Relationship Between the Methionine Tryptic Peptides of

Transcription

1 JOURNAL OF VIROLOGY, Oct. 1977, p Copyright 1977 American Society for Microbiology Vol. 24, No. 1 Printed in U. S.A. Relationship Between the Methionine Tryptic Peptides of Simian Virus 40 and BK Virus Tumor Antigens DANIEL T. SIMMONS,l* KENNETH K. TAKEMOTO,2 AND MALCOLM A. MARTIN' Laboratory of Biology of Viruses' and Laboratory of Viral Diseases,2 National Institute of Allergy and Infectious Diseases, Bethesda, Maryland Received for publication 31 May 1977 The monomer form of BK virus (BKV) tumor antigen (T Ag) was immunoprecipitated from extracts of BKV-transformed cells and had a molecular weight of approximately 113,000. This compared with 97,000 for the molecular weight of either BKV or simian virus 40 (SV40) T Ag from lyrically infected cells. The SV40 and BKV T Ag's from productively infected cells were compared by examining their methionine-labeled tryptic peptides. Out of a total of 20 SV40- and 21 BKV-specific peptides, there were seven pairs of similar peptides on the basis of ion-exchange chromatography. These coeluting peptides contained approximately 25 to 30% of the total methionine radioactivity. Similar results were obtained when the tryptic peptides of SV40 T Ag from lyrically infected cells were compared with those of BKV T Ag from virally transformed cells. Since the initial isolation of BK virus (BKV) from the urine of a renal transplant recipient on immunosuppressive therapy (6), a number of reports have appeared which have shown a relationship between the nucleic acid and protein components of this human papovavirus with those of simian virus 40 (SV40). For example, Mullarkey et al. (12) and Wright and Di- Mayorca (20) demonstrated that the sizes and relative proportion of the structural proteins of BKV were only slightly different from those of SV40. Although the peptide compositions of the capsid proteins of these two papovaviruses differ significantly (20), the structural proteins of these two viruses cross-react weekly, as demonstrated by immunofluorescence (13, 15), antibody neutralization (15), or immunoelectron microscopy (6). However, the tumor antigen (T Ag), induced by SV40 or BKV in virus-infected or -transformed cells, reacts quite strongly with serum directed against the heterologous T Ag (15). Howley et al. (7) showed that the molecular weight of BKV DNA was 3.45 x 10" compared with 3.6 x 106 for SV40 DNA and that the two primate papovavirus DNAs shared approximately 20 to 25% of their base sequences. Khoury et al. (10) demonstrated that this base sequence homology was localized to the regions of the virus DNAs transcribed late in infection (late regions). Recent experiments by P. Howley and M. Martin (manuscript in preparation) indicated a small degree of sequence homology involving 5 to 6% of the early gene regions of SV40 and BKV DNA. Furthermore, T. Kelley 319 and collaborators (personal communications) have detected extensive homology throughout the early regions of these DNAs, using less stringent conditions for hybridization. Because the SV40 and BKV T Ag's crossreact strongly in immunological assays (15) and because some base sequence homology exists between the regions of the virus DNAs that are believed to code for T Ag (the early regions), we have examined the sizes and peptide compositions of the two papovavirus T Ag's. In a subsequent manuscript (submitted for publication), we will report that the molecular weights of the T Ag's isolated by immunoprecipitation from extracts of SV40-infected monkey cells, SV40- transformed cells, or BKV-infected human cells are indistinguishable (97,000) by acrylamide gel electrophoresis. In this study, the size of BKV T Ag isolated from virally transformed hamster cells was examined and shown to be significantly larger (113,000 daltons) than the corresponding protein from lyrically infected cells. Furthermore, the methionine-labeled tryptic peptides of SV40 T Ag from productively infected cells were compared with those of BKV T Ag from lyrically infected and virus-transformed cells. The results indicate that out of a total of 20 SV40- and 21 BKV-specific peptides, there were six or seven pairs of similar peptides on the basis of coelution from Chromobead ionexchange columns. MATERIALS AND METHODS Labeling and cell extraction conditions. Primary cultures of African green monkey kidney cells in-

2 320 SIMMONS, TAKEMOTO, AND MARTIN fected with SV40 (500 PFU/cell) were labeled with 100 /LCi of L-[methyl-3H]methionine per ml (specific activity, 7 Ci/mmol) between 22 and 23.5 h postinfection in methionine-free minimal essential medium containing 2% dialyzed calf serum and 10-4 M L-1-tosylamide-2-phenyl-ethylchloromethyl ketone (TPCK) added to prevent proteolytic degradation of T Ag. Secondary cultures of human embryonic kidney cells infected with BKV (2 to 5 PFU/cell) were labeled with 50 jici of L-[35S]methionine per ml (specific activity 327 Ci/mmol) between 85 and 86.5 h postinfection. Ten minutes before cell harvest, TPCK was added to a final concentration of 10-4 M. BKV-transformed hamster cells (14) growing in roller bottles and at 80% of confluency were labeled with 100,.tCi of L-[35S]methionine per ml for 1.5 h in minimal essential medium lacking unlabeled methionine and supplemented with 10% fetal calf serum. TPCK (10-4 M) was added 30 min before cell harvest. Labeled cells were washed twice with ice-cold 0.02 M Tris (ph 7.4) M Na2HPO M NaCl (Tris-buffered saline) and collected into the same buffer. The cells were pelleted at 1,000 x g for 10 min at 0C and resuspended in a small volume of Trisbuffered saline at ph 8.0 containing 15% glycerol, M dithiothreitol, and 250 ug of phenylmethylsulfonyl fluoride per ml. The material was sonically treated for two periods of 30 s each at 30% maximum output, using a Branson Sonifier. Nonidet P-40 and sodium deoxycholate were added to the sonic extracts to a final concentration of 0.5%. Preparation of Staphylococcus aureus. Heatkilled suspensions of protein A-bearing S. aureus (Cowan I strain, NCTC 8530) were prepared according to the method of Jonsson and Kronval (9). The suspensions were stored in small samples at -70 C. Before use, the bacteria were washed once in 0.02 M Tris (ph 8.0), M Na2HPO4, M NaCl, 15% glycerol, M dithiothreitol, 0.5% Nonidet P-40, and 0.5% sodium deoxycholate and resuspended in the same buffer to a final concentration of 10% (wt/ vol). Conditions for immunoprecipitation and gel electrophoresis. Cell extracts were clarified by spinning at 40,000 rpm for 40 min at 2 C in the Spinco SW 50.1 rotor. Supernatants were carefully removed and incubated for 1 h at 0 C in the presence of 20 1.l of hamster normal serum or anti-t serum (1:320 or 1:640 as assayed by immunofluorescence) per ml of extract. Washed protein A-bearing S. aureus was then added (0.2 ml of a 10% suspension per ml of cell extract) to bind immune complexes to the surface of the bacteria (9), and the material was incubated for an additional 30 min at 0 C. The bacteria were centrifuged at 2,000 x g for 10 min at 2 C and washed twice in ice-cold Tris-buffered saline, ph 8.0, containing 15% glycerol, M dithiothreitol, 0.5% Nonidet-40, and 0.5% sodium deoxycholate and twice in phosphate-buffered saline. The final bacterial pellets were resuspended in a small volume of electrophoresis sample buffer (0.075 M Tris-PO4, ph 8.6, 2% sodium dodecyl sulfate, 2% 2-mercaptoethanol, 0.002% bromophenol blue, and 15% glycerol) and incubated at 60 C for 5 min. After pelleting the J. VIROL. bacteria, the supernatants were carefully removed, heated to 100 C for 7 min, and subjected to electrophoresis through polyacrylamide slab gels (20% acrylamide-0.1% bisacrylamide in the separating gel and 5% acrylamide-0.12% bisacrylamide in the stacking gel) as described by Maurer and Allen (11) and modified by Tegtmeyer et al. (17). Electrophoresis was for 12 to 18 h at 12.5 ma. Preparation and chromatography of tryptic peptides. The methods used for elution of proteins from acrylamide gels, trypsin treatments, and subsequent analysis of peptides by ion-exchange chromatography were modified from those described by Fey and Hirt (5) and Vogt et al. (19). Bands of labeled T Ag proteins identified by autoradiography or fluorography (2) were cut out from acrylamide gels, and the proteins were eluted with 0.1 M (NH4)2CO3-0.1% sodium dodecyl sulfate, ph 8.6, by shaking at 37 C for 48 to 72 h. The eluates were clarified by centrifugation at 25,000 rpm in the Spinco SW 27.1 rotor for 40 min at 23 C. The solutions were lyophilized, and the material was resuspended in 2 ml of water. Radiolabeled protein was precipitated with 25% trichloroacetic acid in the presence of human serum albumin (50,ug/ml) at 0 C for 16 h. The precipitates were collected by centrifugation at 14,000 x g for 20 min, washed once in acetone and resuspended in 0.2 ml of 0.1 M NaOH. After a second trichloroacetic acid precipitation and acetone wash, the protein was washed once in diethyl ether and resuspended in 0.2 ml of 0.1 M NH40H. The protein samples were then precipitated with 25% trichloroacetic acid, washed once in acetone to quantitatively remove the sodium dodecyl sulfate, and resuspended in 0.1 ml of ice-cold, fresh performic acid prepared by incubating 1.9 ml of formic acid and 0.1 ml of 30% H202 for 1 h at 23 C. Oxidation of proteins by performic acid was carried out for 1 h at 0 C, and the reaction was stopped by the addition of 1 ml of water. The protein samples were lyophilized and resuspended in 1 ml of 0.05 M (NH4)2CO3, ph 8.6, and lyophilized a second time. Trypsin (TPCK treated; Worthington Biochemicals Corp.) was added at a final concentration of 310,ug/ ml to protein samples in 0.2 ml of 0.05 M (NH4)2CO3, ph 8.6, and incubated for 4 h at 37 C. The same amounts of trypsin were added, and the mixtures were incubated for an additional 4-h period at 37 C. The trypsin digestions were stopped with 1 ml of 0.01 M acetic acid, and the peptides were lyophilized twice from 0.01 M acetic acid and resuspended in 0.5 ml of water-acetic acid-formic acid-pyridine (2,354:1,264:350:32, ph 1.9). The peptide solutions were clarified by centrifugation at 8,000 x g for 10 min at 23 C and applied to a 40- by 0.8-cm column of P-type Chromobeads (Technicon Chemicals) equilibrated in the above solution at ph 1.9. Chromatography was carried out at 60 C, and the peptides were eluted with an exponentially increasing concentration of pyridine from 0.1 to 2 M. The pyridine gradient was approximately linear in ph from 1.9 to 4.5 and made by connecting three mixing chambers, the first two containing 210 ml of the solution at ph 1.9 and the third containing 210 ml of the solution at ph 4.5 (water-pyridine-acetic acid-formic acid,

3 VOL. 24, :158:140:39). Fractions (2.5 ml) were collected and evaporated to dryness in an 80'C oven. The peptides were resuspended in 0.4 ml of 0.01 M HCl and counted for radioactivity in 10 ml of counting cocktail (Toluene-Triton X-100-water-fluor, 279:- 150:50:21). RESULTS Chromatography of methionine-labeled tryptic peptides of SV40 and BKV T Ag's from lytically infected cells. In a subsequent manuscript (submitted for publication), we will report that the molecular weights of the largest forms of SV40 and BKV T Ag from extracts of lyrically infected cells are both approximately 97,000, as determined by acrylamide gel electrophoresis. Because these proteins have one or more antigenic determinants in common (15), we expected them to have some similar amino acid sequences, and possibly common tryptic peptides. To examine this possibility, [3H]- methionine-labeled SV40 T Ag and [35S]- methionine-labeled BKV T Ag were prepared by immunoprecipitation from extracts of productively infected cells, using the homologous hamster anti-t serum. Neither of these T Ag proteins was precipitated in the presence of normal hamster serum. The incorporation of methionine radioactivity into these proteins was optimized by labeling infected cells at the times of maximal T Ag synthesis (20 to 25 h and 75 to 85 h for infections with SV40 and BKV, respectively) (data not shown) and by adding the chymotrypsin inhibitor TPCK to the cells either at the beginning of the labeling period 14 r_112a C 0 ax it ll 6~8 Al~ 6II 4 ~l ~ '~ Hi Alt PEPTIDES OF SV40 AND BKV TUMOR ANTIGENS 321 (for SV40 infections) or near the end of the labeling period (for BKV infections). The precipitated SV40 and BKV T Ag proteins were preparatively fractionated on acrylamide gels, and those corresponding to a molecular weight of 97,000 were eluted from the gels. The proteins were digested with trypsin as described in Materials and Methods, and their methioninelabeled peptides were compared by ion-exchange chromatography on columns of chromobeads. Figure 1 shows a characteristic 20-peak elution profile (each peptide is numbered) for the methionine tryptic peptides of SV40 T Ag (dashed lines). The solid lines in Fig. 1 indicate the elution profile of the BKV-specific methionine tryptic peptides and its characteristic 21 peaks. Both of these tryptic peptide profiles were reproducible from experiment to experiment and consisted of major and minor peaks. Figure 1 shows that at least seven of the 3Hlabeled SV40-specific peptides coeluted with 35S-labeled BKV-specific tryptic peptides. These pairs of coeluting peptides chromatographed at the positions labeled 3, 7, 10, 13, 14, 17, and 19 (Fig. 1) and represented 25 to 30% of the total methionine radioactivity present in peak fractions only. Other SV40-specific peptides eluted within a single fraction of a BKVspecific peptide (e.g., peptides labeled 2 and 15) and were not included in such a calculation. Since on a totally random basis one or possibly two of the SV40- and BKV-specific peptides which coeluted from the column could have the same ionic charge but different amino acid sequences, these seven pairs of coeluting peptides,,,,, I,,,, I I 25 e, :,, A~~~~~~~~~~~~~~~~~~,A0 150 FRACTION NUMBER FIG. 1. Chromobead ion-exchange chromatography of methionine-labeled tryptic peptides of SV40 and BKV T Ag's from lytically infected cells. [3H]methionine-labeled SV40 (22 to 23.5 h postinfection) and [35S]methionine-labeled BKV (85 to 86.5 h postinfection) T Ag's were prepared by immunoprecipitation from extracts of lytically infected cells as described in the text. The bands corresponding to T Ag proteins with molecular weights of97,000 were excised from preparative acrylamide gels. The proteins were elated from the gels and treated with trypsin. SV40- and BKV-specific T Ag peptides were analyzed together by ion-exchange chromatography on P-type Chromobeads. Peptides were elated with a linear ph gradient from 1.9 to 4.5 consisting ofexponentially increasing concentrations ofpyridine, and the fractions were evaporated to dryness and counted for 3H and 35S radioactivities. Symbols: ( ) 35S-labeled tryptic peptides of BKV T Ag from infected cells; (-----) 3H-labeled tryptic peptides of SV40 T Ag from infected cells. 20 N Cn 15 9 X

4 322 SIMMONS, TAKEMOTO, AND MARTIN represent a maximum estimate for the number of identical methionine tryptic peptides between the T Ag's of these two papovaviruses. Immunoprecipitation of BKV T Ag from transformed cell lysates. A recent report (3) suggested that T Ag from SV40-transformed cells is larger than the T Ag from SV40-infected monkey cells. In our hands, however, these two proteins are indistinguishable in molecular weight (97,000; submitted for publication). To compare the sizes of BKV T Ag's from infected and transformed cells, T Ag was immunoprecipitated from labeled extracts of BKV-transformed hamster cells (Fig. 2). A labeled protein significantly larger than the 97K form of SV40 T Ag (Fig. 2a) was specifically immunoprecipitated from the extracts (Fig. 2c). The size of this protein was estimated to be 113,000 daltons in our acrylamide gels, using as molecular weight standards ovalbumin (45,000), bovine serum 97 K a b C c K FIG. 2. Immunoprecipitation of T Ag from BKVtransformed hamster cells. BKV-transformed cells were labeled with [35S]methionine for 1.5 h when the cells were at approximately 80% of confluence. Total cell extracts were prepared and incubated in the presence of anti-bkv T serum or normal hamster serum as described in the text. Labeled precipitated proteins were subjected to electrophoresis on acrylamide gels and detected in the gel by autoradiography. (a) SV40 97K T Ag marker prepared by immunoprecipitation from extracts of[35s]methionine-labeled monkey cells infected with SV40. (b) Labeled proteins precipitated from extracts of BKV-transformed cells, using normal hamster serum and (c) anti-bkv T serum. albumin (68,000), and phosphorylase A (94,000) (Fig. 3). The immunoreactive 113K protein was not precipitated in the control reaction with normal serum (Fig. 2b). Labeled proteins with molecular weights of 103,000 and 94,000, as well as smaller-sized proteins, were also specifically immunoprecipitated with anti-$kv T serum (see Fig. 4). In this experiment, larger amounts of labeled BKV T Ag, immunoprecipitated from transformed hamster cells, were subjected to acrylamide gel electrophoresis. In other preparations of T Ag from BKV-transformed cells, a 97K form of T Ag was also observed (data not shown). Chromatography of methionine-labeled tryptic peptides of BKV T Ag isolated from transformed cells. In view of the differences in molecular weight of the BKV T Ag's from transformed and lyrically infected cells, methionine-labeled tryptic peptides of BKV T Ag from transformed cells were compared with the peptides of SV40 T Ag from lyrically infected cells. [3H]methionine-labeled SV40 T Ag (97,000 daltons) and [35S]methionine-labeled BKV T Ag (113,000 daltons) were prepared by immunoprecipitation and acrylamide gel electrophore- 0 -j 0 2 J. VIROL DISTANCE MIGRATED (cm) FIG. 3. Molecular weight estimate of BKV T Ag from transformed cells. BKV T Ag prepared by immunoprecipitation from transformed cells using anti- BKV T serum was subjected to acrylamide gel electrophoresis in the presence of unlabeled ovalbumin (45,000 daltons), bovine serum albumin (68,000 daltons), and phosphorylase A (94,000 daltons) as protein markers. Unlabeled proteins were detected by staining the gel with Coomassie brilliant blue, and labeled TAg was detected by exposing the dried gel to X-ray film (autoradiography). The distance the marker proteins migrated into the gel was plotted as a function ofthe logarithm of their molecular weight. The arrow indicates the position in the gel of the largest T Ag protein immunoprecipitated from BKVtransformed cells. From the linear relationship obtained, the molecular weight of BKV T Ag was estimated to be 113,000.

5 VOL. 24, 1977 V- 42-"2iw", 1; -1, -1.w.- -.-I PEPTIDES OF SV40 AND BKV TUMOR ANTIGENS 323 Ag shown in Fig. 1 was absent or present in smaller amounts in the tryptic peptides of T Ag from BKV-transformed cells (Fig. 5). This result did not significantly alter the proportion of methionine counts in coeluting SV40- and BKV-specific peptides oil dii -II... t. I... -i 113 K 103 K 94 K FIG. 4. Detection of 103K and 94K T Ag proteins from BKV-transformed cells. A sample of T Ag immunoprecipitated from [35S]methionine-labeled BKV-transformed cells was subjected to electrophoresis for a longer period oftime than was done in Fig. 2, and the dried gel was exposed to X-ray film for a period of time necessary to visualize minor protein bands. In this autoradiogram, bands of BKV T Ag proteins with molecular weights of 103,000 and 94,000 as well as smaller T Ag proteins were detected. sis. The proteins were digested with trypsin, and the resulting peptides were subjected to ion-exchange chromatography on columns of Chromobeads (Fig. 5). The elution pattern of the BKV-specific peptides from transformed cell T Ag (solid lines) was remarkably similar to the peptide pattern of the corresponding T Ag protein from lyrically infected cells (cf. solid lines of Fig. 1). All but one of the SV40- and BKV-specific peptides that coeluted when BKV T Ag was prepared from productively infected cells also eluted together when the T Ag was obtained from BKV-transformed cells (Fig. 5, peptides 3, 10, 13, 14, 17, and 19). The BKVspecific peptide eluting with peptide 7 of SV40 T DISCUSSION In this report, we have shown that BKV T Ag from transformed hamster cells was significantly larger (113,000 daltons) than the corresponding T Ag isolated from lyrically infected human cells (97,000 daltons). In another line of BKV-transformed hamster cells, the T Ag isolated was also approximately 113,000 daltons in size. On the other hand, the largest species of T Ag isolated from two different lines of SV40- transformed cells (11A8 hamster cells and SV80 human cells) had the same molecular size as SV40 T Ag from lyrically infected cells (97,000 daltons; submitted for publication). This result is in agreement with those of Tegtmeyer et al. (16) and Ito et al. (8), who have found no differences in the size of SV40 or polyoma T Ag isolated from lyrically infected and transformed cells. Carroll and Smith (3) and Ahmad-Zadeh et al. (1) have reported, on the other hand, that the molecular weight of SV40 T Ag is smaller in lyrically infected cells than in acutely infected nonpermissive or transformed cells. At the present time, we do not have a reasonable explanation for the difference in size of the BKV T Ag's from transformed cells (113,000 daltons) and infected human cells (97,000 daltons) and why this molecular weight difference was observed for BK T Ag's and not for SV40 T Ag's (16; our observations) or polyoma T Ag's (8). In addition to the 113K T Ag protein, smaller species of T Ag were isolated from BKV-transformed hamster cells, including three proteins with molecular weights of 103,000, 97,000, and 94,000. In some experiments, the 103K or 97K protein was the predominant species of T Ag isolated from BKV-transformed cells. The various molecular weight classes of BKV T Ag either may represent degradation products of the 113K form or they may each be gene products of different viral DNA molecules integrated within the host chromosome. We believe that the former explanation is more likely since the pattern of immunoprecipitated T Ag proteins from the same BKV-transformed cells was somewhat variable from experiment to experiment and since the protease inhibitor TPCK inhibited the formation of the lowermolecular-weight T Ag proteins (data not shown). The relationship between the SV40- and

6 324 SIMMONS, TAKEMOTO, AND MARTIN J. VIROL. 14 :12 20 bi. I}tI 4 I ~~~~~~~~~~~~~~~0 b ' x, 8:-n FRACTION NUMBER FIG. 5. Ion-exchange chromatography of methionine-labeled tryptic peptides of SV4O T Ag from lytically infected cells and BKV T Ag from transformed cells. [3H]methionine-labeled SV4O T Ag from infected monkey cells and [35S]methionine-labeled BKV T Ag from transformed cells were prepared by immunoprecipitation with anti-t serum as described in the text. The bands corresponding to T Ag proteins of 97K (SV4O) and 113K (BKV) daltons, respectively, were cut out of acrylamide gels, and the proteins were eluted from the gels and treated with trypsin. 3H- and 35S-labeled peptides were analyzed together by ion-exchange chromatography. Symbols: ( ) 35S-labeled tryptic peptides of BKV T Ag from transformed cells; (---- 3H-labeled tryptic peptides of SV4O T Ag from lytically infected cells. BKV-specific T Ag's was studied by comparing their methionine-containing tryptic peptides by ion-exchange chromatography. A quantitative estimate for the degree of homology between the T Ag's of BKV and SV40 was not entirely possible since only methionine-containing peptides were examined. Figures 1 and 5 show that although six or seven of the SV40- and BKVspecific peptides had very similar or identical ionic charges, the majority were clearly different from one another. Further experiments are necessary to show whether the coeluting pairs of peptides with similar ionic charges also had similar molecular weights. Although we identified 20 SV40- and 21 BKV-specific tryptic peptides, several appeared as minor labeled peaks in the chromatograms, and, consequently, it was not known whether each peptide peak represented a separate and unique peptide of each T Ag protein or whether some of the minor peptides were products of a contaminating protease activity in the TPCK-trypsin. Nevertheless, the peptide profiles of the SV40 and BKV T Ag proteins were quite reproducible from experiment to experiment and were essentially equivalent to a peptide "fingerprint" of each T Ag molecule. Our interpretation of only partial homology between the amino acid sequences of the SV40 and BKV T Ag's is compatible with results of T. Kelley and co-workers (personal communications) that extensive homology was found between the early regions of the genomes of SV40 and BKV when the hybridization of the two DNAs was performed under relatively nonstringent conditions. We must point out that when hybridization of SV40 and BKV DNAs is done under standard reaction conditions, little homology can be detected in the early regions of the respective viral genomes (10; P. Howley, unpublished data). Generally, whenever two proteins have similar amino acid sequences, the DNA molecules that encode them have a much smaller degree of homology when tested by standard DNA-DNA hybridization conditions. For example, rabbit and duck hemoglobins share 70% of their amino acid sequences (4), yet the hemoglobin complementary DNAs hybridize with one another to an extent of only 5% (18). By comparing Fig. 1 and 5, it was apparent that the methionine peptides of T Ag from BKV-infected human cells (Fig. 1) were very similar to those of BKV T Ag from transformed cells (Fig. 5). The major peptide peaks in the profile shown in Fig. 1 were also present in the pattern shown in Fig. 5. The differences observed between the two BKV T Ag peptide patterns were relatively small and involved only what we considered minor peptides. For example, the BKV-specific peptides eluting at position 7 and just after position 15 (fraction 178) in Fig. 1 were either absent or found in reduced amounts at the same positions in the peptide profile of T Ag from BKV-transformed cells (Fig. 5). A peptide in T Ag from transformed cells that eluted just before peptide 17 (fraction 213, Fig. 5) was absent from BKV T Ag of lyrically infected cells (Fig. 1). The total difference between the methionine Tryptic peptides of BKV T Ag's from these cells appeared

7 VOL. 24, 1977 to be too small to account for the difference in molecular weight of these proteins (113,000 versus 97,000). It is unlikely that this size difference is due to macromolecules other than peptides, since T Ag from BKV-transformed cells does not appear to contain carbohydrate or nucleic acid moieties (unpublished observations). One possible explanation is that the extra amino acid sequences of T Ag from BKV-transformed cells (equivalent to 16,000 daltons) lack methionine residues. To test this possibility, we are in the process of analyzing all of the tryptic peptides in the BKV T Ag's from lyrically infected and transformed cells by labeling T Ag with a mixture of amino acids. PEPTIDES OF SV40 AND BKV TUMOR ANTIGENS 325 LITERATURE CITED 1. Ahmad-Zadeh, C., B. Allet, J. Greenblatt, and R. Weil Two forms of simian-virus-40-specific T-antigen in abortive and lytic infection. Proc. Natl. Acad. Sci. U.S.A. 73: Bonner, W. M., and R. A. Laskey A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46: Carroll, R. B., and A. E. Smith Monomer molecular weight of T antigen from simian virus 40-infected and transformed cells. Proc. Natl. Acad. Sci. U.S.A. 73: Dayhoff, M. 0. (ed.) Atlas of protein sequence and structure, vol. 5, D53-D54. National Biomedical Research Foundation, Washington, D.C. 5. Fey, G., and B. Hirt Fingerprints of polyoma virus proteins and mouse histones. Cold Spring Harbor Symp. Quant. Biol. 39: Gardner, S. D., A. M. Field, D. V. Coleman, and B. Hulme New human papovavirus (B.K.) isolated from urine after renal transplantation. Lancet i: Howley, P. M., M. F. Mullarkey, K. K. Takemoto, and M. A. Martin Characterization of human papovavirus BK DNA. J. Virol. 15: Ito, Y., N. Spurr, and R. Dulbecco Characterization of polyoma virus T antigen. Proc. Natl. Acad. Sci. U.S.A. 74: Jonsson, S., and G. Kronvall The use of protein A-containing Staphylococcus aureus as a solid phase anti-ig G reagent in radioimmunoassays as exemplified in the quantitation of a-fetoprotein in normal human adult serum. Eur. J. Immunol. 4: Khoury, G., P. M. Howley, C. Garon, M. F. Mullarkey, K. K. Takemoto, and M. A. Martin Homology and relationship between the genomes of papovaviruses, B. K. virus and simian virus 40. Proc. Natl. Acad. Sci. U.S.A. 72: Maurer, H. R., and R. C. Allen Useful buffer and gel systems for acrylamide gel electrophoresis. Z. Klin. Chem. Klin. Biochem. 10: Mullarkey, M. F., J. F. Hruska, and K. K. Takemoto Comparison of two human papovaviruses with simian virus 40 by structural protein and antigenic analysis. J. Virol. 13: Shah, K. V., H. L. Ozer, H. N. Ghazey, and T. J. Kelley, Jr Common structural antigen of papovaviruses of the simian virus 40-polyoma subgroup. J. Virol. 21: Takemoto, K. K., and M. A. Martin Transformation of hamster kidney cells by BK papovavirus DNA. J. Virol. 17: Takemoto, K. K., and M. F. Mullarkey Human papovavirus, BK strain: biological studies including antigenic relationship to simian virus 40. J. Virol. 12: Tegtmeyer, P., K. Rundell, and J. K. Collins Modification of simian virus 40 protein A. J. Virol. 21: Tegtmeyer, P., M. Schwartz, J. K. Collins, and K. Rundell Regulation of tumor antigen synthesis by simian virus gene A. J. Virol. 16: Verna, I. M., G. F. Temple, H. Fan, and D. Baltimore In vitro synthesis of DNA complementary to rabbit reticulocyte 10S RNA. Nature (London) New Biol. 235: Vogt, V. M., R. Eisenman, and H. Diggelmann Generation of avian myeloblastosis virus structural proteins to proteolytic cleavage of precursor polypeptide. J. Mol. Biol. 96: Wright, P. J., and G. DiMayorca Virion polypeptide composition of the human papovavirus BK: comparison with simian virus 40 and polyoma virus. J. Virol. 15: