The general aim of this proposal is the identification and characterization of regulatory sequences internal to the long terminal repeats (LTRs) of avian retroviruses. A cis-acting transcriptional enhancer sequence has been identified within the gag gene of Rous sarcoma virus (RSV) and other avian retroviruses. This enhancer has been localized, by deletion analysis, between nt 786 and -900, downstream from the transcriptional initiation site. The current aim is to assess the biological significance of this sequence for viral replication. Viral mutants will be generated by recombinant DNA techniques. Steady-state levels of viral will be determined by RNase-protection assays and normalized relative to levels of viral DNA in cells. nuclear run-on assays will also be carried out to look at transcription more directly. Transcription from the 3' LTR will be compared with that from the 5' LTR to determine whether the gag enhancer affects choice of LTRs. Tissue- specificity of the gag enhancer sequence will be studied in transgenic mice. Viral or cellular proteins which interact with the gag enhancer sequence will be identified, and their possible interaction with LTR enhancers will be investigated. Retroviral RNA splicing will be studied to try to understand the mechanism regulating the incompleteness of the splicing reaction. This incomplete splicing is necessary so that unspliced RNA is transported to the cytoplasm for translation to yield the gag and pol gene products, as well as for packaging into virions. Deletion of either nt700-800 or 880- 990 from the PR-C RSV genome has been found to cause a 10 to 20-fold increase in the amount of spliced viral RNA in a transient assays. Both of these sequences are necessary for the normal low level of splicing. The mechanism of this inhibition of splicing will be investigated in in vitro systems. The hypothesis that these sequences are directly involved in inhibition of splicing, either through formation of secondary structure or of specific interaction with cellular factors, will be tested. Alternatively, these sequences may be implicated in transport of the viral RNA from the nucleus to the cytoplasm. these cis-acting elements will be further delineated by deletion and base-substitution mutagenesis. Their secondary structure will be probed chemically and with nucleases. The effect of mutations on choice of splice acceptor will be studied. Factors regulating the stability of RSV RNA will also be investigated by generation and analysis of viral mutants.
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