JOURNAL OF VIROLOGY, Aug. 1979, p. 522-536 0022-538X/79/08-0522/15$02.00/0

Vol. 31, No. 2

Cell Surface Location of Simian Virus 40-Specific Proteins on HeLa Cells Infected with Adenovirus Type 2-Simian Virus 40 Hybrid Viruses Ad2+ND1 and Ad2+ND2 WOLFGANG DEPPERT* AND ROBERT PATES Max Planck Institute for Biophysical Chemistry, D-3400 Gottingen, Federal Republic of Germany Received for publication 29 January 1979

HeLa cells infected with the nondefective adenovirus type 2-simian virus 40 hybrid viruses Ad2+ ND1 or Ad2+ND2 were analyzed for cell surface location of the SV40-specific hybrid virus proteins by indirect immunofluorescence microscopy. Two different batches of sera from SV40 tumor-bearing hamsters, serum from SV40 tumor-bearing mice, or two different antisera prepared against purified sodium dodecyl sulfate-denatured SV40 T-antigen, respectively, were used. All sera were shown to exhibit comparable T- and U-antibody titers and to specifically immunoprecipitate the SV40-specific proteins from cell extracts of Ad2+ND2infected cells. Whereas analysis of living, hybrid virus-infected HeLa cells did not yield conclusive results, analysis of Formalin-fixed cells resulted in positive cell surface fluorescence with both Ad2+ND1- and Ad2+ND2-infected HeLa cells when antisera prepared against sodium dodecyl sulfate-denatured SV40 T-antigen were used as first antibody. In contrast, sera from SV40 tumor-bearing animals were not or only very weakly able to stain the surfaces of these cells. The fact that the tumor sera had comparable or even higher T- and U-antibody titers than the antisera against sodium dodecyl sulfate-denatured T-antigen but were not able to recognize SV40-specific proteins on the cell surface suggests that SV40 tumor-specific transplantation antigen may be an antigenic entity different from T- or U-antigen.

Cells infected with the nondefective adenovirus type 2-simian virus 40 (Ad2+SV40) hybrid viruses Ad2+ND1 and Ad2+ND2 express SV40 U-antigen and SV40 tumor-specific transplantation antigen (TSTA) but are T-antigen negative (13, 22, 23). The SV40-specific proteins encoded by these viruses and most probably carrying these antigenic determinants have been identified and shown to correspond to the COOHterminal part of SV40 T-antigen (10, 26). By biochemical cell fractionation procedures the SV40-specific proteins of Ad2+ND1 (SV40 28kilodalton [28K] protein) and AD2+ND2 (SV40 56K and 42K proteins) have been shown to accumulate in the nuclei (7,9) and plasma membranes (7, 9, 10, 13, 14) of infected cells. Immunological analysis demonstrated that the SV40specific proteins in the nuclei of Ad2+ND1- and AD2+ND2-infected cells carry the SV40 U-antigenic determinants (9). Recent experiments by Jay et al. (13) suggested that the SV40-specific 28K protein located in the plasma membranes of cells infected with Ad2+ND1 may exhibit SV40 TSTA activity. These authors showed that not only Ad2+ND1 hybrid virus but also isolated plasma

membranes of Ad2+NDl-infected cells were able to induce SV40 tumor transplantation resistance upon injection into mice and that this TSTA activity could be blocked by preincubation of the membranes of Ad2+ND1-infected cells with serum from SV40 tumor-bearing hamsters before injection. Cells infected with Ad2+ND1 or Ad2+ND2, therefore, may serve as model systems to investigate the expression of the SV40 TSTA. SV40 TSTA, by definition, is recognized by the host immune system. Therefore, a prerequisite for the SV40-specific hybrid virus proteins to exhibit TSTA activity by themselves would be that they are located on the surface of the infected cells. This, however, has not yet been demonstrated. Previous attempts in this and other laboratories (G. Walter, personal communication) have failed in unequivocally demonstrating SV40-specific cell surface fluorescence by immunofluorescence microscopy with sera from SV40 tumor-bearing hamsters. Possible explanations for the failure to detect the SV40specific hybrid virus proteins on the cell surface by immunofluorescence microscopy with sera from SV40 tumor-bearing animals may be: (i) 522

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TSTA may be a cellular immune function, which is not mediated by serologically detectable antibodies (2), and therefore these proteins, though located on the cell surface, may be sterically hindered from reacting with the antibody; or (ii) upon location on the cell surface, the structures of these proteins are altered in such a way that they are no longer recognized by the tumor serum. To investigate these possibilities, we prepared antisera against purified, sodium dodecyl sulfate (SDS)-denatured T-antigen (NaDodSO4-T). In contrast to tumor sera, which probably are directed against native T-antigen released from the nuclei of necrotic tumor material, antisera against NaDodSO4-T are more likely directed against the primary structure of T-antigen rather than against its native conformation. These sera might therefore be able to recognize SV40-specific proteins on the cell surface even if these proteins exhibit different structures from those in the cytoplasm. Here we report that antisera against NaDodSO4-T enable the visualization of SV40-specific proteins on the surfaces of Ad2+ND1- and Ad2+ND2-infected HeLa cells by immunofluorescence microscopy. Sera from SV40 tumorbearing animals, on the other hand, react only very weakly with the surfaces of such cells. Immunofluorescence analyses of two individual anti-NaDodSO4-T sera and of three different pools of sera from SV40 tumor-bearing animals revealed that there is no correlation between the T- and U-antibody titers of these sera and their abilities to stain the surfaces of hybrid virusinfected cells. SV40 TSTA, therefore, may be a separate class of antigenic determinants on the T-antigen polypeptide, which usually is not, or only weakly, recognized by sera from SV40 tumor-bearing animals. A preliminary account of this work has been published (8).

MATERIALS AND METHODS Viruses and cells. Seed stocks of the nondefective Ad2+SV40 hybrid viruses Ad2+ND1 and Ad2+ND2 were obtained from A. M. Lewis, Jr. Stocks were prepared in HeLa cells grown in monolayer culture in Dulbecco modified Eagle medium supplemented with 10% calf serum. The titer of the virus stocks used in these experiments was 109 PFU/ml. Stocks were assayed on HeLa monolayer cultures as described by Williams (33). Infection and labeling of cells. (i) Suspension cultures. HeLa S3 cells grown in suspension culture in minimal essential medium for suspension culture (F-13; Grand Island Biological Co., Grand Island, N.Y.) supplemented with 5% calf serum were infected with 1 ml of undiluted virus stock per 107 cells. After an adsorption period of 20 min the cells were diluted with medium to a final concentration of 2 x 105/ml. At 36

523

h postinfection (p.i.) the cells were harvested and suspended in Eagle medium minus methionine containing 50 liCi of [3S]methionine (Amersham/Buchler, specific activity, 800 mCi/mmol) per ml at a concentration of 1 ml of labeling medium per 5 x 106 cells. After labeling for 1 h the cells were washed twice with 30-mi amounts of phosphate-buffered saline (PBS) (32) and then fractionated into nuclei and a cytoplasmic extract (see below). (ii) Monolayer cultures. HeLa cells in monolayer cultures were removed from the culture dishes with trypsin-EDTA, washed once with growth medium, and infected with 1 ml of undiluted virus stock per 107 cells. After an adsorption period of 20 min the cells were diluted with growth medium and reseeded onto culture dishes. At the times indicated in the figure legends, the cells were labeled with 40,uCi of [3S]methionine per ml in Eagle minimal essential medium minus methionine. A 500-tLI amount of labeling medium was added per culture dish (3.5 cm in diameter). Labeling was for 2 h. The cells were then washed once with PBS and dissolved directly in 250 ld of electrophoresis sample buffer (0.0625 M Tris [pH 6.8], 3% SDS, 5% 2-mercaptoethanol). Preparation of NP40 cytoplasmic extract. A total of 5 x 107 to 10 x 107 HeLa S3 cells grown in spinner culture, infected with virus, and labeled as described above were suspended in 4 ml of reticulocyte standard buffer (11) (0.01 M Tris-hydrochloride, pH 7.2; 0.01 M NaCl; 1.5 mM MgCl2) containing 0.5% Nonidet P-40 (NP40; Shell Oil Co., London, England) and phenylmethyl sulfonyl fluoride at 1 mM concentration. After 20 min of swelling on ice the cells were Dounce homogenized until 80 to 90% of the cells were ruptured as judged by phase microscopy. The nuclei were pelleted by centrifugation at 800 x g for 5 min (IEC, rotor head 269, 2,000 rpm at 4°C). The supernatant was collected and termed NP40 cytoplasmic extract.

Immunoprecipitation of cytoplasmic extracts. Before immunoprecipitation, the salt concentration in the cytoplasmic extracts was raised to 200 mM by addition of NaCl. Aliquots of 300 p1 of NP40 cytoplasmic extract were incubated with 30,ul of serum for 12 h at 4°C. To reduce proteolysis, phenylmethyl sulfonyl fluoride was added at 1 mM concentration. Then 150 Fine Al of preswollen protein A-Sepharosewas(Pharmacia added per asChemicals, Inc., Piscataway, N.J.) say, and the reaction mixture was incubated for a further 90 min at 0°C. The protein A-Sepharose containing the immune complexes was removed by centrifugation. The supernatant was kept for SDS-polyacrylamide gel electrophoresis, and the protein ASepharose was washed extensively with isotonic Tris buffer, pH 7.2 (10), containing 0.2% NP40. The immune complexes were eluted from the protein A-Sepharose with 500 ,ul of 50 mM NH4HCO3, containing 1% SDS. The eluates were lyophilized, and samples of the NP40 cytoplasmic extracts (original reaction mixture), the supernatants, and the eluates were processed for SDS-polyacrylamide gel electrophoresis as described below. Preparation of samples for SDS-polyacrylamide gel electrophoresis. The lyophilized samples of the immunoprecipitation eluates (see above) were

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dissolved directly in electrophoresis sample buffer. Aliquots of the NP40 cytoplasmic extracts, the original immunoprecipitation reaction mixtures, and the immunoprecipitation supernatants (see above) were precipitated with ice-cold trichloroacetic acid (10% [wt/ vol] final concentration), washed twice with ethanol at -20°C, air dried, and dissolved in electrophoresis sample buffer. Solubilization of the samples was facilitated by brief sonication with a Branson Sonifier equipped with a microtip (position 4, three 10-s pulses with 30-s intervals) and by treatment of the samples for 3 min in a boiling water bath. The protein contents of the samples were determined with the Lowry method (24) and adjusted to approximately 15 ,ug/10

AlL

Polyacrylamide gel electrophoresis and fluorography. The polyacrylamide gel system of Laemmli (17) and Maizel (25) was used. A total of 15 ,tg of 10 was applied per slot. protein in approximately lO Electrophoresis was performed at a constant current of 12 mA with slab gels 1 mm thick. After electrophoresis, the gels were prepared for fluorography as described by Bonner and Laskey (3). Sera from tumor-bearing animals. (i) Hamster SV40 tumor serum, pool 1. SV40 tumor sera were produced in 15 5- to 6-week-old male golden Syrian hamsters by subcutaneous inoculation of 106 SV40transformed hamster cells (the SV40-transformed hamster cells [line H65/90B], as well as the procedure for preparing SV40 tumor sera, were kindly provided by V. Defendi). Sera were collected at a tumor size of approximately 3 to 4 cm, at 3 to 4 weeks after inoculation, and pooled. (ii) Hamster SV40 tumor serum, pool 2. Hamster SV40 tumor serum pool 2, prepared in a similar manner to that described above for hamster serum pool 1, was kindly provided by R. Henning, The University of Ulm Medical School, Ulm, Federal Republic of Germany. (iii) Mouse SV40 tumor serum. SV40 tumor sera were produced in 10- to 12-week-old male BALB/c mice by subcutaneous inoculation of 5 x 105 SV40transformned BALB/c mouse cells (the SV40-transformed mouse cells, line VLM [34], were a generous gift from M. Tevethia and S. S. Tevethia). Sera were collected at a tumor size of approximately 1 to 2 cm, at 6 weeks after inoculation, and pooled. Preparation of SV40 T-antigen by preparative immunoaffinity chromatography and SDS-polyacrylamide gel electrophoresis. Hamster SV40 tumor cells (line H65/90B) were grown in monolayer cultures on plastic culture dishes (9 cm in diameter). Batches of 50 confluent cultures were harvested by scraping the cells into 1 ml of PBS per culture dish. After centrifugation, the cells were suspended in 30 ml of extraction buffer (0.1 M Tris-hydrochloride [pH 9.0], 0.1 M NaCl, 0.005 M KCl, 0.001 M CaCl2, 0.0005 M MgCl2, 0.5% NP40 [30; modified]). After three cycles of freeze-thawing, the cell lysate was cleared by centrifugation at 800 x g for 10 min. SV40 T-antigen was prepared by preparative immunoaffinity chromatography with protein A-Sepharose according to Schwyzer (30). To 30 ml of cell extract 1.2 ml of hamster SV40 tumor serum and phenylmethyl sulfonyl fluoride at 1 mM concentration were added, and the reaction

J. VIROL. mixture was incubated overnight at 40C. Five milliliters of protein A-Sepharose, preswollen in isotonic Tris buffer (pH 7.2) was added, and the mixture was agitated for a further 2 h at 4°C. Washing and elution of the protein A-Sepharose were performed as described above. The eluate was processed for SDSpolyacrylamide gel electrophoresis as described above. Preparation of antiserum against detergenttreated HeLa cell nuclei. HeLa S3 cells grown in suspension (5 x 107) were harvested by centrifugation, washed once with 30 ml of PBS, and suspended in 4 ml of reticulocyte standard buffer containing 0.5% NP40. After swelling the cells for 20 min on ice, they were Dounce homogenized, and the nuclei were pelleted by centrifugation at 800 x g for 5 min. The nuclei were then washed three times with isotonic Tris buffer containing 0.5% NP40 and fixed by treatment with 3.7% Formalin in PBS for 10 min. After fixation, the nuclei were washed five times in PBS, resuspended in 500 p1 of PBS, and injected into the ear vein of a rabbit. The rabbit received two additional intravenous injections at 4-weekly intervals before the serum was collected. Preparation of anti-HeLa cell surface antiserum. HeLa S3 cells grown in suspension (5 x 107) were harvested by centrifugation, washed three times with PBS and fixed for 10 min in 10 ml of 3.7% Formalin in PBS. After fixation, the cells were washed five times in PBS, resuspended in 500 ,ul of PBS, and injected into the ear vein of a rabbit. The rabbit received two additional intravenous injections at 4-weekly intervals before the serum was collected. Absorption of sera. Before the determination of T- and U-antibody titers on SV101 cells (see below), sera from tumor-bearing animals and antisera against NaDodSO4-T were absorbed on methanol-fixed mouse 3T3 cells. Confluent monolayers of 3T3 cells grown on plastic petri dishes (9 cm in diameter) were washed twice with PBS and then fixed with methanol for 10 min at room temperature. After air drying the cells, they were rinsed with PBS, and 1-ml aliquots of the sera, diluted 1:10 with PBS, were added to each plate. The sera were absorbed at 40C for 12 h per petri dish three times, for a total of 36 h. For determination of U-antibody titers on hybrid virus-infected cells (see below) the sera were absorbed as described above on Ad2-infected HeLa cells fixed with methanol at approximately 24 h p.i. Sera to be used for analysis of hybrid virus-infected cells for cell surface fluorescence also were absorbed on methanol-fixed, Ad2-infected cells as described above. Second antibodies (fluorescein-labeled immunoglobulin prepared against immunoglobulin of the first antibody, see below) were adjusted to a protein concentration of about 0.5 mg/ml and absorbed as described above on methanol-fixed mouse 3T3 cells for analysis on SV101 cells (SV40-transformed mouse 3T3 cells) or on methanol-fixed, Ad2-infected HeLa cells for analysis on hybrid virus-infected cells. After absorption, all sera were cleared of particulate material by brief (4-min) centrifugation at 20,000 x g in an Eppendorf centrifuge. Absorption of T- and U-antibodies from rabbit anti-NaDodSO4-T serum. For removal of T- and Uantibodies from the rabbit anti-NaDodSO4-T serum,

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a 1:10 dilution of this serum in PBS was absorbed four SV40-specific cell surface fluorescence on hybrid virustimes as described above on methanol-fixed, SV40- infected cells were determined on Ad2+ND1- and transformed mouse cells (clone SV101). The results of Ad2+ND2-infected HeLa cells fixed with Formalin as this absorption are documented in Table 2 (see Re- described above. Serum dilutions of the first antibodies sults). were done with filtered PBS in 1:2 dilution steps. In Immunofluorescence staining. 3T3 and SV101 repeat experiments, titers determined were reproduccells grown on glass cover slips (12 mm in diameter) in ible within ±1 dilution step. Dulbecco modified Eagle medium with 10% calf serum Analysis of unspecific binding of SV40 56K were washed once with PBS and fixed by treatment and 42K proteins on cell surfaces. HeLa cells inwith methanol at -20°C for 5 min followed by acetone fected with Ad2+ND2 (at 40 h p.i.) were washed twice treatment at -200C for 30 s and air drying. Then the with PBS and scraped into 1 ml of PBS with a rubber first antibody (serum from an SV40 tumor-bearing policeman. The cell suspension was Dounce homogeanimal or antisera against purified, SDS-denatured T- nized in a Glass-Douncer (Bellco Glass, Inc., Vineland, antigen, diluted with PBS as indicated) was added, N.J.; tight-fitting pestle) until about 80% of the cells and the cover slips were incubated for 45 min at 37°C. were broken and the nuclei still appeared to be intact After washing them with PBS, fluorescein-labeled im- as judged by phase-contrast microscopy. The cell homunoglobulin prepared against immunoglobulin of the mogenate was then diluted with 9 ml of the original first antibody (see below) was added, and the cover growth medium and cleared by low-spin centrifugation slips were held for a further 45 min at 37°C. After a (800 x g at 40C). This Ad2+ND2 cell extract was then second series of washes with PBS, the cover slips were added to Ad2-infected HeLa cells (at 40 h p.i.) from mounted with Elvanol on microscope slides. The cells which the growth medium had been removed. After 1 were viewed with a Zeiss microscope (Carl Zeiss, Ob- h of incubation at 37°C, the Ad2-infected HeLa cells erkochen, Federal Republic of Germany) equipped were processed for immunofluorescence microscopy as with epifluorescent illumination. Pictures were taken described above. with Planapo x40 and x63 oil immersion objectives. RESULTS For analysis of U-antibody titers on hybrid virusinfected cells, Ad2+NDl- and Ad2+ND2-infected Preparation of antisera against NaHeLa cells grown on cover slips (12 mm in diameter) DodSO4-T. Hamster SV40 tumor cells (line in Dulbecco modified Eagle medium supplemented with 10% calf serum were processed for immunofluo- H65/90B) were grown on plastic petri dishes. rescence microscopy approximately 32 h after infec- SV40 T-antigen was extracted from batches of 50 confluent cultures and prepared by preparation as described above. For analysis of cell surface fluorescence on hybrid tive immunoaffinity chromatography with provirus-infected cells, HeLa cells infected with Ad2+ND1 tein A-Sepharose according to Schwyzer (30) as or Ad2+ND2 were fixed at approximately 40 h p.i. for described in Materials and Methods. A sample 5 min in 3.7% Formalin in PBS on ice, followed by air of the eluate of the protein A-Sepharose immudrying. noaffinity column was analyzed on an analytical Second antibodies. Fluorescein-labeled immuno- SDS-polyacrylamide gel. Upon Coomassie blue globulin prepared against immunoglobulin of the first staining, a single T-antigen band was obtained antibody was from the following sources: (i) goat im- (Fig. lb), which was not detectable in the eluate munoglobulin, fluorescein labeled, prepared against hamster immunoglobulin (Cappel Laboratories, Inc., of the immunoprecipitation with hamster nonCochranville, Pa.); (ii) rabbit immunoglobulin, fluores- immune serum (Fig. ld). The whole eluate was then applied onto a 2cein labeled, prepared against mouse immunoglobulin (Nordig/Byk-Malinckrodt, Dietzenbach, Federal Re- mm preparative SDS-polyacrylamide slab gel, public of Germany); (iii) rabbit immunoglobulin, flu- and electrophoresis was carried out at 20 mA for orescein labeled, prepared against guinea pig immu- 8 h. The T-antigen band was visualized by brief noglobulin (Miles Laboratories, Inc., Frankfurt, Fed- staining with Coomassie blue (3 min) followed eral Republic of Germany); (iv) pig immunoglobulin, by destaining in distilled water for 5 min. For fluorescein labeled, prepared against rabbit immuno- immunization of animals with T-antigen, the globulin (Behringwerke, A. G., Marburg,/Lahn, Fedin processing the T-anmodifications following eral Republic of Germany). Determination of T- and U-antibody titers and tigen slab gel were used. (i) The T-antigen band of antibody titers for SV40-specific cell surface was excised, transferred to a glass tube, and fluorescence. Antibody titers against SV40 T-antigen crushed with a glass rod in 500 ul of PBS conwere determined on methanol-acetone-fixed SV101 taining 0.5% SDS. Complete Freund adjuvant cells as described previously (28). For determination (500 pi) was added, and the antigen was mixed of U-antibody titers in SV101 cells, T-antigen was heat to an emulsion. T-antigen was then injected inactivated (22). SV101 cells grown on cover slips were subcutaneously into a guinea pig. The animal washed twice with PBS and incubated in a wet cham- received three such injections at 4-weekly interber at 50°C for 30 min. The cells were then washed twice with PBS and processed for immunofluorescence vals before collection of the serum. (ii) The Tmicroscopy. Antibody titers against SV40 U-antigen antigen band was excised and electrophoretiin HeLa cells infected with Ad2+ND1 or Ad2+ND2 cally eluted essentially as described by Lazarides were determined as described (22). Antibody titers for and Weber (19). The extracted T-antigen was

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T-Ag 9. .,w .

w

..

t.

'Al

IgG

IgG

a bc FIG. 1. Immunoprecipitation of SV40 T-antigen (T-Ag) from extracts of SV40-transformed hamster cells; staining pattern of Coomassie blue-stained polypeptides. Extracts of SV40-transformed hamster cells were prepared and immunoprecipitated as described in the text. Samples of the cell extracts and of the immunoprecipitates were analyzed on a 10% acrylamide slab gel and run for 6 h at 12 mA. The sample order is: (a) cell extract of SV40-transformed hamster cells; (b) same, immunoprecipitated with hamster SV40 tumor serum, eluate ofprotein A-Sepharose; (c) cell extract of SV40-transformed hamster cells; (d) same, immunoprecipitated with control serum, eluate of protein A-Sepharose. IgG, Immunoglobulin G.

dialyzed against distilled water containing 0.1% SDS and lyophilized. T-antigen was then dissolved in 500 ,ul of PBS, emulsified with 500 ,ul of Freund complete adjuvant, and injected subcutaneously into a rabbit. The animal received three such injections at 3-weekly intervals before collection of the serum. The production of antisera against purified, SDS-denatured SV40 Tantigen by essentially similar procedures has been described by other investigators (4, 18). Characterization of SV40 tumor sera and of antisera against NaDodSO4-T. Sera from SV40 tumor-bearing animals and antisera

against NaDodSO4-T were prepared as described in Materials and Methods. The sera were first characterized by determination of their Tand U-antibody titers on SV40-transformed mouse cells (clone SV101), on heat-treated SV101 cells, and on Ad2+ND1- as well as on Ad2+ND2-infected HeLa cells, respectively. All sera exhibited reasonably high titers both for Tand U-antibodies (Table 1). In addition to the variation of the T- and U-antibody titers, a significant diversity in the antibody specificities of these sera could be observed. With hamster pool 1 serum, the T- and U-antibody titers on SV101 cells were similar; the U-antibody titers on Ad2+ND1- and on Ad2+ND2-infected cells, on the other hand, were considerably lower than the U-antibody titers on heated SV101 cells. Hamster pool 2 serum revealed an approximately fourfold higher titer of T-antibodies when compared with hamster pool 1 serum. On heated SV101 cells the U-antibody titer of hamster pool 2 serum was comparable to the Uantibody titer of hamster pool 1 serum. The Uantibody titers of hamster pool 2 serum on Ad2+ND1- and Ad2+ND2-infected cells, however, were considerably higher than those of hamster pool 1 serum and corresponded well to its U-antibody titer on heated SV101 cells. The mouse SV40 tumor serum, the guinea pig antiNaDodSO4-T serum, as well as the rabbit antiNaDodSO4-T serum exhibited similarly high titers for both T- and U-antibodies on SV101 cells and on heated SV101 cells. The titers for Uantibodies on Ad2+ND1- and on Ad2+ND2-infected cells, however, were generally lower than the U-antibody titers on heated SV101 cells. The SV40 specificities of these sera were then analyzed by immunoprecipitation of the SV40specific proteins from cell extracts of Ad2+ND2infected cells (see Materials and Methods). All sera specifically immunoprecipitated the SV40specific 56K and 42K proteins (Fig. 2). However, differences in the amounts of precipitated 56K and 42K proteins were observed. Whereas with the mouse SV40 tumor serum and the rabbit anti-NaDodSO4-T serum these proteins were almost quantitatively precipitated, with the guinea pig anti-NaDodSO4-T serum and the two pools of hamster SV40 tumor serum only a fraction of these proteins were removed from the cytoplasmic extract. Reprecipitation of the supeinatants of the immunoprecipitations with these sera, using additional fresh serum, however, also resulted in almost quantitative precipitations of the SV40-specific 56K and 42K proteins by these sera (data not shown). The differences in the amounts of precipitated SV40-specific 56K and 42K proteins observed in the first immunoprecipitation reaction, therefore, are

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TABLE 1. T- and U-antibody titers of sera from SV40 tumor-bearing animals and of anti-NaDodSO4-T sera Antibody titera on:

Seumm

SV101 cells (T)

Heated SV101

cells (U)

Ad2fecNteD2-

Ad2+ND1-nfceHIA

HeLa cells

cells (U)

(U) 160 80 600 800 Hamster SV40 tumor serum, pool 1 640 320 800 3,200 Hamster SV40 tumor serum, pool 2 640 1,280 3,200 3,200 Mouse SV40 tumor serum 320 320 Guinea pig anti-NaDodSO4-T serum 1,600 3,200 320 640 1,600 1,600 Rabbit anti-NaDodSO4-T serum a Antibody titers were determined as described in the text. Each titer is given as the reciprocal of the serum dilution at which the staining intensity was 1+ on an arbitrary scale of 4+ = briJliant and ± = very dull. The variation of the titers between separate experiments corresponds to ±1 dilution step.

11 _iw III

lV 56K 42K

_

_

_

_.

_

_

_ _

_ __

_

-

56K

-

-.

a

b

42K

-n*

--

c

d

e

f

g

h

i

j

k

I

m

n

o

p

FIG. 2. Immunoprecipitation of SV4O-specific proteins from NP40 cytoplasmic extracts of Ad2@ND2-infected HeLa cells, using sera from SV40 tumor-bearing animals or antisera prepared against NaDodSO4-T; fluorogram of 3S-polypeptides. NP40 cytoplasmic extracts of HeLa cells infected with Ad2+ND2 and labeled with [3SJmethionine were prepared and immunoprecipitated as described in the text. Samples of the NP40 cytoplasmic extracts, the or4ginal reaction mixture, the supernatants of the immunoprecipitations, and the eluates were analyzed on a 8.5% acrylamide gel and run for 5 h at 12 mA. The sample order is: (a) Ad2+ND2 cytoplasmic extract; (b) Ad2ND2 cytoplasmic extract (original reaction mixture); (c) same, supernatant after immunoprecipitation with hamster SV40 tumor serum, pool 1; (d) same, eluate ofprotein A-Sepharose after immunoprecipitation with hamster SV40 tumor serum, pool 1; (e) Ad2+ND2 cytoplasmic extract (original reaction mixture); (f) same, supernatant after immunoprecipitation with hamster SV40 tumor serum, pool 2; (g) same, eluate of protein A-Sepharose after immunoprecipitation with hamster SV40 tumor serum, pool 2; (h) Ad2+ND2 cytoplasmic extract (original reaction mixture); (i) same, supernatant after immunoprecipitation with mouse SV40 tumor serum; (j) same, eluate ofprotein A-Sepharose after immunoprecipitation with mouse SV40 tumor serum; (k) Ad2+ND2 cytoplasmic extract (original reaction mixture); (1) same, supernatant after immunoprecipitation with guinea pig anti-NaDodS04-T serum; (m) same, eluate ofprotein A-Sepharose after immunoprecipitation with guinea pig anti-NaDodSO4-T serum; (n) Ad2+ND2 cytoplasmic extract (original reaction mixture); (o) same, supernatant after immunoprecipitation with rabbit anti-NaDodSO4-T serum; (p) same, eluate ofprotein A-Sepharose after immunoprecipitation with rabbit anti-NaDodS04-T serum. Immunoprecipitation of Ad2+ND2 cytoplasmic extract with the respective nonimmune sera did not result in immunoprecipitation of SV40-specific proteins (data not shown). II, III, and IV are Ad2-specific proteins; 56K and 42K are the SV40-specific proteins encoded by Ad2+ND2.

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likely to reflect differences either in the affinities of the immunoglobulins of the individual sera to bind the SV40-specific proteins or in the abilities of the antigen-antibody complexes to bind to protein A (16) or both rather than the overall abilities of the individual sera to recognize the SV40-specific proteins. Conditions for analyzing Ad2+ND1- and Ad2'ND2-infected HeLa cells for cell surface fluorescence. Analysis of HeLa cells infected with Ad2+ND1 and Ad2+ND2 hybrid viruses for cell surface fluorescence is complicated for the following reasons. (i) The expression of SV40-specific proteins in hybrid virus-infected cells occurs late in infection (20, 31), i.e., at a time when these cells already exhibit cytopathic effects: the nuclei of these cells are greatly enlarged, the cells are rounded up, and when grown in monolayer (culture dishes, glass cover slips) they begin to lose close contact with the substratum. Furthermore, the cells are fragile and tend to lyse upon handling. Probably for these reasons, during processing of living hybrid virusinfected cells for immunofluorescence microscopy, many cells came off the cover slips and were lost. Alternatively, when cells grown in suspension culture were used, many cells lysed and were no longer suitable for fluorescence analysis. (ii) An additional problem stems from the fact that the SV40-specific proteins are also present in the cytoplasm of Ad2+ND1- and Ad2+ND2-infected cells (SV40 U-antigen; see Fig. 4a). Leakage of these proteins from the inside or permeation of the antibody through the plasma membrane, therefore, could possibly obscure a cell surface location of the SV40-specific proteins. Despite these problems we have tried to analyze living hybrid virus-infected cells, grown either on glass cover slips or in suspension culture, for SV40-specific cell surface fluorescence, but we have obtained inconclusive results: the few cells either left on the glass cover slips or still intact after processing for immunofluorescence microscopy exhibited only a weak and not very reproducible cell surface fluorescence staining (data not shown). One possible explanation for this result could be that only cells which were not infected or which were still at an early stage of infection stayed on the cover slips or survived the handling. Alternatively, however, it seems possible that on living cells SV40-specific proteins on the cell surface are masked by other cell surface components and thereby are hindered from reacting with antibody. Because of these potential difficulties encountered with living cells, we decided to fix hybrid virus-infected cells with formaldehyde before

J. VIROL.

analysis for cell surface fluorescence, a procedure which has been used successfully by other investigators in analyses of cell surface proteins (6, 12, 27). Formaldehyde treatment is believed to stabilize subcellular structures without rendering the cells permeable to antibody molecules. Due to the difficulties outlined above, we have tested whether or not hybrid virus-infected cells are permeable to antibody after Formalin fixation. Formalin-fixed and methanol-acetonefixed Ad2+ND2-infected HeLa cells were analyzed by immunofluorescence microscopy with antiserum directed against purified, Formalinfixed HeLa cell nuclei (see Materials and Methods). After methanol-acetone fixation the nuclei of Ad2+ND2-infected HeLa cells could be stained brilliantly with this antiserum (Fig. 3a). After fixation with Formalin alone, however, no nuclear fluorescence could be detected (Fig. 3b), indicating that, as expected, the antibody was not able to permeate through the plasma membrane of the Formalin-fixed cells. Similar results were obtained when hybrid virus-infected cells were stained with antibodies against tubulin, using cells fixed with either methanol-acetone or Formalin: the cytoplasmic fluorescence observed in methanol-acetone-fixed cells was not detectable in Formalin-fixed cells without a further treatment with organic solvents (data not shown). We therefore conclude that the Formalin fixation procedure did not induce antibody permeability of the cells. The morphology of the cell surface fluorescence of Formalin-fixed, Ad2+ND2-infected HeLa cells was probed with anti-serum directed against intact, Formalin-fixed HeLa cells (see Materials and Methods). The uniform staining of the cell surfaces with this antiserum is documented in Fig. 3c (compare with phase-contrast picture, Fig. 3d). SV40-specific cell surface staining of HeLa cells infected with Ad2+ND2, using rabbit anti-NaDodSO4-T serum. Rabbit antiNaDodSO4-T serum was used to analyze Ad2+ND2-infected HeLa cells for SV40-specific fluorescence. Figure 4a shows the typical cytoplasmic U-antigen fluorescence of Ad2+ND2-infected HeLa cells (21) after methanol-acetone fixation and staining with rabbit antiNaDod-SO4-T serum. No staining was visible in identically treated Ad2-infected HeLa cells, further indicating the SV40 specificity of the rabbit anti-NaDodSO4-T serum (Fig. 4b). When Formalin-fixed, Ad2+ND2-infected HeLa cells were stained with this serum, a typical cell surface staining (compare Fig. 3c) was observed (Fig. 4c; compare with phase-contrast picture of same cells, Fig. 4d). Again, this cell surface staining

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529

d

FIG. 3. Analysis of Ad2+ND2-infected HeLa cells for nuclear and for cell surface fluorescence after Formalin fixation. HeLa cells on cover slips were infected with Ad2+ND2 and processed for immunofluorescence microscopy at 40 h p.i. Cells were fixed with methanol-acetone (a) and with Formalin (b through d) as described in the text. For analysis of cells in (a) and (b), antiserum directed against purified HeLa cell nuclei (see text) at a 1:10 dilution with PBS was used as first antibody; for analysis of cells in (c), antiserum directed against intact, Formalin-fixed HeLa cells (see text) at a 1:100 dilution with PBS was used as first antibody. (d) Corresponding phase-contrast picture of cells in (c). Magnification, x600.

was not detectable with identically treated Ad2infected HeLa cells (data not shown; see Fig. 5). The SV40 specificity of the cell surface staining shown in Fig. 4c was further assessed by analyzing Ad2+ND2-infected HeLa cells for cell surface fluorescence with rabbit antiNaDodSO4-T serum which had been extensively absorbed on methanol-fixed, and therefore permeable, SV101 cells (see Materials and Methods). This absorption resulted in a more than 80% reduction of both the T- and the U-antibody titers in this serum (Table 2). Using this absorbed serum, no cell surface fluorescence could be observed at a 1:10 serum dilution, although cell surface staining of Ad2+ND2-infected HeLa cells with the rabbit anti-NaDodSO4-T serum otherwise was positive down to a serum dilution of 1:160 (Table 3). Analysis of unspecific binding of SV40specific 56K and 42K proteins from lysed

cells. Since Ad2+ND2-infected HeLa cells can be analyzed for cell surface fluorescence only late in infection, the possibility existed that the cell surface fluorescence seen in Fig. 4c was the result of the SV40-specific proteins being released from these cells and then being unspecifically adsorbed onto the cell surfaces. To test this possibility, Ad2-infected HeLa cells were incubated for 1 h with a cell lysate of Ad2+ND2infected HeLa cells and then processed for immunofluorescence microscopy (see Materials and Methods). No cell surface fluorescence was observed with these Ad2-infected cells, indicating that the cell surface fluorescence observed on Ad2+ND2-infected cells was not due to unspecific binding of the SV40-specific 56K and 42K proteins on the cell surface after release from the cells. Analysis of Ad2+ND1- and Ad2+ND2-infected HeLa cells for cell surface fluores-

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FIG. 4. Visualization of SV40-specific proteins within and on the surface of Ad2+ND2-infected HeLa cells by immunofluorescence microscopy, using rabbit anti-NaDodSO4-T serum. HeLa cells grown on cover slips were infected with Ad2+ND2 (a, c, d) or Ad2 (b) and processed for immunofluorescence microscopy at approximately 40 h p.i. Cells were fixed with methanol-acetone (a and b) and with Formalin (c and d) as described in the text. Rabbit anti-NaDodSO4-T serum was used at a 1:100 dilution in (a) and (b) and at a 1: 10 dilution in (c) and (d). (a) An island of cells, two of which are infected and exhibit cytoplasmic U-antigen staining. The uninfected cells do not show detectable U-antigen fluorescence, neither do the Ad2-infected cells in (b). (d) Phase-contrast picture of surface-stained cells in (c). Exposure times were approximately 15 s (a and c) and 2.5 min (b). Magnification, x650.

cence with sera from SV40 tumor-bearing

rescence with the sera described in Table 1 and

animals and antisera against NaDodSO4-T. Fig. 2. Staining with sera from SV40 tumorAd2+ND1-infected and Ad2+ND2-infected bearing animals resulted only in a weak cell HeLa cells were analyzed for cell surface fluo- surface fluorescence, if any. With hamster pool

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TABLE 2. T- and U-antibody titers of rabbit anti-NaDodSO4-T serum before and after absorption on SViOl cells Antibody titera on: Serum

Heated SV1O1 cells SV101 cel

(T)

() SVOUcel

1,600 1,600 Rabbit anti-NaDodSO4-T serum, 3T3 absorbed 200 200 Rabbit anti-NaDodSO4-T serum, SV101 absorbed a Antibody titers were determined as described in the footnote to Table 1.

1 serum and mouse SV40 tumor serum, this cell surface fluorescence staining was just slightly above the background staining observed with Ad2-infected cells (data not shown) and was previously not considered to be unequivocally positive. With hamster pool 2 serum, a weak stAining of the surfaces of Ad2+ND1- and of Ad2+ND2-infected HeLa cells could be obtained (Fig. 5a and c). Although cell surface staining with this serum was weak, it was clearly above background and was judged positive in repeat experiments. In contrast, the guinea pig antiNaDodSO4-T serum as well as the rabbit antiNaDodSO4-T serum both were able to cause bright staining of cell surfaces of Ad2+ND1- and Ad2+ND2-infected cells (Fig. 5). At the same serum dilution (1:10 with PBS) interesting differences were observed with these sera in the intensity of the cell surface staining of either Ad2+ND1- or Ad2+ND2-infected cells: the guinea pig anti-NaDodSO4-T serum more strongly stained the cell surfaces of Ad2+ND1infected cells than the cell surfaces of Ad2+ND2-infected cells, whereas the rabbit antiNaDodSO4-T serum stained the cell surfaces of both Ad2+ND1- and Ad2+ND2-infected HeLa cells equally well. In general, the intensity of cell surface staining was found to be greater with the rabbit anti-NaDodSO4-T serum than with the guinea pig anti-NaDodSO4-T serum. The morphology of the cell surface staining on Ad2+NDl-infected HeLa cells was different from the morphology of the cell surface staining on Ad2+ND2-infected cells: whereas on Ad2+ND2-infected cells the SV40-specific cell surface proteins seemed to be evenly distributed over the plasma membranes (Fig. 5f and i), on Ad2+ND1-infected HeLa cells the SV40-specific proteins seemed to be organized in patches (Fig. 5d and g). No correlation could be found between the Tand U-antibody titers of the individual sera (see Table 1) and their abilities to stain the cell surfaces of Ad2+ND1- and Ad2+ND2-infected HeLa cells (see Fig. 5). Two sera from SV40 tumor-bearing animals, hamster pool 2 serum and the mouse SV40 tumor serum, had compa-

Ad2+ND2~~~~~~Ad2+NDIinfected infected HeLa cells HeLa cells

(U)

(U)

640 80

320 40

TABLE 3. Antibody titers of sera from SV40 tumorbearing animals and of anti-NaDodSO4-T sera for ceU surface staining on Ad2+NDl- and Ad2+ND2infected HeLa cells Antibody titera on:

Serum

Ad2+ND1- Ad2+ND2infected infected HeLa cells HeLa celLs

10 10 Hamster SV40 tumor serum, pool 1 10 80 Hamster SV40 tumor serum, pool 2 10 10 Mouse SV40 tumor serum 320 160 Guinea pig antiNaDodSO4-T serum 320 160 Rabbit anti-NaDodSO4-T serum a Antibody titers were determined as described in the text. Each titer is given as the reciprocal of the serum dilution at which cell surface staining could still be observed on hybrid virus-infected cells when compared with Ad2-infected cells.

rable or even higher T- and U-antibody titers than had the guinea pig or the rabbit antiNaDodSO4-T sera, but they were only very weakly positive on the cell surfaces of Ad2+ND1or Ad2+ND2-infected cells. This may suggest that SV40-specific proteins on the cell surface of Formalin-fixed, hybrid virus-infected cells exhibit a different structure to that in the cytoplasm and, therefore, are only poorly recognized by the SV40 tumor sera. The sera directed against NaDodSO4-T, on the other hand, seemed to recognize both the cytoplasmic and the cell surface SV40-specific proteins equally well. This argument was further substantiated by titrating the individual sera for cell surface fluorescence staining. Table 3 shows that the very weak cell surface staining obtained with sera from SV40 tumor-bearing animals was no longer visible at serum dilutions of lower than 1: 10, with the exception of hamster pool 2 serum, which had a titer for its weak cell surface staining on Ad2+ND2-infected cells of 1:80. The guinea pig and the rabbit anti-NaDodSO4-T sera, on the other hand, both exhibited titers for

I

Ad2+ND1

Ad2

Ad2+ND2

FIG. 5. Analysis of HeLa cells infected with Ad2, Ad2+ND1, or Ad2+ND2 for cell surface fluorescence, using sera from SV40 tumor-bearing animals and antisera directed against NaDodSO4- T. HeLa cells grown on cover slips were infected with Ad2 (b, e, and h), Ad2+ND1 (a, d, and g), or Ad2+ND2 (c, f, and i). Cells were fixed with Formalin and processed for immunofluorescence microscopy as described in the text. The following sera at 1:10 dilutions were used as first antibody: hamster SV40 tumor serum, pool 2 (a through c); guinea pig anti-NaDodSO4-T serum (d through f); rabbit anti-NaDodSO4-T serum (g through i). Exposure times were approximately 90 s (a and c), 30 s (f and i), 15 s (d and g) and 2.5 min (b, e, and h). Magnification, X460. 532

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SV40-SPECIFIC CELL SURFACE PROTEINS

cell surface staining which correlated well with their U-antibody titers in Ad2+ND1- or Ad2+ND2-infected cells. It is important to note that the titers of the individual sera for cell surface staining of Ad2+ND1- and Ad2+ND2-infected cells given in Table 3 did not reflect intensity of staining. So, for instance, hamster pool 2 serum had a titer for cell surface staining of 1:80 on Ad2+ND2infected cells. This titer was about the same as the titer for the rabbit anti-NaDodSO4-T serum. However, the intensity of the cell surface staining with hamster pool 2 serum was already weak at the 1:10 dilution (see Fig. 5c). This weak staining then could be observed down to a serum dilution of 1:80. With the rabbit anti-NaDodSO4T serum, on the other hand, staining at a 1:10 dilution was brightly positive (see Fig. 5i) and became weaker only at serum dilutions higher than 1:40 to 1:80. Expression of SV40-specific proteins, SV40 U-antigen, and SV40-specific cell surface staining during the course of infection of HeLa cells with Ad2+ND1 or Ad2+ND2. The SV40-specific proteins in cells infected with Ad2+SV40 hybrid viruses are expressed late in infection, i.e., together with the late Ad2 proteins (31). SV40-specific cell surface fluorescence on cells infected with these viruses, therefore, should also appear late during the infection cycle. The results of a time course experiment in which the expression of SV40-specific proteins and the appearances of SV40 U-antigen and of SV40-specific cell surface fluorescence were analyzed with Ad2+ND1- and Ad2+ND2-infected HeLa cells are summarized in Table 4 and Fig. 6. At 18 h p.i. host protein synthesis was not yet completely shut off and the SV40-specific proteins could not yet be detected in the cell homogenates of Ad2+ND1- and Ad2+ND2-infected cells (Fig. 6a, b, and c). However, about 10% of the Ad2+ND1-infected cells and about 20 to 30% of the cells infected with Ad2+ND2 were positive for SV40 U-antigen both with the guinea pig and with the rabbit anti-NaDodSO4-T sera. No cell surface fluorescence could be observed at this time of infection (Table 4). At 24 h p.i. between 40 and 60% of the Ad2+ND1- and Ad2+ND2infected cells were positive for SV40 U-antigen. SV40-specific cell surface fluorescence just began to appear on approximately 10 to 20% of the cells (Table 4). SV40-specific proteins now were detectable in the cell homogenates (Fig. 6d, e,

533

TABLE 4. Expression of SV40 U-antigen and of SV40-specific cell surface fluorescence during the course of infection of HeLa cells with Ad2+ND1 and Ad2+ND2a Expression of SV40-specific: Time (hp.i.)

U-antigen fluorescence (% of positive

celUs)

Cell surface fluorescence (% of positive cells)

Ad2+ND1 Ad2+ND2 Ad2+ND1 Ad2+ND2

18 10 27 0 0 20 20 24 46 69 30 75 66 82 85 36 90 85 85 79 42 90 92 92 90 48 91 91 88 85 a Ad2+NDl- and Ad2+ND2-infected HeLa cells were processed for immunofluorescence microscopy at the times indicated as described in the text. Approximately 100 cells per time point were analyzed, and the percentage of positive cells was recorded. Immunofluorescence analyses were perforned with both the guinea pig and the rabbit anti-NaDodSO4-T sera and yielded virtually identical results.

the cell homogenates (Fig. 6g, h, and i). From 36 to 48 h p.i. (the latest time analyzed) the percentage of U-antigen-positive and SV40-specific cell surface fluorescence-positive cells was about the same (ca. 80 to 90% [Table 4]). Expression of SV40-specific cell surface fluorescence, therefore, paralleled the expression of SV40-specific proteins and the expression of SV40 U-antigen in Ad2+NDl- and Ad2+ND2-infected cells with a lag period of about 6 h. Since the SV40-specific proteins of Ad2+ND1 and Ad+ND2 accumulate in the plasma membranes of the infected cells during the course of infection (7, 9, 10, 13-15), this time lag probably represents the time required for these proteins to be accumulated in the plasma membranes in sufficient quantities to give a positive immunofluorescence staining reaction. Interesting differences could be observed in the synthesis of the SV40-specific proteins in Ad2+ND1- and Ad2+ND2-infected cells during the course of infection. The SV40-specific 28K protein of Ad2+ND1 and the SV40-specific 42K and 56K proteins of Ad2+ND2 all became apparent in the homogenates of cells labeled 24 to 26 h p.i. Synthesis of the SV40-specific 28K protein of Ad2+ND1 peaked at around 30 h p.i. and then gradually declined (7). The SV40-speand f [see arrows]). At 30 h p.i. between 80 and cific 56K and 42K proteins of Ad2+ND2, on the 90% of the Ad2+ND1- and Ad2+ND2-infected other hand, were continually synthesized cells were positive for SV40 U-antigen, and ap- throughout the course of infection. proximately 60 to 80% were positive for SV40DISCUSSION specific cell surface fluorescence (Table 4). This study provides evidence that the SV40SV40-specific proteins now were prominent in

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