Walleye dermal sarcoma virus (WDSV) is a complex retrovirus found associated with
tumors that appear and regress on a seasonal basis. There are quantitative and qualitative differences in the amount of virus expression between developing and regressing
tumors. To understand the role of host cell factors in WDSV expression,
DNase I footprint analysis, electrophoretic mobility shift assays (EMSA), and reporter gene assays were employed.
DNase I footprint analysis of the U3 region of the WDSV long terminal repeat with nuclear extract prepared from a walleye cell line revealed protection of an Oct1, AP1, Whn, and two E4BP4 sites. Additionally, three regions that contained no putative
transcription factor binding sites were protected. EMSA confirmed the specific binding of the protected sites and revealed three additional sites, NF1, AP3, and LVa, not protected in
DNase I footprint analysis. Site-directed mutagenesis of the individual sites, in the context of a
luciferase reporter plasmid, revealed that the NF1, Oct1, AP1, E4BP4#2, AP3, and LVa sites contributed to transcription activation driven by the WDSV U3 region. Mutation of Novel#2 resulted in an increase in
luciferase activity, suggesting the Novel#2 site may function to bind a negative regulator of transcription. Anti-Jun and anti-Fos antiserum specifically inhibited
protein-
DNA complex formation, indicating the presence of c-Jun and c-Fos in the walleye cell nuclear extracts and their participation in binding to the AP1 site. Interestingly, degenerative 15-bp repeats found in the U3 region are differentially protected in
DNase I footprint analysis by the walleye cell line nuclear extract and regressing-
tumor nuclear extract. EMSA utilizing the 15-bp repeat probe revealed that there are similarities of binding with
W12 cell and developing-
tumor nuclear extracts and that the binding differs from that observed with regressing-
tumor nuclear extract.