Vol. 15, No. 4 Printed in U.S.A.
JOURNAL OF VIROLOGY, Apr. 1975, p. 828-835 Copyright © 1975 American Society for Microbiology
Virion Polypeptide Composition of the Human Papovavirus BK: Comparison with Simian Virus 40 and Polyoma Virus PETER J. WRIGHT* AND GIAMPIERO DI MAYORCA Department of Microbiology, University of Illinois at the Medical Center, Chicago, Illinois 60680 Received for publication 5 August 1974
The polypeptide composition of labeled BK virus was compared with that of simian virus 40 (SV40) and polyoma virus by co-electrophoresis of disrupted virions in polyacrylamide gels containing sodium dodecyl sulfate. The major polypeptide (VP1) of BK constituted approximately 73% of the capsid protein and had a molecular weight of 39,000. It was smaller than VP1 of SV40 and polyoma virus. The other polypeptides of BK virus were similar in molecular weight to those of SV40. A comparison of the proteins of BK virus and SV40 iodinated with chloramine T before and after disruption in alkaline buffer at pH 10.5 revealed differences between the two viruses in the number and distribution of tyrosines available for iodination. The tryptic peptides of VP1, VP3, VP4, and VP5 combined of SV40 were compared with those of the same polypeptides of BK virus. Among the 19 peptides of VP1 resolved, only two were common to both viruses. The analyses of VP4 and VP5, the histone-like proteins, however, showed more similarity between the viruses, with 6 of 15 resolved peptides in common. The tryptic digests of VP3 were completely different. BK virus, originally isolated by Gardner et al. from the urine of a renal allograft recipient (6), is one of the group of human papovaviruses that have been isolated either from patients with progressive multifocal leukoencephalopathy (15, 22) or from renal allograft recipients (3, 6). An antigenic relationship between BK virus and simian virus 40 (SV40) has been demonstrated by immune electron microscopy (6, 16) and by fluorescent antibody tests (20). A low level of cross-reaction has also been measured by plaque neutralization (20; E. 0. Major and G. di Mayorca, unpublished data). No antigenic similarity was detected by the techniques of complement fixation, immunodiffusion, or immunoelectrophoresis (14). To gain more information on the relationship of BK virus to other papovaviruses, the proteins of the viral particle have been examined. In this paper the virion polypeptides of BK virus are compared by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate (SDS) with those of two well characterized papovaviruses, polyoma and SV40. Polyoma, like BK virus but unlike SV40, is hemagglutinating. A preliminary report of these results has already appeared (P. J. Wright and G. di Mayorca, Abstr. Annu. Meet. Amer. Soc. Microbiol. 1974, V224, p. 238). Also presented are comparisons of the tryptic peptides derived from the virion proteins of SV40 and BK virus. 828
MATERIALS AND METHODS Cells and viruses. BK virus was grown as previously described (13) in primary and secondary cultures of human embryonic kidney cells. The human embryonic kidney cells were grown on Dulbecco's modified Eagle (DME) medium with 10% fetal calf serum before infection, and with 5% horse serum after infection. Cells were infected at a multiplicity of 0.5 to 5 PFU/cell and harvested when virtually all cells showed cytopathic effects, normally 2 weeks after infection. To obtain labeled virus, 8H-labeled reconstituted protein hydrolysate (Schwarz/Mann) or [8H]lysine (Amersham Searle, specific activity of 160 mCi/mmol) at final concentrations of 10 MCi/ml or 0.2 MCi/ml, respectively, were added at the time of infection to cells in DME or DME medium lacking lysine, respectively. Polyoma virus was grown in mouse 3T3 cells (2) and SV40 in BSC-1 cells, both a gift from C. Basilico. Labeled virus was obtained by maintaining infected cells in DME medium containing 1 ACi of "C-labeled reconstituted protein hydrolysate (Schwarz/ Mann) per ml and either 5% horse serum (3T3 cells) or 2% fetal calf serum (BSC-1 cells). Cells were harvested 4 to 7 days after infection when cytopathic effects were evident. Purification of virus. The methods used were essentially those described by Roblin et al. (18). The infected cells, still suspended in medium, were given six cycles of freezing and thawing and treated with 0.2% sodium deoxycholate for 1 h at 37 C, and large debris was removed by centrifugation for 15 min at 10,000 x g. The supernatant was centrifuged at 20 C for 4 h at 64,000 x g in a Spinco SW 25.2 rotor over a
VOL. 15, 1975
POLYPEPTIDES OF BK VIRUS
cushion of 14 ml of saturated KBr solution in 0.05 M Tris-hydrochloride (pH 8.0) plus 0.01 M EDTA. Two, sometimes three, bands were observed; the lowest one was collected and dialyzed against a Tris buffer, TD (135 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 25 mM Tris). The dialyzed material was made up to 7.5 ml with TD and added to 3.45 g of CsCl, and the solution was centrifuged at 20 C for 16 h at 74,000 x g in a Spinco type 50 rotor. The single band of virus obtained was dialyzed against TD and either subjected to a second CsCl gradient as above, or a sucrose gradient (see Fig. IC). The acid-insoluble counts in gradient fractions were assayed on Whatman no. 1 filter paper disks. Disruption and iodination of virus. Virus was disrupted by dialysis for 16 h at 5 C against 0.15 M carbonate-bicarbonate buffer, pH 10.5 (17). lodination (1211, carrier free; Amersham Searle) was done by the chloramine T method (9), which preferentially iodinates tyrosine (8). Each experiment used 0.25 ml of a viral preparation with an absorbance at 260 nm of 0.32 units. Gel electrophoresis. Viral preparations for electrophoresis were dialyzed against 0.05 M phosphate (pH 7.3), adjusted to 2% SDS and 1% 2-mercaptoethanol, heated for 2 min at 100 C, and finally dialyzed against 0.01 M phosphate (pH 7.3), 0.1% SDS, and 0.1% 2-mercaptoethanol. Samples were subject to electrophoresis on 10% gels containing SDS using the continuous gel system described by Maizel (12), the gels were sliced, and the slices were counted in dioxanebased scintillation fluid (24). Tryptic digests. After electrophoresis of iodinated viral preparations, the proteins to be treated with trypsin were eluted from the gel slices into 8 ml of a solution of 0.01 M phosphate (pH 7.3) and 0.1% SDS. Ovalbumin (7.5 mg), followed by trichloroacetic acid to a final concentration of 10%, was added, and the precipitates were washed twice with ethanol and then redissolved in 0.7 ml of a buffer of 0.5 M Tris-hydrochloride (pH 8.8) containing 0.5% SDS. The samples were treated with 20 mg of dithiothreitol for 1 h at 37 C, and subsequently with 70 mg of iodoacetamide for 2 h at 25 C. Thereafter the proteins were prepared for analysis of the tryptic peptides as described by Jacobson et al. (10). The initial treatment with 0.1 mg of trypsin (treated with TPCK; Worthington) was at 37 C for 21 h, followed by a further 0.2 mg for 2 h. The tryptic digests were analyzed on a column (30 by 0.9 cm; Glenco Scientific) of Technicon type P chromobeads ion-exchange resin held at 50 C and developed with a linear gradient of 0.2 M pyridineacetate (pH 3.1) to 2 M pyridine-acetate, pH 5.0 (10, 19). The column was maintained under pressure; the flow rate was approximately 16 ml per h, and the fractions (1.3 ml) were counted in 5 ml of the same dioxan-based scintillation fluid used to count gel slices (24).
RESULTS Purif'ication of virus. The procedures used by Roblin et al. for polyoma virus (18) proved suitable for the purification of BK virus. The
829
typical result from several experiments is displayed in Fig. 1. After centrifugation to equilibrium in CsCl solution, BK virus banded at a similar density (1.34 g/ml). The approximate s value of the purified virus, estimated from Fig. 1C by the method of McEwen (11), was 280S. Published values for other papovaviruses are 240 to 300S (23). The material in fractions 13, 14, and 15 of Fig. 1C was also centrifuged through successive CsCl and sucrose gradients. The density was approximately that of protein (1.29 g/ml), and its s value was about 200S. This 8200 000 -
\
600 i200-
000 3H cpm00 800
I
400
600 Y
300
400-
200 "C cpm
C0 20C
100
~~0
B 3003H
cpm 200
C 600
50C 00c 3H
cpm
-
00 ' 3CDC
200
C
C 5 L 20 FRACT 0N %JM9ER
3c.
FIG. 1. (A) Approximately the bottom third of a tube containing a cushion of saturated KBr solution after centrifugation as described. Prior to centrifugation, infected cell lysate labeled with [3H]lysine was mixed with uninfected cell lysate labeled with "Clabeled reconstituted protein hydrolysate. (B) The virus shown in Fig. IA (fractions 10, 11, 12) after dialysis and centrifugation to equilibrium in CsCI solution. (C) The same viral preparation as shown in Fig. lB after dialysis and centrifugation at 20 C for 1.5 h at 48,000 x g in the Spinco SW41 rotor through a 5 to 20% (wt/wt) sucrose gradient in TD buffer.
J. VIROL.
WRIGHT AND DI MAYORCA
830
material probably corresponded to empty cap- 700 sids. Polypeptide composition. The gel pattern of - 600 BK virus labeled with reconstituted protein hydrolysate is shown in Fig. 2. From this preparation of virus the estimated proportion of 500 the total virion protein in the major polypeptide (VP1) (peak at fraction 28) was 73%, in VP3 - 400 (peak at fraction 46) was 7%, and in the C cpm low-molecular-weight region VP4,VP5 (frac- 3H cpm tions 55 to 70) was 9%. The proportion of protein - 300 at high molecular weight (peak at fraction 10) was 10%, and the remaining 1% corresponded to -200 the small amount of material (VP2) migrating It is 34. unlikely just ahead of VP1 at fraction - 100 that the material at fraction 10 represented aggregated protein, since care was taken to IA-A maintain reducing conditions during the disruption and electrophoresis of viral preparations. The estimated molecular weight of VP1 0 20 40 60 80 was 39,000, calculated by co-electrophoresing FRACTION NUMBER viral samples with the following iodinated stanFIG. 3. Co-electrophoresis of BK virus labeled with dard proteins: pepsin (34,000 daltons), myoglobin (17,300), and cytochrome c (12,500) (24). [3H]lysine and SV40 labeled with "C-labeled reconThe results of co-electrophoresing the poly- stituted protein hydrolysate. peptides of BK virus with those of SV40 and 700polyoma virus are shown in Fig. 3 and 4, respectively. The major virion protein VP1 of 1200 BK virus was smaller than that of SV40 (differ600 ence of one slice) and even smaller than that of -1000 polyoma virus (difference of three slices). These
FO
500 -
-
600
400-
3H
3000-
800
14C
cpm
cpm
400
300-
200
2500-
100
20001
-
3H cpm
-I
B
B
1500-
v
0
Kl
60 80 FRACTION NUMBER 20
40
[3H]lysine and polyoma virus labeled with "4C-labeled reconstituted protein hydrolysate. differences were reproducible in six different
500.
V,
NII/, \-
20 40 60 80 FRACTION NUMBER FIG. 2. Gel electrophoresis of BK virus labeled with 3H-labeled reconstituted protein hydrolysate.
O
0
FIG. 4. Co-electrophoresis of BK virus labeled with
1000-
O0
-
experiments. VP3 of BK virus and of SV40 were identical in molecular weight (peaks at fraction 48; Fig. 3). The two smaller polypeptides, VP4 at fraction 62 and VP5 at fraction 69 in Fig. 3, were not well resolved in these gels. The presumed empty capsids mentioned above were also co-electrophoresed with the same prepara-
831
POLYPEPTIDES OF BK VIRUS
VOL. 15, 1975
tions of SV40 and polyoma virus (gels not shown). There was a small increase in the amount of VP2, slightly lower in molecular weight than VP1, and the proportion of VP5 relative to VP4 decreased markedly. lodinated virus. In further attempts to demonstrate differences between the polypeptide composition of SV40 and BK virus, the gel patterns of the two viruses were obtained following iodination before and after dialysis against a buffer of 0.15 M carbonate-bicarbonate (pH 10.5). The results of one such experiment are described below. The gel patterns of BK virus and SV40 iodinated while still intact are given in Fig. 5A and 5C, respectively. Both gels were subject to electrophoresis at the same time, and the similarity in migration of the polypeptides indicated their similarity in molecular weight. The gel patterns of the viruses iodinated after treatment at pH 10.5 (Fig. 5B and 5D) were subject to electrophoresis for differing time periods and cannot be compared in this manner. In Fig. 5A, VP1, VP3, VP4 and VP5 are at fractions 32, 52, 69 and 77, respectively. Rather more VP2 (fraction 38) was apparent than in the preceding gels. When BK virus was iodinated after dialysis against alkaline buffer (Fig. 5B), there was little change in the relative heights of the peaks. However, VP6, at fraction 72, was iodinated and
resolved on electrophoresis. The same treatment of SV40 yielded a result similar to that reported by Huang et al. (9); there was an increase in the amount of label attached to VP1 relative to the radioactivity in the other virion proteins. These results suggested a difference between the two viruses in the accessibility of the tyrosine residues available for iodination after dialysis against 0.15 M carbonate-bicarbonate (pH 10.5). It is unlikely that this was caused by a failure of the buffer treatment to disrupt both viruses, since the following experiment demonstrated the degradative effect of the alkaline buffer. SV40, BK, and polyoma viruses were iodinated, dialyzed against 0.15 M carbonatebicarbonate (pH 10.5), and then centrifuged in parallel through 5 to 20% (wt/wt) sucrose gradients in the same buffer (Fig. 6). Polyoma virus was apparently unaffected by the treatment, but both SV40 and BK virus were disrupted to material of small s value and to a component of 30-40S (11) containing VP3, VP4, and VP5 (gels not shown), and thus corresponding to the deoxynucleoprotein complex described by Huang et al. (9). A third complex of BK virus, at fraction 16 in Fig. 6B, was observed in several 2000- A 1500 1000-
80 1 A 4 (1t
500
CA)
05 1500 B
B
I
11
125
I
cpm00
40( D
.j
20C(0 11
.
D
C CXT TC ~ 20(
40(
40( C
0 4 U,
6
20 40 8U a:* FRACTION NUMBER
60
80
06
FIG. 5. Gel electrophoresis of BK virus and SV40 iodinated before (A and C) and after (B and D) disruption with 0.15 M carbonate-bicarbonate (pH 10.5).
5
10
15
20
25
FRACTION NUMBER virus (B), and polyoma virus BK FIG. 6. SV40 (A), (C) iodinated and dialyzed against 0.15 M carbonatebicarbonate buffer (pH 10.5) and then centrifuged at 20 C for 1.5 h at 234,000 x g in a Spinco SW50.1 rotor through a 5 to 20% (wt/wt) sucrose gradient in the same buffer.
832
J. VIROL.
WRIGHT AND DI MAYORCA
experiments, but its nature was not further examined. Analysis of tryptic peptides. Since the molecular weights of the component polypeptides of SV40 and BK virions were so similar, the tryptic peptides of individual proteins were compared. Particular interest lay with VP3, which was the same size in both viruses, and with VP1, which was smaller in BK virus and therefore possibly containing a primary amino acid sequence wholly or partially contained in VP1 of SV40. To compare a particular polypeptide from the two viruses, three separate analyses of iodinated tryptic peptides were completed using the ionexchange column described above. The polypeptide from each virus was analyzed alone, followed by a mixture of the polypeptide from the two viruses containing equal amounts of radioactivity from the two sources. The column was eluted with buffer solution from a gradient marker designed to give a linear gradient. Knowing the initial and final concentrations of the buffer of the linear gradient, the molarities at which peptides eluted were calculated. Peptides which were resolved in the analysis of the mixture were assigned numbers or letters. Thus it was possible to identify a peptide resolved in the mixture with one resolved in an analysis of a single polypeptide by two means: the molarity of the buffer at elution from the column of the peptide, and, less importantly, the amount of radioactivity in the peptide. The tryptic peptide analyses are shown in Fig. 7, 8 and 9, and the data derived from these analyses are shown in Table 1. As examples of the identification procedure, consider peptides 2 and 3 in Fig. 7. Peptide 2 eluted at 0.38 in Fig. 7A and at 0.37 in Fig. 7C (from Table 1). Peptide 3 eluted at 0.42 in Fig. 7A, 0.43 in Fig. 7B, and 0.44 in Fig. 7C. The amounts of peptides 2 and 3 in Fig. 7A were also near expected values, since the amounts of radioactivity of SV40 and BK material used in Fig. 7A were one-half those used in Fig. 7B and 7C, respectively. The flow rate was constant during each column run, but was not necessarily the same for each experiment. Thus it was necessary when identifying peptides to use the molarity of elution from Table 1, not the fraction numbers from the figures. It was uncertain what proportion of tryptic peptides containing tyrosine would be iodinated, as the proteins were still associated with other capsid material during the labeling procedure, possibly masking some tyrosine residues. The virion polypeptides of SV40 contain ap-
251 cpm 4,000-
140009
16
2
2Q000
13
1000-
16,000-
18
15
BK
14,000-
2,000 10,000-
8,000
8
6,000-
4,000C 2Q000-1 0
12
19
0 20 40 6U 80 X I3L 14c 160 FRACTION NJMBER
FIG. 7. Analysis of the tryptic peptides of VPI derived from virus iodinated after treatment with 0.15 M carbonate-bicarbonate buffer (pH 10.5). The approximate quantities of radioactivity used are shown in parentheses. (A) Mixture of SV40 (2.3 x 105 counts/min) and BK virus (2.2 x 10O counts/min). (B) SV40 only (4.5 x 10' counts/min). (C) BK virus only (4.3 x 10' counts/min). Refer to Table 1 for explanation of the numbering of the peaks.
proximately three tyrosines per 10,000 daltons (7). On this basis, the number of tyrosines expected in VP1 and VP3 would be roughly 12 and 7, respectively. In Fig. 7B and 7C approximately 12 and 9 peptides, respectively, were
VOL. 15, 1975
833
POLYPEPTIDES OF BK VIRUS
tryptic digests of VP1 of SV40 and BK virus (peptides 3 and 7 only), and none at all between those of VP3 of the two viruses. However, there was substantial similarity between VP4,VP5 from the two sources (peptides b, c, f, i, n, o). DISCUSSION The polypeptides of BK virus are more similar in size to those of SV40 than to those of polyoma. The smaller molecular weight of VP1 of BK virus compared with that of SV40 has also been reported by Mullarkey et al. (14), who observed another polypeptide migrating between VP2 and VP3, possibly corresponding to the very small peak at fraction 42 in Fig. 5A.
14001 '25'
A
cpm
1251 cpm
O
20 40 60 80 100 120 140 FRACTION NUMBER
FIG. 8. Analysis of the tryptic peptides of VP3 obtained from iodinated intact virus. (A) Mixture of SV40 (2.5 x 10' counts/min) and BK virus (2.4 x 10' counts/min). (B) SV40 only (4.9 x 10' counts/min). (C) BK virus only (4.9 x 105 counts/min). Refer to Table 1 for explanation of the lettering of the peaks.
resolved from tryptic digests of VP1 of each virus; in Fig. 8B and 8C approximately 6 and 5 peptides, respectively, were resolved from digests of VP3 of each virus. Thus, the procedure used in these experiments did label the majority of tyrosine-containing peptides. It is clear from the data presented in Fig. 7, 8, 9 and Table 1 that there was little similarity between the
O
20
40 60 80 100 120 140 FRACTION NUMBER
FIG. 9. Analysis of the tryptic peptides of VP4 and VP5 obtained from iodinated intact virus. (A) Mixture of SV40 (1.2 x 10' counts/min) and BK virus (1.1 x 10' counts/min). (B) SV40 only (1.2 x 105 counts! min). (C) BK virus only (1.1 x 10' counts/min). For these columns, the elution gradient was from 0.2 to 1.65 M pyridine-acetate. Refer to Table 1 for explanation of the lettering of the peaks.
834
WRIGHT AND DI MAYORCA
J. VIROL.
TABLE 1. Molarity of the pyridine-acetate buffer at elution from the column of the individual tryptic peptidesa
VP1
DeSig-
nation (Fig. Mixed 7A) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 a
0.33 0.38 0.42 0.51 0.55
0.63 0.68 0.77 0.91 0.97 1.08 1.18 1.25 1.33 1.48 1.52 1.59 1.70 1.93
VP3
SV40 BK (Fig. 7B) (Fig. 7C)
0.34
0.43 0.53
0.37 0.44
0.58 0.64
0.68
0.66 0.75
0.94 0.99 1.06 1.15
DeSig-
nation A B C D E F G H I J K
Mixed
SV40
0.28 0.38 0.43 0.51 0.73 0.91 0.97 1.21 1.37 1.47 1.60
0.27 0.38
VP4 and VP5
DeSi;g
BK
(Fig. 8A) (Fig. 8B) (Fig. 8C)
1.27 1.32 1.46
a b
0.42
c
0.72 0.90
d e f
0.51
0.95 1.21
1.37 1.48
1.60
nation
g h i j k i m n O
BK Mixed SV40 (Fig. 9A) (Fig. 9B) (Fig. 9C) 0.29 0.39 0.54 0.63
0.79 0.82 0.94 1.04 1.09 1.16 1.24 1.36 1.45 1.53 1.57
0.29 0.39 0.54 0.63
0.39 0.56
0.82
0.83 0.87
0.94 1.04 1.09
1.08 1.14 1.24
1.35 1.52 1.58
1.43 1.50 1.58
1.55
1.58 1.69
1.95
Peptides have the same labels as in Fig. 7, 8, and 9. The figure used to calculate the molarity of elution is
indicated.
The proportion of the capsid protein in VP1 (73%) is close to the values estimated for SV40, 70 (4) and 71% (from Fig. 3). Attempts to phosphorylate purified BK virus in a manner similar to that described for SV40 (21) failed, as did an effort to incorporate [3H ]glucosamine into the virus during the growth cycle. The analysis of the tryptic peptides of VP1 and VP3 established that there are major differences between the two viruses in the primary structure of the capsid protein. It is feasible that VP1 is at least partly responsible for the antigenic relatedness between SV40 and BK virus (6, 16, 20), since it is the major component of the capsid and there is limited overlap in tryptic peptides (3 and 7 in Fig. 7). VP3 is unlikely to contribute to this relationship, as the peptide patterns of VP3 from the two viruses are very different (Fig. 8). The similarity between the tryptic digests of VP4,VP5 of the viruses is consistent with the claim that these may be histones, coded for by the cell genome (5, 18). The amino acid sequence of histone proteins is remarkably constant from species to species (1). VP4 and VP5 are associated with the viral DNA (9; Fig. 6), and therefore the degree to which they are exposed on the virion surface, allowing them to contribute to the antigenic relationship, depends on the method of DNA packaging in the virions.
ACKNOWLEDGMENT This work was supported by Public Health Service contract NIH-NCI VCP 43318 from the Virus Cancer Program.
LITERATURE CITED 1. DeLange, R. J., and E. L. Smith. 1971. Histones: structure and function. Annu. Rev. Biochem. 40:279-314. 2. di Mayorca, G., J. Callender, G. Marin, and R. Giordano. 1969. Temperature-sensitive mutants of polyoma virus. Virology 38:126-133. 3. Dougherty, R. M., and H. S. DiStefano. 1974. Isolation and characterization of a papovavirus from human urine. Proc. Soc. Exp. Biol. Med. 146:481-487. 4. Estes, M. K., Huang, E-S., and J. S. Pagano. 1971. Structural polypeptides of simian virus 40. J. Virol. 7:635-641. 5. Frearson, P. M., and L. V. Crawford. 1972. Polyoma virus basic proteins. J. Gen. Virol. 14:141-155. 6. Gardner, S. D., A. M. Field, D. V. Coleman, and B. Hulme. 1971. New human papovavirus (BK) isolated from urine after renal transplantation. Lancet 1:1253-1257. 7. Greenaway, P. J., and D. LeVine. 1973. Amino acid compositions of simian virus 40 structural proteins. Biochem. Biophys. Res. Commun. 52:1221-1227. 8. Greenwood, F. C., W. M. Hunter, and J. S. Glover. 1963. The preparation of '311-labeled human growth hormone of high specific radioactivity. Biochem. J. 89:114-123. 9. Huang, E-S., M. K. Estes, and J. S. Pagano. 1972. Structure and function of the polypeptides in simian virus 40. I. Existence of subviral deoxynucleoprotein complexes. J. Virol. 9:923-929. 10. Jacobson, M. F., J. Asso, and D. Baltimore. 1970. Further evidence on the formation of poliovirus proteins. J. Mol. Biol. 49:657-669.
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POLYPEPTIDES OF BK VIRUS
11. McEwen, C. R. 1967. Tables for estimating sedimentation through linear concentration gradients of sucrose
solution. Anal. Biochem. 20:114-149. 12. Maizel, J. V. 1971. Polyacrylamide gel electrophoresis and viral proteins, p. 180-244, In K. Maramorosch and H. Koprowski (ed.), Methods in virology, vol. 5. Academic Press, New York. 13. Major, E. O., and G. di Mayorca. 1973. Malignant transformation of BHK,, clone 13 cells by BK virus-a human papovavirus. Proc. Nat. Acad. Sci. U.S.A. 70:3210-3212. 14. 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. 15. Padgett, B. L., D. L. Walker, G. M. Zurhein, R. J. Echroade, and B. H. Dessel. 1971. Cultivation of papova-like virus from human brain with progressive multifocal leukoencephalopathy. Lancet 1:1257-1260. 16. Penney, J. B., and 0. Narayan. 1973. Studies of the antigenic relationships of the new human papovaviruses by electron microscopy agglutination. Infect. Immun. 8:299-300. 17. Perry, J. L., C. M. To, and R. A. Consigli. 1969. Alkaline degradation of polyoma virus. J. Gen. Virol. 4:403-411.
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18. Roblin, R., E. Harle, and R. Dulbecco. 1971. Polyoma virus proteins. I. Multiple virion components. Virology 45:555-566. 19. Schroeder, W. A., R. T. Jones, J. Cormick, and K. McCalla. 1962. Chromatographic separation of peptides on ion exchange resins. Anal. Chem. 34:1570-1575. 20. Takemoto, K. K., and M. F. Mullarkey. 1973. Human papovavirus, BK strain: biological studies including antigenic relationship to simian virus 40. J. Virol. 12:625-631. 21. Tan, K. B., and F. Sokol. 1973. Phosphorylation of simian virus 40 proteins in a cell-free system. J. Virol. 12:696-703. 22. Weiner, L. P., R. M. Herndon, 0. Narayan, R. T. Johnson, K. Shah, L. J. Rubinstein, T. J. Preziosi, and F. K. Conley. 1972. Isolation of virus related to SV40 from patients with progressive multifocal leukoencephalopathy. N. Engl. J. Med. 286:385-390. 23. Wildy, P. 1971. Classification and nomenclature of viruses, p. 38-39. In J. L. Melnick (ed.), Monographs in virology, vol. 5. Karger, Basel. 24. Wright, P. J., and P. D. Cooper. 1974. Poliovirus proteins associated with ribosomal structure in infected cells. Virology 59:1-20.
JOURNAL OF VIROLOGY, Apr. 1975, p. 828-835 Copyright © 1975 American Society for Microbiology
Virion Polypeptide Composition of the Human Papovavirus BK: Comparison with Simian Virus 40 and Polyoma Virus PETER J. WRIGHT* AND GIAMPIERO DI MAYORCA Department of Microbiology, University of Illinois at the Medical Center, Chicago, Illinois 60680 Received for publication 5 August 1974
The polypeptide composition of labeled BK virus was compared with that of simian virus 40 (SV40) and polyoma virus by co-electrophoresis of disrupted virions in polyacrylamide gels containing sodium dodecyl sulfate. The major polypeptide (VP1) of BK constituted approximately 73% of the capsid protein and had a molecular weight of 39,000. It was smaller than VP1 of SV40 and polyoma virus. The other polypeptides of BK virus were similar in molecular weight to those of SV40. A comparison of the proteins of BK virus and SV40 iodinated with chloramine T before and after disruption in alkaline buffer at pH 10.5 revealed differences between the two viruses in the number and distribution of tyrosines available for iodination. The tryptic peptides of VP1, VP3, VP4, and VP5 combined of SV40 were compared with those of the same polypeptides of BK virus. Among the 19 peptides of VP1 resolved, only two were common to both viruses. The analyses of VP4 and VP5, the histone-like proteins, however, showed more similarity between the viruses, with 6 of 15 resolved peptides in common. The tryptic digests of VP3 were completely different. BK virus, originally isolated by Gardner et al. from the urine of a renal allograft recipient (6), is one of the group of human papovaviruses that have been isolated either from patients with progressive multifocal leukoencephalopathy (15, 22) or from renal allograft recipients (3, 6). An antigenic relationship between BK virus and simian virus 40 (SV40) has been demonstrated by immune electron microscopy (6, 16) and by fluorescent antibody tests (20). A low level of cross-reaction has also been measured by plaque neutralization (20; E. 0. Major and G. di Mayorca, unpublished data). No antigenic similarity was detected by the techniques of complement fixation, immunodiffusion, or immunoelectrophoresis (14). To gain more information on the relationship of BK virus to other papovaviruses, the proteins of the viral particle have been examined. In this paper the virion polypeptides of BK virus are compared by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate (SDS) with those of two well characterized papovaviruses, polyoma and SV40. Polyoma, like BK virus but unlike SV40, is hemagglutinating. A preliminary report of these results has already appeared (P. J. Wright and G. di Mayorca, Abstr. Annu. Meet. Amer. Soc. Microbiol. 1974, V224, p. 238). Also presented are comparisons of the tryptic peptides derived from the virion proteins of SV40 and BK virus. 828
MATERIALS AND METHODS Cells and viruses. BK virus was grown as previously described (13) in primary and secondary cultures of human embryonic kidney cells. The human embryonic kidney cells were grown on Dulbecco's modified Eagle (DME) medium with 10% fetal calf serum before infection, and with 5% horse serum after infection. Cells were infected at a multiplicity of 0.5 to 5 PFU/cell and harvested when virtually all cells showed cytopathic effects, normally 2 weeks after infection. To obtain labeled virus, 8H-labeled reconstituted protein hydrolysate (Schwarz/Mann) or [8H]lysine (Amersham Searle, specific activity of 160 mCi/mmol) at final concentrations of 10 MCi/ml or 0.2 MCi/ml, respectively, were added at the time of infection to cells in DME or DME medium lacking lysine, respectively. Polyoma virus was grown in mouse 3T3 cells (2) and SV40 in BSC-1 cells, both a gift from C. Basilico. Labeled virus was obtained by maintaining infected cells in DME medium containing 1 ACi of "C-labeled reconstituted protein hydrolysate (Schwarz/ Mann) per ml and either 5% horse serum (3T3 cells) or 2% fetal calf serum (BSC-1 cells). Cells were harvested 4 to 7 days after infection when cytopathic effects were evident. Purification of virus. The methods used were essentially those described by Roblin et al. (18). The infected cells, still suspended in medium, were given six cycles of freezing and thawing and treated with 0.2% sodium deoxycholate for 1 h at 37 C, and large debris was removed by centrifugation for 15 min at 10,000 x g. The supernatant was centrifuged at 20 C for 4 h at 64,000 x g in a Spinco SW 25.2 rotor over a
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POLYPEPTIDES OF BK VIRUS
cushion of 14 ml of saturated KBr solution in 0.05 M Tris-hydrochloride (pH 8.0) plus 0.01 M EDTA. Two, sometimes three, bands were observed; the lowest one was collected and dialyzed against a Tris buffer, TD (135 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, 25 mM Tris). The dialyzed material was made up to 7.5 ml with TD and added to 3.45 g of CsCl, and the solution was centrifuged at 20 C for 16 h at 74,000 x g in a Spinco type 50 rotor. The single band of virus obtained was dialyzed against TD and either subjected to a second CsCl gradient as above, or a sucrose gradient (see Fig. IC). The acid-insoluble counts in gradient fractions were assayed on Whatman no. 1 filter paper disks. Disruption and iodination of virus. Virus was disrupted by dialysis for 16 h at 5 C against 0.15 M carbonate-bicarbonate buffer, pH 10.5 (17). lodination (1211, carrier free; Amersham Searle) was done by the chloramine T method (9), which preferentially iodinates tyrosine (8). Each experiment used 0.25 ml of a viral preparation with an absorbance at 260 nm of 0.32 units. Gel electrophoresis. Viral preparations for electrophoresis were dialyzed against 0.05 M phosphate (pH 7.3), adjusted to 2% SDS and 1% 2-mercaptoethanol, heated for 2 min at 100 C, and finally dialyzed against 0.01 M phosphate (pH 7.3), 0.1% SDS, and 0.1% 2-mercaptoethanol. Samples were subject to electrophoresis on 10% gels containing SDS using the continuous gel system described by Maizel (12), the gels were sliced, and the slices were counted in dioxanebased scintillation fluid (24). Tryptic digests. After electrophoresis of iodinated viral preparations, the proteins to be treated with trypsin were eluted from the gel slices into 8 ml of a solution of 0.01 M phosphate (pH 7.3) and 0.1% SDS. Ovalbumin (7.5 mg), followed by trichloroacetic acid to a final concentration of 10%, was added, and the precipitates were washed twice with ethanol and then redissolved in 0.7 ml of a buffer of 0.5 M Tris-hydrochloride (pH 8.8) containing 0.5% SDS. The samples were treated with 20 mg of dithiothreitol for 1 h at 37 C, and subsequently with 70 mg of iodoacetamide for 2 h at 25 C. Thereafter the proteins were prepared for analysis of the tryptic peptides as described by Jacobson et al. (10). The initial treatment with 0.1 mg of trypsin (treated with TPCK; Worthington) was at 37 C for 21 h, followed by a further 0.2 mg for 2 h. The tryptic digests were analyzed on a column (30 by 0.9 cm; Glenco Scientific) of Technicon type P chromobeads ion-exchange resin held at 50 C and developed with a linear gradient of 0.2 M pyridineacetate (pH 3.1) to 2 M pyridine-acetate, pH 5.0 (10, 19). The column was maintained under pressure; the flow rate was approximately 16 ml per h, and the fractions (1.3 ml) were counted in 5 ml of the same dioxan-based scintillation fluid used to count gel slices (24).
RESULTS Purif'ication of virus. The procedures used by Roblin et al. for polyoma virus (18) proved suitable for the purification of BK virus. The
829
typical result from several experiments is displayed in Fig. 1. After centrifugation to equilibrium in CsCl solution, BK virus banded at a similar density (1.34 g/ml). The approximate s value of the purified virus, estimated from Fig. 1C by the method of McEwen (11), was 280S. Published values for other papovaviruses are 240 to 300S (23). The material in fractions 13, 14, and 15 of Fig. 1C was also centrifuged through successive CsCl and sucrose gradients. The density was approximately that of protein (1.29 g/ml), and its s value was about 200S. This 8200 000 -
\
600 i200-
000 3H cpm00 800
I
400
600 Y
300
400-
200 "C cpm
C0 20C
100
~~0
B 3003H
cpm 200
C 600
50C 00c 3H
cpm
-
00 ' 3CDC
200
C
C 5 L 20 FRACT 0N %JM9ER
3c.
FIG. 1. (A) Approximately the bottom third of a tube containing a cushion of saturated KBr solution after centrifugation as described. Prior to centrifugation, infected cell lysate labeled with [3H]lysine was mixed with uninfected cell lysate labeled with "Clabeled reconstituted protein hydrolysate. (B) The virus shown in Fig. IA (fractions 10, 11, 12) after dialysis and centrifugation to equilibrium in CsCI solution. (C) The same viral preparation as shown in Fig. lB after dialysis and centrifugation at 20 C for 1.5 h at 48,000 x g in the Spinco SW41 rotor through a 5 to 20% (wt/wt) sucrose gradient in TD buffer.
J. VIROL.
WRIGHT AND DI MAYORCA
830
material probably corresponded to empty cap- 700 sids. Polypeptide composition. The gel pattern of - 600 BK virus labeled with reconstituted protein hydrolysate is shown in Fig. 2. From this preparation of virus the estimated proportion of 500 the total virion protein in the major polypeptide (VP1) (peak at fraction 28) was 73%, in VP3 - 400 (peak at fraction 46) was 7%, and in the C cpm low-molecular-weight region VP4,VP5 (frac- 3H cpm tions 55 to 70) was 9%. The proportion of protein - 300 at high molecular weight (peak at fraction 10) was 10%, and the remaining 1% corresponded to -200 the small amount of material (VP2) migrating It is 34. unlikely just ahead of VP1 at fraction - 100 that the material at fraction 10 represented aggregated protein, since care was taken to IA-A maintain reducing conditions during the disruption and electrophoresis of viral preparations. The estimated molecular weight of VP1 0 20 40 60 80 was 39,000, calculated by co-electrophoresing FRACTION NUMBER viral samples with the following iodinated stanFIG. 3. Co-electrophoresis of BK virus labeled with dard proteins: pepsin (34,000 daltons), myoglobin (17,300), and cytochrome c (12,500) (24). [3H]lysine and SV40 labeled with "C-labeled reconThe results of co-electrophoresing the poly- stituted protein hydrolysate. peptides of BK virus with those of SV40 and 700polyoma virus are shown in Fig. 3 and 4, respectively. The major virion protein VP1 of 1200 BK virus was smaller than that of SV40 (differ600 ence of one slice) and even smaller than that of -1000 polyoma virus (difference of three slices). These
FO
500 -
-
600
400-
3H
3000-
800
14C
cpm
cpm
400
300-
200
2500-
100
20001
-
3H cpm
-I
B
B
1500-
v
0
Kl
60 80 FRACTION NUMBER 20
40
[3H]lysine and polyoma virus labeled with "4C-labeled reconstituted protein hydrolysate. differences were reproducible in six different
500.
V,
NII/, \-
20 40 60 80 FRACTION NUMBER FIG. 2. Gel electrophoresis of BK virus labeled with 3H-labeled reconstituted protein hydrolysate.
O
0
FIG. 4. Co-electrophoresis of BK virus labeled with
1000-
O0
-
experiments. VP3 of BK virus and of SV40 were identical in molecular weight (peaks at fraction 48; Fig. 3). The two smaller polypeptides, VP4 at fraction 62 and VP5 at fraction 69 in Fig. 3, were not well resolved in these gels. The presumed empty capsids mentioned above were also co-electrophoresed with the same prepara-
831
POLYPEPTIDES OF BK VIRUS
VOL. 15, 1975
tions of SV40 and polyoma virus (gels not shown). There was a small increase in the amount of VP2, slightly lower in molecular weight than VP1, and the proportion of VP5 relative to VP4 decreased markedly. lodinated virus. In further attempts to demonstrate differences between the polypeptide composition of SV40 and BK virus, the gel patterns of the two viruses were obtained following iodination before and after dialysis against a buffer of 0.15 M carbonate-bicarbonate (pH 10.5). The results of one such experiment are described below. The gel patterns of BK virus and SV40 iodinated while still intact are given in Fig. 5A and 5C, respectively. Both gels were subject to electrophoresis at the same time, and the similarity in migration of the polypeptides indicated their similarity in molecular weight. The gel patterns of the viruses iodinated after treatment at pH 10.5 (Fig. 5B and 5D) were subject to electrophoresis for differing time periods and cannot be compared in this manner. In Fig. 5A, VP1, VP3, VP4 and VP5 are at fractions 32, 52, 69 and 77, respectively. Rather more VP2 (fraction 38) was apparent than in the preceding gels. When BK virus was iodinated after dialysis against alkaline buffer (Fig. 5B), there was little change in the relative heights of the peaks. However, VP6, at fraction 72, was iodinated and
resolved on electrophoresis. The same treatment of SV40 yielded a result similar to that reported by Huang et al. (9); there was an increase in the amount of label attached to VP1 relative to the radioactivity in the other virion proteins. These results suggested a difference between the two viruses in the accessibility of the tyrosine residues available for iodination after dialysis against 0.15 M carbonate-bicarbonate (pH 10.5). It is unlikely that this was caused by a failure of the buffer treatment to disrupt both viruses, since the following experiment demonstrated the degradative effect of the alkaline buffer. SV40, BK, and polyoma viruses were iodinated, dialyzed against 0.15 M carbonatebicarbonate (pH 10.5), and then centrifuged in parallel through 5 to 20% (wt/wt) sucrose gradients in the same buffer (Fig. 6). Polyoma virus was apparently unaffected by the treatment, but both SV40 and BK virus were disrupted to material of small s value and to a component of 30-40S (11) containing VP3, VP4, and VP5 (gels not shown), and thus corresponding to the deoxynucleoprotein complex described by Huang et al. (9). A third complex of BK virus, at fraction 16 in Fig. 6B, was observed in several 2000- A 1500 1000-
80 1 A 4 (1t
500
CA)
05 1500 B
B
I
11
125
I
cpm00
40( D
.j
20C(0 11
.
D
C CXT TC ~ 20(
40(
40( C
0 4 U,
6
20 40 8U a:* FRACTION NUMBER
60
80
06
FIG. 5. Gel electrophoresis of BK virus and SV40 iodinated before (A and C) and after (B and D) disruption with 0.15 M carbonate-bicarbonate (pH 10.5).
5
10
15
20
25
FRACTION NUMBER virus (B), and polyoma virus BK FIG. 6. SV40 (A), (C) iodinated and dialyzed against 0.15 M carbonatebicarbonate buffer (pH 10.5) and then centrifuged at 20 C for 1.5 h at 234,000 x g in a Spinco SW50.1 rotor through a 5 to 20% (wt/wt) sucrose gradient in the same buffer.
832
J. VIROL.
WRIGHT AND DI MAYORCA
experiments, but its nature was not further examined. Analysis of tryptic peptides. Since the molecular weights of the component polypeptides of SV40 and BK virions were so similar, the tryptic peptides of individual proteins were compared. Particular interest lay with VP3, which was the same size in both viruses, and with VP1, which was smaller in BK virus and therefore possibly containing a primary amino acid sequence wholly or partially contained in VP1 of SV40. To compare a particular polypeptide from the two viruses, three separate analyses of iodinated tryptic peptides were completed using the ionexchange column described above. The polypeptide from each virus was analyzed alone, followed by a mixture of the polypeptide from the two viruses containing equal amounts of radioactivity from the two sources. The column was eluted with buffer solution from a gradient marker designed to give a linear gradient. Knowing the initial and final concentrations of the buffer of the linear gradient, the molarities at which peptides eluted were calculated. Peptides which were resolved in the analysis of the mixture were assigned numbers or letters. Thus it was possible to identify a peptide resolved in the mixture with one resolved in an analysis of a single polypeptide by two means: the molarity of the buffer at elution from the column of the peptide, and, less importantly, the amount of radioactivity in the peptide. The tryptic peptide analyses are shown in Fig. 7, 8 and 9, and the data derived from these analyses are shown in Table 1. As examples of the identification procedure, consider peptides 2 and 3 in Fig. 7. Peptide 2 eluted at 0.38 in Fig. 7A and at 0.37 in Fig. 7C (from Table 1). Peptide 3 eluted at 0.42 in Fig. 7A, 0.43 in Fig. 7B, and 0.44 in Fig. 7C. The amounts of peptides 2 and 3 in Fig. 7A were also near expected values, since the amounts of radioactivity of SV40 and BK material used in Fig. 7A were one-half those used in Fig. 7B and 7C, respectively. The flow rate was constant during each column run, but was not necessarily the same for each experiment. Thus it was necessary when identifying peptides to use the molarity of elution from Table 1, not the fraction numbers from the figures. It was uncertain what proportion of tryptic peptides containing tyrosine would be iodinated, as the proteins were still associated with other capsid material during the labeling procedure, possibly masking some tyrosine residues. The virion polypeptides of SV40 contain ap-
251 cpm 4,000-
140009
16
2
2Q000
13
1000-
16,000-
18
15
BK
14,000-
2,000 10,000-
8,000
8
6,000-
4,000C 2Q000-1 0
12
19
0 20 40 6U 80 X I3L 14c 160 FRACTION NJMBER
FIG. 7. Analysis of the tryptic peptides of VPI derived from virus iodinated after treatment with 0.15 M carbonate-bicarbonate buffer (pH 10.5). The approximate quantities of radioactivity used are shown in parentheses. (A) Mixture of SV40 (2.3 x 105 counts/min) and BK virus (2.2 x 10O counts/min). (B) SV40 only (4.5 x 10' counts/min). (C) BK virus only (4.3 x 10' counts/min). Refer to Table 1 for explanation of the numbering of the peaks.
proximately three tyrosines per 10,000 daltons (7). On this basis, the number of tyrosines expected in VP1 and VP3 would be roughly 12 and 7, respectively. In Fig. 7B and 7C approximately 12 and 9 peptides, respectively, were
VOL. 15, 1975
833
POLYPEPTIDES OF BK VIRUS
tryptic digests of VP1 of SV40 and BK virus (peptides 3 and 7 only), and none at all between those of VP3 of the two viruses. However, there was substantial similarity between VP4,VP5 from the two sources (peptides b, c, f, i, n, o). DISCUSSION The polypeptides of BK virus are more similar in size to those of SV40 than to those of polyoma. The smaller molecular weight of VP1 of BK virus compared with that of SV40 has also been reported by Mullarkey et al. (14), who observed another polypeptide migrating between VP2 and VP3, possibly corresponding to the very small peak at fraction 42 in Fig. 5A.
14001 '25'
A
cpm
1251 cpm
O
20 40 60 80 100 120 140 FRACTION NUMBER
FIG. 8. Analysis of the tryptic peptides of VP3 obtained from iodinated intact virus. (A) Mixture of SV40 (2.5 x 10' counts/min) and BK virus (2.4 x 10' counts/min). (B) SV40 only (4.9 x 10' counts/min). (C) BK virus only (4.9 x 105 counts/min). Refer to Table 1 for explanation of the lettering of the peaks.
resolved from tryptic digests of VP1 of each virus; in Fig. 8B and 8C approximately 6 and 5 peptides, respectively, were resolved from digests of VP3 of each virus. Thus, the procedure used in these experiments did label the majority of tyrosine-containing peptides. It is clear from the data presented in Fig. 7, 8, 9 and Table 1 that there was little similarity between the
O
20
40 60 80 100 120 140 FRACTION NUMBER
FIG. 9. Analysis of the tryptic peptides of VP4 and VP5 obtained from iodinated intact virus. (A) Mixture of SV40 (1.2 x 10' counts/min) and BK virus (1.1 x 10' counts/min). (B) SV40 only (1.2 x 105 counts! min). (C) BK virus only (1.1 x 10' counts/min). For these columns, the elution gradient was from 0.2 to 1.65 M pyridine-acetate. Refer to Table 1 for explanation of the lettering of the peaks.
834
WRIGHT AND DI MAYORCA
J. VIROL.
TABLE 1. Molarity of the pyridine-acetate buffer at elution from the column of the individual tryptic peptidesa
VP1
DeSig-
nation (Fig. Mixed 7A) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 a
0.33 0.38 0.42 0.51 0.55
0.63 0.68 0.77 0.91 0.97 1.08 1.18 1.25 1.33 1.48 1.52 1.59 1.70 1.93
VP3
SV40 BK (Fig. 7B) (Fig. 7C)
0.34
0.43 0.53
0.37 0.44
0.58 0.64
0.68
0.66 0.75
0.94 0.99 1.06 1.15
DeSig-
nation A B C D E F G H I J K
Mixed
SV40
0.28 0.38 0.43 0.51 0.73 0.91 0.97 1.21 1.37 1.47 1.60
0.27 0.38
VP4 and VP5
DeSi;g
BK
(Fig. 8A) (Fig. 8B) (Fig. 8C)
1.27 1.32 1.46
a b
0.42
c
0.72 0.90
d e f
0.51
0.95 1.21
1.37 1.48
1.60
nation
g h i j k i m n O
BK Mixed SV40 (Fig. 9A) (Fig. 9B) (Fig. 9C) 0.29 0.39 0.54 0.63
0.79 0.82 0.94 1.04 1.09 1.16 1.24 1.36 1.45 1.53 1.57
0.29 0.39 0.54 0.63
0.39 0.56
0.82
0.83 0.87
0.94 1.04 1.09
1.08 1.14 1.24
1.35 1.52 1.58
1.43 1.50 1.58
1.55
1.58 1.69
1.95
Peptides have the same labels as in Fig. 7, 8, and 9. The figure used to calculate the molarity of elution is
indicated.
The proportion of the capsid protein in VP1 (73%) is close to the values estimated for SV40, 70 (4) and 71% (from Fig. 3). Attempts to phosphorylate purified BK virus in a manner similar to that described for SV40 (21) failed, as did an effort to incorporate [3H ]glucosamine into the virus during the growth cycle. The analysis of the tryptic peptides of VP1 and VP3 established that there are major differences between the two viruses in the primary structure of the capsid protein. It is feasible that VP1 is at least partly responsible for the antigenic relatedness between SV40 and BK virus (6, 16, 20), since it is the major component of the capsid and there is limited overlap in tryptic peptides (3 and 7 in Fig. 7). VP3 is unlikely to contribute to this relationship, as the peptide patterns of VP3 from the two viruses are very different (Fig. 8). The similarity between the tryptic digests of VP4,VP5 of the viruses is consistent with the claim that these may be histones, coded for by the cell genome (5, 18). The amino acid sequence of histone proteins is remarkably constant from species to species (1). VP4 and VP5 are associated with the viral DNA (9; Fig. 6), and therefore the degree to which they are exposed on the virion surface, allowing them to contribute to the antigenic relationship, depends on the method of DNA packaging in the virions.
ACKNOWLEDGMENT This work was supported by Public Health Service contract NIH-NCI VCP 43318 from the Virus Cancer Program.
LITERATURE CITED 1. DeLange, R. J., and E. L. Smith. 1971. Histones: structure and function. Annu. Rev. Biochem. 40:279-314. 2. di Mayorca, G., J. Callender, G. Marin, and R. Giordano. 1969. Temperature-sensitive mutants of polyoma virus. Virology 38:126-133. 3. Dougherty, R. M., and H. S. DiStefano. 1974. Isolation and characterization of a papovavirus from human urine. Proc. Soc. Exp. Biol. Med. 146:481-487. 4. Estes, M. K., Huang, E-S., and J. S. Pagano. 1971. Structural polypeptides of simian virus 40. J. Virol. 7:635-641. 5. Frearson, P. M., and L. V. Crawford. 1972. Polyoma virus basic proteins. J. Gen. Virol. 14:141-155. 6. Gardner, S. D., A. M. Field, D. V. Coleman, and B. Hulme. 1971. New human papovavirus (BK) isolated from urine after renal transplantation. Lancet 1:1253-1257. 7. Greenaway, P. J., and D. LeVine. 1973. Amino acid compositions of simian virus 40 structural proteins. Biochem. Biophys. Res. Commun. 52:1221-1227. 8. Greenwood, F. C., W. M. Hunter, and J. S. Glover. 1963. The preparation of '311-labeled human growth hormone of high specific radioactivity. Biochem. J. 89:114-123. 9. Huang, E-S., M. K. Estes, and J. S. Pagano. 1972. Structure and function of the polypeptides in simian virus 40. I. Existence of subviral deoxynucleoprotein complexes. J. Virol. 9:923-929. 10. Jacobson, M. F., J. Asso, and D. Baltimore. 1970. Further evidence on the formation of poliovirus proteins. J. Mol. Biol. 49:657-669.
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POLYPEPTIDES OF BK VIRUS
11. McEwen, C. R. 1967. Tables for estimating sedimentation through linear concentration gradients of sucrose
solution. Anal. Biochem. 20:114-149. 12. Maizel, J. V. 1971. Polyacrylamide gel electrophoresis and viral proteins, p. 180-244, In K. Maramorosch and H. Koprowski (ed.), Methods in virology, vol. 5. Academic Press, New York. 13. Major, E. O., and G. di Mayorca. 1973. Malignant transformation of BHK,, clone 13 cells by BK virus-a human papovavirus. Proc. Nat. Acad. Sci. U.S.A. 70:3210-3212. 14. 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. 15. Padgett, B. L., D. L. Walker, G. M. Zurhein, R. J. Echroade, and B. H. Dessel. 1971. Cultivation of papova-like virus from human brain with progressive multifocal leukoencephalopathy. Lancet 1:1257-1260. 16. Penney, J. B., and 0. Narayan. 1973. Studies of the antigenic relationships of the new human papovaviruses by electron microscopy agglutination. Infect. Immun. 8:299-300. 17. Perry, J. L., C. M. To, and R. A. Consigli. 1969. Alkaline degradation of polyoma virus. J. Gen. Virol. 4:403-411.
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18. Roblin, R., E. Harle, and R. Dulbecco. 1971. Polyoma virus proteins. I. Multiple virion components. Virology 45:555-566. 19. Schroeder, W. A., R. T. Jones, J. Cormick, and K. McCalla. 1962. Chromatographic separation of peptides on ion exchange resins. Anal. Chem. 34:1570-1575. 20. Takemoto, K. K., and M. F. Mullarkey. 1973. Human papovavirus, BK strain: biological studies including antigenic relationship to simian virus 40. J. Virol. 12:625-631. 21. Tan, K. B., and F. Sokol. 1973. Phosphorylation of simian virus 40 proteins in a cell-free system. J. Virol. 12:696-703. 22. Weiner, L. P., R. M. Herndon, 0. Narayan, R. T. Johnson, K. Shah, L. J. Rubinstein, T. J. Preziosi, and F. K. Conley. 1972. Isolation of virus related to SV40 from patients with progressive multifocal leukoencephalopathy. N. Engl. J. Med. 286:385-390. 23. Wildy, P. 1971. Classification and nomenclature of viruses, p. 38-39. In J. L. Melnick (ed.), Monographs in virology, vol. 5. Karger, Basel. 24. Wright, P. J., and P. D. Cooper. 1974. Poliovirus proteins associated with ribosomal structure in infected cells. Virology 59:1-20.
