Comparative pathogenesis of epsilonretroviruses.
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Characterization of the protease of a fish retrovirus, walleye dermal sarcoma virus.
Three fish retroviruses infecting walleyes constitute the recently recognized genus called epsilonretrovirus. The founding member of this group, walleye dermal sarcoma virus (WDSV), induces benign skin tumors in the infected fish and replicates near 4 degrees C. While the viral genomic sequence is known, biochemical characterization of the virus has been limited to the identification of the mature structural and envelope proteins present in virions. We undertook this study to determine the cleavage sites in the WDSV Pro and Pol proteins and to characterize the viral protease (PR) in vitro. A recombinant PR was expressed in and purified from Escherichia coli as a larger fusion with additional nucleocapsid and reverse transcriptase residues flanking the PR domain. Autocleavage produced a functional, mature PR. Autocleavage as well as cleavage of peptides and of Gag protein by the mature PR occurred at a pH optimum of 7.0, higher than that of other retroviral proteases. Analysis of the cleavage sites identified a glutamine residue in the P2 position of all WDSV sites, both in Gag and in Pol. Amino acid sequence alignments of Gag-Pro-Pol from WDSV, walleye epidermal hyperplasia virus type 1, and walleye epidermal hyperplasia virus type 2 showed the P2 glutamine to be conserved in all cleavage sites in these three viruses. Such conservation is unprecedented in other retroviruses.
Amino Acid Sequence↗
Walleye dermal sarcoma virus reverse transcriptase is temperature sensitive.
Walleye dermal sarcoma virus (WDSV) is a piscine retrovirus that replicates naturally in fish at temperatures near 4 degrees 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 microM 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 degrees C. The ratio of RT activity at 15 degrees C to that at 4 degrees 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.
Animals↗
Walleye dermal sarcoma virus Orf C is targeted to the mitochondria.
Walleye dermal sarcomas are associated with the presence of a complex retrovirus, walleye dermal sarcoma virus (WDSV). These sarcomas develop and regress seasonally in naturally infected fish. In addition to gag, pol and env, WDSV contains three open reading frames (ORFs), designated orf a, orf b and orf c. orf c is located between the 5' long terminal repeat and gag. Developing tumours contain low levels of orf a and orf b transcripts, whereas regressing tumours contain high levels of genomic transcripts and virus particles. Orf C protein is encoded by the full-length, genomic transcript and can be detected in tumour extracts with anti-Orf C-specific antisera. To determine the subcellular location of WDSV Orf C, cultured cells were transfected with an expression vector encoding haemagglutinin-tagged Orf C and examined by immunofluorescence. Orf C was observed throughout the cytoplasm and accumulated in cytoplasmic organelles. Dual-antibody staining for Orf C and mitochondrial cytochrome c demonstrated colocalization of Orf C with mitochondria and loss of the normal distribution of mitochondria in the cytoplasm. Cells transiently expressing Orf C exhibited apoptotic morphology and increased levels of surface phosphatidylserine and were unable to retain MitoTracker Orange, a dye that accumulates in active mitochondria. These results imply a functional role for WDSV Orf C in an alteration of mitochondrial function that results in apoptosis contributing to tumour regression.
Animals↗
Walleye dermal sarcoma virus cyclin interacts with components of the mediator complex and the RNA polymerase II holoenzyme.
Walleye dermal sarcoma virus (WDSV) encodes an accessory protein, OrfA, with sequence homology to cyclins (retrovirus cyclin). In cells transfected with an expression construct, OrfA was localized to the nucleus and was concentrated in interchromatin granule clusters (IGCs), sites where splicing factors are concentrated. Other proteins identified in IGCs include transcription factors, the large subunit of RNA polymerase II (Pol II), and cyclin-dependent kinase 8 (cdk8). cdk8 is the kinase partner of cyclin C and a component of the mediator complex, associated with the Pol II holoenzyme. cdk8 and cyclin C can regulate transcription via phosphorylation of cyclin H and the carboxy-terminal domain of Pol II. OrfA in transfected HeLa cells was found to colocalize and copurify with hyperphosphorylated forms of Pol II (Pol IIO) in IGCs, and OrfA was coimmunoprecipitated from lysates of transfected cells with an antibody against Pol IIO. Likewise, Pol IIO could be coprecipitated with an antibody against OrfA. A survey with antibodies against several different cdks resulted in coimmunoprecipitation of OrfA with anti-cdk8, and antiserum against OrfA was able to coprecipitate cdk8 from lysates of cells that express OrfA. Coprecipitation of OrfA with anti-cyclin C demonstrated that it was included in complexes with OrfA and cdk8. OrfA has sequence and structural similarities to cyclin C, and, functionally, OrfA appears to have the capacity to both enhance and inhibit the activity of promoters in a cell-specific manner, similar to functions of the mediator complex. These data suggest that WDSV OrfA functions through its interactions with these large, transcription complexes. Further investigations will clarify the role of the retrovirus cyclin in control of virus expression and transformation.
3T3 Cells↗
Identification and characterization of cis-acting elements residing in the walleye dermal sarcoma virus promoter.
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.
Animals↗