RETROVIRUS REGULATION IN VITRO AND DURING DEVELOPMENT

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The long range goal of the proposed experiments is to understand how retroviral sequences mediate control of gene expression under permissive conditions such as in plasmid molecules introduced into cultured cells and how such sequences overcome developmental signals to induce chromatin structural changes and aberrant expression of genes in flanking cellular DNA after insertion into the genome. These questions are important in understanding both viral regulation and viral pathogenesis, particularly the ability of integrated proviruses to modulate the expression of adjacent cellular oncogenes in provirus- induced tumor-derived cells. Previous work has focused on the expression of avain endogenous viruses during development, and the interactions between integrated proviruses and adjacent cellular DNA. The focus in this proposal is on virus expression during avian erythorpoiesis and the cis-effects induced by integrated proviruses on the chormatin structure and transcriptional activity of flanking host sequences in these cells. This will be accomplished using the endogenous provirus ev-6, which is drived off of a cellular promoter. Analysis of this virus and adjacent host sequences will therefore allow the investigation of the effects of provirus integration on the activity of a known promoter element. Previous results indicate that differentiating erythrocytes are an interesting system to investigate the relationship between sequence content, chromatin structure and transcription due to the fact that, while these cells undergo a global condensation and transcriptional inactivation of most cellular genes, proviral DNA is able to escape both of these processes. The second set of experiments seeks to complement and extend the in vivo experiments described above by directly analyzing the role of three viral sequences from pathogenic, exogenous viruses and non-pathogenic, endogenous viruses in transcriptional regulation of linked LTRs. The aim of these studies is to define sequences involved in transcription regulation of viral and adjacent host-specific sequences. The three regions from endogenous and exogenous viruses that will be tested are teh U3 regions from proviral LTRs, a region in the env gene which, as with other proviral and cellular transcriptional regulatory regions, is included in a nuclease hypersensitive site and is specifically protected from de novo methylation during development and the 3' untranslated region from endogenous viruses, which has been implicated in inhibiting the enhancer/promoter activity of linked LTRs. These experiments will be conducted by analyzing RNA expression of recombinant, transfected plasmids by transient expression assays.