We propose to continue studies on the molecular genetic analysis of the early gene blocks of human adenovirus 2 (Ad2). Our studies will focus on the transforming early genes E1a (map position (mp) 1.2-4.4), and E1b (mp 5.1-11.2), and early gene block L1 (mp 30-39) whose function is unknown. Our earlier studies have shown that a single E1a protein (243R) coded by a 12S mRNA can immortalize and fully transform rat embryo fibroblasts (REF) in cooperation with E1b. We propose experiments to gain insights into the mechanism of the 243R protein-mediated cell transformation. A detailed mutational analysis of the 12S gene using deletion and point mutants will reveal the critical domains and the various forms of the 243R protein involved in cell transformation. Small in-phase deletion and point mutations will be introduced into the 243R T antigen coding sequence. The effect of the mutations on cell transformation will be assessed using the defective M.MuLV vector or by DNA transfection via protoplast fusion. The cellular genes whose expression is altered by the 243R T antigen will be identified by cloning cDNA of cellular transcripts. The identity of the cellular genes will be determined by direct DNA sequence analysis and by comparison with known protein coding sequences. The effect of the various mutations on the regulation of expression of cellular genes will also be determined. We will investigate whether the Ad2 E1a 289R T antigen or the N-terminal fragments of this protein can alone immortalize primary REF cells. Using the immortalized cells, we will investigate the alterations in expression of cellular genes and identify those cellular genes by cDNA cloning and by DNA sequence analysis. The role of the Ad2 E1b 495R T antigen and related proteins in cell transformation will be determined by transfection with DNA fragments in which the ATG initiation codon for the 495R protein will be mutagenized. The fuctions of the newly identified Ad2 E1b 155R protein in cell transformation and in productive viral infection will be determined by using a specific viral mutant defective in the biogenesis of the mRNA coding for the protein. Attempts will be made to identify the putative proteins coded by open reading frames (URFs) present on the 1 strand of the E1 region. The functions of the URF-coded proteins will be assessed using specific viral mutants. The functions of the 52-55 kd proteins coded by the L1 region in regulation of gene expression of viral early and late genes will be determined using suppressible nonsense mutants or host range deletion mutants, or by inducing synthesis of antisense L1 RNA.
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