The goal of this project is to elucidate in molecular terms the mechanisms governing the expression of retroviral transcription units endogenous to the murine genome. Although these retroviral sequences do not result in infectious virus they do encode viral antigens, including a distinct class of the glycoprotein, gp70. Recombination between these sequences and that of infectious ecotropic virus results in the de novo generation of leukemogenic MCF virus in which replacement of ecotropic viral sequence with endogenous information accounts for expanded host range and tissue tropism. Expression of the endogenous retroviral genes is controlled by two distinct, mendelian loci, Gv-1 and Gv-2, requiring positive alleles at both loci for expression of the multiple transcription units. Transcription of individual endogenous retroviral sequences is also independently regulated at the tissue-specific level. The coordinate regulation of endogenous retroviral genes indicates that Gv-1 and Gv-2 encode a trans-acting factor affecting the expression of multiple transcription units. The mechanisms of this gene regulation will be examined in the congeneic partner strains 129Gix+ and Gix-. These strains bear positive alleles at the Gv-2 locus but differ for alternative positive and negative alleles at Gv-1. The genomic DNA containing actively transcribed endogenous retroviral genes will be isolated as recombinant DNA. The cis-acting elements which determine tissue-specific and genetic, Gv-1, responsiveness will be analysed. First, indirect assays of DNase 1 hypersensitive sites in different tissues of Gix+ and Gix- mice will indicate possible binding sites of proteins that confer gene regulation. These studies will distinguish between different transcription units by 5' unique flanking DNA sequence. Second direct analysis of the regulatory sequences will be conducted by measuring the promotor activity in transient transfection assays. Progressive deletion and replacement experiments will locate the position and orientation independent enhancer regions and factor binding sites at the promotor. Finally, strategies are described for identifying and isolating the genetically defined Gv-1 regulatory locus. These studies will lead to further investigation to determine how the Gv-1 product governs transcriptional activity in terms of protein structure and its possible relationship to other eukaryote trans-acting regulatory factors.
