CLONING OF ROUS-SARCOMA VIRUS ENHANCER FACTOR GENES .2. RSV-EF-II, ABUNDANTLY EXPRESSED IN FIBROBLASTS AND MUSCLE-TISSUE, BINDS TO AN OCTAMER SEQUENCE, 5'-GTACCACC-3', IN THE NONCODING STRAND OF RSV ENHANCER

Rous sarcoma virus (RSV) mainly replicates in avian fibroblasts, and t he U3 enhancer region of the long terminal repeats of RSV contains the determinants for its tissue-tropic expression. We describe the clonin g and characterization of an avian gene that encodes a protein capable of binding to the enhancer region of Rous sarcoma virus. A PCR-derive d probe corresponding to the U3 region of RSV was used to isolate a cD NA clone by screening a chicken cDNA expression library. The cDNA is p redicted to encode a polypeptide of 298 amino acids that is homologous to the Y-box (inverted CCAAT) family of DNA-binding transcription fac tors. This factor, which we refer to as Rous sarcoma virus enhancer fa ctor-it (RSV-EF-II), shows 99% aa identity over a 105-amino-acid stret ch that is highly conserved in all Y-box proteins, and is commonly ref erred to as the cold shock domain. RSV-EF-II selectively binds to sing le-stranded DNA, and the binding site, as determined by electrophoreti c mobility shift assays, consists of the sequence 5' GTACCACC 3' locat ed between nucleotides -112 to -119 in the noncoding strand of the RSV enhancer. Although RSV-EF-II shares considerable homology with the Y- box family of proteins, it does not bind to the inverted CCAAT boxes a t positions -65 to -69 and -129 to -133 in the RSV LTR. Northern analy sis indicates that RSV-EF-II-specific transcripts are expressed predom inantly in avian fibroblasts and muscle tissue. The results of these b inding and mRNA expression experiments suggest that RSV-EF-II may play an important role in tissue- and host-specific expression of RSV LTR- driven gene expression. Further, we show that RSV-EF-II acts as a repr essor of transcription. (C) 1996 Academic Press, Inc.