ADENOVIRUS GENOME EXPRESSION: PHYSICAL MAPPING STUDIES

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The proposed research is designed to elucidate the roles of adenovirus DNA sequences in the regulation of gene expression, DNA encapsidation and evolutionary diversification. Recently described autoregulation by the adenovirus E1A gene provides a model system for the investigation. E1A promoter sequences, or derivative variants, have been joined to probe genes such as chloramphenicol acetyltransferase to conveniently measure extents of gene expression in transfected human cells in culture. The effects of cotransfecting wild type or mutant E1A genes on expression of the E1A-probe is readily determined. Evidence indicates negative autorepression and two modes of positive feedback in E1A autoregulation. These assays will be used to localize cis-targets for promoter response to E1A and functional domains within the E1A peptide(s). A cis-element specifying polar DNA encapsidation lies within the transcriptional control region of the E1A gene and this will be further characterized. The adenovirus E1A is one of the two adenovirus genes involved in the process of adenovirus induced oncogenic transformation of cells. The tumorigenicity of adenoviruses is also related to the E1A gene, in a fashion which varies with the particular subgroup of human adenovirus serotypes. This enhances interest in the regulatory properties and evolution of the adenovirus E1A gene. The B subgroup of human adenoviruses, moderately oncogenic, is comprised of two closely related but epidemiologically and pathogenically distinct clans or sub-subgroups. The B1 viruses can cause serious epidemic respiratory disease while the B2 viruses are generally recovered from asymptomatic patients subject to immunosuppression. DNA sequence analysis of the E1A and fiber genes will be performed for representative serotypes of each sub-subgroup to characterize their phylogeny and evolutionary constraints on regulatory and structural genes. This may provide further insights on the functional domains within the genes and their fate through evolution. Sequences from B and C subgroup adenoviruses will be cloned into bacterial expression vectors and screened using sera from individuals having been infected or vaccinated with specific adenoviruses. Immunopositive clones will be mapped by hybridization and DNA sequence to the respective viral genomes to identify DNA sequences which encode antigenic determinants. This background will be useful in the further efforts to develop a practical adenovirus vaccine vector.