Our continued long term objective is to gain an understanding of the molecular basis of the selective and sequential regulation of eukaryotic gene expression. Several novel features of frog virus 3 (FV3) replication in eukaryotic cells make it an unusual and attractive model in progressing towards our objective. We will investigate mechanisms of virus imposed controls on macromolecular synthesis both in infected cells and in vitro from two perspectives: (1) the selective effect of the virus on host cell macromolecular synthesis, and (2) the regulation by the virus of its own macromolecular synthesis. This will be accomplished by meeting the following specific aims. 1. To determine the structural and functional organization of the FV3 genome by genetic, transcriptional and translational mapping. 2. To define the mechanisms of four separate and sequential transcriptional controls in FV3-infected cells: (a) switch-off of host cell transcription; (b) switch-on of delayed early and late FV3 transcription; (c) switch-off of immediate early transcripts late in infection; and (d) transcription of methylated FV3 DNA. These will be studied by analyzing (i) regulatory DNA promoter sequences, (ii) cellular and viral RNA polymerases, and (iii) virus-specific transcription co-factors. 3. To define the mechanisms of three selective translational controls of protein synthesis in FV3-infected cells: (a) switch-off of host cell protein synthesis; (b) enhancement of translation of late FV3 mRNAs; and (c) inhibition of translation of early FV3 mRNAs late in infection. These will be studied by (i) identification of the viral proteins involved, (ii) analysis of modification of the cellular translational machinery, and (iii) analysis of sequences at the 5 feet end of mRNAs. Cancer is a state of uncontrolled cell growth where the precise regulation of gene expression is disrupted. We expect our studies on regulatory mechanisms of FV3 genes to extend to elucidation of mechanisms controlling gene expression in normal and malignant cells. The highly methylated FV3 genome offers a unique opportunity to gain insight into the role of DNA methylation during differentiation and in neoplasia. Our studies on the switch-off of host-cell macromolecular synthesis should provide clues to mechanisms by which viruses produce cytopathology, knowledge needed for a rational approach to treatment of viral diseases.
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