hampered by a lack of appropriate genetic tools. These include adenoviruses in which the major late promoter (MLP) is under inducible control, so that the mechanisms by which products of the major late transcription unit (MLTU) repress the expression of other viral genes can be more easily investigated. Methods to create such a virus include the use of an altered specificity version of the TATA-box binding protein (TBP) to inducibly suppress the lethality of an MLP containing a mutant TATA box. The altered specificity TBP wll be induced using the """"""""tet-on"""""""" system of Gossen and Bujard. Alternatively, a modified MLP dependent on the binding of the NF-AT1 transcription factor to the small but highly responsive NF-AT1 element will be created and tested in cell lines inducibly expressing NF-AT1. The inducible viruses will be used to measure changes in gene expression contingent on MLTU expression under various uninduced conditions. In further experiments, two possible mechanisms for late phase negative regulation will be tested. The MLTU may down-regulate expression of the E2 and E4 transcription units transcribed from the opposite DNA strand by an in vivo anti-sense mechanism. The resulting double-stranded RNA should be deaminated by Double-Stranded RNA Adenosine Deaminase, as inferred from analysis of RT-PCR products. Alternatively, down-regulation may reflect promoter competition in which the """"""""strong"""""""" MLP out-competes """"""""weaker"""""""" viral promoters. A viral test system for examining this concept is proposed. Turning to positive regulation, the MLP may be activated by the virus IVa2 gene product, which binds to two elements in the first intron of the MLTU. The phenotypic effects of a recently created null mutation in the IVa2 gene and of multiple mutations in the DNA binding sites to which IVa2 protein binds, will be examined in detail. Further genetic analysis of the structure and function of the IVa2 protein is also proposed, targeting specific protein domains and residues postulated to be important in function. The effects of these mutations will be examined in reconstructed virus genomes.
