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Rescue of chemically inactivated Rous sarcoma virus transforming activity by avion leukosis virus
VIROLOGY
63, 463-467 (1973)
Rescue of Chemically
Inactivated
Activity
Rous Sarcoma
by Avian
Since the discovery of reverse transcriptase (1, Z’), the central role of this enzyme has been assumed to be the synthesis of a DNA provirus as proposed by Temin (3). Unambiguous experimental evidence that infection and transformation of cells by RNA tumor viruses require the activity of reverse transcriptase is not yet available. Circumstantial evidence implicating reverse transcriptase in transformation is provided basically by the following observations : (A) A variant of Rous sarcoma virus RSVrv (0) which appeared to be completely uninfectious and failed to transform chick fibroblasts, lacked the RNA-dependent DNA polymerase activity (4, 5). (B) The degree of inhibition of reverse transcriptase by derivatives of rifamycin correlated with the blocking of RNA tumor viruses in infection and transformation (6). In this study we show that N-ethylmaleimide destroyed both the reverse transcriptase and transforming activities of RSV(RAV-2). However, the chemically inactivated virus recovered its transforming activity when coinfected with a nontransforming leukosis virus, RAV-1, which possessed active reverse transcriptase. These observations suggest that reverse transcriptase is one of t,he requirements in transformation of chick cells by Rous sarcoma virus. The Bryan high-titer strain of Rous sarcoma virus RSV(RAV-2) was grown in type C/A or C/O chick embryo fibroblasts as described by Robinson et al. (7). Rous virus titers in the medium, determined by focus formation in tissue culture (8) were about 1 X lo6 focus-forming units (FFU) per milliliter. Raus associated virus, RAV-I , was grown similarly in C/B or C/O cells with a titer of about 2 X 106 infectious units/ml. Virus was concentrated and purified from la.rge volumes of culture medium as described previously (9). In order to en463 Copyright All rights
0 1973 by Academic Press, of reproduction in any form
Inc. reserved.
Virus Transforming
Leukosis Virus hance reverse transcriptase activity (I), virus was first incubated at 0 C for 10 min with a solution to disrupt virus (0.2% Nonidet P-40, 0.1 M dithiothreitol, 0.01 M Tris-HCl, pH 8.3). Incorporation of [3H] thymidine triphosphate (56 Ci/mmole) by the virus or by a partially purified reverse transcriptase was carried out according to Spiegelman’s method (10). Virus preparations in standard buffer (0.01 M Tris-HCl, pH 7.4; 0.1 M NaCl; 0.001 M EDTA) were reacted with N-ethylmaleimide (NEM) at room temperature for 30 min by adding 1 M solution of NEM (in dimethylsulfoxide) to final concentrations of 1O-2 or 10F3 M. The reaction was stopped by either adding the solution for virus disruption containing excess amount of dithiothreitol, or removing unreacted NEM with a Sephadex G25 column (1 X 6 cm) in standard buffer. DEAEcellulose chromatography of reverse transcriptase was carried out as follows: A total of 35 Azso units of purified RSV-(RAV-2) in standard buffer was dissociated by incubation at 37 C for 30 min with 1% Brij 35, 0.1% mercaptoethanol, 0.001 M EDTA, and 0.2 M NaHC03 buffer, pH 9.5. The mixture was diluted to 240 ml with distilled water and passed through a prewashed DEAE-cellulose column (2.5 X 4.0 cm, 0.05 M Tris-HCl, pH 7.2). The adsorbed material was eluted into l-ml fractions with a linear gradient of sodium chloride in the Tris buffer. Aliquots of 100 pliters were analyzed with the reverse transcriptase assay. Subgroup classification of progeny virus was analyzed in the following way: Transformed cells together with uninfected cells in the assay plate (NEM-inactivated RSV(RAV-2) rescued by RAV-1, Table 3) were trypsinized, and lo6 cells were passed with an equal number of secondary C/O fibroblasts. Ten days after the passage, media from the plate were assayed for
464
SHORT COMMUNICATIONS
focus-forming units in the presence or absence of anti-Rous antibodies. Anti-RSV (RAV-2) was generously provided by Drs. W. S. and H. L. Robinson. Medium (0.2 ml) containing virus was mixed with 0.2 ml of the diluted antibody (1: 50) and incubated at 37 C for 30 min in a CO, incubator before infecting C/O fibroblasts for focus formation. After purification and concentration, preparations of Rous sarcoma virus RSV(RAV-2) as well as avian Ieukosis virus RAV-1 demonstrated high activities of reverse transcriptase as shown by the [3H]TTP incorporation in Table 1. Without addition of the exogenous template of dTrA, [;(H]TTP incorporations (not shown here) were about one-fourth of those shown in Table 1. Nethylmaleimide rapidly inactivated reverse transcriptase of RSV (RAV-2). Table 2 shows that little or no reverse transcriptase in the virion remained active after the treatment with N-ethylmaleimide for 30 min at 10-2M. Although not shown here, N-ethylmaleimide also inactivated the enzyme activities of RSV(RAV-I), SR-RSV, and RAV-1 under similar conditions. Further evidence of the enzyme inactivation by NEM is provided by isolation of RSV(RAV2) reverse transcriptase by chromatography. Figure 1 (A) shows a control experiment in which an active reverse transcriptase from TABLE 1 REVERSE TRANSCRIPTASE ACTIVITIES OF AV~AN TUMOR VIFLUSES~ Preparations
[3H]TTP incorporation kpm/hr/ &so)
RSV(RAV-2) RAV-1 RAV-1 without dATP, dGTP, dCTP, and dTrA Buffer
66,500 92,500 500 150
a Oligo dT:poly rA (5 fig) was used as the template for [3H]TTP incorporation under standard conditions for DNA polymerase assay. The concentrations of purified RSV(RAV-2) and RAV-1 were 15.5 and 15.0 A&ml, respectively. Standard buffer was used to serve as background incorporation of [3H]TTP without virus preparation.
TABLE 2 INACTIVATION OF REVERSE TRANSCRIPTASE IN Rons VIRION BY N-ETHYLMALEIMIDE” Preparations
[aH]TTP Percent incorporation (cpm/hr/0.4 Amd
RSV(RAV-2) Buffer RSV(RAV-2)
+ NEM
33,900 560 1370
100 2 4
= N-ethylmaleimide (1 M in dimethylsulfoxide) was reacted at room temperature for 30 min with the virus in standard buffer at a final concentration of W2M. The reaction was stopped by adding the solution for virus disruption containing excess dithiothreitol. Oligo dT:poly rA was used as the template for DNA polymerase assay as described in Table 1.
RSV(RAV-2) was eluted at 0.3 M NaCl in DEAE-cellulose chromatography. The enzyme incorporated PH]TTP into DNA only in the presence of oligo dT:poly rA, suggesting separation of the enzyme from viral RNA. Figure 1 (B) shows the failure to detect active reverse transcriptase after the NEM treatment. Sometimes a very small incorporation of [3H]TTP could be observed at, fractions around 0.3 M NaCl elution. N-ethylmaleimide inactivated not only reverse transcriptase but also transforming activity of RSV(RAV-2). As shown in Table 3, NEM at lO+M completely destroyed transforming activity of RSV(RAV-2) and at 10-3M greatly decreased the focus-forming units of the virus. However, ing activity was recovered totally
transformwhen cells
were coinfected with RAV-1 and the inactivated transforming virus. RAV-1 was used as the rescuing virus for RSV(RAV-2) to avoid interference phenomena (11). It is well known that the avian leukosis virus RAV-1, which belongs to another subgroup, greatly enhances the infectivity of RSV(RAV-2) (12). Under the experimental eonditions used, RAV-1 which caused no focus-forming units itself, enhanced the transforming activity of the controI virus RsV(RAV-2) by about threefold. The transformed cells from the assay plate of NEM (10FM) inactivated Rous virus coinfected with RAV-1 (Table 3) were
465
SHORT COMMUNICATIONS
2000-
-z n
1500-
v * t >
,
c d E
/’ IOOO-
,’
2
500
/’
.’
0’
:
: : :
. / .A. +ujyham, R’
0
I
10
20
4$iknmm¶rn 30
0
10
20
30
O
FRACTION NO. Fig. 1. (A). RSV(RAV-‘2) was dissociated and chromatographed with DEAE-cellulose. Closed circles show [3H]TTP incorporation when oligo dT:poly rA (5 fig) was used as the template. Closed triangles represent [3H]TTP incorporation without addition of the exogenous template. (B). RSV(RAV-2) was first reacted with 10-W N-ethylmaleimide before it was dissociated, chromatographed, and assayed for [3H]TTP incorporation as described in Fig. 1 (A).
passed with chick secondary cells to obtain progeny virus produced by the transformed cells. The progeny virus was analyzed with specific antibodies for subgroup classification. Table 4 shows that the transformed cells produced infectious ROW sarcoma virus after 10 days of subculturing and, that the rescued transforming virus had a mixed progeny of RSV(RAV-1) and RSV(RAV2). Since anti-RSV(RAV-2) partially neutralized RSV(RAV-1) in a control experiment, it appeared that more RSV(RAV-1) than RSV(RAV-2) was present in the progeny. This observation suggests that RAV-1 rescued not only the inactivated transforming virus RsV(RAV-2), but also the inactivated RAV-2 in the Bryan high-titer stock culture which is known to be a sarcomaleukemia complex (11).
The biological function of reverse transcriptase in RNA tumor viruses is not fully understood, but the enzyme may be a prerequisite for integration of viral information into host DNA by the production of a DNA copy of the viral RNA. In the early stages of RSV infection synthesis of both cellular and viral DNA is required for the establishment of transformation (13, 14). In this study we showed that RSV lost transforming activity when its reverse transcriptase became inactivated. Transforming activity was recovered when cells were coinfected with inactivated RSV and RAV-1 which possessed active reverse transcriptase. This suggests the dependence of transforming activity on functional reverse transcriptase. The reverse transcriptase from avian tumor viruses was found to be easily
SHORT COMMUNICATIONS
466
inactivated by the alkylating agent of sulfhydryl groups, N-ethylmaleimide. This is not unexpected becausereverse transcriptase activity is stimulated by sulfhydryl reducing TABLE 3 RESCUE OF INACTIVATED RSV(RAV-2) BY RAV-I Preparations
Buffer RSV(RAV-2) RSV (RAV-2) RSV (RAV-2) RSV (RAV-2) RSV (RAVS) RSV (RAV-2)
NEM treatmenta
RAV-lb
lo-2M lo-2M IO-3M 10-3&f
+ + + +
FFU/plate
0, 125, 380, 0, 387, 16, 322,
0 151 408 0 414 22 369
a Purified virus preparations in standard buffer (0.4 A& were reacted with N-ethylmaleimide at the concentration of 1O-2 or IO-%4 for 30 min at room temperature. The unreacted N-ethylmaleimide was removed by gel filtration with a sterile Sephadex G25 column (1 X 6 cm in standard buffer). The combined virus fractions (2 ml) were diluted 1: 10 and assayed for focus-forming units by taking aliquots (0.2 ml). A control virus preparation was treated identically except that it was not reacted with NEM. b When RAV-1 was used, it was added at the same time as RSV(RAV-2) to chick embryo fibroblasts (4 X 106) at a multiplicity of infection of 4.
agents (I, 2). Unequivocal interpretation of the results is difficult because of probable unknown reactions of N-ethylmaleimide with RSV components in addition to the inactivation of reverse transcriptase. Using 1YJN-ethylmaleimide, we analyzed the Iabeled products of the RSV virion by SDSgel electrophoresis and found that some group specific proteins, but not glycoproteins, were labeled (15). Although the rescuing virus, RAV-1, might complement some inactivated functions of unknown nature in RSV, it is possible that the rescue of the inactivated virus was due at least partially to the presence of functional reverse transcriptase in RAV-1. Alkylation by *Vethylmaleimide apparently had little or no effect on the integrity of the RSV genome. Progeny of the rescued virus was capable of normal replication and in turn produced infectious virus progeny (Table 4). N-ethylmaleimide treatment did not destroy the entry mechanism of the virus, since it would be highly unlikely for two virions to complement outside the cell. Perhaps one of the most unambiguous ways to prove the absolute requirement of reverse transcriptase in transformation would be the rescue of t,ransformation by a polymerase-negative virus by purified reverse transcriptase, delivered into the cells by other means.
TABLE 4 PROGENY OF TEE RESCUED RSV Antibody treatmentb
Preparations? RSV(RAV-1) RSV(RAV-1) RSV (RAV-1) RSV(RAV-2) RSV(RAV-2) RSV (RAV-2) Progeny of rescued Progeny of rescued Progeny of rescued Progeny of rescued
Anti RSV(RAV-1) Anti RSV (RAV-2) Anti RSV (RAV-1) Anti RSV(RAV-2) RSV RSV RSV RSV
-
Anti RSV (RAV-1) Anti RSV(RAV-2) Anti RSV(RAV-1 + Anti RSV(RAV-2)
FFU/plate 202, 14, 170, 263, 263, 10, 259, 100, 173, 10,
277 14 206 274 268 14 273 157 194 12
a Media containing virus were diluted either 500- or lOOO-fold to give about 2-300 focus-forming units per plate. 6 Heat-inactivated antibodies, diluted 50-fold with growth medium 1 (8), were incubated with the virus to be tested at 37 C for 30 min in a CO2 incubator before infecting fibroblasts.
SHORT
COMMUNICATIONS
ACKNOWLEDGMENTS The author expresses appreciation to Dr. L. R. Overby for discussions and support. Technical assistance of Carlos Maldonado and Lelia M. Straube is acknowledged. REFERENCES 1. TEMIN, H. M. ~~~MIZUTANI, S., Nature (London) 226, 1211-1213 (1970). d. BALTIMORE, D., Nature (London) 226,12QQ-1211 (1970), 9. TEMIN, H. M., Nat. Cancer Inst. Monogr. 17, 557-570 (1964). 4. HANAFUSA, H. and HANAFUSA, T., Virology 43, 313316 (1971). 5. ROBINSON, W. S. and ROBINSON, H. L., Virology 44,457462 (1971). 6. TING, R. C., YANG, S. S., and GALLO, R. C., Nature (London) New Biol. 236, 163-166 (1972). 7. ROBINSON, W. S., PITKANEN, A., and RUBIN, H., Proc. Nat. Acad. Sci. USA 54, 137-144 (1965). 8. HOBOM-SCHNEGG, B., ROBINSON, H. L., and
467
ROBINSON, W. S., J. Gen. Viral. 7, 85-93 (1970). 9. HUNG, P. P., ROBINSON, H. L., and ROBINSON, w. s., virology 43,251-266 (1971). 10, SPIEGELMAN, S., BURNY, A., DAS, M. R., KEYDAR, J., SCHLOM, J., TRAVNICEK, M., and WATSON, K., Nature (London) 227,563567 (1970). 11. RUBIN, H. and VOGT, P. K., Virology 17, 1% 194 (1962). lb. HANAFUSA, T. and HANAFUSA, H., Proc. Nut. Acad. Sci. USA 58,818-825 (1967). IS. NAKATA, Y. and BADER, J. P., Virology 36, 401410 (1968). 14. BOETTIGER, D. and TEMIN, H. M., Nature (London) 228, 622-624 (1970). i6. HUNG, P. P. and STRAUBE, L. M. Fed. Proc. 30, 1099 (1971). P. P. HUNG Department of Virology Abbott Laboratories North Chicago, Illinois 60064 Accepted March 5, 197s
63, 463-467 (1973)
Rescue of Chemically
Inactivated
Activity
Rous Sarcoma
by Avian
Since the discovery of reverse transcriptase (1, Z’), the central role of this enzyme has been assumed to be the synthesis of a DNA provirus as proposed by Temin (3). Unambiguous experimental evidence that infection and transformation of cells by RNA tumor viruses require the activity of reverse transcriptase is not yet available. Circumstantial evidence implicating reverse transcriptase in transformation is provided basically by the following observations : (A) A variant of Rous sarcoma virus RSVrv (0) which appeared to be completely uninfectious and failed to transform chick fibroblasts, lacked the RNA-dependent DNA polymerase activity (4, 5). (B) The degree of inhibition of reverse transcriptase by derivatives of rifamycin correlated with the blocking of RNA tumor viruses in infection and transformation (6). In this study we show that N-ethylmaleimide destroyed both the reverse transcriptase and transforming activities of RSV(RAV-2). However, the chemically inactivated virus recovered its transforming activity when coinfected with a nontransforming leukosis virus, RAV-1, which possessed active reverse transcriptase. These observations suggest that reverse transcriptase is one of t,he requirements in transformation of chick cells by Rous sarcoma virus. The Bryan high-titer strain of Rous sarcoma virus RSV(RAV-2) was grown in type C/A or C/O chick embryo fibroblasts as described by Robinson et al. (7). Rous virus titers in the medium, determined by focus formation in tissue culture (8) were about 1 X lo6 focus-forming units (FFU) per milliliter. Raus associated virus, RAV-I , was grown similarly in C/B or C/O cells with a titer of about 2 X 106 infectious units/ml. Virus was concentrated and purified from la.rge volumes of culture medium as described previously (9). In order to en463 Copyright All rights
0 1973 by Academic Press, of reproduction in any form
Inc. reserved.
Virus Transforming
Leukosis Virus hance reverse transcriptase activity (I), virus was first incubated at 0 C for 10 min with a solution to disrupt virus (0.2% Nonidet P-40, 0.1 M dithiothreitol, 0.01 M Tris-HCl, pH 8.3). Incorporation of [3H] thymidine triphosphate (56 Ci/mmole) by the virus or by a partially purified reverse transcriptase was carried out according to Spiegelman’s method (10). Virus preparations in standard buffer (0.01 M Tris-HCl, pH 7.4; 0.1 M NaCl; 0.001 M EDTA) were reacted with N-ethylmaleimide (NEM) at room temperature for 30 min by adding 1 M solution of NEM (in dimethylsulfoxide) to final concentrations of 1O-2 or 10F3 M. The reaction was stopped by either adding the solution for virus disruption containing excess amount of dithiothreitol, or removing unreacted NEM with a Sephadex G25 column (1 X 6 cm) in standard buffer. DEAEcellulose chromatography of reverse transcriptase was carried out as follows: A total of 35 Azso units of purified RSV-(RAV-2) in standard buffer was dissociated by incubation at 37 C for 30 min with 1% Brij 35, 0.1% mercaptoethanol, 0.001 M EDTA, and 0.2 M NaHC03 buffer, pH 9.5. The mixture was diluted to 240 ml with distilled water and passed through a prewashed DEAE-cellulose column (2.5 X 4.0 cm, 0.05 M Tris-HCl, pH 7.2). The adsorbed material was eluted into l-ml fractions with a linear gradient of sodium chloride in the Tris buffer. Aliquots of 100 pliters were analyzed with the reverse transcriptase assay. Subgroup classification of progeny virus was analyzed in the following way: Transformed cells together with uninfected cells in the assay plate (NEM-inactivated RSV(RAV-2) rescued by RAV-1, Table 3) were trypsinized, and lo6 cells were passed with an equal number of secondary C/O fibroblasts. Ten days after the passage, media from the plate were assayed for
464
SHORT COMMUNICATIONS
focus-forming units in the presence or absence of anti-Rous antibodies. Anti-RSV (RAV-2) was generously provided by Drs. W. S. and H. L. Robinson. Medium (0.2 ml) containing virus was mixed with 0.2 ml of the diluted antibody (1: 50) and incubated at 37 C for 30 min in a CO, incubator before infecting C/O fibroblasts for focus formation. After purification and concentration, preparations of Rous sarcoma virus RSV(RAV-2) as well as avian Ieukosis virus RAV-1 demonstrated high activities of reverse transcriptase as shown by the [3H]TTP incorporation in Table 1. Without addition of the exogenous template of dTrA, [;(H]TTP incorporations (not shown here) were about one-fourth of those shown in Table 1. Nethylmaleimide rapidly inactivated reverse transcriptase of RSV (RAV-2). Table 2 shows that little or no reverse transcriptase in the virion remained active after the treatment with N-ethylmaleimide for 30 min at 10-2M. Although not shown here, N-ethylmaleimide also inactivated the enzyme activities of RSV(RAV-I), SR-RSV, and RAV-1 under similar conditions. Further evidence of the enzyme inactivation by NEM is provided by isolation of RSV(RAV2) reverse transcriptase by chromatography. Figure 1 (A) shows a control experiment in which an active reverse transcriptase from TABLE 1 REVERSE TRANSCRIPTASE ACTIVITIES OF AV~AN TUMOR VIFLUSES~ Preparations
[3H]TTP incorporation kpm/hr/ &so)
RSV(RAV-2) RAV-1 RAV-1 without dATP, dGTP, dCTP, and dTrA Buffer
66,500 92,500 500 150
a Oligo dT:poly rA (5 fig) was used as the template for [3H]TTP incorporation under standard conditions for DNA polymerase assay. The concentrations of purified RSV(RAV-2) and RAV-1 were 15.5 and 15.0 A&ml, respectively. Standard buffer was used to serve as background incorporation of [3H]TTP without virus preparation.
TABLE 2 INACTIVATION OF REVERSE TRANSCRIPTASE IN Rons VIRION BY N-ETHYLMALEIMIDE” Preparations
[aH]TTP Percent incorporation (cpm/hr/0.4 Amd
RSV(RAV-2) Buffer RSV(RAV-2)
+ NEM
33,900 560 1370
100 2 4
= N-ethylmaleimide (1 M in dimethylsulfoxide) was reacted at room temperature for 30 min with the virus in standard buffer at a final concentration of W2M. The reaction was stopped by adding the solution for virus disruption containing excess dithiothreitol. Oligo dT:poly rA was used as the template for DNA polymerase assay as described in Table 1.
RSV(RAV-2) was eluted at 0.3 M NaCl in DEAE-cellulose chromatography. The enzyme incorporated PH]TTP into DNA only in the presence of oligo dT:poly rA, suggesting separation of the enzyme from viral RNA. Figure 1 (B) shows the failure to detect active reverse transcriptase after the NEM treatment. Sometimes a very small incorporation of [3H]TTP could be observed at, fractions around 0.3 M NaCl elution. N-ethylmaleimide inactivated not only reverse transcriptase but also transforming activity of RSV(RAV-2). As shown in Table 3, NEM at lO+M completely destroyed transforming activity of RSV(RAV-2) and at 10-3M greatly decreased the focus-forming units of the virus. However, ing activity was recovered totally
transformwhen cells
were coinfected with RAV-1 and the inactivated transforming virus. RAV-1 was used as the rescuing virus for RSV(RAV-2) to avoid interference phenomena (11). It is well known that the avian leukosis virus RAV-1, which belongs to another subgroup, greatly enhances the infectivity of RSV(RAV-2) (12). Under the experimental eonditions used, RAV-1 which caused no focus-forming units itself, enhanced the transforming activity of the controI virus RsV(RAV-2) by about threefold. The transformed cells from the assay plate of NEM (10FM) inactivated Rous virus coinfected with RAV-1 (Table 3) were
465
SHORT COMMUNICATIONS
2000-
-z n
1500-
v * t >
,
c d E
/’ IOOO-
,’
2
500
/’
.’
0’
:
: : :
. / .A. +ujyham, R’
0
I
10
20
4$iknmm¶rn 30
0
10
20
30
O
FRACTION NO. Fig. 1. (A). RSV(RAV-‘2) was dissociated and chromatographed with DEAE-cellulose. Closed circles show [3H]TTP incorporation when oligo dT:poly rA (5 fig) was used as the template. Closed triangles represent [3H]TTP incorporation without addition of the exogenous template. (B). RSV(RAV-2) was first reacted with 10-W N-ethylmaleimide before it was dissociated, chromatographed, and assayed for [3H]TTP incorporation as described in Fig. 1 (A).
passed with chick secondary cells to obtain progeny virus produced by the transformed cells. The progeny virus was analyzed with specific antibodies for subgroup classification. Table 4 shows that the transformed cells produced infectious ROW sarcoma virus after 10 days of subculturing and, that the rescued transforming virus had a mixed progeny of RSV(RAV-1) and RSV(RAV2). Since anti-RSV(RAV-2) partially neutralized RSV(RAV-1) in a control experiment, it appeared that more RSV(RAV-1) than RSV(RAV-2) was present in the progeny. This observation suggests that RAV-1 rescued not only the inactivated transforming virus RsV(RAV-2), but also the inactivated RAV-2 in the Bryan high-titer stock culture which is known to be a sarcomaleukemia complex (11).
The biological function of reverse transcriptase in RNA tumor viruses is not fully understood, but the enzyme may be a prerequisite for integration of viral information into host DNA by the production of a DNA copy of the viral RNA. In the early stages of RSV infection synthesis of both cellular and viral DNA is required for the establishment of transformation (13, 14). In this study we showed that RSV lost transforming activity when its reverse transcriptase became inactivated. Transforming activity was recovered when cells were coinfected with inactivated RSV and RAV-1 which possessed active reverse transcriptase. This suggests the dependence of transforming activity on functional reverse transcriptase. The reverse transcriptase from avian tumor viruses was found to be easily
SHORT COMMUNICATIONS
466
inactivated by the alkylating agent of sulfhydryl groups, N-ethylmaleimide. This is not unexpected becausereverse transcriptase activity is stimulated by sulfhydryl reducing TABLE 3 RESCUE OF INACTIVATED RSV(RAV-2) BY RAV-I Preparations
Buffer RSV(RAV-2) RSV (RAV-2) RSV (RAV-2) RSV (RAV-2) RSV (RAVS) RSV (RAV-2)
NEM treatmenta
RAV-lb
lo-2M lo-2M IO-3M 10-3&f
+ + + +
FFU/plate
0, 125, 380, 0, 387, 16, 322,
0 151 408 0 414 22 369
a Purified virus preparations in standard buffer (0.4 A& were reacted with N-ethylmaleimide at the concentration of 1O-2 or IO-%4 for 30 min at room temperature. The unreacted N-ethylmaleimide was removed by gel filtration with a sterile Sephadex G25 column (1 X 6 cm in standard buffer). The combined virus fractions (2 ml) were diluted 1: 10 and assayed for focus-forming units by taking aliquots (0.2 ml). A control virus preparation was treated identically except that it was not reacted with NEM. b When RAV-1 was used, it was added at the same time as RSV(RAV-2) to chick embryo fibroblasts (4 X 106) at a multiplicity of infection of 4.
agents (I, 2). Unequivocal interpretation of the results is difficult because of probable unknown reactions of N-ethylmaleimide with RSV components in addition to the inactivation of reverse transcriptase. Using 1YJN-ethylmaleimide, we analyzed the Iabeled products of the RSV virion by SDSgel electrophoresis and found that some group specific proteins, but not glycoproteins, were labeled (15). Although the rescuing virus, RAV-1, might complement some inactivated functions of unknown nature in RSV, it is possible that the rescue of the inactivated virus was due at least partially to the presence of functional reverse transcriptase in RAV-1. Alkylation by *Vethylmaleimide apparently had little or no effect on the integrity of the RSV genome. Progeny of the rescued virus was capable of normal replication and in turn produced infectious virus progeny (Table 4). N-ethylmaleimide treatment did not destroy the entry mechanism of the virus, since it would be highly unlikely for two virions to complement outside the cell. Perhaps one of the most unambiguous ways to prove the absolute requirement of reverse transcriptase in transformation would be the rescue of t,ransformation by a polymerase-negative virus by purified reverse transcriptase, delivered into the cells by other means.
TABLE 4 PROGENY OF TEE RESCUED RSV Antibody treatmentb
Preparations? RSV(RAV-1) RSV(RAV-1) RSV (RAV-1) RSV(RAV-2) RSV(RAV-2) RSV (RAV-2) Progeny of rescued Progeny of rescued Progeny of rescued Progeny of rescued
Anti RSV(RAV-1) Anti RSV (RAV-2) Anti RSV (RAV-1) Anti RSV(RAV-2) RSV RSV RSV RSV
-
Anti RSV (RAV-1) Anti RSV(RAV-2) Anti RSV(RAV-1 + Anti RSV(RAV-2)
FFU/plate 202, 14, 170, 263, 263, 10, 259, 100, 173, 10,
277 14 206 274 268 14 273 157 194 12
a Media containing virus were diluted either 500- or lOOO-fold to give about 2-300 focus-forming units per plate. 6 Heat-inactivated antibodies, diluted 50-fold with growth medium 1 (8), were incubated with the virus to be tested at 37 C for 30 min in a CO2 incubator before infecting fibroblasts.
SHORT
COMMUNICATIONS
ACKNOWLEDGMENTS The author expresses appreciation to Dr. L. R. Overby for discussions and support. Technical assistance of Carlos Maldonado and Lelia M. Straube is acknowledged. REFERENCES 1. TEMIN, H. M. ~~~MIZUTANI, S., Nature (London) 226, 1211-1213 (1970). d. BALTIMORE, D., Nature (London) 226,12QQ-1211 (1970), 9. TEMIN, H. M., Nat. Cancer Inst. Monogr. 17, 557-570 (1964). 4. HANAFUSA, H. and HANAFUSA, T., Virology 43, 313316 (1971). 5. ROBINSON, W. S. and ROBINSON, H. L., Virology 44,457462 (1971). 6. TING, R. C., YANG, S. S., and GALLO, R. C., Nature (London) New Biol. 236, 163-166 (1972). 7. ROBINSON, W. S., PITKANEN, A., and RUBIN, H., Proc. Nat. Acad. Sci. USA 54, 137-144 (1965). 8. HOBOM-SCHNEGG, B., ROBINSON, H. L., and
467
ROBINSON, W. S., J. Gen. Viral. 7, 85-93 (1970). 9. HUNG, P. P., ROBINSON, H. L., and ROBINSON, w. s., virology 43,251-266 (1971). 10, SPIEGELMAN, S., BURNY, A., DAS, M. R., KEYDAR, J., SCHLOM, J., TRAVNICEK, M., and WATSON, K., Nature (London) 227,563567 (1970). 11. RUBIN, H. and VOGT, P. K., Virology 17, 1% 194 (1962). lb. HANAFUSA, T. and HANAFUSA, H., Proc. Nut. Acad. Sci. USA 58,818-825 (1967). IS. NAKATA, Y. and BADER, J. P., Virology 36, 401410 (1968). 14. BOETTIGER, D. and TEMIN, H. M., Nature (London) 228, 622-624 (1970). i6. HUNG, P. P. and STRAUBE, L. M. Fed. Proc. 30, 1099 (1971). P. P. HUNG Department of Virology Abbott Laboratories North Chicago, Illinois 60064 Accepted March 5, 197s