An activation domain within the walleye dermal sarcoma virus retroviral cyclin protein is essential for inhibition of the viral promoter

ABSTRACT Walleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with dermal sarcomas in walleye fish. A WDSV accessory gene encodes a cyclin homolog or retroviral cyclin (rv-cyclin). WDSV rv-cyclin was found to be associated with transcription complexes and to affect transcription in a cell-type and promoter-dependent manner. It inhibited the WDSV promoter in walleye fibroblasts and activated transcription from GAL4 promoters when fused to the GAL4 DNA binding domain, and an activation domain (AD) has been localized to 30 amino acids in the carboxyl region. rv-cyclin can block the pulldown of transcription coactivators by the AD of VP16, and the isolated rv-cyclin AD interferes specifically with the interaction between the carboxyl halves of the VP16 AD, VP16C, and TATA-binding protein-associated factor 9 (TAF9). The carboxyl region and isolated AD can bind TAF9 directly in assays of protein-protein interaction in vitro. Furthermore, rv-cyclin and the isolated rv-cyclin AD interfere specifically with the function of VP16C in transcription assays. A previously identified motif within the VP16C sequence mediates TAF9 binding, and this motif is present in the activation domains of a variety of TAF9-binding transcriptional activators. A similar motif is present in the rv-cyclin AD, and point mutations within this motif affect rv-cyclin function and protein-protein interactions. The results support a model of transcription regulation by direct interaction with TAF9.

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The retroviral cyclin of walleye dermal sarcoma virus binds cyclin-dependent kinases 3 and 8

Virology ◽

10.1016/j.virol.2010.10.022 ◽

2011 ◽

Vol 409 (2) ◽

pp. 299-307 ◽

Cited By ~ 11

Author(s):

Connie D. Brewster ◽

Claire H. Birkenheuer ◽

Megan B. Vogt ◽

Sandra L. Quackenbush ◽

Joel Rovnak

Keyword(s):

Cyclin Dependent Kinases ◽

Walleye Dermal Sarcoma Virus ◽

Sarcoma Virus ◽

Retroviral Cyclin ◽

Dermal Sarcoma

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Ether Sensitivity of the Walleye Dermal Sarcoma Virus

Journal of Aquatic Animal Health ◽

10.1577/1548-8667(1994)006<0178:esotwd>2.3.co;2 ◽

1994 ◽

Vol 6 (2) ◽

pp. 178-179 ◽

Cited By ~ 1

Author(s):

Paul R. Bowser ◽

Gregory A. Wooster

Keyword(s):

Walleye Dermal Sarcoma Virus ◽

Sarcoma Virus ◽

Dermal Sarcoma

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Walleye dermal sarcoma virus reverse transcriptase is temperature sensitive

Journal of General Virology ◽

10.1099/0022-1317-83-6-1361 ◽

2002 ◽

Vol 83 (6) ◽

pp. 1361-1365 ◽

Cited By ~ 6

Author(s):

Sharon K. Fodor ◽

Volker M. Vogt

Keyword(s):

Human Immunodeficiency Virus ◽

Reverse Transcriptase ◽

Temperature Sensitive ◽

Leukaemia Virus ◽

Virus Particles ◽

Walleye Dermal Sarcoma Virus ◽

Immunodeficiency Virus ◽

Sarcoma Virus ◽

Dermal Sarcoma

Walleye dermal sarcoma virus (WDSV) is a piscine retrovirus that replicates naturally in fish at temperatures near 4 °C. The reverse transcriptase (RT) protein from virus particles isolated from walleye tumours was purified and biochemically characterized. Like the RT of the distantly related murine leukaemia virus, WDSV RT sediments as a monomer in the absence of template. It exhibits a K m of 22 μM for TTP in an assay with poly(rA) as a template and oligo(dT) as a primer. The enzyme is rapidly inactivated at temperatures greater than 15 °C. The ratio of RT activity at 15 °C to that at 4 °C is similar for WDSV and recombinant human immunodeficiency virus type 1, suggesting that, at least with this template, the fish enzyme is not specially adapted to function more efficiently in the cold.

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Walleye Dermal Sarcoma Virus: OrfA N-Terminal End Inhibits the Activity of a Reporter Gene Directed by Eukaryotic Promoters and Has a Negative Effect on the Growth of Fish and Mammalian Cells

Journal of Virology ◽

10.1128/jvi.73.10.8884-8889.1999 ◽

1999 ◽

Vol 73 (10) ◽

pp. 8884-8889 ◽

Cited By ~ 14

Author(s):

Z. Zhang ◽

D. Martineau

Keyword(s):

Reporter Gene ◽

Mammalian Cells ◽

Full Length ◽

Skin Tumor ◽

Accessory Gene ◽

Walleye Dermal Sarcoma Virus ◽

Negative Effect ◽

Sarcoma Virus ◽

Eukaryotic Promoters ◽

Dermal Sarcoma

ABSTRACT Walleye dermal sarcoma virus (WDSV) is a fish retrovirus causing a skin tumor termed walleye dermal sarcoma, which develops and regresses on a seasonal basis. The WDSV genome contains three short open reading frames designated orfA, orfB, andorfC in addition to the viral structural genes,gag, pol, and env. orfAand orfB transcripts are detected in tumors by reverse transcription-PCR. Recently, OrfA, whose amino acid sequence is similar to that of cyclins A and D, has been shown to complement a cyclin-deficient yeast strain. We report that expression of the accessory gene orfA inhibited nonspecifically the activity of a reporter gene directed by various eukaryotic promoters. In addition, stable transfection with the wild-type orfAgenerated substantially fewer G418-resistant colonies in both fish and mammalian cells than the parent vector. An orfA mutant expressing only the first N-terminal 49 residues of the full-length protein had the same negative effect on the activity of the reporter gene and on the number of stably transfected colonies as the full-length OrfA. Thus, OrfA inhibits cell growth and/or causes cell death, and the first 49 N-terminal residues of this protein are sufficient to cause these negative effects.

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

Journal of Virology ◽

10.1128/jvi.78.14.7590-7601.2004 ◽

2004 ◽

Vol 78 (14) ◽

pp. 7590-7601 ◽

Cited By ~ 7

Author(s):

Brett W. Hronek ◽

Ashley Meagher ◽

Joel Rovnak ◽

Sandra L. Quackenbush

Keyword(s):

Cell Line ◽

Nuclear Extract ◽

Dnase I ◽

Negative Regulator ◽

Luciferase Reporter ◽

Walleye Dermal Sarcoma Virus ◽

Nuclear Extracts ◽

Sarcoma Virus ◽

Footprint Analysis ◽

Dermal Sarcoma

ABSTRACT 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.

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