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Journal of Virology, July 2004, p. 7590-7601, Vol. 78, No. 14
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.14.7590-7601.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identification and Characterization of cis-Acting Elements Residing in the Walleye Dermal Sarcoma Virus Promoter

Brett W. Hronek, Ashley Meagher, Joel Rovnak, and Sandra L. Quackenbush*

Department of Molecular Biosciences, University of Kansas, Lawrence, Kansas 66045

Received 5 November 2003/ Accepted 17 March 2004

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.

* Corresponding author. Mailing address: Department of Molecular Biosciences, 7047 Haworth Hall, 1200 Sunnyside Ave., The University of Kansas, Lawrence, KS 66045. Phone: (785) 864-4022. Fax: (785) 864-5294. E-mail: squack{at}ku.edu.

Journal of Virology, July 2004, p. 7590-7601, Vol. 78, No. 14
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.14.7590-7601.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.





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