Vol. 19, No. 2 Printed in U.S.A.
JOURNAL OF VIROLOGY, Aug. 1976, p. 750-755 Copyright © 1976 American Society for Microbiology
Radioisotopic Labeling of Human Papovavirus (BK) by Iodination and Reductive Alkylation PETER J. WRIGHT' AND GIAMPIERO DI MAYORCA* Department of Microbiology, University ofIllinois at the Medical Center, Chicago, Illinois 60680
Received for publication 14 January 1976
Purified virions of the GS strain of the BK group of human papovaviruses labeled with 125I using chloramine T or lactoperoxidase or with tritium using sodium borohydride. All viral polypeptides were labeled. Tryptic digests of iodinated VP1 were analyzed. were
The polypeptide composition of purified BK virus, a human papovavirus derived from urine, has been examined in several laboratories (1, 6, 8, 9). The molecular weights of the capsid proteins are more similar to those of simian virus 40 (SV40) than to those of polyoma virus. The major virion protein VP1, constituting approximately 75% of the capsid protein, is smaller than VP1 of both SV40 and polyoma virus and has a tryptic peptide pattern different from that of VP1 of SV40 (9). In this communication we describe attempts using lactoperoxidase-catalyzed iodination and reductive alkylation to determine which polypeptide or polypeptides are exposed on the surface of the human papovavirus. Tryptic digests of iodinated VP1 were also examined for evidence of selective labeling of peptides by different methods of iodination. The GS strain of the BK group of human papovaviruses isolated from urine was used in all experiments. Serological analyses and examination of the tryptic peptides of GS virus and BK virus have indicated that they are identical or very closely related (8). Virus was grown in human embryonic kidney cells and purified in CsCl gradients as previously described (9). Three different methods were used for iodinating purified preparations of intact virions. (i) A 35-,ug sample of virus in TD buffer (135 mM NaCl-5 mM KCl-0.7 mM Na2HPO4-25 mM Tris) was iodinated with 0.5 mCi of carrier-free 125I (New England Nuclear) using chloramine T (9). The final 125I concentration was 0.5 ,uM. (ii) A 0.1-ml portion of virus (130 ,ug) in TD was added to 0.3 ml of 0.5 M Tris buffer (pH 6.8) followed by 0.15 ml of carrierfree 12'I (1.5 mCi) in NaOH, 24 jig of lactoperoxidase (Calbiochem) dissolved in 0.02 ml of '
Present address: Department of Microbiology, Monash
University Medical School, Alfred Hospital, Prahran, Victoria, 3181, Australia.
buffer, and finally by 0.025 ml of a 10-4 dilution of 50% H202, (7). The final pH of the reaction mixture was 7.5, and the 12"I concentration was 1.2 ,uM. After 30 min at 25 C the mixture was dialyzed against TD. (iii) The third method of iodinating virus used lactoperoxidase bound to Sepharose (Worthington; specific activity, 300 units/g [dry weight]). The procedure followed was that of Montelaro and Rueckert (5). Virus (60 gg) was iodinated using a 0.4-ml bed volume of lactoperoxidase-Sepharose and 1.0 mCi of '25I. The final iodide concentration was 15 ,uM. Labeled virus was separated from the other components of the reaction mixture as described by Gibson (2). Disrupted virions were also iodinated; 0.175 ml of 4 M urea and 0.2% sodium dodecyl sulfate in TD were added to 35 ,g of virus in 0.025 ml of TD, the mixture was heated at 100 C for 2 min, and then the viral polypeptides were iodinated using chloramine T (method i). Intact virions were labeled with L3H]formaldehyde by a method identical to that described by McMillen and Consigli for labeling polyoma virus (4). Viral preparations were disrupted with sodium dodecyl sulfate and 2-mercaptoethanol before electrophoresis (9). Tryptic digests were analyzed on a column of Technicon type P chromobeads ion-exchange resin. Conditions for developing the column have been described elsewhere (8). The column was developed with a linear gradient of 0.2 M pyridine-acetate, pH 3.1 (100 ml) to 2 M pyridine-acetate, pH 5.0 (100 ml). Fractions of equal volume were collected, and the molarity at which each peptide eluted from the column was estimated as previously described (8). The molarity of elution of a peptide is marked adjacent to its corresponding peak in Fig. 3-5. To determine the effect of iodination on the structural integrity of virions, viral preparations were centrifuged through sucrose gra-
750
VOL. 19, 1976
NOTES
751
FIG. 1. Analysis in linear sucrose gradients of viral preparations iodinated using (A) chloramine T and (B) free lactoperoxidase. Sucrose gradients (5 to 20%, wtlwt) in TD were centrifuged for1.5 h at 48,000 x g in a Spinco SW41 rotor at 20 t. 15
A
'251 cpm
ID
i~~~~VP,
P
I'IzIII-3 VPI
'
25 ,cpm
FIG. 2. Gel electrophoresis of labeled GS virus. (A) Virus disrupted with urea and sodium dodecyl sulfate before iodination with chloramine T. Intact virus labeled using chloramine T (B), using free lactoperoxidase (C), using Sepharose-bound lactoperoxidase (D), and using [3H]formaldehyde (E). Gels in (A), (B), and (C) were subjected to electrophoresis in parallel, as were those in (D) and (E). The histone-like proteins are in fractions 55 to 75.
752
NOTES
J. VIROL.
125i
FIG. 3. Analysis of the tryptic digests of VP1 obtained from iodinated intact virus. (A) Virus iodinated using chloramine T; 5.0 x 105 counts/min were analyzed. (B) Virus iodinated using free lactoperoxidase; 6.0 x 105 countslmin were analyzed. (C) Mixture of the above two digests; each contributing 1.5 x 105 counts/min .
VOL. 19, 1976
NOTES
dients after treatment with chloramine T or with lactoperoxidase. A typical result following iodination with chloramine T is shown in Fig. 1A. Some breakdown occurred, although the major proportion of the label (75%) sedimented in a position corresponding to that of intact virus. lodination using lactoperoxidase (method ii) (Fig. 1B) did not change the sedimentation properties of the virus (9). The gel patterns after electrophoresis of labeled viral preparations are shown in Fig. 2. Each of the three methods used for iodinating intact virus gave preparations remarkably similar in the composition of their labeled polypeptides. There was no evidence from the gels that lactoperoxidase labeled polypeptides more selectively than chloramine T. However, analysis of the tryptic digests of iodinated VP1 (Fig. 3 and 4) demonstrated that the degree of labeling of individual peptides depended upon the method used. Some peptides were not present in all preparations (e.g., those at 1.26-1.27 and 1.58 in Fig. 3B and 4 and that at 1.14 in Fig. 3A). Significant quantitative differences in labeling were apparent in the three peptides in the region from 0.36 to 0.51 and in the peptide at 1.58 (Fig. 3A, 3B, and 4). Figure 5 compares the tryptic peptides of VP1 '251
753
derived from iodinated, disrupted virus with those of the high-molecular-weight protein of the same preparation (fractions 14 and 15 in Fig. 2A). Both polypeptides contained the same iodinated peptides in the same proportions, suggesting that their amino acid sequences are identical or at least very closely related. A similar result was reported for polyoma virus, in which the larger protein is a dimer of VP1 (3). With the exception of the two peptides at 0.930.96 and at 1.58 (Fig. 3B and 4), and of that at 0.88 (Fig. 4), all the iodinated peptides of VP1 from intact virus were found in VP1 from disrupted virus (Fig. 5A). Neither iodination nor reductive alkylation of intact virus selectively labeled only one or two virion proteins. This implies that all polypeptides, including the histone-like proteins, are exposed on the surface of the virion, or that the reaction conditions disrupted the capsid structure (2). The data in Fig. 1 argues against the latter possibility in reactions with lactoperoxidase. The labeling of tryptic peptides of VP1 was dependent on the method of iodination. The concentration of iodide, as well as the nature of the iodinating agent, was probably an important factor in determining the degree of labeling of each peptide (5).
cDmI
0
40
90
120
160
FRACTION NO
FIG. 4. Analysis of the tryptic digests of VPI obtained from intact virus iodinated using Sepharose-bound lactoperoxidase (1.7 x 1O5 counts/min were analyzed).
754
J. VIROL.
NOTES 12511
I1cpm A
144
160460
040
20
80 116
068 046
0 75
40 0 51
082
108 92 ~~~~~~~~~1
127
B
040
146
32
24'Is
07
16
0 66
045 0 52
8
00
0 82
~~~107 0
40
80
1 92
FRACTION NO.
120
160
200
FIG. 5. Analysis of the tryptic peptides ofpolypeptides derived from virus iodinated after disruption with sodium dodecyl sulfate and urea. (A) VP1; approximately 6.3 x 105 counts/min were analyzed. (B) Largemolecular-weight-protein (fractions 14 and 15 in Fig. 2A); 1.8 x 105 counts/mmn were analyzed. ACKNOWLEDGMENTS This work was supported by Public Health Service conthe Virus Cancer Program tract NIH-NCI VCP 43318 from Cancer
son with the structural proteins of simian virus 40. Virology 64:269-271. 2. Gibson, W. 1974. Polyoma virus proteins: a description of the structural proteins of the virion based on polyacrylamide gel electrophoresis and peptide analysis.
LITERATURE CITED 1. Barbanti-Brodano, G., G. P. Minelli, M. Portolani, L. Lambertini, and M. Toppini. 1975. Structural proteins of a human papovavirus (BK virus): a compari-
3. Hewick, R. M., M. Fried, and M. D. Waterfield. 1975. Nonhistone virion proteins of polyoma: characterization of the particle proteins by tryptic peptide analysis by use of ion-exchange columns. Virology 66:408-
Inre tracte NatiHnCaVP438fo
Virology 62:319-336.
VOL. 19, 1976 419.
4. McMillen, J., and R. A. Consigli. 1974. In vitro radioisotopic labeling of proteins associated with purified polyoma virions. J. Virol. 14:1627-1629. 5. Montelaro, R. C., and R. R. Rueckert. 1975. On the use of chloramine T to iodinate specifically the surface proteins of intact enveloped viruses. J. Gen. Virol. 29:127-131. 6. Mullarkey, M. F., J. F. Hruska, and K. K. Takemoto. 1974. Comparison of two human papovaviruses with simian virus 40 by structural protein and antigenic analysis. J. Virol. 13:1014-1019.
NOTES
755
7. Phillips, D. R., and M. Morrison. 1970. The arrangement of proteins in the human erythrocyte membrane. Biochem. Biophys. Res. Commun. 40:284-289. 8. Wright, P. J., G. Bernhardt, E. 0. Major, and G. di Mayorca. 1976. Comparison of the serology, transforming ability and polypeptide composition of human papovaviruses isolated from urine. J. Virol. 17:762-775. 9. Wright, P. J., and G. di Mayorca. 1975. Virion polypeptide composition of the human papovavirus BK: comparison with simian virus 40 and polyoma virus. J. Virol. 15:828-835.
JOURNAL OF VIROLOGY, Aug. 1976, p. 750-755 Copyright © 1976 American Society for Microbiology
Radioisotopic Labeling of Human Papovavirus (BK) by Iodination and Reductive Alkylation PETER J. WRIGHT' AND GIAMPIERO DI MAYORCA* Department of Microbiology, University ofIllinois at the Medical Center, Chicago, Illinois 60680
Received for publication 14 January 1976
Purified virions of the GS strain of the BK group of human papovaviruses labeled with 125I using chloramine T or lactoperoxidase or with tritium using sodium borohydride. All viral polypeptides were labeled. Tryptic digests of iodinated VP1 were analyzed. were
The polypeptide composition of purified BK virus, a human papovavirus derived from urine, has been examined in several laboratories (1, 6, 8, 9). The molecular weights of the capsid proteins are more similar to those of simian virus 40 (SV40) than to those of polyoma virus. The major virion protein VP1, constituting approximately 75% of the capsid protein, is smaller than VP1 of both SV40 and polyoma virus and has a tryptic peptide pattern different from that of VP1 of SV40 (9). In this communication we describe attempts using lactoperoxidase-catalyzed iodination and reductive alkylation to determine which polypeptide or polypeptides are exposed on the surface of the human papovavirus. Tryptic digests of iodinated VP1 were also examined for evidence of selective labeling of peptides by different methods of iodination. The GS strain of the BK group of human papovaviruses isolated from urine was used in all experiments. Serological analyses and examination of the tryptic peptides of GS virus and BK virus have indicated that they are identical or very closely related (8). Virus was grown in human embryonic kidney cells and purified in CsCl gradients as previously described (9). Three different methods were used for iodinating purified preparations of intact virions. (i) A 35-,ug sample of virus in TD buffer (135 mM NaCl-5 mM KCl-0.7 mM Na2HPO4-25 mM Tris) was iodinated with 0.5 mCi of carrier-free 125I (New England Nuclear) using chloramine T (9). The final 125I concentration was 0.5 ,uM. (ii) A 0.1-ml portion of virus (130 ,ug) in TD was added to 0.3 ml of 0.5 M Tris buffer (pH 6.8) followed by 0.15 ml of carrierfree 12'I (1.5 mCi) in NaOH, 24 jig of lactoperoxidase (Calbiochem) dissolved in 0.02 ml of '
Present address: Department of Microbiology, Monash
University Medical School, Alfred Hospital, Prahran, Victoria, 3181, Australia.
buffer, and finally by 0.025 ml of a 10-4 dilution of 50% H202, (7). The final pH of the reaction mixture was 7.5, and the 12"I concentration was 1.2 ,uM. After 30 min at 25 C the mixture was dialyzed against TD. (iii) The third method of iodinating virus used lactoperoxidase bound to Sepharose (Worthington; specific activity, 300 units/g [dry weight]). The procedure followed was that of Montelaro and Rueckert (5). Virus (60 gg) was iodinated using a 0.4-ml bed volume of lactoperoxidase-Sepharose and 1.0 mCi of '25I. The final iodide concentration was 15 ,uM. Labeled virus was separated from the other components of the reaction mixture as described by Gibson (2). Disrupted virions were also iodinated; 0.175 ml of 4 M urea and 0.2% sodium dodecyl sulfate in TD were added to 35 ,g of virus in 0.025 ml of TD, the mixture was heated at 100 C for 2 min, and then the viral polypeptides were iodinated using chloramine T (method i). Intact virions were labeled with L3H]formaldehyde by a method identical to that described by McMillen and Consigli for labeling polyoma virus (4). Viral preparations were disrupted with sodium dodecyl sulfate and 2-mercaptoethanol before electrophoresis (9). Tryptic digests were analyzed on a column of Technicon type P chromobeads ion-exchange resin. Conditions for developing the column have been described elsewhere (8). The column was developed with a linear gradient of 0.2 M pyridine-acetate, pH 3.1 (100 ml) to 2 M pyridine-acetate, pH 5.0 (100 ml). Fractions of equal volume were collected, and the molarity at which each peptide eluted from the column was estimated as previously described (8). The molarity of elution of a peptide is marked adjacent to its corresponding peak in Fig. 3-5. To determine the effect of iodination on the structural integrity of virions, viral preparations were centrifuged through sucrose gra-
750
VOL. 19, 1976
NOTES
751
FIG. 1. Analysis in linear sucrose gradients of viral preparations iodinated using (A) chloramine T and (B) free lactoperoxidase. Sucrose gradients (5 to 20%, wtlwt) in TD were centrifuged for1.5 h at 48,000 x g in a Spinco SW41 rotor at 20 t. 15
A
'251 cpm
ID
i~~~~VP,
P
I'IzIII-3 VPI
'
25 ,cpm
FIG. 2. Gel electrophoresis of labeled GS virus. (A) Virus disrupted with urea and sodium dodecyl sulfate before iodination with chloramine T. Intact virus labeled using chloramine T (B), using free lactoperoxidase (C), using Sepharose-bound lactoperoxidase (D), and using [3H]formaldehyde (E). Gels in (A), (B), and (C) were subjected to electrophoresis in parallel, as were those in (D) and (E). The histone-like proteins are in fractions 55 to 75.
752
NOTES
J. VIROL.
125i
FIG. 3. Analysis of the tryptic digests of VP1 obtained from iodinated intact virus. (A) Virus iodinated using chloramine T; 5.0 x 105 counts/min were analyzed. (B) Virus iodinated using free lactoperoxidase; 6.0 x 105 countslmin were analyzed. (C) Mixture of the above two digests; each contributing 1.5 x 105 counts/min .
VOL. 19, 1976
NOTES
dients after treatment with chloramine T or with lactoperoxidase. A typical result following iodination with chloramine T is shown in Fig. 1A. Some breakdown occurred, although the major proportion of the label (75%) sedimented in a position corresponding to that of intact virus. lodination using lactoperoxidase (method ii) (Fig. 1B) did not change the sedimentation properties of the virus (9). The gel patterns after electrophoresis of labeled viral preparations are shown in Fig. 2. Each of the three methods used for iodinating intact virus gave preparations remarkably similar in the composition of their labeled polypeptides. There was no evidence from the gels that lactoperoxidase labeled polypeptides more selectively than chloramine T. However, analysis of the tryptic digests of iodinated VP1 (Fig. 3 and 4) demonstrated that the degree of labeling of individual peptides depended upon the method used. Some peptides were not present in all preparations (e.g., those at 1.26-1.27 and 1.58 in Fig. 3B and 4 and that at 1.14 in Fig. 3A). Significant quantitative differences in labeling were apparent in the three peptides in the region from 0.36 to 0.51 and in the peptide at 1.58 (Fig. 3A, 3B, and 4). Figure 5 compares the tryptic peptides of VP1 '251
753
derived from iodinated, disrupted virus with those of the high-molecular-weight protein of the same preparation (fractions 14 and 15 in Fig. 2A). Both polypeptides contained the same iodinated peptides in the same proportions, suggesting that their amino acid sequences are identical or at least very closely related. A similar result was reported for polyoma virus, in which the larger protein is a dimer of VP1 (3). With the exception of the two peptides at 0.930.96 and at 1.58 (Fig. 3B and 4), and of that at 0.88 (Fig. 4), all the iodinated peptides of VP1 from intact virus were found in VP1 from disrupted virus (Fig. 5A). Neither iodination nor reductive alkylation of intact virus selectively labeled only one or two virion proteins. This implies that all polypeptides, including the histone-like proteins, are exposed on the surface of the virion, or that the reaction conditions disrupted the capsid structure (2). The data in Fig. 1 argues against the latter possibility in reactions with lactoperoxidase. The labeling of tryptic peptides of VP1 was dependent on the method of iodination. The concentration of iodide, as well as the nature of the iodinating agent, was probably an important factor in determining the degree of labeling of each peptide (5).
cDmI
0
40
90
120
160
FRACTION NO
FIG. 4. Analysis of the tryptic digests of VPI obtained from intact virus iodinated using Sepharose-bound lactoperoxidase (1.7 x 1O5 counts/min were analyzed).
754
J. VIROL.
NOTES 12511
I1cpm A
144
160460
040
20
80 116
068 046
0 75
40 0 51
082
108 92 ~~~~~~~~~1
127
B
040
146
32
24'Is
07
16
0 66
045 0 52
8
00
0 82
~~~107 0
40
80
1 92
FRACTION NO.
120
160
200
FIG. 5. Analysis of the tryptic peptides ofpolypeptides derived from virus iodinated after disruption with sodium dodecyl sulfate and urea. (A) VP1; approximately 6.3 x 105 counts/min were analyzed. (B) Largemolecular-weight-protein (fractions 14 and 15 in Fig. 2A); 1.8 x 105 counts/mmn were analyzed. ACKNOWLEDGMENTS This work was supported by Public Health Service conthe Virus Cancer Program tract NIH-NCI VCP 43318 from Cancer
son with the structural proteins of simian virus 40. Virology 64:269-271. 2. Gibson, W. 1974. Polyoma virus proteins: a description of the structural proteins of the virion based on polyacrylamide gel electrophoresis and peptide analysis.
LITERATURE CITED 1. Barbanti-Brodano, G., G. P. Minelli, M. Portolani, L. Lambertini, and M. Toppini. 1975. Structural proteins of a human papovavirus (BK virus): a compari-
3. Hewick, R. M., M. Fried, and M. D. Waterfield. 1975. Nonhistone virion proteins of polyoma: characterization of the particle proteins by tryptic peptide analysis by use of ion-exchange columns. Virology 66:408-
Inre tracte NatiHnCaVP438fo
Virology 62:319-336.
VOL. 19, 1976 419.
4. McMillen, J., and R. A. Consigli. 1974. In vitro radioisotopic labeling of proteins associated with purified polyoma virions. J. Virol. 14:1627-1629. 5. Montelaro, R. C., and R. R. Rueckert. 1975. On the use of chloramine T to iodinate specifically the surface proteins of intact enveloped viruses. J. Gen. Virol. 29:127-131. 6. Mullarkey, M. F., J. F. Hruska, and K. K. Takemoto. 1974. Comparison of two human papovaviruses with simian virus 40 by structural protein and antigenic analysis. J. Virol. 13:1014-1019.
NOTES
755
7. Phillips, D. R., and M. Morrison. 1970. The arrangement of proteins in the human erythrocyte membrane. Biochem. Biophys. Res. Commun. 40:284-289. 8. Wright, P. J., G. Bernhardt, E. 0. Major, and G. di Mayorca. 1976. Comparison of the serology, transforming ability and polypeptide composition of human papovaviruses isolated from urine. J. Virol. 17:762-775. 9. Wright, P. J., and G. di Mayorca. 1975. Virion polypeptide composition of the human papovavirus BK: comparison with simian virus 40 and polyoma virus. J. Virol. 15:828-835.
