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Distinct nonstructural polypeptides in polyoma and simian virus 40 DNA-protein complexes
77, 418-420
VIROLOGY
Distinct
(1977)
Nonstructural
ATEEF Dkpartement
Polypeptides in Polyoma DNA-Protein Complexes A. QURESHI
de Microbiologic,
PIERRE
AND
Faculte’ de MPdecine, Universitt! Quebec, Canada JlH 5N4 Accepted
November
and Simian Virus 40
BOURGAUX’ de Sherbrooke,
Sherbrooke,
3,1976
As already reported for polyoma virus [Qureshi, A. A., and Bourgaux, P., Virology 74, 377-385 (1976)], a polypeptide absent from the virion was detected in a viral DNAprotein complex isolated using Triton X-100 from cells productively infected with simian virus 40 (SV40). Migration in sodium dodecyl sulfate-polyacrylamide gels indicated molecular weights of 76,000 and 86,000, respectively, for the SV40 and the polyoma virus polypeptide.
It has been known for some time that viral DNA accumulates as a DNA-protein complex distinct from the virus in cells productively infected with either simian virus 40 (SV40) or polyoma virus (Py) (I 7). It has further been shown that such complexes include either replicating or mature DNA in association with several polypeptides (3, 4, 6, 8, 9). Recently, we reported that the 55 S-sedimenting viral complex extracted with Triton from Pyinfected cells contains, besides the viruscoded structural polypeptides, a polypeptide absent from the virion, with an apparent molecular weight of 70,000 or more (I 0). In this communication, we document the presence of an analogous nonstructural polypetide in a viral complex similarly isolated from SV40-infected cells. As a rule, the procedures used to prepare, label, and characterize the polypeptides of the SV40 complex were those already employed for the Py complex (10). Confluent monolayers of CV, cells were exposed for 90 min to SV40 (obtained from Dr. P. Tegtmeyer, Stony Brook Medical Center, Stony Brook, New York), used at a multiplicity of 20 plaque-forming units per cell, and covered thereafter with DMEM containing 10% fetal calf serum. Thirtyfour hours later, [“Hlthymidine (53.2 Ci/ ’ Address
reprint
requests
to Dr.
Bourgaux.
mmol, I.C.N.) was added to this medium (5 @i/ml), and incubation was continued for another 6 hr. The viral DNA-protein complex was then extracted using a Triton X-100 solution in 0.01 M EDTA, 0.01 M Tris-HCl, pH 7.9, and purified by velocity sedimentation through a neutral sucrose gradient (10). As already observed for the Py complex (IO), the SV40 complex was found to sediment at approximately 55-60 S in relation to a 21 S marker, in this instance purified SV40-DNA form I. This rapidly sedimenting material was dialyzed and concentrated using an Amicon ultrafilter (XM50) and further purified by chromatography on a QAE-Sephadex A-25 ionexchange column (10). Under these conditions, 70 to 80% of the total acid-precipitable counts loaded on the column were reproducibly eluted at 0.78 M KCl, as expected for a DNA-protein complex (10). After this 0.78 M KC1 eluate had been dialyzed and concentrated by ultrafiltration, its protein was labeled with l’s’I, following a procedure already used for the iodination of the polypeptides of Py complex (10) and Py virions (II). In the first experiment, ““I-labeled SV40 complex and 12~I-labeled SV40 particles were mixed, denatured and subjected to electrophoresis through 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels (12). As shown in Fig. la, the distribution
418 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reseWed.
ISSN
0042-6822
SHORT
419
COMMUNICATIONS
of “‘1 through the gel revealed the various structural polypeptides characteristic of SV40 (13-18). The IX’1 distribution suggested that the complex contained the same polypeptides as well as a component of lower electrophoretic mobility. The latter was very prominent in all of several preparations of iodinated SV40-DNA-protein complex analyzed. When electrophoresed under a variety of conditions (see below), this material always migrated as one well-defined peak, suggesting it represented a single polypeptide. As already observed in the case of Py, this polypeptide was found to copurify with the complex during both chromatography on QAESephadex and exclusion chromatography on Sephadex G-200 and was also observed to sediment with the purified complex during centrifugation through sucrose gradients. We considered the possibility that the unidentified polypeptide detected in both the Py (10) and the SV40 complexes, was actually the same trivial contaminant originating either from the host cell or from the serum used for cell propagation. Therefore, ‘:“I-labeled SV40 complex and ““I-labeled Py complex (10) were mixed, denatured, and electrophoresed through 10% SDS-polyacrylamide gels (Fig. lb). In such experiments, the unidentified polypeptide of the SV40 complex reproducibly migrated at a faster rate than that of the Py complex. With the aim of assessing relative mobilities more accurately, these two polypeptides were first separated from the other protein components of the complexes (manuscript in preparation) before being mixed and electrophoresed through 7.5% SDS-polyacrylamide gels in Trisglycine buffer (12) at the same time as various polypeptides of known molecular weight (y-globulin, 150,000; bovine serum albumin, 68,000; ovalbumin, 45,000, and cytochrome c, 13,000 daltons). In spite of the change in the gel system, the relative mobilities of the two nonstructural polypeptides were not detectably modified (not shown). Thus, calculated molecular weights were 86,000 for the Py and 76,000 for the SV40 polypeptide. Both SV40 (20-23) and Py (24) have
a 9
18
0
10
20
30 40 50 Migration (mm)
60
70
FIG. 1. SDS-polyacrylamide-gel electrophoresis of polypeptides from purified Py and SV40 complexes. Remazol blue-stained (19) ovalbumin (0) and histone (H), SV40 complex labeled with Ia11 after QAE-Sephadex chromatography, and Iy51 labeled SV40 particles or Py complex were mixed, denatured, and subjected to electrophoresis (12). Slices from the gels were cut, air dried, and counted in a Nuclear Chicago gamma counter. In addition to the distribution of radioactivity through the gel, the figure shows the position of the stained markers. (a), Coelectrophoresis of 13’I-labeled SV40 complex and lZ”I-labeled SV40 particles. (b), Coelectrophoresis of ‘311-labeled SV40 complex and 1”51-labeled Py complex.
been reported to induce the synthesis of polypeptides of 70,000 daltons or more in either productively infected or transformed cells. Yet, such nonstructural polypeptides have never been detected so far in viral DNA-protein complexes. This apparent inconsistency between our observations and those of others is likely to reflect differences between the various procedures used for the purification of such complexes. In this context, it should be pointed out that our finding of virus-coded structural polypeptides in the complex is in keeping with several published reports (8, 91, except one that describes a similarly extracted SV40 complex containing viral DNA and histone exclusively (7). The detection of polypeptides with molecular
420
SHORT
COMMUNICATIONS
weights between 70,000 and 100,000 in Pyand SV40-DNA-protein complexes is of interest, as both T antigen, a viral gene product that binds strongly to DNA (ZZ), and the unwinding activity (25) already detected in the SV40 complex (26) represent polypeptides with molecular weights falling in that range (27,281. We thus feel that our observations might help to define a practical approach for the purification and characterization of some of the proteins, either cellular or viral, involved in virus replication. ACKNOWLEDGMENTS This investigation was supported by the Medical Research Council of Canada. One of us (A.A.Q.) was a Research Fellow of the Cancer Research Society Inc., Montreal, P.Q. REFERENCES 1.
2. 3. 4. 5. 6. 7.
8. 9. 10.
M. H., MILLER, H. I., and HENDLER, S., Proc. Nut. Acad. Sci. USA 68, 1032-1036 (1971). GREEN, M. H., J. Virol. 10, 32-41 (1972). GOLDSTEIN, D. A., HALL, M. R., and MEINKE, W., J. Virol. 12, 887-900 (1973). HALL, M. R., MEINKE, W., and GOLDSTEIN, D. A., J. Virol. 12, 901-908 (1973). FROST, E., and BOURGAUX, P., Canad. J. Biothem. 51, 1225-1228 (1973). WHITE, M., and EASON, R., J. Virol. 8, 363-371 (1971). MEINKE, W., HALL, M. R., and GOLDSTEIN, D. A., J. Viral. 15, 439-448 (1975). SEN, A., HANCOCK, R., and LEVINE, A. J., Virology 61, 11-21 (1974). MCMILLEN, J., and CONSIGLI, R. A., J. Virol. 14, 1326-1336 (1974). QURESHI, A. A., and BOURGAUX, P., Virology 74, GREEN,
377-385 (1976). E., and BOURGAUX, P., Virology 68, 245 255 (1975). 12. MAIZEL, J. V., JR., In “Methods in Virology” (K. Maramorosch and H. Koprowski, eds.), Vol. 5, pp. 179-246. Academic Press, New York, 1971. 13. BARBAN, S., and GOOR, R. S., J. Virol. 7, 19% 203 (1971). 14. ESTES, M. K., HUANG, E. S., and PAGANO, J. S.. J. Viral. 7, 635-641 (1971). 15. HUANG, E. S., ESTES, M. K., and PAGANO, J. S., J. Virol. 9, 923-929 (1972). 16. LAKE, R. S., BARBAN, S., and SALZMAN, N. P., Biochem. Biophys. Res. Commun. 54, 640-647 (1973). 17. WALTER, G., ROBLIN, R., and DULBECCO, R., Proc. Nat. Acad. Sci. USA 69, 921-924 (1972). 18. CRAWFORD, L. V., Brit. Med. Bull. 29, 253-258 (1973). 19. GRIFFITH, I. P., Anal. Biochem. 46, 402-412 (1972). 20. Ho, L., and COHEN, A., Arch. Virol. 48,327-333 (1975). 21. TEGTMEYER, P., Cold Spring Harbor Symp. Quant. Biol. 39, 9-15 (1974). 22. CARROLL, R. B., HAGER, L., and DULBECCO, R., Cold Spring Harbor Symp. Quant. Biol. 39, 291-293 (1974). 23. POTTER, C. W., MCLAUGHLIN, B. C., and OxFORD, J. S., J. Virol. 4, 574-579 (1969). 24. CUZIN, F., In “Viruses, Evolution and Cancer” (E. Kurstak and K. Maramorosch, eds.), pp. 151-165. Academic Press, New York, 1974. 25. CHAMPOUX, J. J., and DULBECCO, R., Proc. Nat. Acad. Sci. USA 69, 143-146 (1972). 26. SEN, A., and LEVINE, A. J., Nature (London) 249, 343-344 (1974). 27. DEL VILLANO, B. D., and DEFENDI, V., Virology 51, 34-46 (1973). 28. KELLER, W., and WENDEL, I., Cold Spring Harbor Symp. Quant. Biol. 39, 199-208 (1974). Il.
FROST,
VIROLOGY
Distinct
(1977)
Nonstructural
ATEEF Dkpartement
Polypeptides in Polyoma DNA-Protein Complexes A. QURESHI
de Microbiologic,
PIERRE
AND
Faculte’ de MPdecine, Universitt! Quebec, Canada JlH 5N4 Accepted
November
and Simian Virus 40
BOURGAUX’ de Sherbrooke,
Sherbrooke,
3,1976
As already reported for polyoma virus [Qureshi, A. A., and Bourgaux, P., Virology 74, 377-385 (1976)], a polypeptide absent from the virion was detected in a viral DNAprotein complex isolated using Triton X-100 from cells productively infected with simian virus 40 (SV40). Migration in sodium dodecyl sulfate-polyacrylamide gels indicated molecular weights of 76,000 and 86,000, respectively, for the SV40 and the polyoma virus polypeptide.
It has been known for some time that viral DNA accumulates as a DNA-protein complex distinct from the virus in cells productively infected with either simian virus 40 (SV40) or polyoma virus (Py) (I 7). It has further been shown that such complexes include either replicating or mature DNA in association with several polypeptides (3, 4, 6, 8, 9). Recently, we reported that the 55 S-sedimenting viral complex extracted with Triton from Pyinfected cells contains, besides the viruscoded structural polypeptides, a polypeptide absent from the virion, with an apparent molecular weight of 70,000 or more (I 0). In this communication, we document the presence of an analogous nonstructural polypetide in a viral complex similarly isolated from SV40-infected cells. As a rule, the procedures used to prepare, label, and characterize the polypeptides of the SV40 complex were those already employed for the Py complex (10). Confluent monolayers of CV, cells were exposed for 90 min to SV40 (obtained from Dr. P. Tegtmeyer, Stony Brook Medical Center, Stony Brook, New York), used at a multiplicity of 20 plaque-forming units per cell, and covered thereafter with DMEM containing 10% fetal calf serum. Thirtyfour hours later, [“Hlthymidine (53.2 Ci/ ’ Address
reprint
requests
to Dr.
Bourgaux.
mmol, I.C.N.) was added to this medium (5 @i/ml), and incubation was continued for another 6 hr. The viral DNA-protein complex was then extracted using a Triton X-100 solution in 0.01 M EDTA, 0.01 M Tris-HCl, pH 7.9, and purified by velocity sedimentation through a neutral sucrose gradient (10). As already observed for the Py complex (IO), the SV40 complex was found to sediment at approximately 55-60 S in relation to a 21 S marker, in this instance purified SV40-DNA form I. This rapidly sedimenting material was dialyzed and concentrated using an Amicon ultrafilter (XM50) and further purified by chromatography on a QAE-Sephadex A-25 ionexchange column (10). Under these conditions, 70 to 80% of the total acid-precipitable counts loaded on the column were reproducibly eluted at 0.78 M KCl, as expected for a DNA-protein complex (10). After this 0.78 M KC1 eluate had been dialyzed and concentrated by ultrafiltration, its protein was labeled with l’s’I, following a procedure already used for the iodination of the polypeptides of Py complex (10) and Py virions (II). In the first experiment, ““I-labeled SV40 complex and 12~I-labeled SV40 particles were mixed, denatured and subjected to electrophoresis through 10% sodium dodecyl sulfate (SDS)-polyacrylamide gels (12). As shown in Fig. la, the distribution
418 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reseWed.
ISSN
0042-6822
SHORT
419
COMMUNICATIONS
of “‘1 through the gel revealed the various structural polypeptides characteristic of SV40 (13-18). The IX’1 distribution suggested that the complex contained the same polypeptides as well as a component of lower electrophoretic mobility. The latter was very prominent in all of several preparations of iodinated SV40-DNA-protein complex analyzed. When electrophoresed under a variety of conditions (see below), this material always migrated as one well-defined peak, suggesting it represented a single polypeptide. As already observed in the case of Py, this polypeptide was found to copurify with the complex during both chromatography on QAESephadex and exclusion chromatography on Sephadex G-200 and was also observed to sediment with the purified complex during centrifugation through sucrose gradients. We considered the possibility that the unidentified polypeptide detected in both the Py (10) and the SV40 complexes, was actually the same trivial contaminant originating either from the host cell or from the serum used for cell propagation. Therefore, ‘:“I-labeled SV40 complex and ““I-labeled Py complex (10) were mixed, denatured, and electrophoresed through 10% SDS-polyacrylamide gels (Fig. lb). In such experiments, the unidentified polypeptide of the SV40 complex reproducibly migrated at a faster rate than that of the Py complex. With the aim of assessing relative mobilities more accurately, these two polypeptides were first separated from the other protein components of the complexes (manuscript in preparation) before being mixed and electrophoresed through 7.5% SDS-polyacrylamide gels in Trisglycine buffer (12) at the same time as various polypeptides of known molecular weight (y-globulin, 150,000; bovine serum albumin, 68,000; ovalbumin, 45,000, and cytochrome c, 13,000 daltons). In spite of the change in the gel system, the relative mobilities of the two nonstructural polypeptides were not detectably modified (not shown). Thus, calculated molecular weights were 86,000 for the Py and 76,000 for the SV40 polypeptide. Both SV40 (20-23) and Py (24) have
a 9
18
0
10
20
30 40 50 Migration (mm)
60
70
FIG. 1. SDS-polyacrylamide-gel electrophoresis of polypeptides from purified Py and SV40 complexes. Remazol blue-stained (19) ovalbumin (0) and histone (H), SV40 complex labeled with Ia11 after QAE-Sephadex chromatography, and Iy51 labeled SV40 particles or Py complex were mixed, denatured, and subjected to electrophoresis (12). Slices from the gels were cut, air dried, and counted in a Nuclear Chicago gamma counter. In addition to the distribution of radioactivity through the gel, the figure shows the position of the stained markers. (a), Coelectrophoresis of 13’I-labeled SV40 complex and lZ”I-labeled SV40 particles. (b), Coelectrophoresis of ‘311-labeled SV40 complex and 1”51-labeled Py complex.
been reported to induce the synthesis of polypeptides of 70,000 daltons or more in either productively infected or transformed cells. Yet, such nonstructural polypeptides have never been detected so far in viral DNA-protein complexes. This apparent inconsistency between our observations and those of others is likely to reflect differences between the various procedures used for the purification of such complexes. In this context, it should be pointed out that our finding of virus-coded structural polypeptides in the complex is in keeping with several published reports (8, 91, except one that describes a similarly extracted SV40 complex containing viral DNA and histone exclusively (7). The detection of polypeptides with molecular
420
SHORT
COMMUNICATIONS
weights between 70,000 and 100,000 in Pyand SV40-DNA-protein complexes is of interest, as both T antigen, a viral gene product that binds strongly to DNA (ZZ), and the unwinding activity (25) already detected in the SV40 complex (26) represent polypeptides with molecular weights falling in that range (27,281. We thus feel that our observations might help to define a practical approach for the purification and characterization of some of the proteins, either cellular or viral, involved in virus replication. ACKNOWLEDGMENTS This investigation was supported by the Medical Research Council of Canada. One of us (A.A.Q.) was a Research Fellow of the Cancer Research Society Inc., Montreal, P.Q. REFERENCES 1.
2. 3. 4. 5. 6. 7.
8. 9. 10.
M. H., MILLER, H. I., and HENDLER, S., Proc. Nut. Acad. Sci. USA 68, 1032-1036 (1971). GREEN, M. H., J. Virol. 10, 32-41 (1972). GOLDSTEIN, D. A., HALL, M. R., and MEINKE, W., J. Virol. 12, 887-900 (1973). HALL, M. R., MEINKE, W., and GOLDSTEIN, D. A., J. Virol. 12, 901-908 (1973). FROST, E., and BOURGAUX, P., Canad. J. Biothem. 51, 1225-1228 (1973). WHITE, M., and EASON, R., J. Virol. 8, 363-371 (1971). MEINKE, W., HALL, M. R., and GOLDSTEIN, D. A., J. Viral. 15, 439-448 (1975). SEN, A., HANCOCK, R., and LEVINE, A. J., Virology 61, 11-21 (1974). MCMILLEN, J., and CONSIGLI, R. A., J. Virol. 14, 1326-1336 (1974). QURESHI, A. A., and BOURGAUX, P., Virology 74, GREEN,
377-385 (1976). E., and BOURGAUX, P., Virology 68, 245 255 (1975). 12. MAIZEL, J. V., JR., In “Methods in Virology” (K. Maramorosch and H. Koprowski, eds.), Vol. 5, pp. 179-246. Academic Press, New York, 1971. 13. BARBAN, S., and GOOR, R. S., J. Virol. 7, 19% 203 (1971). 14. ESTES, M. K., HUANG, E. S., and PAGANO, J. S.. J. Viral. 7, 635-641 (1971). 15. HUANG, E. S., ESTES, M. K., and PAGANO, J. S., J. Virol. 9, 923-929 (1972). 16. LAKE, R. S., BARBAN, S., and SALZMAN, N. P., Biochem. Biophys. Res. Commun. 54, 640-647 (1973). 17. WALTER, G., ROBLIN, R., and DULBECCO, R., Proc. Nat. Acad. Sci. USA 69, 921-924 (1972). 18. CRAWFORD, L. V., Brit. Med. Bull. 29, 253-258 (1973). 19. GRIFFITH, I. P., Anal. Biochem. 46, 402-412 (1972). 20. Ho, L., and COHEN, A., Arch. Virol. 48,327-333 (1975). 21. TEGTMEYER, P., Cold Spring Harbor Symp. Quant. Biol. 39, 9-15 (1974). 22. CARROLL, R. B., HAGER, L., and DULBECCO, R., Cold Spring Harbor Symp. Quant. Biol. 39, 291-293 (1974). 23. POTTER, C. W., MCLAUGHLIN, B. C., and OxFORD, J. S., J. Virol. 4, 574-579 (1969). 24. CUZIN, F., In “Viruses, Evolution and Cancer” (E. Kurstak and K. Maramorosch, eds.), pp. 151-165. Academic Press, New York, 1974. 25. CHAMPOUX, J. J., and DULBECCO, R., Proc. Nat. Acad. Sci. USA 69, 143-146 (1972). 26. SEN, A., and LEVINE, A. J., Nature (London) 249, 343-344 (1974). 27. DEL VILLANO, B. D., and DEFENDI, V., Virology 51, 34-46 (1973). 28. KELLER, W., and WENDEL, I., Cold Spring Harbor Symp. Quant. Biol. 39, 199-208 (1974). Il.
FROST,