With regard to oncology, it is critical to know how the same viral gene performs a transforming function in one cell species and yet a regulatory function during infection in another. This has been shown to be true for the EIA gene of adenovirus. The long-term objective is to understand how expression of adenovirus genes is regulated in cells infected and transformed by the virus. An immediate goal is to learn how the large acidic protein of the EIA gene activates expression of early viral gene transcription. Two approaches will be taken. The first is to study the biochemical features of the EIA protein and the second is to construct and analyze specific EIA viral mutants. The objective of these combined approaches is to define the functional properties and domains of the EIA protein required for modulation of transcription and transformation.
The specific aims will be to purify EIA proteins using our recently produced monospecific antibodies. Both purified EIA proteins and EIA antibodies will be used to determine whether EIA proteins: bind to DNA; associate with other cellular proteins; enhance in vitro transcription; as well as to determine: the size of native EIA proteins; the steady state levels of EIA proteins throughout infection; and the identity of those amino acids of EIA which are phosphorylated.
We aim to produce more EIA monoclonal antibodies to be used in these assays. Secondly, we intend to further resolve the functions of EIA by genetic analysis. The mutation within the EIA DNA of the putatively missense Ad5 host range mutants have been functionally located by analysis of wild-type/host range EIA hybrid genes; the defective region of DNA will now be sequenced. We are furthermore constructing specific EIA point mutations in Ad5 and Ad12 by oligonucleotide-directed mutagenesis. In addition, we will pursue our most recent finding that expression of class I major histocompatibility (MHC) antigens is abolished on the surface of Ad12 transformed mouse cells (highly oncogenic) but not Ad5 transformed cells (weakly oncogenic) and that only Ad12 transformed cells resist being killed in a cell-mediated cytotoxicity assay. We will investigate why H-2 class I surface antigens fail to be expressed in Ad12 transformed cells and study whether the EIA or the EIB genes of Ad12 may independently achieve MHC restriction in mouse cells.
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