scholarly journals 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

1999 ◽  
Vol 73 (10) ◽  
pp. 8884-8889 ◽  
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.

scholarly journals Walleye Dermal Sarcoma Virus Retroviral Cyclin Directly Contacts TAF9

2006 ◽  
Vol 80 (24) ◽  
pp. 12041-12048 ◽  
Author(s):  
Joel Rovnak ◽  
Sandra L. Quackenbush
Keyword(s):  
Protein Interactions ◽  
Point Mutations ◽  
Direct Interaction ◽  
Dependent Manner ◽  
Accessory Gene ◽  
Walleye Dermal Sarcoma Virus ◽  
Sarcoma Virus ◽  
Retroviral Cyclin ◽  
Dermal Sarcoma

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.

Cloning and characterization of dominant negative splice variants of the human histamine H4 receptor

2008 ◽  
Vol 414 (1) ◽  
pp. 121-131 ◽  
Author(s):  
Richard M. van Rijn ◽  
André van Marle ◽  
Paul L. Chazot ◽  
Ellen Langemeijer ◽  
Yongjun Qin ◽  
...  
Keyword(s):  
Mast Cells ◽  
Mammalian Cells ◽  
Splice Variants ◽  
Inflammatory Diseases ◽  
Dominant Negative ◽  
Full Length ◽  
Dominant Negative Effect ◽  
Histamine H4 Receptor ◽  
Negative Effect ◽  
H4 Receptor

The H4R (histamine H4 receptor) is the latest identified member of the histamine receptor subfamily of GPCRs (G-protein-coupled receptors) with potential functional implications in inflammatory diseases and cancer. The H4R is primarily expressed in eosinophils and mast cells and has the highest homology with the H3R. The occurrence of at least twenty different hH3R (human H3R) isoforms led us to investigate the possible existence of H4R splice variants. In the present paper, we report on the cloning of the first two alternatively spliced H4R isoforms from CD34+ cord blood-cell-derived eosinophils and mast cells. These H4R splice variants are localized predominantly intracellularly when expressed recombinantly in mammalian cells. We failed to detect any ligand binding, H4R–ligand induced signalling or constitutive activity for these H4R splice variants. However, when co-expressed with full-length H4R [H4R(390) (H4R isoform of 390 amino acids)], the H4R splice variants have a dominant negative effect on the surface expression of H4R(390). We detected H4R(390)–H4R splice varianthetero-oligomers by employing both biochemical (immunoprecipitation and cell-surface labelling) and biophysical [time-resolved FRET (fluorescence resonance energy transfer)] techniques. mRNAs encoding the H4R splice variants were detected in various cell types and expressed at similar levels to the full-length H4R(390) mRNA in, for example, pre-monocytes. We conclude that the H4R splice variants described here have a dominant negative effect on H4R(390) functionality, as they are able to retain H4R(390) intracellularly and inactivate a population of H4R(390), presumably via hetero-oligomerization.

scholarly journals Walleye dermal sarcoma virus reverse transcriptase is temperature sensitive

2002 ◽  
Vol 83 (6) ◽  
pp. 1361-1365 ◽  
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.

scholarly journals Establishment of productively infected walleye dermal sarcoma explant cells

2007 ◽  
Vol 88 (9) ◽  
pp. 2583-2589 ◽  
Author(s):  
Joel Rovnak ◽  
Rufina N. Casey ◽  
Connie D. Brewster ◽  
James W. Casey ◽  
Sandra L. Quackenbush
Keyword(s):  
Tumour Regression ◽  
Accessory Gene ◽  
Fish Virus ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Sarcoma Virus ◽  
Gene Transcripts ◽  
Virus Expression ◽  
Dermal Sarcoma

Walleye dermal sarcoma virus (WDSV) is a complex retrovirus associated with dermal sarcomas in walleye fish. Virus expression is tightly regulated and limited to accessory gene transcripts throughout tumour development. During tumour regression, this regulation is lost and the replication of virus is greatly enhanced. Cultured walleye fibroblasts infected in vitro do not produce significant quantities of infectious virus. Tissue culture cells established by explantation of tumour cells were found to harbour WDSV provirus and to express accessory and structural proteins. The sequence of the provirus showed little variation from a previous WDSV isolate. Retroviral particles were isolated from supernatants from these cells and were able to transfer infection to uninfected walleye fibroblasts. In addition to the virus present in supernatants, much of the virus was cell associated and liberated only by sonication. This virus was found at internal cellular membranes, including mitochondria, and was infectious.

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

2004 ◽  
Vol 78 (14) ◽  
pp. 7590-7601 ◽  
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.

scholarly journals Transcriptional Analysis of Walleye Dermal Sarcoma Virus (WDSV)

Virology ◽  
1997 ◽  
Vol 237 (1) ◽  
pp. 107-112 ◽  
Author(s):  
Sandra L. Quackenbush ◽  
Donald L. Holzschu ◽  
Paul R. Bowser ◽  
James W. Casey
Keyword(s):  
Transcriptional Analysis ◽  
Walleye Dermal Sarcoma Virus ◽  
Sarcoma Virus ◽  
Dermal Sarcoma
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