A major focus of this proposal is to elucidate the mechanism by which the large number of alternatively spliced HIV mRNAs are generated. HIV has three size classes of mRNAs, all generated from a single primary RNA transcript: 9kb unspliced mRNA, 4-5kb singly-spliced mRNAs, and 2kb multiply-spliced mRNAs. The hypothesis to be tested is that each of the individual splicing events must be either inefficient or regulated in some way. The efficiency of individual HIV 5' and 3' splice sites will be tested in vivo in combination with known, efficient splice sites. These experiments will be carried out with and without Rev (and the RRE). If some of the HIV splice sites are observed to be used efficiently in the chimeric constructs, we will search for cis-acting sequences that could moderate their splicing efficiency (either in the vicinity of the splice site or at some distance from it). HIV cis-acting elements involved in control of RNA processing will be compared to elements previously studied in RSV, including cis-acting negative regulators of splicing (NRS) within the gag gene and near the src 3' splice site, as well as an inefficient env 3' splice site. A cis-acting sequence has been identified in the env region of HIV that inhibits splicing of a heterologous intron in an orientation-dependent manner. Function of this HIV element will be further compared to that of the RSV gag NRS in in vitro splicing assays. If pre- mRNA splicing is blocked by this element, spliceosome formation will be monitored in native gels and by immune precipitation using antibodies to splicing factors. Factors binding to the sequence will also be identified using UV-crosslinking or gel shift analysis. Mutagenesis studies will be carried out to determine the critical sequences in the element, and its secondary and tertiary structure will be determined. It has been proposed that cis repressive sequences (CRS) play a role in nuclear retention of HIV unspliced and singly-spliced RNA in the absence of Rev, although the mechanism is unknown. The potential role of CRS elements in splicing will be studied both in vivo, using a heterologous intron construct, and in an in vitro splicing system. The HIV env sequence that inhibits splicing will also be tested for CRS activity. Unspliced, RSV NRS-containing heterologous RNA is confined to the nucleus in association with components of the splicing apparatus; we will determine if Rev in trans and the RRE in cis will elicit cytoplasmic appearance of this RNA. Lastly, the effect of premature translation termination within the gag region of HIV on unspliced RNA stability will be probed. Insertion of termination codons within the analogous region of RSV results in a marked decrease in unspliced RNA accumulation, due to decreased RNA stability. Any role of the Rev protein in RNA stability of these mutants will also be evaluated.