Walleye dermal sarcoma virus reverse transcriptase is temperature sensitive

Abstract

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 ...

Characterization of the Protease of a Fish Retrovirus, Walleye Dermal Sarcoma Virus

“…As discussed above, the assignment of the WDSV PR-RT The predicted mass of RT is consistent with size estimates of enzymatically active WDSV RT purified from virions, about 70 kDa (6). In some retroviruses Pro-Pol is processed at other sites in addition to those at the PR-RT and RT-IN junctions.…”

Biochemical Characterization of Novel Retroviral Integrase Proteins

“…Epsilonretrovirus is the only retroviral genus for which an IN protein has not previously been characterized. It has been suggested that WDSV reverse transcriptase is temperature sensitive, displaying optimal activity at 4–15°C, which might reflect the natural habitat of the fish host [31]. We can hypothesize that WDSV IN might also be temperature sensitive, and that our tested conditions did not satisfy its parameters for proper folding when expressed in bacteria.…”

Cloning and characterization of a cDNA encoding walleye (Sander vitreum) beta-2 microglobulin

“…In order to understand the effect of temperature on teleost immune function, we have been characterizing the immune systems of teleosts which exhibit temperature dependent disease susceptibility. Walleye develop a retrovirally-induced dermal sarcoma in a seasonally dependent manner [4]. We have cloned the b 2 m gene since temperature dependent regulation of that gene has been previously demonstrated in carp [5].…”

“…The prevalence of the tumours can vary form 1% to 27% of the individuals in a given lake, depending on the density of the fish [3]. Several researchers have investigated the temperature dependence of the virus and its proteins, for example Fodor and Vogt [4] have recently examined the reverse transcriptase, in order to explain its seasonal nature. Interestingly the fish may also have a role in this temperature dependence as it has been shown that carp subjected to low temperatures down regulate the cell surface expression of class I major histocompatibility (MH) receptors by specifically down regulating expression of the beta 2-microglobulin (b 2 m) gene [5].…”