The overall objective of this research project is to understand the host transcription factors involved in the determination of species-specific and tissue-specific expression of Rous sarcoma virus genome. The long terminal repeats (LTR) of the virus contain powerful cis-acting elements to which host factors bind and activate transcription. For some time we have been studying in detail the molecular reactions between host factors and cis- sequences in the promoter-enhancer region (U3) of the RSV lTR. We, and others, have found that at least three well-defined cis elements are required for maximal expression of LTR-linked genes and three different factors interact with these motifs. We have also succeeded in isolating three different yet related cDNA clones encoding polypeptides that specifically bind to distinct sequence motifs. We have shown that one of these factors, mainly present in the avian fibroblasts, and in muscle tissue, binds to a specific site, E3/E3-1, localized at nucleotides -151 to -171. The importance of this cis-acting motif for viral LTR function has not ben examined. The aims of the current study are; (i) to define the role of the cis-acting sequence E3/E3-1 (-151 to -171) in LTR function and characterize the protein binding to this sequence as it appears that this factor is present only in avian fibroblasts, the target tissue for efficient expression of viral genome; (ii) to determine the functional domains of the cloned factors and their cognate cis-acting sequences; (iii) to establish the role of the cloned cDNA products in RSV gene expression. In-frame deletions and point mutations will be made in the cDNAs to identify the various DNA binding domains of the factors. We will also address the issue of whether phosphorylation and homo- or heterodimerization of various factors are required for their interaction, using antibodies raised against the purified factors. These studies will provide further insights into the regulation of viral gene expression and the contribution of host factors in determining tissue tropism and species- specific expression of viral genes. Preliminary results indicated that the GT-rich sequence of the U5 region of the lTR contributes to transcription termination and/or the 3' end processing of the RNA. We intend to continue these studies with an objective of identifying precisely the nucleotides involved in the 3' end maturation of the mRNA, which will be determined by S1 protection assays, oligonucleotide-directed mutagenesis and by studies using polymerase chain reaction.