JouRNAL OF VIROLOGY, Mar. 1979, p. 881-887 0022-538X/79/03-0881/07$02.00/0
Vol. 29, No. 3
Multiple Forms of Polyoma Virus Tumor Antigens from Infected and Transformed Cells DANEL T. SIMMONS,t* CHUNGMING CHANG,4 AND MALCOLM A. MARTIN Laboratory of Biology of Viruses, National Institute of Allergy and Infectious Dzseases, and Immunology Program, National Cancer Institute, Bethesda, Maryland 20014 Received for publication 21 September 1978 At least three distinct forms of
polyoma
virus tumor
antigens
were
isolated
from productively infected and transformed hamster cells by inmunoprecipitation with anti-T serum. These proteins had approximate molecular weights of 105,000 (large T antigen), 63,000 (middle T antigen), and 20,000 (small T antigen) as estimated by acrylamide gel electrophoresis. An examination of the appearance of these antigens in polyoma-infected mouse cells showed that all three polypeptides were synthesized maximally at approximately the same time after infection. Analysis of the methionine-containing tryptic peptides of these proteins indicated that the large, middle, and small forms of polyoma T antigens contained five similar or identical peptides. In addition, the 63,000- and 20,000-dalton antigens contained two other methionine peptides absent from the large T-antigen species. Other methionine peptides were found only in the large or middle T-antigen forms. These results and results obtained previously suggested that the three Tantigen species have the same NH2-terminal end regions but different COOH termini. A model is presented describing the synthesis of these polypeptides from different regions of the polyoma virus genome. The region of the polyoma virus genome which is first expressed early in lytic infection (before the onset of DNA replication) consists of at least two genes (denoted hr-t and ts-a) which are required for virus-induced cellular transformation (6, 7). The products of these two genes appear to be proteins with molecular weights of 95 to 108,000 (large T antigen [T Ag]) and 20 to 22,000 (small T Ag), as estimated by acrylamide gel electrophoresis (11, 13, 17), although the true size of the large T Ag may be closer to 80,000 daltons (17). These proteins can be isolated from infected mouse cells or virus-transformed cells by immunoprecipitation with serum directed against polyoma-induced tumors (anti-T serum). Although the product of the hr-t gene does not seem to be required for lytic growth of polyoma virus in pennissive celLs (9, 20), a functional ts-a gene product is absolutely required to replicate the viral DNA in productively infected cells (4, 8, 22). In addition to the large and small T-Ag species, other polypeptides can be specifically immunoprecipitated from polyoma-infected cells
(11, 13, 17). These have molecular weights between 72,000 and 28,000. Ito et al. (13) described one such immunoreactive protein (55,000 daltons) in preparations of plasma membranes from infected cells. This protein was missing from plasma membrane fractions when the cells had been infected with a host range (hr-t) deletion mutant of polyoma (NG-18). Schaffhausen et al. (17) determined that the NG-18 mutation caused the disappearance of proteins with molecular weights of 63,000, 56,000, 36,000, and 22,000 from infected cells, while having no measurable effect on the large T Ag species. In this report we have examined the relationship between some of these proteins by analyzing their methionine-containing tryptic peptides. Our data indicate that the large polyoma T Ag (105,000 daltons), the small T Ag (20,000 daltons), and a 63,000-dalton protein (middle T Ag) isolated from productively infected or transformed cells share some amino acid sequences. The middle and small T-Ag species appear to share other sequences which are different from any present in the large T-Ag molecule. Furthermore, each of the middle and large T Ag's contains unique sequences not found in either of t Present address: School of Life and Health Sciences, the other two proteins. We conclude from these University of Delaware, Newark, DE 19711. t Present address: Department of Mdicrobiology, National results that these three polypeptides are distinct Yang-Ming Medical College, and Taipei Veteran General Hos- products of the early gene region of polyoma virus. pital, Sheh-Pei, Taipei, Taiwan. 881
882
SIMMONS, CHANG, AND MARTIN
J. VIROL.
MATERILS AND METHODS Infection and labeling conditions. Primary mouse kidney cells were infected with polyoma virus (large plaque strain originally isolated by Vogt and Dulbecco) at a multiplicity of 25 to 50 PFU/cell. Infected cells or polyoma-transformed hamster cells (obtained from K. K. Takemoto) were labeled with 200 uCi of L-[methyl-3H]methionine per ml (specific activity, 7 Ci/mmol) for 3 h or with 150 ,uCi of L[35S]methionine per ml (specific activity, 327 Ci/ mmol) for 1 hr in Eagle minimal essential medium lacking unlabeled methionine. Extraction and immunoprecipitation. Labeled cells were washed twice with ice-cold 0.02 M Tris (pH 7.4)-0.001 M Na2HPO4-0.137 M NaCl (Tris-buffered saline), collected into the same buffer, and centrifuged at 1,000 x g for 10 min at 00C. The pelleted cells were suspended in a small volume of a solution containing 0.01 M Tris (pH 9.0), 10% glycerol, 10-4 M phenylmethylsulfonyl fluoride, 10-4 M L-1-tosylamide-2phenylethylchloromethyl ketone, 0.5% Triton X-100, 0.5% sodium deoxycholate (extraction buffer) and incubated at 00C for 3 h. The material was sedimented at 40,000 rpm for 40 min at 2°C in an SW50.1 rotor. The supernatant was incubated for 1 h at 0°C with 10 to 20 pl of either normal nonimmune hamster serum or hamster anti-T serum (with a titer of 1:320 or 1:640 as assayed by complement fixation). A suspension (100 to 200 pl) of protein A-bearing Staphylococcus aureus, washed and prepared as described by Simmons et al. (19) was added to the reaction. After 1 h at 00C, the bacteria were centrifuged at 2,000 x g for 10 min at 0°C, washed twice in 5 ml of extraction buffer, three times in 0.1 M Tris (pH 9.0)-0.5 M LiCl-1% 2-mercaptoethanol and once in 0.008 M Na2HPO4-0.0015 M KH2PO4 (pH 7.4)-0.137 M NaCl-0.0027 M KCL. The final bacterial pellet was suspended in a small volume of electrophoresis sample buffer (0.075 M Tris-sulfate [pH 8.6], 2% sodium dodecyl sulfate, 2% 2-mercaptoethanol, 0.002% bromophenol blue, 15% glycerol). Gel electrophoresis and chromatography of tryptic peptides. Protein samples were heated and subjected to acrylamide gel electrophoresis as previously described (19). Protein bands to be characterized further were excised from the gel, and the proteins were eluted and digested with trypsin as described previously (19). Tryptic peptides were analyzed by ion-exchange chromatography on columns of Chromobeads (Technicon Chemicals) (19).
induced tumors (anti-T serum). To identify the species of T antigen made in polyoma-transformed hamster cells (which were T antigen positive as assayed by immunofluorescence [21]), [35S]methionine-labeled extracts of these cells were incubated with anti-T serum in an immunoprecipitation reaction, and the labeled proteins in the precipitates were examined by acrylamide gel electrophoresis (Fig. la). This gel shows that several major proteins with molecular weights of 105,000, 63,000, 56,000, and 20,000 were precipitated with hamster anti-T serum but not with normal hamster serum. Two minor proteins (72,000 and 34,000 daltons) were also specifically immunoprecipitated (Fig. la). In a parallel experiment, immunoprecipitated proteins of labeled polyoma-infected cells were pre-
RESULTS Identification of several forms of polyoma T Ag's in virus-infected and transformed cells. Ito et al. (11, 13) and Schaffhausen et al. (17) have detected several forms of virus-specific antigens in mouse cells infected with polyoma virus. These antigens included proteins with molecular weights of approximately 100,000 (large T Ag) and 20,000 (small T Ag) as well as several other proteins of intermediate sizes (molecular weights, 28,000 to 72,000). All of these proteins were immunoprecipitated with serum directed against polyoma-
FIG. 1. Acrylamide gel electrophoresis of anti-T reactive proteins isolated from polyoma-infected or transformed cells. [3S]methionine-labeled extracts of polyoma virus-infected mouse cells or polyomatransformed hamster cells were reacted with either normal, nonimmune hamster serum or anti-polyoma T hamster serum. Labeledproteins in the precipitates were subjected to acrylamide gel electrophoresis and detected in the gel by fluorography (3). (a) Labeled proteins from transformned cellsprecipitated with normal serum (N) or anti- T serum (T). (b) Labeled proteins from infected cells precipitated with normal (N) or anti-T serum (T). Molecular weight values, expressed in thousands (K), were estimated as previously described (19).
N
N
..
-T".
..
.".
..
KK K IK 34 K
A-
_
4e
105 72 63 56
N
......
.:-s
; 4mm%
20 K-
VOL. 29, 1979
pared and examined on the same gel (Fig. lb). All of the immunoreactive polypeptides detected in transformed hamster cells were also identified in infected mouse cells. The major difference between the antigens of these two cell types was that infected cells apparently synthesized smaller amounts of the 56,000-dalton protein. It is worth noting that most of the polyoma-transformed hamster cells examined to date cow tained all of the immunoreactive proteins described above with the exception of the large TAg species (D. T. Simmons, M. Israel, and M. A. Martin unpublished data). In contrast, infected mouse cells always synthesized large T Ag. To determine the rate of synthesis of these various proteins during the virus life cycle, polyoma-infected mouse cells were labeled for 1 h with [3S]methionine at various times. Figure 2 shows that at least three immunoreactive proteins (105,000, 63,000, and 20,000 daltons) were made during the course of the infection. The synthesis of these three polypeptides was initially detected between 18 and 19 h (Fig. 2a), was maximal between 41 and 42 h (Fig. 2c), and decreased at later times (Fig. 2d and e). We observed no striking differences in the optimal times at which each protein was synthesized. Relationship between the methionine-labeled tryptic peptides of the large and small T Ag's. Recent data indicate that the simian virus 40 (SV 40)-specific large and small T Ag's contain five to seven common methionine-labeled tryptic peptides (16, 18). Furthermore, the small T-Ag species contains two additional peptides not found in the larger molecule. The relationship between the correspond-
POLYOMA TUMOR ANTIGENS
883
ing polyoma T Ag's was determined by examining their methionine tryptic peptides by ionexchange chromatography (Fig. 3). At least 11
b
a
e
4 T :2.
63 K-
d
I
!
105 K-'
c
-9-
I
A.
"A LA
20 KFIG. 2. Synthesis of various T-antigen forms at different times during polyoma infection of mouse cells. Primary mouse kidney cells were infected with polyoma virus and labeled with [3S]methionine for 1 hr at various times. Extracts of those cells were incubated with anti-T serum, and the precipitated proteins were examined by gel electrophoresis. Immunoprecipitatedproteins from cells labeled between (a) 18 and 19, (b) 30 and 31, (c) 41 and 42, (d) 54 and 55, and (e) 66 and 67 h postinfection. Molecular weight values are expressed in thousands (K).
-'20 X~15 10 I
FRACTION NUMBER FIG. 3. Comparison of the methionine-labeled tryptic peptides of the large- and small-molecular-weight species of polyoma T Ag. ['S]methionine-labeled small T Ag (20,000 daltons) and [3H]methionine-labeled large TAg (105,000 daltons) were prepared by immunoprecipitation from polyoma-transformed hamster cells and purified by gel electrophoresis. The proteins were treated with trypsin, and the resulting peptides were subjected to ion-exchange chromatography on columns of Chromobeads as previously described (19). [3'S]methionine-labeled trypticpeptides ofpolyoma 20,000-dalton TAg; - - -, [3H]methionine-labeled tryptic peptides ofpolyoma 105,000-dalton T Ag. Numbers and letters on figure refer to peptides (see text).
J. VIROL.
SIMMONS, CHANG, AND MARTIN
884
methionine-labeled tryptic peptides were detected in the large polyoma T Ag by this technique, of which 5 (peptides 1, 4, 5, 6, and 10) were constituents of the small T Ag as well. In addition, the 20,000-dalton polyoma antigen contained two peptides (peptides a and b) absent from the large T Ag. Thus, in a situation similar to that of SV40, the polyoma small T Ag appears to share amino acid sequences with the large T Ag and to contain sequences missing from the larger protein. Analysis of the methionine peptides of the middle T Ag's. Because a 63,000-dalton protein was immunoprecipitated from both productively infected and transformed cells, the possibility existed that these proteins were related to the large and/or small forms of polyoma T Ag. Figure 4 shows that the 63,000-dalton protein isolated from infected mouse cells contains at least 11 methionine-labeled tryptic peptides, of which 5 (1, 4, 5, 6, and 10) eluted with peptides in the large T Ag. Thus, the same five peptides shared by the large and small T Ag's were also present in the 63,000-dalton protein (middle T Ag). The middle T Ag consisted as well of two peptides which eluted at positions similar to those of peptides a and b in the chromatogram shown in Fig. 3. The remaining four peptides did not correspond to any methionine peptides constituting the large or small T Ag. We identified peptides a and b in the middle T-Ag protein by comparing the methionine peptides of the 63,000- and 20,000-dalton antigens on the same chromatogram. The source of the 63,000-dalton protein in this experiment was from polyoma-transformed cells. Peptides a and b from each of these two proteins eluted at the
same positions as shown in Fig. 5. Thus, the middle and small T-Ag species contained apparently the same seven methionine peptides. Furthernore, all of the resolvable methionine peptides in the small T Ag were contained in the middle T-Ag protein as well. The tryptic peptide patterns of the 63,000-dalton protein isolated from infected or transformed cells were quite -similar (Fig. 4 and 5). At least one other protein (56,000 daltons) was isolated by immunoprecipitation in relatively large quantities from labeled polyoma-transformed hamster cells (Fig. 1). A similarly sized protein was also immunoprecipitated from polyoma-infected cells. To examine any possible relationship of the sequences in the 56,000-dalton antigen from transformed cells to that of large T Ag, we compared their methionine-labeled peptides by ion-exchange chromatography (Fig. 6). Our data indicated that this protein had no peptides in common with the large T Ag. Similarly, we could not detect any peptides common to the 56,000-dalton protein and the middle or small T-Ag forms and therefore consider it likely that this protein is cellularly coded. We were not able to examine the peptides of the corresponding protein made in polyoma-infected mouse cells due to the limited quantities synthesized. DISCUSSION The data contained in this paper indicate that polyoma virus codes for at least three proteins made early in lytic infection and in transformed cells. These are called the large (105,000 daltons), middle (63,000 daltons), and small (20,000 daltons) forms of T Ag. The small T Ag of polyoma probably shares sequences with the N10
.8 C
001111
E
12-
200 250 300 150 FRACTION NUMBER FIG. 4. Comparison of the methionine tryptic peptides of the large and middle species ofpolyoma T Ag. [3'SJmethionine-labeled middle T Ag (63,000 daltons) and [3HJmethionine-labeled large T Ag were isolated by iunmunoprecipitation from polyoma-infected and transformed cells, respectively. Tryptic peptides of gel, [3'SJmethionine-labeled tryptic purified proteins were analyzed by ion-exchange chromatography. peptides of middle TAg (63,000 daltons); - - -, 3HJmethionine-labeled trypticpeptides of large TAg (105,000 daltons). Numbers and letters on figure refer to peptides (see text). -
0
50
100
VOL. 29, 1979
885
POLYOMA TUMOR ANTIGENS
120~
16
159
,,.; 4
Ii
ii
0 0
50
10
x
1
0
FRACTION NUMBER FIG. 5. Comparison of the methionine tryptic peptides of middle and smaU TAg's.[35S]methionine-labeled small T Ag and [3H]methionine-labeled middle T Ag were purified firom polyoma- transformed hamster cells by imnmunoprecipitation and gel electrophoresis. Trypticpeptides of the proteins were analyzed by Chromobead ion-exchange chromatography. ~, [35Slmethionine-labeled tryptic peptides of small T Ag (20,000 daltons); - - -, [3H]methionine-labeled tryptic peptides of middle T Ag (63,000 daltons). Numbers and letters refer to
peptides (see text).
25 aJ
Ix
w
c)
0
15 -u .,
I
x 10
O
5
FRACTION NUMBER
FIG. 6. Analysis of the methionine trypticpeptides of a 56,000-dalton immunoreactive protein frompolyomatransformed hamster cells. Polyoma-transformed hamster cells were labeled with [3H]methionine, and the extracts were incubated with anti-T serum. A 56,000-dalton protein (Fig. 1) immunoprecipitated from these cells was purified by gel electrophoresis. Tryptic peptides of this protein were compared with [3S]methioninelabeled tryptic peptides of the large polyoma T antigen isolated from transformed cells by ion-exchange -, [3H]methionine-labeled chromatography. [3S]methionine-labeled trypticpeptides of large TAg; tryptic peptides of the 56,000-dalton protein. Numbers and letters refer to peptides (see text). - -
,
0.00 0.25 0.73 0.79 0.86 terminal portion of large T Ag (5, 12). This is l I I supported by the experiment of Paucha et al. 105K (15) who showed that the large and small T Ag's 63K V\AA of SV40 have a common NH2 terminus as deter20K mined by sequence analysis. Because the middle T Ag of polyoma contains the same tryptic pep- PEPTIDES 1,4,5 a,b tides that are present in the large and small T 6,10 Ag's, these three proteins appear to contain the FIG. 7. Model describing the region of the polyoma same amino-terminal regions. A model consistgenome coding for large, middle, and small T Ag's. ent with our observations is presented in Fig. 7. For explanation, see text. Molecular weights are exRecently, similar models have been suggested pressed in thousands (K). independently by Hutchinson et al. (10) and by Smart and Ito (unpublished). The relative map early RNA by the Berk and Sharp technique positions shown in Fig. 7 were derived from the (2). The model predicts that peptides 1, 4, 5, 6, estimates of R. Kamen and collaborators (un- and 10 are coded by DNA mapping between 0.73 published data) who have mapped the species of and 0.79 because these peptides are common to I
I
886
J. VIROL.
SIMMONS, CHANG, AND MARTIN
all three T Ag's and because the proximal end of the early gene region (coding for the NH2 termini of these proteins) maps at position 0.73 (14). Similarly, the two peptides (a and b) which are present in the 63,000- and 20,000-dalton proteins but are missing from the 105,000-dalton protein are encoded by DNA mapping between 0.79 and 0.86 units. This is consistent with the effect of hr-t deletion on the size of the middle and small forms of polyoma T Ag; the hr-t deletion has no effect on the large T Ag (11, 17). We hypothesize that the C-terminal portion of the 63,000-dalton protein is encoded by polyoma DNA mapping from a position close to 0.86 to approximately the EcoRI cleavage site (0.00). Sequences which map distal to the EcoRI site are apparently not involved in the synthesis of middle T Ag (M. Israel, D. T. Simmons, and M. A. Martin, unpublished data). Because some of the methionine peptides in middle T Ag are not found in large T Ag, the region of middle T Ag containing those peptides (the C-terminal region) could be coded by the cell genome. A more likely possibility is that middle T Ag is coded entirely by the early viral gene region but that the message specifying this protein is translated in a different reading frame from the large T-Ag message between 0.86 and 0.00 map units. Indeed, B. Griffin and co-workers (unpublished data) have sequenced this portion of polyoma DNA and have detected a second open reading frame in the early message spanning from 0.86 to 0.99. Consequently, this stretch of DNA could code for two entirely different polypeptide chains depending on the reading frame used. One must assume that the stop (i.e., UAG) condon is removed from the messenger RNA which codes for the 63,000-dalton protein, possibly by a mechanism such as RNA splicing (1). Another possible interpretation of our data is that the mRNA's coding for the large and middle T Ag's are translated in the same reading frame between 0.86 and 0.00 and that one protein is modified within this region (i.e., phosphorylated, acetylated) while the other one is not. indicated that a protein corresponding to the 63,000-dalton protein of polyoma is not made in SV40-infected or transformed cells (unpublished data). Proteins with molecular weights between 60,000 and 56,000 could be immunoprecipitated from various SV40-transformed cells (C. Chang, D. T. Simmons, M. A. Martin, and P. T. Mora, submitted for publication). However, the evidence indicated that these proteins are organized differently from the middle T Ag of polyoma in that they appear to share amino acid sequences with at most only a portion of the region in common to both large and small SV40 T Ag's.
The nature of the 56,000-dalton protein which was immunoprecipitated from polyoma-transformed hamster cells remains puzzling. This protein appears not to be coded by the viral genome because none of its methionine-containing peptides matched any of the peptides present in the T-antigen species. However, all of the polyomatransformed rat and hamster cells examined to date contained an immunoprecipitable protein of that size (unpublished data). Thus, this protein may be cellularly coded and induced in cells transformed by the virus. Furthermore, it appears that this protein bears a relationship to proteins with the same approximate size (56,000 daltons) isolated from SV40-transformed cells in that they are all synthesized in relatively large quantities in cells transformed by these viruses but are either not made or made in much smaller quantities in infected cells. Ito et al. (11) have identified an immunoprecipitable protein (55,000 daltons) in membrane fractions of infected mouse cells. Analysis of the tryptic peptides of that protein has indicated that it is the same protein which we have called middle T Ag (63,000 daltons). It seems reasonable that the middle T-Ag form has a functional role on the surface of infected or transformed cells. Whether this protein is related to the cell surface tumor-specific transplantation antigen remains to be determined. LITERATURE CITED 1. Berget, S. M., C. Moore, and P. H. Sharp. 1977. Spliced segments at the 5' terminus of adenovirus 2 late mRNA. Proc. Natl. Acad. Sci. U.S.A. 74:3171-3175. 2. Berk, A. J., and P. A. Sharp. 1978. Spliced early mRNAs of simian virus 40. Proc. Natl. Acad. Sci. U.S.A. 75: 1274-1278. 3. Bonner, W. M., and R. A. Laskey. 1974. A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83-88. 4. Chou, J. Y., J. Avila, and R. G. Martin. 1974. Viral DNA synthesis in cells infected by temperature-sensitive mutants of simian virus 40. J. Virol. 14:116-124. 5. Crawford, L V., C. N. Cole, A. E. Smith, E. Paucha, P. Tegtmeyer, K. Rundell, and P. Berg. 1978. Organization and expression of early genes of simian virus 40. Proc. Natl. Acad. Sci. U.S.A. 75:117-121. 6. Eckhart, W. 1977. Complementation between temperature-sensitive (ts) and host range nontransforming (hrt) mutants of polyoma virus. Virology 77:589-597. 7. Fluck, M., R. Staneloni, and T. Benjamin. 1977. Hr-t and ts-a: two early gene functions of polyoma virus. Virology 77:610-624. 8. Francke, B., and W. Eckhart. 1973. Polyoma gene function required for viral DNA synthesis. Virology 55: 127-135. 9. Goldman, E., and T. L. Benjamin. 1975. Analysis of host range of nontransforming polyoma virus mutants. Virology 66:372-384. 10. Hutchinson, M. A., T. Hunter, and W. Eckhart. 1978. Characterization of T-antigens in polyoma-infected and transformed cells. Cell 15:65-77. 11. Ito, Y., J. R. Brocklehurst, and R. Dulbecco. 1977.
POLYOMA TUMOR ANTIGENS
VOL. 29, 1979 Virus-specific proteins in the plasma membrane of cells lytically infected or transformed by polyoma virus. Proc. Natl. Acad. Sci. U.S.A. 74:4666-4670. 12. Ito, Y., J. R. Brochlehurst, N. Spurr, M. Griffiths, J. Hurst, and M. Fried. 1978. Polyoma virus wild type and mutant T-antigens. INSERM 69:145-156. 13. Ito, Y., N. Spurr, and R. Dulbecco. 1977. Characterization of polyoma virus T antigen. Proc. Natl. Acad. Sci. U.S.A. 74:1259-1263. 14. Kamen, R., J. Sedat, and E. Ziff. 1976. Orientation of the complementary strands of polyoma virus DNA with respect to the DNA physical map. J. Virol. 17:212-218. 15. Paucha, E., M. Andrew, R. Harvey, A. E. Smith, R. M. Hervich, and A. D. Waterfield. 1978. Large and small tumor antigens from simian virus 40 have identical amino termini mapping at 0.65 map units. Proc. Natl. Acad. Sci. U.S.A. 75:2165-2169. 16. Prives, C., and Y. Beck. 1978. Characterization of SV40 T-antigen polypeptide synthesized in vivo and in vitro. INSERM 69:175-187. 17. Schaffhausen, B. S., J. E. Silver, and T. L Benjamin.
18.
19.
20. 21.
22.
887
1978. Tumor antigen(s) in cells productively infected by wild-type polyoma virus and mutant NG-18. Proc. Natl. Acad. Sci. U.S.A. 75:79-83. Simmons, D. T., and M. A. Martin. 1978. Common methionine-tryptic peptides near the amino-terminal end of primate papovavirus tumor antigens. Proc. Natl. Acad. Sci. U.S.A. 75:1131-1135. Simmons, D. T., K. K. Takemoto, and M. A. Martin. 1977. Relationship between the methionine tryptic peptides of simian virus 40 and BK virus tumor antigens. J. Virol. 24:319-325. Staneloni, R., M. Fluck, and T. Benjamin. 1977. Host range selection of transformation-defective hr-t mutants of polyoma virus. Virology 77:598-609. Takemoto, K. K., R. A. Malmgren, and K. Habel. 1966. Immunofluorescence of polyoma tumor antigens: a heat-labile factor in serum required for optimal activity. Science 153:1122-1123. Tegtmeyer, P. 1972. Simian virus 40 deoxyribonucleic acid synthesis: the viral replicon. J. Virol. 10:591-598.
Vol. 29, No. 3
Multiple Forms of Polyoma Virus Tumor Antigens from Infected and Transformed Cells DANEL T. SIMMONS,t* CHUNGMING CHANG,4 AND MALCOLM A. MARTIN Laboratory of Biology of Viruses, National Institute of Allergy and Infectious Dzseases, and Immunology Program, National Cancer Institute, Bethesda, Maryland 20014 Received for publication 21 September 1978 At least three distinct forms of
polyoma
virus tumor
antigens
were
isolated
from productively infected and transformed hamster cells by inmunoprecipitation with anti-T serum. These proteins had approximate molecular weights of 105,000 (large T antigen), 63,000 (middle T antigen), and 20,000 (small T antigen) as estimated by acrylamide gel electrophoresis. An examination of the appearance of these antigens in polyoma-infected mouse cells showed that all three polypeptides were synthesized maximally at approximately the same time after infection. Analysis of the methionine-containing tryptic peptides of these proteins indicated that the large, middle, and small forms of polyoma T antigens contained five similar or identical peptides. In addition, the 63,000- and 20,000-dalton antigens contained two other methionine peptides absent from the large T-antigen species. Other methionine peptides were found only in the large or middle T-antigen forms. These results and results obtained previously suggested that the three Tantigen species have the same NH2-terminal end regions but different COOH termini. A model is presented describing the synthesis of these polypeptides from different regions of the polyoma virus genome. The region of the polyoma virus genome which is first expressed early in lytic infection (before the onset of DNA replication) consists of at least two genes (denoted hr-t and ts-a) which are required for virus-induced cellular transformation (6, 7). The products of these two genes appear to be proteins with molecular weights of 95 to 108,000 (large T antigen [T Ag]) and 20 to 22,000 (small T Ag), as estimated by acrylamide gel electrophoresis (11, 13, 17), although the true size of the large T Ag may be closer to 80,000 daltons (17). These proteins can be isolated from infected mouse cells or virus-transformed cells by immunoprecipitation with serum directed against polyoma-induced tumors (anti-T serum). Although the product of the hr-t gene does not seem to be required for lytic growth of polyoma virus in pennissive celLs (9, 20), a functional ts-a gene product is absolutely required to replicate the viral DNA in productively infected cells (4, 8, 22). In addition to the large and small T-Ag species, other polypeptides can be specifically immunoprecipitated from polyoma-infected cells
(11, 13, 17). These have molecular weights between 72,000 and 28,000. Ito et al. (13) described one such immunoreactive protein (55,000 daltons) in preparations of plasma membranes from infected cells. This protein was missing from plasma membrane fractions when the cells had been infected with a host range (hr-t) deletion mutant of polyoma (NG-18). Schaffhausen et al. (17) determined that the NG-18 mutation caused the disappearance of proteins with molecular weights of 63,000, 56,000, 36,000, and 22,000 from infected cells, while having no measurable effect on the large T Ag species. In this report we have examined the relationship between some of these proteins by analyzing their methionine-containing tryptic peptides. Our data indicate that the large polyoma T Ag (105,000 daltons), the small T Ag (20,000 daltons), and a 63,000-dalton protein (middle T Ag) isolated from productively infected or transformed cells share some amino acid sequences. The middle and small T-Ag species appear to share other sequences which are different from any present in the large T-Ag molecule. Furthermore, each of the middle and large T Ag's contains unique sequences not found in either of t Present address: School of Life and Health Sciences, the other two proteins. We conclude from these University of Delaware, Newark, DE 19711. t Present address: Department of Mdicrobiology, National results that these three polypeptides are distinct Yang-Ming Medical College, and Taipei Veteran General Hos- products of the early gene region of polyoma virus. pital, Sheh-Pei, Taipei, Taiwan. 881
882
SIMMONS, CHANG, AND MARTIN
J. VIROL.
MATERILS AND METHODS Infection and labeling conditions. Primary mouse kidney cells were infected with polyoma virus (large plaque strain originally isolated by Vogt and Dulbecco) at a multiplicity of 25 to 50 PFU/cell. Infected cells or polyoma-transformed hamster cells (obtained from K. K. Takemoto) were labeled with 200 uCi of L-[methyl-3H]methionine per ml (specific activity, 7 Ci/mmol) for 3 h or with 150 ,uCi of L[35S]methionine per ml (specific activity, 327 Ci/ mmol) for 1 hr in Eagle minimal essential medium lacking unlabeled methionine. Extraction and immunoprecipitation. Labeled cells were washed twice with ice-cold 0.02 M Tris (pH 7.4)-0.001 M Na2HPO4-0.137 M NaCl (Tris-buffered saline), collected into the same buffer, and centrifuged at 1,000 x g for 10 min at 00C. The pelleted cells were suspended in a small volume of a solution containing 0.01 M Tris (pH 9.0), 10% glycerol, 10-4 M phenylmethylsulfonyl fluoride, 10-4 M L-1-tosylamide-2phenylethylchloromethyl ketone, 0.5% Triton X-100, 0.5% sodium deoxycholate (extraction buffer) and incubated at 00C for 3 h. The material was sedimented at 40,000 rpm for 40 min at 2°C in an SW50.1 rotor. The supernatant was incubated for 1 h at 0°C with 10 to 20 pl of either normal nonimmune hamster serum or hamster anti-T serum (with a titer of 1:320 or 1:640 as assayed by complement fixation). A suspension (100 to 200 pl) of protein A-bearing Staphylococcus aureus, washed and prepared as described by Simmons et al. (19) was added to the reaction. After 1 h at 00C, the bacteria were centrifuged at 2,000 x g for 10 min at 0°C, washed twice in 5 ml of extraction buffer, three times in 0.1 M Tris (pH 9.0)-0.5 M LiCl-1% 2-mercaptoethanol and once in 0.008 M Na2HPO4-0.0015 M KH2PO4 (pH 7.4)-0.137 M NaCl-0.0027 M KCL. The final bacterial pellet was suspended in a small volume of electrophoresis sample buffer (0.075 M Tris-sulfate [pH 8.6], 2% sodium dodecyl sulfate, 2% 2-mercaptoethanol, 0.002% bromophenol blue, 15% glycerol). Gel electrophoresis and chromatography of tryptic peptides. Protein samples were heated and subjected to acrylamide gel electrophoresis as previously described (19). Protein bands to be characterized further were excised from the gel, and the proteins were eluted and digested with trypsin as described previously (19). Tryptic peptides were analyzed by ion-exchange chromatography on columns of Chromobeads (Technicon Chemicals) (19).
induced tumors (anti-T serum). To identify the species of T antigen made in polyoma-transformed hamster cells (which were T antigen positive as assayed by immunofluorescence [21]), [35S]methionine-labeled extracts of these cells were incubated with anti-T serum in an immunoprecipitation reaction, and the labeled proteins in the precipitates were examined by acrylamide gel electrophoresis (Fig. la). This gel shows that several major proteins with molecular weights of 105,000, 63,000, 56,000, and 20,000 were precipitated with hamster anti-T serum but not with normal hamster serum. Two minor proteins (72,000 and 34,000 daltons) were also specifically immunoprecipitated (Fig. la). In a parallel experiment, immunoprecipitated proteins of labeled polyoma-infected cells were pre-
RESULTS Identification of several forms of polyoma T Ag's in virus-infected and transformed cells. Ito et al. (11, 13) and Schaffhausen et al. (17) have detected several forms of virus-specific antigens in mouse cells infected with polyoma virus. These antigens included proteins with molecular weights of approximately 100,000 (large T Ag) and 20,000 (small T Ag) as well as several other proteins of intermediate sizes (molecular weights, 28,000 to 72,000). All of these proteins were immunoprecipitated with serum directed against polyoma-
FIG. 1. Acrylamide gel electrophoresis of anti-T reactive proteins isolated from polyoma-infected or transformed cells. [3S]methionine-labeled extracts of polyoma virus-infected mouse cells or polyomatransformed hamster cells were reacted with either normal, nonimmune hamster serum or anti-polyoma T hamster serum. Labeledproteins in the precipitates were subjected to acrylamide gel electrophoresis and detected in the gel by fluorography (3). (a) Labeled proteins from transformned cellsprecipitated with normal serum (N) or anti- T serum (T). (b) Labeled proteins from infected cells precipitated with normal (N) or anti-T serum (T). Molecular weight values, expressed in thousands (K), were estimated as previously described (19).
N
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..
KK K IK 34 K
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20 K-
VOL. 29, 1979
pared and examined on the same gel (Fig. lb). All of the immunoreactive polypeptides detected in transformed hamster cells were also identified in infected mouse cells. The major difference between the antigens of these two cell types was that infected cells apparently synthesized smaller amounts of the 56,000-dalton protein. It is worth noting that most of the polyoma-transformed hamster cells examined to date cow tained all of the immunoreactive proteins described above with the exception of the large TAg species (D. T. Simmons, M. Israel, and M. A. Martin unpublished data). In contrast, infected mouse cells always synthesized large T Ag. To determine the rate of synthesis of these various proteins during the virus life cycle, polyoma-infected mouse cells were labeled for 1 h with [3S]methionine at various times. Figure 2 shows that at least three immunoreactive proteins (105,000, 63,000, and 20,000 daltons) were made during the course of the infection. The synthesis of these three polypeptides was initially detected between 18 and 19 h (Fig. 2a), was maximal between 41 and 42 h (Fig. 2c), and decreased at later times (Fig. 2d and e). We observed no striking differences in the optimal times at which each protein was synthesized. Relationship between the methionine-labeled tryptic peptides of the large and small T Ag's. Recent data indicate that the simian virus 40 (SV 40)-specific large and small T Ag's contain five to seven common methionine-labeled tryptic peptides (16, 18). Furthermore, the small T-Ag species contains two additional peptides not found in the larger molecule. The relationship between the correspond-
POLYOMA TUMOR ANTIGENS
883
ing polyoma T Ag's was determined by examining their methionine tryptic peptides by ionexchange chromatography (Fig. 3). At least 11
b
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20 KFIG. 2. Synthesis of various T-antigen forms at different times during polyoma infection of mouse cells. Primary mouse kidney cells were infected with polyoma virus and labeled with [3S]methionine for 1 hr at various times. Extracts of those cells were incubated with anti-T serum, and the precipitated proteins were examined by gel electrophoresis. Immunoprecipitatedproteins from cells labeled between (a) 18 and 19, (b) 30 and 31, (c) 41 and 42, (d) 54 and 55, and (e) 66 and 67 h postinfection. Molecular weight values are expressed in thousands (K).
-'20 X~15 10 I
FRACTION NUMBER FIG. 3. Comparison of the methionine-labeled tryptic peptides of the large- and small-molecular-weight species of polyoma T Ag. ['S]methionine-labeled small T Ag (20,000 daltons) and [3H]methionine-labeled large TAg (105,000 daltons) were prepared by immunoprecipitation from polyoma-transformed hamster cells and purified by gel electrophoresis. The proteins were treated with trypsin, and the resulting peptides were subjected to ion-exchange chromatography on columns of Chromobeads as previously described (19). [3'S]methionine-labeled trypticpeptides ofpolyoma 20,000-dalton TAg; - - -, [3H]methionine-labeled tryptic peptides ofpolyoma 105,000-dalton T Ag. Numbers and letters on figure refer to peptides (see text).
J. VIROL.
SIMMONS, CHANG, AND MARTIN
884
methionine-labeled tryptic peptides were detected in the large polyoma T Ag by this technique, of which 5 (peptides 1, 4, 5, 6, and 10) were constituents of the small T Ag as well. In addition, the 20,000-dalton polyoma antigen contained two peptides (peptides a and b) absent from the large T Ag. Thus, in a situation similar to that of SV40, the polyoma small T Ag appears to share amino acid sequences with the large T Ag and to contain sequences missing from the larger protein. Analysis of the methionine peptides of the middle T Ag's. Because a 63,000-dalton protein was immunoprecipitated from both productively infected and transformed cells, the possibility existed that these proteins were related to the large and/or small forms of polyoma T Ag. Figure 4 shows that the 63,000-dalton protein isolated from infected mouse cells contains at least 11 methionine-labeled tryptic peptides, of which 5 (1, 4, 5, 6, and 10) eluted with peptides in the large T Ag. Thus, the same five peptides shared by the large and small T Ag's were also present in the 63,000-dalton protein (middle T Ag). The middle T Ag consisted as well of two peptides which eluted at positions similar to those of peptides a and b in the chromatogram shown in Fig. 3. The remaining four peptides did not correspond to any methionine peptides constituting the large or small T Ag. We identified peptides a and b in the middle T-Ag protein by comparing the methionine peptides of the 63,000- and 20,000-dalton antigens on the same chromatogram. The source of the 63,000-dalton protein in this experiment was from polyoma-transformed cells. Peptides a and b from each of these two proteins eluted at the
same positions as shown in Fig. 5. Thus, the middle and small T-Ag species contained apparently the same seven methionine peptides. Furthernore, all of the resolvable methionine peptides in the small T Ag were contained in the middle T-Ag protein as well. The tryptic peptide patterns of the 63,000-dalton protein isolated from infected or transformed cells were quite -similar (Fig. 4 and 5). At least one other protein (56,000 daltons) was isolated by immunoprecipitation in relatively large quantities from labeled polyoma-transformed hamster cells (Fig. 1). A similarly sized protein was also immunoprecipitated from polyoma-infected cells. To examine any possible relationship of the sequences in the 56,000-dalton antigen from transformed cells to that of large T Ag, we compared their methionine-labeled peptides by ion-exchange chromatography (Fig. 6). Our data indicated that this protein had no peptides in common with the large T Ag. Similarly, we could not detect any peptides common to the 56,000-dalton protein and the middle or small T-Ag forms and therefore consider it likely that this protein is cellularly coded. We were not able to examine the peptides of the corresponding protein made in polyoma-infected mouse cells due to the limited quantities synthesized. DISCUSSION The data contained in this paper indicate that polyoma virus codes for at least three proteins made early in lytic infection and in transformed cells. These are called the large (105,000 daltons), middle (63,000 daltons), and small (20,000 daltons) forms of T Ag. The small T Ag of polyoma probably shares sequences with the N10
.8 C
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200 250 300 150 FRACTION NUMBER FIG. 4. Comparison of the methionine tryptic peptides of the large and middle species ofpolyoma T Ag. [3'SJmethionine-labeled middle T Ag (63,000 daltons) and [3HJmethionine-labeled large T Ag were isolated by iunmunoprecipitation from polyoma-infected and transformed cells, respectively. Tryptic peptides of gel, [3'SJmethionine-labeled tryptic purified proteins were analyzed by ion-exchange chromatography. peptides of middle TAg (63,000 daltons); - - -, 3HJmethionine-labeled trypticpeptides of large TAg (105,000 daltons). Numbers and letters on figure refer to peptides (see text). -
0
50
100
VOL. 29, 1979
885
POLYOMA TUMOR ANTIGENS
120~
16
159
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Ii
ii
0 0
50
10
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FRACTION NUMBER FIG. 5. Comparison of the methionine tryptic peptides of middle and smaU TAg's.[35S]methionine-labeled small T Ag and [3H]methionine-labeled middle T Ag were purified firom polyoma- transformed hamster cells by imnmunoprecipitation and gel electrophoresis. Trypticpeptides of the proteins were analyzed by Chromobead ion-exchange chromatography. ~, [35Slmethionine-labeled tryptic peptides of small T Ag (20,000 daltons); - - -, [3H]methionine-labeled tryptic peptides of middle T Ag (63,000 daltons). Numbers and letters refer to
peptides (see text).
25 aJ
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15 -u .,
I
x 10
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FRACTION NUMBER
FIG. 6. Analysis of the methionine trypticpeptides of a 56,000-dalton immunoreactive protein frompolyomatransformed hamster cells. Polyoma-transformed hamster cells were labeled with [3H]methionine, and the extracts were incubated with anti-T serum. A 56,000-dalton protein (Fig. 1) immunoprecipitated from these cells was purified by gel electrophoresis. Tryptic peptides of this protein were compared with [3S]methioninelabeled tryptic peptides of the large polyoma T antigen isolated from transformed cells by ion-exchange -, [3H]methionine-labeled chromatography. [3S]methionine-labeled trypticpeptides of large TAg; tryptic peptides of the 56,000-dalton protein. Numbers and letters refer to peptides (see text). - -
,
0.00 0.25 0.73 0.79 0.86 terminal portion of large T Ag (5, 12). This is l I I supported by the experiment of Paucha et al. 105K (15) who showed that the large and small T Ag's 63K V\AA of SV40 have a common NH2 terminus as deter20K mined by sequence analysis. Because the middle T Ag of polyoma contains the same tryptic pep- PEPTIDES 1,4,5 a,b tides that are present in the large and small T 6,10 Ag's, these three proteins appear to contain the FIG. 7. Model describing the region of the polyoma same amino-terminal regions. A model consistgenome coding for large, middle, and small T Ag's. ent with our observations is presented in Fig. 7. For explanation, see text. Molecular weights are exRecently, similar models have been suggested pressed in thousands (K). independently by Hutchinson et al. (10) and by Smart and Ito (unpublished). The relative map early RNA by the Berk and Sharp technique positions shown in Fig. 7 were derived from the (2). The model predicts that peptides 1, 4, 5, 6, estimates of R. Kamen and collaborators (un- and 10 are coded by DNA mapping between 0.73 published data) who have mapped the species of and 0.79 because these peptides are common to I
I
886
J. VIROL.
SIMMONS, CHANG, AND MARTIN
all three T Ag's and because the proximal end of the early gene region (coding for the NH2 termini of these proteins) maps at position 0.73 (14). Similarly, the two peptides (a and b) which are present in the 63,000- and 20,000-dalton proteins but are missing from the 105,000-dalton protein are encoded by DNA mapping between 0.79 and 0.86 units. This is consistent with the effect of hr-t deletion on the size of the middle and small forms of polyoma T Ag; the hr-t deletion has no effect on the large T Ag (11, 17). We hypothesize that the C-terminal portion of the 63,000-dalton protein is encoded by polyoma DNA mapping from a position close to 0.86 to approximately the EcoRI cleavage site (0.00). Sequences which map distal to the EcoRI site are apparently not involved in the synthesis of middle T Ag (M. Israel, D. T. Simmons, and M. A. Martin, unpublished data). Because some of the methionine peptides in middle T Ag are not found in large T Ag, the region of middle T Ag containing those peptides (the C-terminal region) could be coded by the cell genome. A more likely possibility is that middle T Ag is coded entirely by the early viral gene region but that the message specifying this protein is translated in a different reading frame from the large T-Ag message between 0.86 and 0.00 map units. Indeed, B. Griffin and co-workers (unpublished data) have sequenced this portion of polyoma DNA and have detected a second open reading frame in the early message spanning from 0.86 to 0.99. Consequently, this stretch of DNA could code for two entirely different polypeptide chains depending on the reading frame used. One must assume that the stop (i.e., UAG) condon is removed from the messenger RNA which codes for the 63,000-dalton protein, possibly by a mechanism such as RNA splicing (1). Another possible interpretation of our data is that the mRNA's coding for the large and middle T Ag's are translated in the same reading frame between 0.86 and 0.00 and that one protein is modified within this region (i.e., phosphorylated, acetylated) while the other one is not. indicated that a protein corresponding to the 63,000-dalton protein of polyoma is not made in SV40-infected or transformed cells (unpublished data). Proteins with molecular weights between 60,000 and 56,000 could be immunoprecipitated from various SV40-transformed cells (C. Chang, D. T. Simmons, M. A. Martin, and P. T. Mora, submitted for publication). However, the evidence indicated that these proteins are organized differently from the middle T Ag of polyoma in that they appear to share amino acid sequences with at most only a portion of the region in common to both large and small SV40 T Ag's.
The nature of the 56,000-dalton protein which was immunoprecipitated from polyoma-transformed hamster cells remains puzzling. This protein appears not to be coded by the viral genome because none of its methionine-containing peptides matched any of the peptides present in the T-antigen species. However, all of the polyomatransformed rat and hamster cells examined to date contained an immunoprecipitable protein of that size (unpublished data). Thus, this protein may be cellularly coded and induced in cells transformed by the virus. Furthermore, it appears that this protein bears a relationship to proteins with the same approximate size (56,000 daltons) isolated from SV40-transformed cells in that they are all synthesized in relatively large quantities in cells transformed by these viruses but are either not made or made in much smaller quantities in infected cells. Ito et al. (11) have identified an immunoprecipitable protein (55,000 daltons) in membrane fractions of infected mouse cells. Analysis of the tryptic peptides of that protein has indicated that it is the same protein which we have called middle T Ag (63,000 daltons). It seems reasonable that the middle T-Ag form has a functional role on the surface of infected or transformed cells. Whether this protein is related to the cell surface tumor-specific transplantation antigen remains to be determined. LITERATURE CITED 1. Berget, S. M., C. Moore, and P. H. Sharp. 1977. Spliced segments at the 5' terminus of adenovirus 2 late mRNA. Proc. Natl. Acad. Sci. U.S.A. 74:3171-3175. 2. Berk, A. J., and P. A. Sharp. 1978. Spliced early mRNAs of simian virus 40. Proc. Natl. Acad. Sci. U.S.A. 75: 1274-1278. 3. Bonner, W. M., and R. A. Laskey. 1974. A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83-88. 4. Chou, J. Y., J. Avila, and R. G. Martin. 1974. Viral DNA synthesis in cells infected by temperature-sensitive mutants of simian virus 40. J. Virol. 14:116-124. 5. Crawford, L V., C. N. Cole, A. E. Smith, E. Paucha, P. Tegtmeyer, K. Rundell, and P. Berg. 1978. Organization and expression of early genes of simian virus 40. Proc. Natl. Acad. Sci. U.S.A. 75:117-121. 6. Eckhart, W. 1977. Complementation between temperature-sensitive (ts) and host range nontransforming (hrt) mutants of polyoma virus. Virology 77:589-597. 7. Fluck, M., R. Staneloni, and T. Benjamin. 1977. Hr-t and ts-a: two early gene functions of polyoma virus. Virology 77:610-624. 8. Francke, B., and W. Eckhart. 1973. Polyoma gene function required for viral DNA synthesis. Virology 55: 127-135. 9. Goldman, E., and T. L. Benjamin. 1975. Analysis of host range of nontransforming polyoma virus mutants. Virology 66:372-384. 10. Hutchinson, M. A., T. Hunter, and W. Eckhart. 1978. Characterization of T-antigens in polyoma-infected and transformed cells. Cell 15:65-77. 11. Ito, Y., J. R. Brocklehurst, and R. Dulbecco. 1977.
POLYOMA TUMOR ANTIGENS
VOL. 29, 1979 Virus-specific proteins in the plasma membrane of cells lytically infected or transformed by polyoma virus. Proc. Natl. Acad. Sci. U.S.A. 74:4666-4670. 12. Ito, Y., J. R. Brochlehurst, N. Spurr, M. Griffiths, J. Hurst, and M. Fried. 1978. Polyoma virus wild type and mutant T-antigens. INSERM 69:145-156. 13. Ito, Y., N. Spurr, and R. Dulbecco. 1977. Characterization of polyoma virus T antigen. Proc. Natl. Acad. Sci. U.S.A. 74:1259-1263. 14. Kamen, R., J. Sedat, and E. Ziff. 1976. Orientation of the complementary strands of polyoma virus DNA with respect to the DNA physical map. J. Virol. 17:212-218. 15. Paucha, E., M. Andrew, R. Harvey, A. E. Smith, R. M. Hervich, and A. D. Waterfield. 1978. Large and small tumor antigens from simian virus 40 have identical amino termini mapping at 0.65 map units. Proc. Natl. Acad. Sci. U.S.A. 75:2165-2169. 16. Prives, C., and Y. Beck. 1978. Characterization of SV40 T-antigen polypeptide synthesized in vivo and in vitro. INSERM 69:175-187. 17. Schaffhausen, B. S., J. E. Silver, and T. L Benjamin.
18.
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20. 21.
22.
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1978. Tumor antigen(s) in cells productively infected by wild-type polyoma virus and mutant NG-18. Proc. Natl. Acad. Sci. U.S.A. 75:79-83. Simmons, D. T., and M. A. Martin. 1978. Common methionine-tryptic peptides near the amino-terminal end of primate papovavirus tumor antigens. Proc. Natl. Acad. Sci. U.S.A. 75:1131-1135. Simmons, D. T., K. K. Takemoto, and M. A. Martin. 1977. Relationship between the methionine tryptic peptides of simian virus 40 and BK virus tumor antigens. J. Virol. 24:319-325. Staneloni, R., M. Fluck, and T. Benjamin. 1977. Host range selection of transformation-defective hr-t mutants of polyoma virus. Virology 77:598-609. Takemoto, K. K., R. A. Malmgren, and K. Habel. 1966. Immunofluorescence of polyoma tumor antigens: a heat-labile factor in serum required for optimal activity. Science 153:1122-1123. Tegtmeyer, P. 1972. Simian virus 40 deoxyribonucleic acid synthesis: the viral replicon. J. Virol. 10:591-598.
