Walleye dermal sarcoma virus (WDSV) is a fish retrovirus associated with the development of tumors in walleyes. We have determined the complete nucleotide sequence of a DNA clone of WDSV, the N-terminal amino acid sequences of the major proteins, and the start site for transcription. The long terminal repeat is 590 bp in length, with the U3 region containing consensus sequences likely to be involved in viral gene expression. A predicted histidyl-tRNA binding site is located 3 nucleotides distal to the 3 end of the long terminal repeat. Virus particles purified by isopycnic sedimentation followed by rate zonal sedimentation showed major polypeptides with molecular sizes of 90, 25, 20, 14, and 10 kDa. N-terminal sequencing of these allowed unambiguous assignment of the small polypeptides as products of the gag gene, including CA and NC, and the large polypeptide as the TM product of env. The 582-amino-acid (aa) Gag protein precursor is predicted to be myristylated as is found for most retroviruses. NC contains a single Cys-His motif like those found in all retroviruses except spumaviruses. The WDSV pro and pol genes are in the same translational reading frame as gag and thus apparently are translated after termination suppression. The env gene encodes a surface (SU) protein of 469 aa predicted to be highly glycosylated and a large transmembrane (TM) protein of 754 aa. The sequence of TM is unusual in that it ends in a very hydrophobic segment of 65 residues containing a single charged residue . Following the env gene are two nonoverlapping long open reading frames of 290 aa (orf-A) and 306 aa (orf-B), neither of which shows significant sequence similarity with known genes. A third open reading frame of 119 aa (orf-C) is located in the leader region preceding gag. The predicted amino acid sequence of reverse transcriptase would place WDSV phylogenetically closest to the murine leukemia virus-related genus of retroviruses. However, other members of this genus do not have accessory genes, suggesting that WDSV acquired orf-A, orf-B, and perhaps orf-C late in its evolution. We hypothesize by analogy with other complex retroviruses that the accessory genes of WDSV function in the regulation of transcription and in RNA processing and also in the induction of walleye dermal sarcoma.

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°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.

Walleye dermal sarcoma virus (WDSV) is a piscine retrovirus that replicates naturally in fish at temperatures near 4 SC. 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 SC. The ratio of RT activity at 15 SC to that at 4 SC 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.Walleye dermal sarcoma virus (WDSV) is a piscine retrovirus associated with skin tumours in walleyes (Bowser et al., 1988). WDSV is the prototype of the Epsilonretrovirus genus, which includes the closely related walleye epidermal hyperplasia viruses types 1 and 2 (WEHV-1 and -2) (Holzschu et al., 1995 ; LaPierre et al., 1999 ;van Regenmortel et al., 2000). Based on sequence similarity in the reverse transcriptase (RT) gene, the walleye viruses are most closely related to the mammalian C-type viruses or gammaretroviruses (van Regenmortel et al., 2000). WDSV and murine leukaemia virus (MuLV) share 45 % sequence identity within the polymerase domain of RT. In addition, these viruses are similar in that they both utilize termination suppression as the mechanism for production of the Gag-Pol polyprotein and both have only a single Cys-His motif in the non-coding domain of Gag. However, MuLV and the other members of this genus are simple retroviruses, while WDSV is a complex retrovirus, exhibiting differential mRNA splicing and containing three open reading frames (ORFs) in addition to gag, pol and env. One of these ORFs encodes a cyclin D homologue believed to be responsible for the oncogenic potential of the virus (Holzschu et al., 1995 ; Lairmore et al., 2000 ; LaPierre et al., 1998 ;Quackenbush et al., 1997).The walleye, the host species for WDSV, is a poikilotherm and its body temperature depends on the temperature of the environment. WDSV-induced tumours exhibit seasonality : tumours develop on the fish in the fall, persist through the winter and subsequently regress and fall off in the spring. The fall tumours do not contain infectious virus, and viral genomic RNA and proteins are not detectable, although subgenomic RNA is present. In contrast, spring tumours contain large quantities of virus (Martineau et al., 1992). Infection of walleyes is believed to take place during the spring spawning season when the fish are in close contact, at which time the water temperature is approximately 4 mC. For infection to occur at this time, RT, a DNA polymerase that reverse transcribes the viral RNA genome to yield a double-stranded DNA copy, must be able to synthesize proviral DNA at low temperatures. The goal of the present study was ...