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Inactivation of murine sarcoma and leukemia viruses by ultra-violet irradiation
VIROLOGY
42, 1133-1135
Inactivation
(180)
of Murine Sarcoma and leukemia
Murine sarcoma virus preparations contain both transforming (sarcoma) virions (MSV) and nontransforming (leukemia) virions (MuLV) with the latter usually present in large excess. Initial studies by Hartley and Rowe (1) have shown that MuLV is required for the detection of foci of transformed cells produced by MSV in mouse embryo cells; however, in other cell systems it has been shown that transformation can occur independently of MuLV replication @, 31. The present study was undertaken in an attempt to distinguish the transforming function of MSV from the replicating function of MuLV by comparing their rates of inactivation by ult,raviolet light. The results revealed that, the inactivation of both functions followed single-hit kinetics and exhibited very similar inactivation rates. Three sarcoma virus stocks were studied: the Kirsten strain of murine sarcoma virus (Ki-MSV), and Rauscher and BALB/c leukemia virus pseudotypes (4) of the Moloney sarcoma virus termed, respectively, M-MSV(RLV) and M-MSV(BALB/c T-l). The source of the M-MSV genome for the latter two viruses was the nonproducer hamster tumor cell, HT-1 (5). A laboratory stock of Rauscher leukemia virus (RLV) was also studied. Ultraviolet light irradiation was performed with a General Electric germicidal lamp (Glsl8) producing a dose rate of 3,000 erg/cm*/sec at the surface of the virus suspension, 32 cm from the lamp, as measured by an ultraviolet meter (Ultra-Violet Products, San Gabriel, Calif.). Virus suspended in 2 ml of medium was irradiated for various time periods in open SO-mm plastic petri dishes with continuous agitation. Cultures of BALB/3T3 and NIH/3T3 cells were prepared according to published methods (6). For the MSV focus assay, cells were grown in Dulbecco’s modification of Eagle’s medium supplemented with 10 % calf serum. Cells were inoculated 24 hours
Viruses by Ultra-violet
irradiation
prior to infection at 2.0 X lo5 cells per petri dish. The cells were pretreated with diethylaminoethyl dextran for 1 hour preceding infection (7), then exposed to 0.4 ml of virus in complete medium for 1 hour with frequent shaking, and then 4 ml of fresh medium was added. The medium was changed once at 4 days, and foci of transformed cells were counted at 7 days. For titrating MuLV, the XC test (8), modified for plaque assay (<9), was used. Virus-infected cultures were overlaid with 1.0 X lo6 XC cells (10) on the 6-7 days after MuLV infection or immediately after focus enumeration when YISV and MuLV were to be assayed simultaneously. Plaques were scored in cultures fixed in methanol and stained with hematoxylin 3 days after XC overlay. By this procedure it was possible to determine focus formation and infectious virus production in the same petri dishes. The data were transformed into a linear form by the least squares method and the dose of irradiation giving 37% survivors (D3,), expressed in seconds, was directly calculated from the linear equation. As shown in Fig. 1, the rates of inactivation of both the focus-forming and XC plaque-forming activities of the MSV stock containing a mixture of N-MSV(RLV) and RLV were linear. The inactivation curves showed “one-hit” kinetics providing evidence that activity resulted from infection by single particles. Within the limit,s of bhe assaysemployed, no differences were seenin the inactivation rates of t’he transforming and replicating functions. The calculated D37 times were 22.8 and 24.6 seconds, respectively (Table 1). The UV survival curves for transformation and plaque formation of &I-MSV(BALB/c Tl) and Ki-MSV were compared to those obtained with M-MSV(RLV). As shown in Table 1, the Da? values of these three sarcoma viruses were not distinguishable from one another. To establish that the inact’ivation of t,he
1133
1134
SHORT COMMUNICATIONS If this were the case, a falsely high UV inactivation rate of focus-forming activity might result. To test this possibility, focus formation by UV-irradiated MMSV(BALB/c Tl) was tested in the presence or absence of 104 PFU of unirradiated RLV. In this experiment, the slopes of the survival curves for focus formation by MSV in the presence or absence of added MuLV mere essentially identical; D37 times were 21.5 and 23.0 seconds, respectively. An additional experiment was performed to confirm the independence of transformation by MSV from the spread of infectious virus. RLV antisera were obtained from hamsters bearing transplant tumors originally induced by cells transformed by RLV (11). The serum pool used completely neutralized 100 focus-forming units (FFU) of
0.L
.OOL
.cxm 15’
I 30’
I I’
:’ TIME OF IRRADIATION
0 I 3’
FIG. 1. Inactivation of plaque-forming and transforming activities of M-MSV(RLV) by ultraviolet light. Sarcoma virus preparations containing lo5 PFU and 104 FFU were irradiated as described in the text. At the indicated times, virus from separate petri dishes was titrated for both activities. The data were transformed into a linear form by the least squares method and the dose of irradiation giving 37% survivors (Ds~), expressed in seconds, was directly calculated from the linear equation. In the figure, the inactivation curves are drawn through the calculated Dz? times, 22.8 seconds for FFU and 24.6 seconds for PFU, the remainder of the points shown are the actual experimental values expressed as a percentage of the unirradiated controls. FFU a---@; PFU o-o.
plaque-forming activity of MuLV was not somehow distorted by the presence of the MSV in the sarcoma virus stocks, the UV survival of plaque formation was measured in an MuLV stock, RLV. Table 1 shows that the D37 value for RLV, 22.5 seconds, was essentially the same as t,hat of RLV in the stock of YI-MSV(RLV). In addition, it was important to completely rule out the possibility that the similar inactivation rates of MSV and MuLV were based on a requirement for the presence of helper MuLV for focus formation.
TABLE
1
DOSE OF IRRADIATION REQUIRED TO PRODUCE 37yo SURVIVORSD~~) FORTRANSFORMING (FFU) AND REPLIClTIVE (PFU) FUNCTIONS INMURINE S.~RCOM~ VIRUS STOCKS Virus M-MSV M-MSV Ki-MSV RLV
(RLV) (BALB/c
0 Da7 in seconds. as described in the
FFU
PFU
22.8” 19.2 22.2 -
24.6 21.0 24.6 22.5
values were to Fig. 1.
calculated
Tl)
These legend TABLE
CELL
MSV
TR~NSFORM.J.TION VIRUS-NEUTRBLIZING
2 IN
THE PRESENCE ANTISERUM
(RLV)Dilution
Normal serum 1: 80 dilution
Anti-RLV 1: 80 dilution
10-l 1o-‘.5 10-2.0
TNT@ 145 106 56 19 8 2
TNTC 138 115 39 13 7 2
10-2.5 10-3.0 lo-3 5 10-4.0
OF
a M-MSV (RLV), at the indicated dilutions, was added to cultures of BALB/3T3. Virus-neutralizing antiserum was then added as described in the text. Transformed-cell foci were counted at 7 days postinfection. Focus-forming units per plate: TNTC = too numerous to count.
SHORT
COMMUNICATIONS
M-MSV(RLV) at a dilution of 1: 320. Focus titrations of M-MSV(RLV) were carried out with or without this anti-RLV serum in the medium at a final dilution of 1:80. Antiserum was added to the medium 20 hours after infection and was dso present in fresh medium added 4 days later. Table 2 shows that the presence of antibody had no appreciable effect on the titer or the “one-hit” kinetics of the titration pattern for focus production by MSV. To prove that the amount of antibody used was effective in preventing spread of infectious virus, supernatant fluids from antibody-containing and control plates receiving the highest concentration of virus were assayed at 4 and 7 days for focus-forming activity (FFA). No FFA was found in the supernatant fluids contraining antiserum, suggesting that all virus that had been releasedhad been neutralized. The control fluids contained as much as 10” FFU/O.4 ml. Previous studies using contact inhibited mouse cells have shown a one-hit titration pattern for transformation by MSV @,I@. This strongly suggests that foci can result from infection by single MSV virions independent of coinfection by MuLV. The isolation of non-producer foci which contain the sarcoma genome but no detectable leukemia virus strongly supports this hypothesis (13). On this basis, it was predicted that inactivation of MSV by ultraviolet light would follow one-hit kinetics becauseif two distinct particles were required for focus formation, complex inactivation curves would be obtained. Our results clearly show single-hit inactivation kinetics for the three sarcoma viruses tested and the antiserum experiments also provide evidence that MSV focus formation can proceed in the absence of virus spread. The observation that inactivation rates for MuLV and MSV were indistinguishable suggeststhat both viruses have very similar target sizesfor expression of their respective replication and transformation functions. Whether the actual geometric sizes of the geneswhich subserve these two functions are
1135
also simila.r can neither be proven or disproven with the current data and remains a topic for speculation. ACKNOWLEDGMENT This work was partially supported by Contracts PH43-66-641, PH43-67-1396, and NIH69-67 from the Special Virus Cancer Program, National Cancer Institute, National Institutes of Health, Bethesda, Maryland. We thank Mrs. Claire Weaver and Mr. Nam Ho Chang for technical assistance. REFERENCES 1. HARTLEY, J. W., and ROWE, W. P., Proc. Nut. Acad. Sci. U.S.A. 55,780-786 (1966). 2. AARONSON, S. A., JAINCHILL, J. L., and TODARO, G. J., Proc. Natl. Acad. Sci. U.S.A., 66, 1236-1243 (1970). 3. PARKMAN, R., LEVY, J. A., and TING, R. C., Science 168, 387-389 (1970). 4. HARTLEY, J. W., ROWE, W. P., and HUEBNER, R. J., J. Viral. 5,221-225 (1970). 6. HUEBNER, R. J., HARTLEY, J. W., ROWE, W. P., LANE, W. T., and CAPPS, W. I., Proc. Nat. Acad. Sci. U.S.A. 56, 11641169 (1966). 6. JAINCHILL, J. L., AARONSON, S. A., and ToDARO, G. J., J. Virol. 4, 549-553 (1969). 7. VOGT, P. K., Virology 33, 175-177 (1967). 8. KLEMENT, V., ROWE, W. P., HARTLEY, J. W., PUGH, W. E., Proc. Nat. Acad. Sci. U.S.A. 63, 753-758 (1969). 9. ROWE, W. P., PUGH, W. E., and HARTLEY, J. W., Virology, in press. 10. SVOBODA, J., CHYLE, P. P., SIMKOVIC, D ., and HILGERT, I., Folia Biol. Prague 9, 77-81
(1963). J. S., HUEBNER, R. J., BASTING, R. C., J. Nat. Cancer Inst. 42, 1053-1060 (1969). 1%‘. TODARO, G. J., and AARONSON, S. A., Virology 38, 174-202 (1969). 1.3. AARONSON, S. A., and ROWE, W. P., Virology 42, 9-19 (1970).
11. RHIM,
GARY KELLOFF STUART A. AARONSON
Carcinogenesis Branch Cancer Institute National Institutes of Health Bethesda, Maryland 2001/f Viral National
RAYMOND Flou, Laboratories, Inc. Rockville, Maryland 20852 Accepted September 22, 1970
V. GILDEN
42, 1133-1135
Inactivation
(180)
of Murine Sarcoma and leukemia
Murine sarcoma virus preparations contain both transforming (sarcoma) virions (MSV) and nontransforming (leukemia) virions (MuLV) with the latter usually present in large excess. Initial studies by Hartley and Rowe (1) have shown that MuLV is required for the detection of foci of transformed cells produced by MSV in mouse embryo cells; however, in other cell systems it has been shown that transformation can occur independently of MuLV replication @, 31. The present study was undertaken in an attempt to distinguish the transforming function of MSV from the replicating function of MuLV by comparing their rates of inactivation by ult,raviolet light. The results revealed that, the inactivation of both functions followed single-hit kinetics and exhibited very similar inactivation rates. Three sarcoma virus stocks were studied: the Kirsten strain of murine sarcoma virus (Ki-MSV), and Rauscher and BALB/c leukemia virus pseudotypes (4) of the Moloney sarcoma virus termed, respectively, M-MSV(RLV) and M-MSV(BALB/c T-l). The source of the M-MSV genome for the latter two viruses was the nonproducer hamster tumor cell, HT-1 (5). A laboratory stock of Rauscher leukemia virus (RLV) was also studied. Ultraviolet light irradiation was performed with a General Electric germicidal lamp (Glsl8) producing a dose rate of 3,000 erg/cm*/sec at the surface of the virus suspension, 32 cm from the lamp, as measured by an ultraviolet meter (Ultra-Violet Products, San Gabriel, Calif.). Virus suspended in 2 ml of medium was irradiated for various time periods in open SO-mm plastic petri dishes with continuous agitation. Cultures of BALB/3T3 and NIH/3T3 cells were prepared according to published methods (6). For the MSV focus assay, cells were grown in Dulbecco’s modification of Eagle’s medium supplemented with 10 % calf serum. Cells were inoculated 24 hours
Viruses by Ultra-violet
irradiation
prior to infection at 2.0 X lo5 cells per petri dish. The cells were pretreated with diethylaminoethyl dextran for 1 hour preceding infection (7), then exposed to 0.4 ml of virus in complete medium for 1 hour with frequent shaking, and then 4 ml of fresh medium was added. The medium was changed once at 4 days, and foci of transformed cells were counted at 7 days. For titrating MuLV, the XC test (8), modified for plaque assay (<9), was used. Virus-infected cultures were overlaid with 1.0 X lo6 XC cells (10) on the 6-7 days after MuLV infection or immediately after focus enumeration when YISV and MuLV were to be assayed simultaneously. Plaques were scored in cultures fixed in methanol and stained with hematoxylin 3 days after XC overlay. By this procedure it was possible to determine focus formation and infectious virus production in the same petri dishes. The data were transformed into a linear form by the least squares method and the dose of irradiation giving 37% survivors (D3,), expressed in seconds, was directly calculated from the linear equation. As shown in Fig. 1, the rates of inactivation of both the focus-forming and XC plaque-forming activities of the MSV stock containing a mixture of N-MSV(RLV) and RLV were linear. The inactivation curves showed “one-hit” kinetics providing evidence that activity resulted from infection by single particles. Within the limit,s of bhe assaysemployed, no differences were seenin the inactivation rates of t’he transforming and replicating functions. The calculated D37 times were 22.8 and 24.6 seconds, respectively (Table 1). The UV survival curves for transformation and plaque formation of &I-MSV(BALB/c Tl) and Ki-MSV were compared to those obtained with M-MSV(RLV). As shown in Table 1, the Da? values of these three sarcoma viruses were not distinguishable from one another. To establish that the inact’ivation of t,he
1133
1134
SHORT COMMUNICATIONS If this were the case, a falsely high UV inactivation rate of focus-forming activity might result. To test this possibility, focus formation by UV-irradiated MMSV(BALB/c Tl) was tested in the presence or absence of 104 PFU of unirradiated RLV. In this experiment, the slopes of the survival curves for focus formation by MSV in the presence or absence of added MuLV mere essentially identical; D37 times were 21.5 and 23.0 seconds, respectively. An additional experiment was performed to confirm the independence of transformation by MSV from the spread of infectious virus. RLV antisera were obtained from hamsters bearing transplant tumors originally induced by cells transformed by RLV (11). The serum pool used completely neutralized 100 focus-forming units (FFU) of
0.L
.OOL
.cxm 15’
I 30’
I I’
:’ TIME OF IRRADIATION
0 I 3’
FIG. 1. Inactivation of plaque-forming and transforming activities of M-MSV(RLV) by ultraviolet light. Sarcoma virus preparations containing lo5 PFU and 104 FFU were irradiated as described in the text. At the indicated times, virus from separate petri dishes was titrated for both activities. The data were transformed into a linear form by the least squares method and the dose of irradiation giving 37% survivors (Ds~), expressed in seconds, was directly calculated from the linear equation. In the figure, the inactivation curves are drawn through the calculated Dz? times, 22.8 seconds for FFU and 24.6 seconds for PFU, the remainder of the points shown are the actual experimental values expressed as a percentage of the unirradiated controls. FFU a---@; PFU o-o.
plaque-forming activity of MuLV was not somehow distorted by the presence of the MSV in the sarcoma virus stocks, the UV survival of plaque formation was measured in an MuLV stock, RLV. Table 1 shows that the D37 value for RLV, 22.5 seconds, was essentially the same as t,hat of RLV in the stock of YI-MSV(RLV). In addition, it was important to completely rule out the possibility that the similar inactivation rates of MSV and MuLV were based on a requirement for the presence of helper MuLV for focus formation.
TABLE
1
DOSE OF IRRADIATION REQUIRED TO PRODUCE 37yo SURVIVORSD~~) FORTRANSFORMING (FFU) AND REPLIClTIVE (PFU) FUNCTIONS INMURINE S.~RCOM~ VIRUS STOCKS Virus M-MSV M-MSV Ki-MSV RLV
(RLV) (BALB/c
0 Da7 in seconds. as described in the
FFU
PFU
22.8” 19.2 22.2 -
24.6 21.0 24.6 22.5
values were to Fig. 1.
calculated
Tl)
These legend TABLE
CELL
MSV
TR~NSFORM.J.TION VIRUS-NEUTRBLIZING
2 IN
THE PRESENCE ANTISERUM
(RLV)Dilution
Normal serum 1: 80 dilution
Anti-RLV 1: 80 dilution
10-l 1o-‘.5 10-2.0
TNT@ 145 106 56 19 8 2
TNTC 138 115 39 13 7 2
10-2.5 10-3.0 lo-3 5 10-4.0
OF
a M-MSV (RLV), at the indicated dilutions, was added to cultures of BALB/3T3. Virus-neutralizing antiserum was then added as described in the text. Transformed-cell foci were counted at 7 days postinfection. Focus-forming units per plate: TNTC = too numerous to count.
SHORT
COMMUNICATIONS
M-MSV(RLV) at a dilution of 1: 320. Focus titrations of M-MSV(RLV) were carried out with or without this anti-RLV serum in the medium at a final dilution of 1:80. Antiserum was added to the medium 20 hours after infection and was dso present in fresh medium added 4 days later. Table 2 shows that the presence of antibody had no appreciable effect on the titer or the “one-hit” kinetics of the titration pattern for focus production by MSV. To prove that the amount of antibody used was effective in preventing spread of infectious virus, supernatant fluids from antibody-containing and control plates receiving the highest concentration of virus were assayed at 4 and 7 days for focus-forming activity (FFA). No FFA was found in the supernatant fluids contraining antiserum, suggesting that all virus that had been releasedhad been neutralized. The control fluids contained as much as 10” FFU/O.4 ml. Previous studies using contact inhibited mouse cells have shown a one-hit titration pattern for transformation by MSV @,I@. This strongly suggests that foci can result from infection by single MSV virions independent of coinfection by MuLV. The isolation of non-producer foci which contain the sarcoma genome but no detectable leukemia virus strongly supports this hypothesis (13). On this basis, it was predicted that inactivation of MSV by ultraviolet light would follow one-hit kinetics becauseif two distinct particles were required for focus formation, complex inactivation curves would be obtained. Our results clearly show single-hit inactivation kinetics for the three sarcoma viruses tested and the antiserum experiments also provide evidence that MSV focus formation can proceed in the absence of virus spread. The observation that inactivation rates for MuLV and MSV were indistinguishable suggeststhat both viruses have very similar target sizesfor expression of their respective replication and transformation functions. Whether the actual geometric sizes of the geneswhich subserve these two functions are
1135
also simila.r can neither be proven or disproven with the current data and remains a topic for speculation. ACKNOWLEDGMENT This work was partially supported by Contracts PH43-66-641, PH43-67-1396, and NIH69-67 from the Special Virus Cancer Program, National Cancer Institute, National Institutes of Health, Bethesda, Maryland. We thank Mrs. Claire Weaver and Mr. Nam Ho Chang for technical assistance. REFERENCES 1. HARTLEY, J. W., and ROWE, W. P., Proc. Nut. Acad. Sci. U.S.A. 55,780-786 (1966). 2. AARONSON, S. A., JAINCHILL, J. L., and TODARO, G. J., Proc. Natl. Acad. Sci. U.S.A., 66, 1236-1243 (1970). 3. PARKMAN, R., LEVY, J. A., and TING, R. C., Science 168, 387-389 (1970). 4. HARTLEY, J. W., ROWE, W. P., and HUEBNER, R. J., J. Viral. 5,221-225 (1970). 6. HUEBNER, R. J., HARTLEY, J. W., ROWE, W. P., LANE, W. T., and CAPPS, W. I., Proc. Nat. Acad. Sci. U.S.A. 56, 11641169 (1966). 6. JAINCHILL, J. L., AARONSON, S. A., and ToDARO, G. J., J. Virol. 4, 549-553 (1969). 7. VOGT, P. K., Virology 33, 175-177 (1967). 8. KLEMENT, V., ROWE, W. P., HARTLEY, J. W., PUGH, W. E., Proc. Nat. Acad. Sci. U.S.A. 63, 753-758 (1969). 9. ROWE, W. P., PUGH, W. E., and HARTLEY, J. W., Virology, in press. 10. SVOBODA, J., CHYLE, P. P., SIMKOVIC, D ., and HILGERT, I., Folia Biol. Prague 9, 77-81
(1963). J. S., HUEBNER, R. J., BASTING, R. C., J. Nat. Cancer Inst. 42, 1053-1060 (1969). 1%‘. TODARO, G. J., and AARONSON, S. A., Virology 38, 174-202 (1969). 1.3. AARONSON, S. A., and ROWE, W. P., Virology 42, 9-19 (1970).
11. RHIM,
GARY KELLOFF STUART A. AARONSON
Carcinogenesis Branch Cancer Institute National Institutes of Health Bethesda, Maryland 2001/f Viral National
RAYMOND Flou, Laboratories, Inc. Rockville, Maryland 20852 Accepted September 22, 1970
V. GILDEN