A simple retrovirus, avian sarcoma virus (ASV), will be used to study how viral RNA processing is regulated in permissive and nonpermissive cells as a modal for understanding latent human retroviral infections and viral oncogenesis. The ASV-infected cell is an excellent model system for investigating cis-acting elements in permissive chicken and nonpermissive mammalian cells that determine alternative RNA splicing responses, 3' RNA cleavage and polyadenylation, and RNA transport from the nucleus to the cytoplasm.
The aims of the proposal are: (1) to investigate aberrant splicing of ASV RNA in nonpermissive mammalian cell lines and to identify cis-acting signals that interact with negative host factors in chicken embryo fibroblasts. The methods to be used include analysis of RNA splicing in mammalian-avian cell heterokaryons, deletion and site-specific mutagenesis of cis-acting sites, identification of RNA binding proteins by mobility shift assays, UV crosslinking, and in vitro splicing assays; (2) to determine if the presence of RNA splicing signals increase the efficiency of processing at the viral 3' cleavage and polyadenylation site. We will correlate the effects of mutations in cis splicing signals with effects on 3' processing to investigate the possible coupling between the two processing events; (3) to identify cis-acting signals in the ASV genome that are necessary and sufficient for transport of unspliced RNA from the nucleus to the cytoplasm. We will develop a method, using cytoplasmic expression of Beta-galactosidase, to screen for mutants defective in RNA transport. Cis-acting fragments from the viral genome will be cloned into heterologous genes to test whether their presence is sufficient to allow transport of unspliced RNA. The information obtained in the proposed studies will add to our knowledge of the role of RNA processing in the maintenance of latent retrovirus infections. It will also add to the understanding of how cellular RNA processing is regulated.
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