ED HARLOW,* PETER WHYTE, B. ROBERT FRANZA, JR., AND CAROL SCHLEY. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724.
MOLECULAR AND CELLULAR BIOLOGY, May 1986, p. 1579-1589 0270-7306/86/051579-11$02.00/0 Copyright © 1986, American Society forNficrobiology
Vol. 6. No. S
Association of Adenovirus Early-Region 1A Proteins with Cellular Polypeptides ED HARLOW,* PETER WHYTE, B. ROBERT FRANZA, JR.,
AND
CAROL SCHLEY
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724 Received 25 October 1985/Accepted 3 February 1986
Extracts from adenovirus-transformed human 293 cells were immunoprecipitated with monoclonal antibodies specific for the early-region 1A (ElA) proteins. In addition to the ElA polypeptides, these antibodies precipitated a series of proteins with relative molecular weights of 28,000, 40,000, 50,000, 60,000, 80,000, 90,000, 110,000, 130,000, and 300,000. The two most abundant of these polypeptides are the 110,000molecular-weight protein (110K protein) and 300K protein. Three experimental approaches have suggested that the 110K and 300K polypeptides are precipitated because they form stable complexes with the ElA proteins. The 110K and 300K polypeptides do not share epitopes with the ElA proteins, they copurify with a subset of the ElA proteins, and they bind to the ElA proteins following mixing in vitro. The 110K and 300K polypeptides are not adenoviral proteins, but are encoded by cellular DNA. Both the 12S and the 13S ElA proteins bind to the 110K and 300K species, and these complexes are found in adenovirus-transformed and -infected ceUls.
One of the best-studied models of eucaryotic gene regulation is the activation of adenovirus genes by the protein products of the viral early-region 1A (ElA). The ElA mRNAs are the first viral transcripts to be synthesized following infection of host cells (36, 42). Shortly after the appearance of the ElA polypeptides, the E1B, E2, E3, E4, and Li genes are transcribed (42, 50). Studies with adenoviruses that carry mutations in the ElA region have shown that one of the functions of the ElA proteins is to activate transcription from these viral genes (1, 27, 40, 45). As well as regulating other early viral transcription units, the ElA proteins can activate transcription from a number of celular genes (16, 20, 30, 41, 52, 53, 55). The ElA proteins also can act to decrease the level of transcription of certain cellular or viral genes (4, 56). In addition to their role in transcriptional control, several studies have shown that the ElA proteins together with the E1B polypeptides are essential for adenovirus transformation (13, 14, 17, 19, 28). While the roles of the ElA and E1B proteins in transformation are not known, one of the functions of ElA appears to be extending the growth potential of normal cells to overcome senescence (25, 47). Cell lines that are established by ElA may be transformed by introducing any of a number of other oncogenes including E1B, polyoma middle-T, or an activated ras gene into the ElA-containing cells (47). The ElA gene may also be replaced in these assays by the myc, p53, or polyoma large-T gene (10, 26, 34, 43, 47). The studies of the collaboration between different classes of genes suggest that there may be functional similarities between the proteins of each group, but little is known about the intrinsic activities of any of the nuclear oncogenes that comprise the myc, p53, polyoma large-T, and ElA set. In both transformed and infected cells, the primary transcript from the ElA gene is differentially spliced to produce a series of related mRNAs (2, 7, 32). The two most abundant of these RNAs are the 12S and 13S mRNAs. The 13S mRNA codes for a protein of 289 amino acids, while the 12S *
polypeptide is 243 amino acids (44). Because of the structure of the splice sites for the 12S and 13S mRNAs, the protein products of these mRNAs have identical amino and carboxy termini, but the 13S protein is larger by an internal addition of 46 amino acids. Both of these proteins migrate more slowly on polyacrylamide gels than would be expected for their predicted molecular weights. The 12S proteins have relative molecular weights of approximately 40,000 to 50,000 and the 13S proteins are approximately 45,000 to 55,000 in molecular weight (11, 21, 24, 45, 46, 48, 51, 58). When these proteins are analyzed by two-dimensional isoelectric focusing polyacrylamide gel electrophoresis, it can be shown that the products of the 12S and 13S mRNAs are heterogeneous in size and charge (23). These multiple polypeptides species appear to be generated by post-translational modification. Although several functions of the ElA proteins are known, the mechanisms by which the ElA polypeptides act are unknown. We have used a set of monoclonal antibodies specific for the ElA proteins to begin studies of the ElA proteins themselves and report here that both the 12S and the 13S proteins form stable protein complexes with cellular polypeptides. MATERIALS AND METHODS Cells and viruses. All cells were grown in Dulbecco modified Eagle media supplemented with 10%o fetal bovine serum. HeLa and 293 cells were from the Cold Spring Harbor Laboratory cell culture facility and were supplied by B. Ahrens. Wild-type adenovirus type 5 was grown and its titer was determined on HeLa cells. The recombinant adenoviruses Ad5.12S and Ad5.13S were kindly supplied by T. Grodzicker and were grown on 293 cells. In these viruses the wild-type ElA region has been replaced with a cDNA copy of either the 12S or the 13S mRNA (39a). Immunoprecipitation and electrophoresis. Cultures were radiolabeled with approximately 500 ,uCi of [35S]methionine per 2 x 106 cells for 4 h in Dulbecco modified Eagle media without methionine. Cell monolayers (approximately 2 x 106 cells) were lysed with 1.0 ml of either ElA (250 mM NaCl, 0.1% Nonidet P-40 [NP40], 50 mM HEPES [N-2-
Corresponding author. 1579
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HARLOW ET AL.
hydroxyethylpiperaxine-N'-2-ethanesulfonic acid], pH 7.0) or RIPA (150 mM NaCl, 1.0% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], 50 mM Tris, pH 8.0) lysis buffer for 30 min on ice. The lysates were cleared of nuclei and debris by centrifugation for 2 min. The supernatants were incubated with 50 ,u l of normal rabbit sera for 1 h on ice. These lysates were then used to suspend a 50-pl pellet of prewashed Staphylococcus aureus Cowan I (31). After 30 min on ice the lysates were centrifuged for 2 min, and the supernatants were used to suspend a second 50-S1. S. aureus pellet. Following the 30-min incubation the extracts were centrifuged for 10 min, and the supernatants were collected for immunoprecipitation. Samples of these lysates were incubated with 50 ,ul of tissue culture supernatant from the appropriate hybridoma for 1 h on ice (PAb 416, described in reference 22; anti-ElA described in reference 23). Protein A-Sepharose (Pharmacia Fine Chemicals) (100 pl of a 3% [dry wt/vol] suspension) was added and incubated at 4°C for 1 h with rocking. The protein A-Sepharose beads were washed three times, and the proteins bound to the beads were separated by electrophoresis on 8 or 10% SDSpolyacrylamide gels (33). Depending on which lysis buffer was used to prepare the detergent extracts, either the ElA or the RIPA buffer was used to rehydrate the protein ASepharose beads and to wash the immune complexes. All gels were prepared for fluorography as described by Bonner and Laskey (3). Samples for two-dimensional isoelectric focusing were prepared as described for the immunoprecipitation except that, following the third wash, the immune complexes were solubilized in 0.3% SDS-9.95 M urea-4% NP40-2% ampholytes (pH 3.5 to 10)-100 mM dithiothreitol. The isoelectric focusing and SDS-polyacrylamide electrophoresis were conducted as described by Garrels (15). Immunoblots. Extracts of 293 cells were prepared in ElA lysis buffer as described above. Samples containing 150 p,g of total protein were run in each lane of a 10% SDSpolyacrylamide gel. After electrophoresis the proteins were transferred to nitrocellulose by electroblotting and prepared for immunoanalysis as described by Burnette (6). Strips from one lane of the gel were incubated with 0.5 ml of the appropriate hybridoma tissue culture supernatant for 30 min at 20°C, washed briefly in NET/GEL buffer (150 mM NaCl, 5 mM EDTA, 0.25% gelatin, 0.05% NP40, 50 mM Tris, pH 7.5), and probed with 1 pCi of 125I-labeled rabbit anti-mouse immunoglobulin (New England Nuclear Corp.) per strip. After 30 min each strip was washed extensively in NET/GEL and autoradiographed. Radioimmunoassays. A fusion protein between the trpE gene of Escherichia coli and the ElA 13S cDNA was purified and bound to nitrocellulose as described previously (23). One-millimeter disks were cut with a paper punch and incubated with 2.0 ml of hybridoma tissue culture supernatant that contained approximately 50,000 cpm of radiolabeled monoclonal antibody. The antibodies were labeled with 1251 in vitro as described before (54) or were prepared by in vivo synthesis. The in vivo labeled antibodies were isolated by affinity chromatography on protein A-Sepharose (12) from tissue culture supernatants of hybridoma cells that were grown overnight in Dulbecco modified Eagle media without methionine but containing 2% fetal bovine serum and [35S]methionine. Sucrose gradient centrifugation. Radiolabeled 293 cells were extracted with ElA lysis buffer, and samples of these lysates were layered onto 5 to 20%o sucrose gradients. All solutions were prepared in ElA lysis buffer. Sedimentation
MOL. CELL. BIOL.
was for 18 h at 200,000 x g. The gradients were fractionated, and each fraction was prepared for immunoprecipitation as described above.
RESULTS Monoclonal antibodies specific for ElA precipitate additional polypeptides. To analyze the ElA polypeptides found in the adenovirus 5-transformed human cell line 293 (18), monoclonal antibodies specific for the ElA proteins (23) were used to immunoprecipitate proteins from detergent extracts of [35S]methionine-labeled cells (Fig. 1). The ElA proteins from cells infected with wild-type adenovirus 5 run as a series of bands with relative molecular weights of approximately 40,000 to 50,000. A similar series of ElA polypeptides was specifically precipitated from the 293 cells. Surprisingly, in addition to the ElA products, a number of other polypeptides from 293 cells were precipitated by the anti-ElA antibodies. These proteins have relative molecular weights of approximately 60,000, 80,000, 90,000, 110,000, 130,000 and 300,000. The 100,000-molecular-weight protein (110K protein) and 300K protein often appear as single bands on 10% and higher percentage polyacrylamide gels; however, when these polypeptides were separated on 8% polyacrylamide gels (Fig. 1), both the 110K and the 300K polypeptides were resolved into several bands. Further analysis of the 110K proteins has shown that the two species
HeLa
293 I
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130K J IOK - 90K - 80K -
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30KFIG. 1. Immunoprecipitation of ElA polypeptides from human cells. HeLa or 293 cells were labeled with [35S]methionine, and detergent lysates from these cells were immunoprecipitated with PAb416, a monoclonal antibody specific for the SV40 large-T antigen, or a mixture of the Ml, M2, M37, and M73 monoclonal antibodies specific for the ElA proteins. The immune complexes were collected on protein A-Sepharose, washed, and separated on an 8% SDS-polyacrylamide gel.
VOL. 6, 1986
with relative molecular weights of 105,000 and 110,000 were closely related (see below). We do not know if the multiple 300K species are related. During the course of these experiments, the optimal conditions for the extraction of the ElA, 60K, 80K, 90K, 110K, 130K, and 300K proteins were determined. Our original experiments used RIPA buffer, while in later experiments cells were extracted with ElA lysis buffer. Notably, the 300K polypeptides were easier to detect when the NaCl concentration was kept above 200 mM. If 293 cells were extracted in buffers with lower concentrations of salt, the amount of the 300K protein, as judged from fluorographs of comparable amounts of starting material, was reduced. This dependence on relatively higher salt concentrations than are normally used to extract nuclear proteins such as simian virus 40 (SV40) large-T antigen was also required in the wash buffers. If the immune complexes were washed in low salt, the 300K proteins were removed from the precipitates. In addition, the presence of 80K polypeptide was sensitive to an ionic detergent concentration in excess of 0.1% for SDS or sodium deoxycholate. Nonionic detergents such as NP40 were shown to be sufficient for efficient extraction when used at concentrations as low as 0.1%. Changes in the pH of the lysis buffer did not drastically alter the levels of the ElA, 60K, 80K, 90K, 110K, 130K, and 300K proteins as long as the pH was between 6.5 and 8.0. We also assayed the effects of divalent cation concentrations on the extraction of these proteins and found that, as long as the NaCl concentrations were kept above 200 mM, the presence of divalent cations or chelating agents had little effect. Although the levels of the ElA, 60K, 80K, 90K, 110K, 130K, and 300K proteins immunoprecipitated from extracts prepared in the two lysis buffers did differ slightly, the conclusions based on these data were in agreement. The two most prominent of the 60K, 80K, 90K, 110K, 130K, and 300K polypeptides were the 110K and 300K species. This was true both for radiolabeled preparations (Fig. 1) and for silver-stained gels (P. Whyte, unpublished observation). The 60K polypeptide was also a relatively strong band, but this polypeptide migrated on onedimensional SDS-polyacrylamide gels just slightly more slowly than the higher-molecular-weight forms of ElA and was often difficult to distinguish from these ElA bands. The 80K and 130K proteins were present at relatively low levels and were often only visible on long exposures. The 90K polypeptide was the most difficult to detect. It was seen reproducibly on all immunoprecipitations in which the background was particularly clean; however, in many of the experiments presented here this polypeptide cannot be detected. Therefore, we have not included this protein in any of the following discussions. The presence of the 60K, 80K, 110K, 130K, and 300K polypeptides in immunoprecipitations with the anti-ElA monoclonal antibodies can be explained by several different mechanisms: (i) they may be nonspecifically trapped in the immune complexes formed by the antibodies and the ElA polypeptides, (ii) they may be recognized directly by one or more of the monoclonal antibodies and therefore share epitopes with the ElA proteins, or (iii) they may be precipitated because they are found in a stable complex with one or more of the ElA polypeptides. Because the 60K, 80K, 110K, 130K, and 300K polypeptides were not detected in the control lanes in this or any other of the immunoprecipitations, they are not likely to be nonspecific contaminants. Distinguishing between the last two possibilities is more difficult, and a series of experimental approaches were used
ElA-HOST PROTEIN COMPLEXES
1581
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to resolve these issues. We have compared the immunochemical properties of the ElA, 60K, 80K, 110K, 130K, and 300K proteins, checked for any physical evidence of complex formation by velocity gradient sedimentation and gel filtration chromatography, and analyzed the ability of these proteins to associate in vitro. In addition, we have performed experiments designed to determine whether these proteins were encoded by host or viral DNA. The 60K, 80K, 11OK, 130K, and 300K polypeptides do not share epitopes with the ElA proteins. To determine whether the anti-ElA monoclonal antibodies will bind directly to the 60K, 80K, 110K, 130K, and 300K polypeptides, three experimental approaches were used. Lysates from a number of human cell lines that do not express the ElA proteins were immunoprecipitated with the anti-ElA monoclonal antibodies. If the 60K, 80K, 110K, 130K, or 300K protein was precipitated from these lysates, it would provide strong evidence for shared epitopes between these polypeptides and the ElA proteins. Figure 1 shows that the 60K, 80K, 110K, 130K, and 300K polypeptides were not immunoprecipitated from the HeLa cell extracts even though we have been able to show that at least the 110K and 300K proteins were present in these lysates (see below). In addition to assaying human cell lines directly for the coprecipitating proteins, the immunochemical properties of these polypeptides were compared with the ElA proteins. Because all of these antigens were present in detergent lysates of 293 cells, we compared the ability of the mono-
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NATIVE 293
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14 K FIG. 3. Immunoprecipitation of native oir denatured proteins from 293 cells. 293 cells were labeled with [35S]methionine, and RIPA lysates were prepared. The extracts w ere divided, and onehalf was made 2% with SDS. The SDS-treated sample was boiled for 10 min and then diluted 20-fold with NET/G EL buffer. Aliquotes from both the untreated and the denatured sa were immunoprecipitated with PAb416, a monoclonal anti body specific for the SV40 large-T antigen, or the anti-ElA monoc immune complexes were collected on protein i washed and separated on a 10%o SDS-polyacrylamide gel. '
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clonal antibodies to bind directly to the 60K, 80K, 110K, 130K, 300K, and ElA polypeptides folkowing denaturation of the antigens and separation by SDS-p(olyacrylamide electrophoresis. All of the anti-ElA monoclo onal antibodies used in these studies were raised against a denLatured, gel-purified antigen (23), and we have previously shown that these antibodies will bind directly to the ElA polypeptides after denaturation (C. Schley and E. Harlow, unpublished observation). Figure 2 shows the binding of tihe same mixture of monoclonal antibodies used in Fig 1 to the ElA polypeptides from the 293 cells after e lectrophoresis and transfer to nitrocellulose. As seen on thiss exposure and also on longer exposures (data not shown), tl here was no detectable binding of these antibodies to the 60K, 80K, 110K, 130K, or 300K polypeptides. Similar findings can be seen when the antigens present in the 293 lysates were denatured prior to innmunoprecipitation (Fig. 3). In this experiment, the proteins were denatured by boiling in 2% SDS, diluted with NET/GI EL buffer, and then immunoprecipitated with the individual nfnonoclonal antibodies. When samples of the native Ilysates were immunoprecipitated with the anti-ElA monioclonal antibodies, the 60K, 80K, 110K, 130K, and 300K polypeptides were precipitated (left panel). However, wher the antigens were denatured prior to immunoprecipitatic mn, only the ElA polypeptides were specifically precipitat(ed (right panel). The data shown in Fig. 3 also suggessted that individual monoclonal antibodies may precipitate dlifferent amounts of the individual, radiolabeled 60K, 80K, 11 [OK, 130K, or 300K
polypeptides. To test this more quantitatively, anti-ElA antibodies of either the G2a or the G2b subclass were assayed for their ability to immunoprecipitate proteins from lysates of 293 cells. Immunoglobulins from these subclasses were chosen because they have the highest affinities for protein A and can be used without the addition of an intermediate layer of rabbit anti-mouse antibodies. Figure 4 presents a series of competition radioimmunoassays that show that the Ml, M2, M37, and M73 antibodies recognized independent epitopes on ElA. The Ml, M37, and M73 antibodies bind to products of the 12S and 13S mRNA, while M2 is specific for the 13S polypeptide (23). The ElA molecules specifically immunoprecipitated from lysates of 293 cells are shown in Fig. 5. The identification of the 12S and 13S polypeptides was confirmed by separation on twodimensional isoelectric focusing polyacrylamide gels (data not shown). The M2, M37, and M73 antibodies precipitated the 300K species, while Ml, M2, and M73 precipitated the 110K species. The reasons for the immunological subsets are unknown at present. It is clear that monoclonal antibodies that bind to distinct epitopes on ElA (Fig. 4) will precipitate members of the 60K, 80K, 110K, 130K, and 300K polypeptide family. Possible explanations for the different patterns immunoprecipitated by the individual monoclonal antibodies are discussed below. Together these three experimental approaches suggested that the epitopes recognized by the monoclonal antibodies used in these studies were present only on the ElA polypeptides and not on the 60K, 80K, 110K, 130K, or 300K antigens. These proteins were not recognized by any of the anti-ElA antibodies when the proteins were denatured prior to exposure to the immunoglobulins even though the ElA polypeptides were recognized under similar conditions. None of the 60K, 80K, 110K, 130K, or 300K proteins were
immunoprecipitated from native lysates in which at least the 110K and 300K species were present. These proteins were only immunoprecipitated when the ElA proteins were also present in the cell lysates. The precipitation of the 60K, 80K, 110K, 130K, and 300K polypeptides in the presence of ElA was not limited to a single monoclonal antibody, but could be achieved by antibodies that bind to different epitopes on the ElA polypeptide. The 110K and 300K polypeptides copurify with a subset of the ElA proteins. To determine whether the ElA, 60K, 80K, 110K, 130K, and 300K polypeptides copurify, samples of detergent lysates from 293 cells were fractionated by sucrose gradient centrifugation. The gradients were fractionated, and samples of each fraction were immunoprecipitated with a control monoclonal antibody or with the anti-ElA monoclonal antibody M73 (Fig. 6). In this experiment approximately 60% of the radiolabeled ElA proteins sedimented at about 4S, while the remaining ElA sedimented across a broad peak at approximately 10S. The 10S ElA fractions also contained the 110K, 130K, and 300K polypeptides, although the peak of each of these proteins was found in different fractions. The 60K and 80K proteins were not detected in this experiment. If similar lysates were fractionated by gel filtration chromatography prior to immunoprecipitation, the pattern of separation was similar to that obtained by sucrose gradient sedimentation. We also noted that the patterns of the ElA proteins found in the 4S and lOS forms were different. Further studies are under way to characterize these polypeptides. In vitro complex formation between the ElA and the 110K and 300K host polypeptides. The experiments described above suggested that the 60K, 80K, 110K, 130K, and 300K
ElA-HOST PROTEIN COMPLEXES
VOL. 6, 1986 18,000
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FIG. 4. Competition radioimmunoassay for cross-blocking of the anti-ElA monoclonal antibodies. A purified preparation of a fusion protein between the trpE product of E. coli and the ElA 13S polypeptide was bound to nitrocellulose sheets. The remaining protein-binding sites on the nitrocellulose were blocked by incubating in a 3% bovine serum albumin solution. One-millimeter disks were cut from these sheets and incubated with a saturating amount of anti-ElA or control monoclonal antibody. Approximately 50,000 cpm of a high-specific-activity, radiolabeled anti-ElA monoclonal antibody was added, and the mixture was incubated overnight. The nitrocellulose disks were washed and dried, and the amount of radioactivity bound was determined. The results were plotted as an average of either duplicate or triplicate samples. The control antibody in all tests was PAb416, a monoclonal antibody specific for SV40 large-T antigen.
polypeptides were immunoprecipitated with anti-ElA monoclonal antibodies from lysates of 293 cells because they formed stable complexes with the ElA proteins. To test this suggestion directly, extracts from [35S]methionine-labeled HeLa cells were mixed with unlabeled lysates of 293 cells, and the proteins were immunoprecipitated with the anti-ElA monoclonal antibody M73 (Fig. 7). These in vitro experiments have shown that the 110K and 300K polypeptides were present in the HeLa cell lysates, but they were only seen when the HeLa cell lysates were mixed with the 293 extracts prior to immunoprecipitation. In addition, if the lysates from 293 cells were precleared of ElA proteins prior to mixing, the 110K and 300K proteins were not seen in the immunoprecipitations. In the experiment shown in Fig. 7 the 60K, 80K, and 130K polypeptides were not detected. In other experiments we have shown that the 60K and 130K polypeptides will bind to ElA in vitro (Whyte, unpublished observation), but they were difficult to detect because of the lower levels of these proteins and the increased background in the mixing experiments. This was consistent with our previous observations that the 110K and 300K polypeptides were the most abundant of the coprecipitated proteins. The three lines of evidence discussed above for the association of the ElA, 110K, and 300K polypeptides argue
very strongly that these polypeptides are physically associated when they are studied in vitro. However, these arguments leave open the possibility that the complexes actually form in vitro and may not exist within the cell. As with other host/virus complexes, it is difficult to address the issue of the significance of these complexes in vivo, particularly at this stage in the development of the experimental approach. However, two experiments suggest that the complexes seen in lysates of 293 cells do not form in vitro. First, formation of the complex in vitro requires a minimum of 30 min before the 110K and 300K polypeptides can be detected. There is no such lag for detection of the complex from 293 cell lysates; the complex from 293 cells can be detected immediately after lysis (data not shown). Second, when radiolabeled 293 cells are lysed in buffers that contain a 30-fold excess of cold 110K and 300K proteins from HeLa cells, the radiolabeled 110K and 300K polypeptides are not competed by the high levels of the cold proteins (Fig. 8). In this experiment the addition of a lysate containing an excess of cold protein is sufficient to block the nonspecific precipitation of the 70K heat shock proteins, indicating that the blocking is working, but does not lower the level of radiolabeled 110K or 300K polypeptides. These experiments do not show that the ElA-cellular protein complexes have any functional role,
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but they do suggest that it is unlikely that complex formation is an in vitro artifact. The presence of the 110K and 300K proteins in HeLa cell lysates (Fig. 7) demonstrated that these polypeptides were not encoded by the adenovirus DNA sequences. Because proteins with similar immunoprecipitation properties and relative molecular weights were found in a variety of cells (Fig. 9 and 10), they are most likely cellular proteins. The formation of virus-host protein complexes is not restricted to the ElA proteins from 293 cells. All of the experiments described above used ElA proteins found in 293 cells. These cells were originally isolated following calcium phosphate transfections and have been in tissue culture for a number of years (18). To ensure that the ElA proteins in these cells were not mutated or modified in such a way as to enable complex formation, similar precipitations were done on a number of other ElA-containing cells. A plasmid that carries the ElA gene along with a selectable neomycin gene was transfected into hamster BHK-21, rat 2, and mouse BALB/c 3T3 A31 cells. Colonies were selected on the basis of their resistance to G418, and these cells were assayed for the presence of the ElA-host protein complexes by precipitation with M73 antibodies. Figure 9 shows the results of these experiments. All cell lines we have tested expressed the ElA proteins, although at varying levels, and all were positive for the 110K and 300K polypeptides. To test whether the formation of these complexes were specific for adenovirus-transformed or immortalized cells, extracts from HeLa cells infected with adenovirus 5 were immunoprecipitated with the anti-ElA antibodies (Fig. 10). Extracts from cells infected with wild-type adenovirus 5 or recombinant adenoviruses in which the wild-type ElA sequences have been replaced with cDNA copies of the 12S or 13S mRNA all contain the ElA-host protein complexes.
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FIG. 6. Sucrose gradient centrifugation of extracts from 293 cells. 293 cells were labeled with [35S]methionine, and detergent lysates were prepared. The extracts were layered on 5 to 20% sucrose gradients and centrifuged for 18 h at 200,000 x g. The gradients were fractionated, and each fraction was immunoprecipitated with either PAb416, a monoclonal antibody specific for the SV40 large-T antigen, or the M73 anti-ElA monoclonal antibody. The immune complexes were collected on protein A-Sepharose, washed, and separated on 10%t SDS-polyacrylamide gels.
ElA-HOST PROTEIN COMPLEXES
VOL. 6, 1986
Both the 110K and the 300K polypeptides were precipitated from lysates of these infected cultures. These data also suggested that both the 12S and the 13S ElA proteins were fully capable of forming complexes with at least the 110K and 300K host polypeptides. Analysis of the ElA-host protein complexes. To further characterize the individual host proteins, these polypeptides were separated on two-dimensional isoelectric focusing polyacrylamide gels. Figure 11 shows a comparison of the 293 proteins precipitated by either M73 or control antibodies. Only the 60K, 80K, and 110K polypeptides have been identified by these types of analysis. We have compared the patterns of immunoprecipitations using the anti-ElA antibodies with immunoprecipitations that use antibodies specific for the human p53, myc, and fos nuclear oncogenes, topoisomerase I, and RNA polymerase II. At least at this level of resolution, there were no spots in common with these immunoprecipitations. Comparison with an existing protein data base (15) has shown that the 60K, 80K, or 110K species are not among the previously identified cytoskeletal
200K-
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1 4K FIG. 7. In vitro complex formation. HeLa cells were labeled with [35S]methionine, and detergent lysates were either precipitated with the M73 anti-ElA monoclonal antibody or mixed with detergent lysates of unlabeled 293 cells prior to immunoprecipitation. The lysates of 293 cells were also either used directly or precleared first with the M73 antibody. Lane 1, HeLa extracts precleared with M73 and then immunoprecipitated with M73; lane 2, HeLa extracts immunoprecipitated with M73; lane 3, HeLa cell extracts mixed with 293 extracts and then immunoprecipitated with M73; lane 4, HeLa cell extracts precleared with M73, mixed with 293 extracts, and then immunoprecipitated with M73; lane 5, HeLa cell extracts precleared with M73 and mixed with 293 extracts that had been precleared with M73, and the mixture was immunoprecipitated with M73; lane 6, HeLa cell extracts mixed with 293 extracts that had been precleared with M73, and the mixture was immunoprecipitated with M73. The immune complexes were collected on protein ASepharose, washed, and separated on a 10%o SDS-polyacrylamide
gel.
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-30K
FIG. 8. In vitro competition. Three parallel cultures of 293 cells were radiolabeled with [35S]methionine, and cells were lysed in ElA lysis buffer containing different amounts of cold HeLa cell lysates. Lane 1 contained 30 cell equivalents of cold HeLa cell lysate; lane 2, 1 cell equivalent of cold HeLa cell lysate; lane 3, standard lysis buffer. Following extraction, the samples were immunoprecipitated with the anti-ElA monoclonal antibody M73. The immune complexes were collected on protein A-Sepharose, washed, and separated on a 10% polyacrylamide gel.
or traditional heat shock proteins. These two-dimensional gels also have resolved the 105K and 110K species shown in Fig. 1. Because the pattern of these two polypeptides were similar, we assume that they are closely related. The two-dimensional analysis has also revealed a series of polypeptides that are specifically precipitated with the antiElA antibodies but were not seen on the one-dimensional gels. These proteins have relative molecular weights of approximately 28,000, 40,000 and 50,000 and were probably missed on one-dimensional gels because they have mobilities very similar to that of ElA. They are indicated on Fig. 11 with arrows. It should be noted that the molecular size designation of the complexed, cellular polypeptides was made prior to the analysis on two-dimensional gels. In particular, the 60K species actually migrated faster than vimentin (60K) and just slightly more slowly than the human p53 (55K) and appears to have a relative molecular weight of approximately 56,000 in this gel system. DISCUSSION One of the interesting aspects of the interactions between viruses and their host cells is the different mechanisms used by viruses to reprogram the endogenous cell metabolism to perform tasks that lead to enhanced viral growth. One mechanism that has been successfully utilized by both procaryotic and eucaryotic viruses is the formation of heteropolymers between viral regulatory proteins and host
1586
HARLOW ET AL.
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