Control of Rous sarcoma virus (RSV) RNA splicing will be studied both in vivo and in vitro. RSV RNA splicing is incomplete, like that of all retroviruses, generating unspliced primary transcripts that serve both as packaged, genomic RNAs and as mRNAs for the gag and pol polyproteins. When splicing does occur, a common 5' splice site is alternatively spliced to either the env or src 3' splice site to generate these subgenomic mRNAs. The mechanism of action of a Negative Regulator of Splicing (NRS) present in the gag gene of the unspliced RNA (part of the intron of the spliced mRNAs) will be studied in detail. The functional significance of binding of Ui, U2, and Ui 11U12 small nuclear ribonucleoproteins (snRNPs) will be assessed. Binding sites for these snRNPs on the NRS will be mapped and subsequently mutated. For these experiments, biotinylated RNA substrates will be incubated in a HeLa nuclear extract, and the complexes will be affinity selected. The composition of the complexes will be determined by Northern analysis. The effects of these mutations will be assessed in in vitro splicing experiments and in transfected cells, using both heterologous NRS-containing constructs and complete viral genomes. If binding of specific snRNPs correlates with NRS activity, we will attempt to restore activity to mutated NRS constructs, by making compensatory mutations in the snRNAs. Protein factors interacting with the NRS will be identified by cross-linking experiments. Structure of the NRS RNA will be determined. Cellular mutants resistant to NRS activity will be selected, using a Neo construct bearing an NRS-containing intron. The possible role of the NRS in polyadenylation of RSV RNA will be investigated; if such a role is confirmed, its mechanism of action will be studied. We will also study the role of an NRS deletion in recombinant avian leukosis viruses that induce early B cell lymphomas. Finally, transport of viral RNA from the nucleus to the cytoplasm will be studied, and the subcellular localization of viral RNA will be determined by in situ hybridization.
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