Department of Molecular Biology and Genetics, Biotechnology Building, Cornell University, Ithaca, NY 14853, USA1
Author for correspondence: Volker Vogt. Fax +1 607 255 2428. e-mail vmv1{at}cornell.edu
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Abstract |
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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 °C. 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 to carry out an initial biochemical characterization of WDSV RT and to investigate the effect of temperature on the enzyme.
The starting material for RT purification was WDSV from walleye tumours. The virus was isolated from mashed tumours submitted to differential centrifugation, followed by isopycnic centrifugation in sucrose gradients (Holzschu et al., 1995 ; Martineau et al., 1991
). RT was purified from virions by affinity chromatography using either poly(U)Sepharose or heparinSepharose followed by rate-zonal sedimentation through a 2040% glycerol gradient using the protocol of Grandgenett et al. (1978)
. Purification of RT using either heparinSepharose or poly(U)Sepharose yielded similar results, as determined by SDSPAGE analysis of chromatography fractions (data not shown). RT activity was monitored using a poly(rA)/oligo(dT) assay (Martineau et al., 1991
; Telesnitsky et al., 1995
). Purified RT was dialysed into storage buffer (50% glycerol, 50 mM Tris, pH 7·5, 0·1 mM EDTA, 2 mM DTT, 0·02% NP-40 and 100 mM KCl). The concentration of heparin-purified RT polypeptide was estimated, by silver staining, to be 10 ng/µl (data not shown). The final purity was estimated to be 3050% (Fig. 1A
) and this enzyme preparation was used in all subsequent enzymatic assays. Limited quantities of virus available as starting material precluded further purification steps.
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The predicted molecular mass of the WDSV RT polypeptide, based on protease cleavage sites in Pol, is 72·5 kDa (Fodor & Vogt, 2002 ). The polymerase domain has a predicted molecular mass of approximately 50 kDa, the RNase H domain is approximately 20 kDa and the integrase domain is approximately 40 kDa. Thus, an RT heterodimer should be about 120 or 180 kDa if it had a subunit composition like that of HIV-1 or ASLV, respectively, while an RT monomer would be about 70 kDa if it were similar to MuLV. To experimentally address the subunit composition of WDSV RT, we compared the sedimentation of RT in a glycerol gradient with the sedimentation of known standards. Rate-zonal sedimentation of RT eluted from poly(U)Sepharose was performed in parallel with the markers phosphorylase B (97 kDa), BSA (69 kDa), ovalbumin (46 kDa) and carbonic anhydrase (30 kDa). The activity peak coincided with the migration of the BSA marker (Fig. 1B
, lane 11). Since RTs are approximately globular, we interpret this result to mean that WDSV RT is a monomer in solution, similar to MuLV RT.
In its natural environment, WDSV infects fish at a temperature near 4 °C, raising the possibility that WDSV RT is specially adapted to function at low temperature. To address this question, we analysed RT activity at 4, 16, 25 and 37 °C. Purified HIV-1 RT obtained from Escherichia coli cells overexpressing the His-tagged p66 subunit was used as a control (Le Grice et al., 1995 ). HIV-1 p66 and WDSV RT exhibited similar rates of TTP incorporation at 4 °C, normalized for amount of enzyme, and both enzymes demonstrated increasing rates of activity with increasing temperature (Fig. 2A
). However, a comparison of the specific activity of both polymerases at temperatures greater than 4 °C demonstrated that the specific activity of WDSV was lower than that of HIV-1. Unlike HIV-1, WDSV showed maximum activity at 25 °C, while activity was neglible at 37 °C, suggesting that the enzyme is thermosensitive. This apparent inactivation could represent instability of the protein because of purification or an inherent characteristic of the enzyme. To distinguish these possibilities, RT activity in crude lysates of WDSV virions was compared with the activity of purified enzyme (Fig. 2B
). Thermosensitivity was found to be the same for both, suggesting that temperature inactivation was not due to loss of stabilizing factors during purification.
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To further characterize WDSV RT, its requirements for pH, salt and divalent cation were determined in assays carried out at 16 °C. The enzyme showed activity over a broad pH range, with a maximum near pH 8 (Fig. 3A). The effect of ionic strength on RT activity was measured at 50, 100, 150, 200, 250 and 300 mM KCl (Fig. 3B
). As reported for other retroviral RTs (Roth et al., 1985
; Taube et al., 1998
), increasing ionic strength inhibited the enzyme. WDSV RT activity was highest at 50 mM KCl and decreased with increasing KCl concentrations. Retroviral RTs require divalent cations for polymerization, with most RTs preferring Mg++. However, MuLV RT is distinct in that it displays maximal activity on a poly(rA) template in the presence of Mn++ (Roth et al., 1985
; Verma, 1975
). Activity assays in the presence of these divalent metal ions showed WDSV RT to be most active with Mg++, over a wide range of concentrations (Fig. 3C
). In contrast, use of Mn++ as the divalent cation did not stimulate polymerization but rather inhibited activity with increasing concentration. It should be noted that the range of concentrations tested (210 mM) was above the optimal manganese concentration (0·5 mM) determined for MuLV (Roth et al., 1985
; Verma, 1975
). However, divalent cation preference of WDSV RT at a similar concentration (0·6 mM) was determined at room temperature using lysed virions (data not shown) and exhibited 1·5 times more activity in Mg++ compared to Mn++; in contrast, MuLV RT was 50 times more active in 0·5 mM Mn++ than in 0·5 mM Mg++ (Roth et al., 1985
). These data clearly demonstrate that WDSV RT shows optimal activity in magnesium and is distinct from MuLV in this characteristic.
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The results presented in this study represent an initial biochemical characterization of WDSV RT. The fact that the enzyme is a monomer in solution like MuLV RT further supports the evolutionary relatedness of WDSV and the C-type mammalian viruses. Comparison of the specific activities of WDSV and HIV-1 showed that the former is much lower. This difference could reflect suboptimal assay conditions, an inherently slower polymerization rate of WDSV RT, or it could merely indicate that the population of purified RT included inactive molecules. An active site titration of purified enzyme with inhibitor could address this question, but insufficient quantities of purified RT unfortunately prevented further testing. The thermosensitivity of WDSV could be explained by the lack of evolutionary pressure for function above about 10 °C, since infection takes place during the spawning season when the lake water is cold. Interestingly, another fish RT was reported long ago to exhibit a low temperature optimum. RT activity associated with C-type virus particles in lymphosarcoma tissue from Northern pike, a fish that inhabits lakes similar to those inhabited by WDSV, was partially purified and tested for activity at temperatures ranging from 5 to 40 °C (Papas et al., 1976 , 1977
). That enzyme was optimally active at 20 °C. Although the published studies did not directly distinguish heat inactivation from catalytic efficiency, the results can be interpreted to mean that the pike retrovirus RT was inactivated by temperatures above 20 °C, similar to WDSV RT. Analysis of RTs of other retroviruses that replicate in the cold, like WEHV-1 and -2, will be needed to establish the generality of RT thermosensitivity. A priori, it seems plausible that for an RT that works naturally in a cold environment, the catalytic activity might be adapted to be most efficient at low temperatures. However, the observation that the ratios of activity at 4 and 15 °C were similar for WDSV and HIV-1 contradicts this notion. Nevertheless, the data do not exclude the possibility that WDSV RT might have an especially high processivity and ability to synthesize through secondary structure at low temperature, compared with mammalian RTs. Further studies using natural templates with secondary structure are needed to address this possibility.
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Acknowledgments |
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Footnotes |
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References |
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Received 21 August 2001;
accepted 4 February 2002.