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 the U3 enhancer region of the long terminal repeats of RSV contains the determinants for its tissue-tropic expression. We describe the cloning and characterization of an avian gene that encodes a protein capable of binding to the enhancer region of Rous sarcoma virus. A PCR-derived probe corresponding to the U3 region of RSV was used to isolate a cDNA clone by screening a chicken cDNA expression library. The cDNA is predicted to encode a polypeptide of 298 amino acids that is homologous to the Y-box (inverted CCAAT) family of DNA-binding transcription factors. This factor, which we refer to as Rous sarcoma virus enhancer factor-it (RSV-EF-II), shows 99% aa identity over a 105-amino-acid stretch that is highly conserved in all Y-box proteins, and is commonly referred to as the cold shock domain. RSV-EF-II selectively binds to single-stranded DNA, and the binding site, as determined by electrophoretic mobility shift assays, consists of the sequence 5' GTACCACC 3' located 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 at positions -65 to -69 and -129 to -133 in the RSV LTR. Northern analysis indicates that RSV-EF-II-specific transcripts are expressed predominantly in avian fibroblasts and muscle tissue. The results of these binding 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 repressor of transcription. (C) 1996 Academic Press, Inc.