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Electrophoretic analysis of the structural polypeptides of polyoma virus mutants
VIROLOGY
65, 286-288 (19751
Electrophoretic
Analysis
of the Structural
Polyoma ERIC FROST’ Dkpartement
de Microbiologic,
Polypeptides
of
Virus Mutants PIERRE
AND
BOURGAUX
Centre Hospitalier Universitaire, Universith Qukbec, Canada JlH 5N4 Accepted
January
de Sherbrooke,
Sherbrooke,
9, 1975
We have analysed the elxtrophoretic mobility of the structural polypeptides of wild-type polyoma virus (Py) and some of its temperature-sensitive (ts) mutants. Mutant ts-IO was found to contain two polypeptides migrating slightly slower than their wild-type counterparts. We suggest that these two structural polypeptides of Py are either coded for or dependent on a single viral gene and possibly are cleavage products from a precursor molecule.
At least six polypeptides have been found in purified polyoma virus (Py) preparations (1-5). The major polypeptide of the capsid (VPl) has always been assumed to be coded for by the virus genome. In vitro coupled transcription and translation of the viral genome (6) and, more recently, genetic evidence (7) back up this assumption. The three or four small molecular weight proteins, VP4-6, are probably host cell histones. Indeed they are synthesized prior to as well as during infection (3, 5), have high lysine to valine ratios (I), lack tryptophan (I, 5) and coelectrophorese with host cell histones (3, 5; and E. Frost, unpublished observations). Recently, two reports have shown that VP2 and VP3 yield similar tryptic peptide maps, which may (8) or may not (9) be similar to that of VPl. Using polyacrylamide-gel electrophoresis, we have compared the structural polypeptides of Py with those of some of its temperature-sensitive (ts) mutants. We report here some of our results which show that a single mutation in the viral genome results in a change in mobility of both VP2 and VP3. Temperature-sensitive mutants of Py I Present address: Institute of Virology, of Glasgow, Glasgow, Scotland GlI 5JR.
University 286
Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
have been grouped into three (10) or four (II) complementation groups. In this study, we have limited ourselves to the early mutant, ts-a (12), two complementing late mutants, ts-10 (10) and ts-1260 (IO), the noncomplementing h-3 (13) and the TSP-1 isolate (14) of the wild type. For virus production, secondary cultures of whole mouse embryos infected at low multiplicity (0.5 plaque-forming units per cell) were incubated for 2 weeks at 33”. The virus was harvested using receptor destroying enzyme (15) and purified by a procedure involving extraction with Freon (16), centrifugation for 1 hr at 38,000 rpm in an SB283 rotor of a B60 International ultracentrifuge, treatment of the sedimented virus with enzymes followed by banding in CsCl, as described by Thorne and Wardle (17), and suspension of the final virus pellet in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA). This purified virus was labeled with either 1311or lz51 using chloramine-T (18). Twenty-five microliters of virus were added to a mixture of 10 ~1 each of 1 M Na phosphate buffer, pH 6.9, 0.1 N HCl, and carrier-free radioactive iodine (300-500 PCi; ICN) in 0.1 N NaOH. Iodination was initiated by addition of 4 pg of chloramine-T and stopped with 12.5 pg of potas-
SHORT
287
COMMUNICATIONS
sium meta-bisulfite after 45 set of incubation at room temperature. Labeled virus was separated from unreacted iodine and iodine-labeled BSA by velocity centrifugation in a sucrose solution (in 0.1 M NaCl, 0.001 M EDTA, 0.01 M Tris-HCl, pH 7.4). The detailed iodination method will be presented elsewhere (E. Frost, in preparation). The 13’1-labeled mutants were mixed with ‘2”I-labeled wild-type virus prior to dissociation and electrophoretic separation of the polypeptides. As shown in Fig. 1, no differences are found in the electrophoretic mobilities of the polypeptides from wildtype, ts-a, h-3 and ts-1260. In the pattern observed for h-10, however, the two polypeptides corresponding to VP2 and VP3 have reduced mobilities, suggesting that they have a higher molecular weight than their wild-type counterparts. It is noteworthy that VP1 of ts-10 and of wild-type virus comigrate. Electrophoresis for longer times failed to show a difference in the mobility of VP1 between wild-type virus, b-10, or any of the other mutants mentioned. Results identical to those shown in Fig. 1 were obtained with several preparations of iodinated virus. Since there is no reason to suspect that the genome of ts-10 contains more than one mutation, our data provide evidence that VP2 and VP3 are either coded for by the same viral gene or, alternatively, dependent on the same viral gene product. Although other explanations are possible, they may also indicate that both polypeptides are derived from a precursor, or precursors, of higher molecular weight. Mutants of the same complementation group as ts-10, have been shown by twodimensional peptide analysis to have alterations in VP1 (7). Possibly, ts-10 has a similar alteration. A mutation causing a change in the sequence of VP1 could affect the size of VP2 and VP3 for one of two obvious reasons: Either VP1 plays a role in the posttranslational cleavage yielding VP2 and VP3; or a portion of the precursor molecule which is cleaved to yield VP2 and VP3 is also present in VPl. Our data therefore neither imply nor exclude the translation of the same polynucleotidic
v’i’“1”
Vr
VT-6
2400
2000
1200
1000
-0
O-
1000
I500 -
-7 I
500
750
L E e
i -
E
o-
-0
F-T 1500
.2000
750
1000
Ln N
-0
0.
200
I400
100
700
-0
0.
60
20 f,,Ctr’,9,
FIG. 1. Electrophoretic analysis of the polypeptides of Py mutants. ‘311-labeled mutants and lz51labeled wild type, iodinated and purified as described in the text, were mixed and heated at 100” for 5 min in 2% Na dodecyl sulfate (SDS), 2% mercaptoethanol, 0.001 A4 Na phosphate buffer, pH 6.9. Electrophoresis through 10% polyacrylamide cylindrical gels was carried out using the SDS-phosphate system described by Maize1 (19). Gels were frozen at -90” and fractionated with a Hoefer gel slicer. The dried slices were double-counted in a Nuclear Chicago Gamma counter. The figure shows the radioactive patterns obtained for the various gels together with the position of the different polypeptides. Migration was from left to right. The 1311patterns correspond to mutants ts-a (a), ts-3 (b), ts-10 (cl and ts-1260 (d).
288
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sequence into these three virion polypeptides, a theoretical possibility for which positive (8) and negative (9) evidence is available at present. Together with consistent results showing similarity in the tryptic maps of VP2 and VP3 (8, 9), our data however establish that these two polypeptides are under common genetic control. ACKNOWLEDGMENTS We thank Dr. W. Eckhart for kindly supplying us with Py mutants and Dr. W. Gibson for communicating his data in advance of publication. This investigation was supported by the Medical Research Council of Canada. One of us (E.F.) was the recipient of a Research Fellowship from the National Cancer Institute of Canada. Note. One of the referees of Virology has reminded us that lack of complementation between ts-10 and mutants having alterations in VP1 need not he taken to indicate that ts-10 is altered in VPl. In the case of SV40, both complementing and noncomplementing mutants map in the same cistron (201, and lack of complementation by ts-10 could result from protein incompatibilities for assembly rather than from mutations in the same cistron as the VP1 mutants. REFERENCES 1. ROBLIN, R., HXRLE, E., and DULBECCO, R., Virology 45, 555-566 (1971). 2. HIRT, B., and GESTELAND, R. F., Erperientia 27,
736 (1971). 3. FREARSON, P. M., and CRAWFORD, L. V., J. Gen. Viral. 14, 141-155 (1972).
4. FRIEDMANN, T., and DAVID, D., J. Viral. 10, 776-782 (1972). 5. SEEHAFER,J. G., and WEIL, R., Virology 58,75-85 (1974).
6. CRAWFORD, L. V., and GESTELAND, R. F., J. Mol. Biol. 74, 627-634 (1973). 7. FRIEDMANN, T., and ECKHART, W., Cold Spring Harbor Symp. Quant. Biol., in press. 8. FRIEDMANN, T., Proc. Nat. Acad. Sci. USA 71, 257-259 (1974).
9. GIBSON, W., Virology 62, 319-336 (1974). 10. ECKHART, W., Cold Spring Harbor Symp. Qua&. Biol., in press. 11. DI MAYORCA, G., CALLENDER, J., MARIN, G., and GIORDANO, R., Virology 38, 126-133 (1969). 12. FRIED, M., Virology 25, 669-671 (1965). 13. DULBECCO,R., and ECKHART, W., hoc. Not. Acad. Sci. USA 67, 1775-1781 (1970). 14. STANNERS, C. P., Virology 21, 464-476 (1963). 15. CRAWFORD,L. V., Virology 18, 177-181 (1962). 16. GIRARDI, A. J., Virology 9, 488-489 (1959). 17. THORNE, H. V., and WARDLE, A. F., Can. J. Microbial. 19, 291-293 (1973). 18. HUNTER, W. M., and GREENWOOD,F. C., Nature (London) 194, 495-496 (1962). (K. 19. MAIZEL, J. V., In “Methods in Virology” Maramorosch and H. Koprowski, eds), Vol. V, pp. 179-246. Academic Press, New York, 1971. 20. LAI, C. J., and NATHANS, D., Virology 60,466-475 (1974).
65, 286-288 (19751
Electrophoretic
Analysis
of the Structural
Polyoma ERIC FROST’ Dkpartement
de Microbiologic,
Polypeptides
of
Virus Mutants PIERRE
AND
BOURGAUX
Centre Hospitalier Universitaire, Universith Qukbec, Canada JlH 5N4 Accepted
January
de Sherbrooke,
Sherbrooke,
9, 1975
We have analysed the elxtrophoretic mobility of the structural polypeptides of wild-type polyoma virus (Py) and some of its temperature-sensitive (ts) mutants. Mutant ts-IO was found to contain two polypeptides migrating slightly slower than their wild-type counterparts. We suggest that these two structural polypeptides of Py are either coded for or dependent on a single viral gene and possibly are cleavage products from a precursor molecule.
At least six polypeptides have been found in purified polyoma virus (Py) preparations (1-5). The major polypeptide of the capsid (VPl) has always been assumed to be coded for by the virus genome. In vitro coupled transcription and translation of the viral genome (6) and, more recently, genetic evidence (7) back up this assumption. The three or four small molecular weight proteins, VP4-6, are probably host cell histones. Indeed they are synthesized prior to as well as during infection (3, 5), have high lysine to valine ratios (I), lack tryptophan (I, 5) and coelectrophorese with host cell histones (3, 5; and E. Frost, unpublished observations). Recently, two reports have shown that VP2 and VP3 yield similar tryptic peptide maps, which may (8) or may not (9) be similar to that of VPl. Using polyacrylamide-gel electrophoresis, we have compared the structural polypeptides of Py with those of some of its temperature-sensitive (ts) mutants. We report here some of our results which show that a single mutation in the viral genome results in a change in mobility of both VP2 and VP3. Temperature-sensitive mutants of Py I Present address: Institute of Virology, of Glasgow, Glasgow, Scotland GlI 5JR.
University 286
Copyright 0 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
have been grouped into three (10) or four (II) complementation groups. In this study, we have limited ourselves to the early mutant, ts-a (12), two complementing late mutants, ts-10 (10) and ts-1260 (IO), the noncomplementing h-3 (13) and the TSP-1 isolate (14) of the wild type. For virus production, secondary cultures of whole mouse embryos infected at low multiplicity (0.5 plaque-forming units per cell) were incubated for 2 weeks at 33”. The virus was harvested using receptor destroying enzyme (15) and purified by a procedure involving extraction with Freon (16), centrifugation for 1 hr at 38,000 rpm in an SB283 rotor of a B60 International ultracentrifuge, treatment of the sedimented virus with enzymes followed by banding in CsCl, as described by Thorne and Wardle (17), and suspension of the final virus pellet in phosphate-buffered saline (PBS) containing 0.1% bovine serum albumin (BSA). This purified virus was labeled with either 1311or lz51 using chloramine-T (18). Twenty-five microliters of virus were added to a mixture of 10 ~1 each of 1 M Na phosphate buffer, pH 6.9, 0.1 N HCl, and carrier-free radioactive iodine (300-500 PCi; ICN) in 0.1 N NaOH. Iodination was initiated by addition of 4 pg of chloramine-T and stopped with 12.5 pg of potas-
SHORT
287
COMMUNICATIONS
sium meta-bisulfite after 45 set of incubation at room temperature. Labeled virus was separated from unreacted iodine and iodine-labeled BSA by velocity centrifugation in a sucrose solution (in 0.1 M NaCl, 0.001 M EDTA, 0.01 M Tris-HCl, pH 7.4). The detailed iodination method will be presented elsewhere (E. Frost, in preparation). The 13’1-labeled mutants were mixed with ‘2”I-labeled wild-type virus prior to dissociation and electrophoretic separation of the polypeptides. As shown in Fig. 1, no differences are found in the electrophoretic mobilities of the polypeptides from wildtype, ts-a, h-3 and ts-1260. In the pattern observed for h-10, however, the two polypeptides corresponding to VP2 and VP3 have reduced mobilities, suggesting that they have a higher molecular weight than their wild-type counterparts. It is noteworthy that VP1 of ts-10 and of wild-type virus comigrate. Electrophoresis for longer times failed to show a difference in the mobility of VP1 between wild-type virus, b-10, or any of the other mutants mentioned. Results identical to those shown in Fig. 1 were obtained with several preparations of iodinated virus. Since there is no reason to suspect that the genome of ts-10 contains more than one mutation, our data provide evidence that VP2 and VP3 are either coded for by the same viral gene or, alternatively, dependent on the same viral gene product. Although other explanations are possible, they may also indicate that both polypeptides are derived from a precursor, or precursors, of higher molecular weight. Mutants of the same complementation group as ts-10, have been shown by twodimensional peptide analysis to have alterations in VP1 (7). Possibly, ts-10 has a similar alteration. A mutation causing a change in the sequence of VP1 could affect the size of VP2 and VP3 for one of two obvious reasons: Either VP1 plays a role in the posttranslational cleavage yielding VP2 and VP3; or a portion of the precursor molecule which is cleaved to yield VP2 and VP3 is also present in VPl. Our data therefore neither imply nor exclude the translation of the same polynucleotidic
v’i’“1”
Vr
VT-6
2400
2000
1200
1000
-0
O-
1000
I500 -
-7 I
500
750
L E e
i -
E
o-
-0
F-T 1500
.2000
750
1000
Ln N
-0
0.
200
I400
100
700
-0
0.
60
20 f,,Ctr’,9,
FIG. 1. Electrophoretic analysis of the polypeptides of Py mutants. ‘311-labeled mutants and lz51labeled wild type, iodinated and purified as described in the text, were mixed and heated at 100” for 5 min in 2% Na dodecyl sulfate (SDS), 2% mercaptoethanol, 0.001 A4 Na phosphate buffer, pH 6.9. Electrophoresis through 10% polyacrylamide cylindrical gels was carried out using the SDS-phosphate system described by Maize1 (19). Gels were frozen at -90” and fractionated with a Hoefer gel slicer. The dried slices were double-counted in a Nuclear Chicago Gamma counter. The figure shows the radioactive patterns obtained for the various gels together with the position of the different polypeptides. Migration was from left to right. The 1311patterns correspond to mutants ts-a (a), ts-3 (b), ts-10 (cl and ts-1260 (d).
288
SHORT
COMMUNICATIONS
sequence into these three virion polypeptides, a theoretical possibility for which positive (8) and negative (9) evidence is available at present. Together with consistent results showing similarity in the tryptic maps of VP2 and VP3 (8, 9), our data however establish that these two polypeptides are under common genetic control. ACKNOWLEDGMENTS We thank Dr. W. Eckhart for kindly supplying us with Py mutants and Dr. W. Gibson for communicating his data in advance of publication. This investigation was supported by the Medical Research Council of Canada. One of us (E.F.) was the recipient of a Research Fellowship from the National Cancer Institute of Canada. Note. One of the referees of Virology has reminded us that lack of complementation between ts-10 and mutants having alterations in VP1 need not he taken to indicate that ts-10 is altered in VPl. In the case of SV40, both complementing and noncomplementing mutants map in the same cistron (201, and lack of complementation by ts-10 could result from protein incompatibilities for assembly rather than from mutations in the same cistron as the VP1 mutants. REFERENCES 1. ROBLIN, R., HXRLE, E., and DULBECCO, R., Virology 45, 555-566 (1971). 2. HIRT, B., and GESTELAND, R. F., Erperientia 27,
736 (1971). 3. FREARSON, P. M., and CRAWFORD, L. V., J. Gen. Viral. 14, 141-155 (1972).
4. FRIEDMANN, T., and DAVID, D., J. Viral. 10, 776-782 (1972). 5. SEEHAFER,J. G., and WEIL, R., Virology 58,75-85 (1974).
6. CRAWFORD, L. V., and GESTELAND, R. F., J. Mol. Biol. 74, 627-634 (1973). 7. FRIEDMANN, T., and ECKHART, W., Cold Spring Harbor Symp. Quant. Biol., in press. 8. FRIEDMANN, T., Proc. Nat. Acad. Sci. USA 71, 257-259 (1974).
9. GIBSON, W., Virology 62, 319-336 (1974). 10. ECKHART, W., Cold Spring Harbor Symp. Qua&. Biol., in press. 11. DI MAYORCA, G., CALLENDER, J., MARIN, G., and GIORDANO, R., Virology 38, 126-133 (1969). 12. FRIED, M., Virology 25, 669-671 (1965). 13. DULBECCO,R., and ECKHART, W., hoc. Not. Acad. Sci. USA 67, 1775-1781 (1970). 14. STANNERS, C. P., Virology 21, 464-476 (1963). 15. CRAWFORD,L. V., Virology 18, 177-181 (1962). 16. GIRARDI, A. J., Virology 9, 488-489 (1959). 17. THORNE, H. V., and WARDLE, A. F., Can. J. Microbial. 19, 291-293 (1973). 18. HUNTER, W. M., and GREENWOOD,F. C., Nature (London) 194, 495-496 (1962). (K. 19. MAIZEL, J. V., In “Methods in Virology” Maramorosch and H. Koprowski, eds), Vol. V, pp. 179-246. Academic Press, New York, 1971. 20. LAI, C. J., and NATHANS, D., Virology 60,466-475 (1974).