Vol. 16, No. 5 Printed in U.S.A.
JOURNAL OF VIROLOGY, Nov. 1975, p. 1236-1247 Copyright © 1975 American Society for Microbiology
Simian Virus 40-Specific Proteins in HeLa Cells Infected with Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses GERNOT WALTER* AND HAWLEY MARTIN
Tumor Virology Laboratory, The Salk Institute, Post Office Box 1809, San Diego, California 92112 Received for publication 17 June 1975
The synthesis of simian virus 40 (SV40)-specific proteins in HeLa cells infected with the nondefective adenovirus 2 (Ad2)-SV40 hybrid viruses, Ad2+ ND2, Ad2+ ND3, Ad2+ ND4, and Ad2+ ND5, was investigated. Infected-cell proteins were labeled with radioactive amino acids late after infection, when host protein synthesis was shut off, and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. All polypeptides normally seen in Ad2infected cells were found in cells infected by the hybrid viruses. In addition to the Ad2-specific proteins, cells infected with Ad2+ ND2 contain two SV40-specific proteins with apparent molecular weights of 42,000 and 56,000, cells infected with Ad2+ ND4 contain one protein with an apparent molecular weight of 56,000, and cells infected with Ad2+ ND5 contain one protein with an apparent molecular weight of 42,000. Cells infected with Ad2+ ND3 do not contain detectable amounts of proteins not seen during Ad2 infection. Pulse-chase experiments demonstrate that the SV40-specific proteins induced by Ad2+ ND2, Ad2+ ND4, and Ad2+ ND5 are metabolically unstable. These proteins are not present in purified virions. Two nonstructural Ad2-specific proteins have been demonstrated in Ad2 and hybrid virus-infected cells which have a smaller apparent molecular weight after a short pulse than after a pulse followed by a chase. The molecular weight increase during the chase may be caused by the addition of carbohydrate to a polypeptide backbone.
The expression of the simian virus 40 (SV40) genome during productive infection occurs in two phases. The early phase is characterized by the induction of the SV40-specific antigens, T (13), tumor-specific transplantation antigen (TSTA) (8), and U (26), and by the induction of cellular DNA synthesis (10, 40). The late phase begins with the synthesis of viral DNA and structural proteins. Little is known about the chemical and biological properties of the early antigens, T, TSTA, and U. They could be either proteins coded by the SV40 genome (early proteins) or, alternatively, cellular products induced or modified by the early viral proteins. The major obstacle to the detection and characterization of early SV40 proteins and early antigens is the continuation of host protein synthesis in SV40infected cells. At present, no methods are available for selectively inhibiting host protein synthesis and making the clear detection of early proteins possible. The nondefective adenovirus 2 (Ad2)-SV40 hybrid viruses, isolated by Lewis and co-workers, are a useful tool for the study of early SV40 proteins. They have the following properties:
each of the five nondefective hybrid viruses, Ad2+ ND1 (24), Ad2+ ND2, Ad2+ ND3, Ad2+ ND4, and Ad2+ ND5 (25), contains a single continuous segment of SV40 DNA covalently inserted at the same site in the Ad2 genome (3, 12, 16, 21, 32). The SV40 DNA segments differ in size: Ad2+ ND1 contains 17 to 20%; Ad2+ ND2, 32 to 36.5%; Ad2+ ND3, 7%; Ad2+ ND4, 43 to 48%; and Ad2+ ND5, 28% of the SV40 genome (16, 20, 31, 32). The segments are completely overlapping and colinear with SV40 DNA (16, 20) (Fig. 1). They have a common end point which is located in the Haemophilus influenzae restriction endonuclease fragment G of SV40 DNA and is 0.11 map units of the SV40 genome away from the cleavage site of EcoRl restriction endonuclease (16, 20, 31, 32). All SV40 DNA segments contain at their common end point a major fraction of the Hin G fragment (20) which is located in the late region of the SV40 genome. During productive infection with the nondefective hybrid viruses, the SV40 DNA segment contained in each hybrid virus is fully transcribed (18, 23). The transcripts are complementary to the minus (early) strand of SV40 DNA and consist of both early and
1236
VOL. 16, 1975
SV40-SPECIFIC PROTEINS
Ad2+ND
U U
Ad2+ ND2
1237
.
TST A
Ad2+ND3 U
TSTA
T
Ad2 ND5
Hin F
J
G
B
|
I
Late Regi
0
0.1
Restriction Endonuclease Cleavage Map C HI A D
Early Region
0.2
0.3
E
I
I
K F
Late Region
0.4 0.5 0.6 0.7 0.8 FRACTIONAL LENGTH OF SV40 DNA
0.9
1.0
FIG. 1. SV40 DNA segments in nondefective Ad2-SV40 hybrid viruses in relation to the Haemophilus influenza restriction endonuclease cleavage map of SV40. The position of the early and late gene regions in SV40 DNA is derived from the results of Khoury et al. (17) and Dhar et al. (5). Arrows indicate the direction of transcription. The dotted lines indicate the degree of variation in the length of the SV40 DNA segments as determined by different investigators (16, 20, 31, 32). The Haemophilus influenza cleavage map is derived from Danna et al. (4).
antilate sequences (18). Ad2+ ND4, containing the largest segment of the SV40 genome, induces the synthesis of three SV40-specific antigens: T, TSTA, and U (25, 27). Ad2+ ND2 induces TSTA and U, and Ad2+ ND1 induces only U antigen (25-27). The SV40 DNA regions in these hybrid viruses, which determine the expression of the early SV40 antigens, have been mapped on the SV40 genome (16, 20). The expression of the SV40 information in the nondefective hybrid viruses is controlled by the Ad2 genome and takes place at a late time during productive infection, that is, after hybrid viral DNA synthesis has been initiated (23). It is this property of the hybrid viruses which facilitates the identification and characterization of early SV40 proteins, since late during infection host protein synthesis is shut off by the adenovirus genome. As a consequence, it may be possible to detect early SV40 proteins or SV40-specific antigens in hybrid virus-infected cells by labeling with radioactive amino acids late in the infection cycle and then analyzing the labeled proteins by gel electrophoresis and autoradiography. It has recently been shown that HeLa cells infected with Ad2+ ND1 synthesize a polypeptide of molecular weight 28,000 which is not present in Ad2-infected cells (9, 28). In this report, we demonstrate the presence of specific proteins in cells infected by Ad2+ ND2, Ad2+ ND4, and Ad2+ ND5. We have provisionally designated these proteins as "SV40 specific," since their expression is caused by the SV40 information contained in the hybrid virus genomes.
MATERIALS AND METHODS Cells. HeLa S3 cells were grown in suspension in Eagle minimum essential medium supplemented with 5% calf serum or in monolayer in Dulbecco's modification of Eagle medium (DMEM) with 10% calf serum. HeLa S3 cells were only used for labeling experiments. For adenovirus plaque assays, a different type of HeLa cell with a flatter morphology was used. These cells were obtained from the Cold Spring Harbor Laboratory and grown in DMEM supplemented with 10% calf serum. CV1 cells, a line of African green monkey kidney cells (AGMK), were obtained from the American Type Culture Collection and grown in DMEM supplemented with 10% fetal calf serum. Primary AGMK cells were purchased from Microbiological Associates, Inc., and grown in DMEM with 10% fetal calf serum. Viruses. Seed stocks of nondefective Ad2-SV40 hybrid viruses Ad2+ ND1, Ad2+ ND2, Ad2+ ND3, Ad2+ ND4, and Ad2+ ND5 were obtained from A. M. Lewis, Jr. Stocks of Ad2+ ND1, Ad2+ ND2, and Ad2+ ND4 were prepared in CV, and HeLa S3 cells. Stocks of Ad2, Ad2+ ND3, and Ad2+ ND5 were prepared in HeLa S3 cells. Hybrid stocks of Ad2+ ND1, Ad2+ ND2, Ad2+ ND3, and Ad2+ ND5 were shown to be free of adeno-associated viruses by cesium chloride density centrifugation, high multiplicity infections, and heat sensitivity assays which will be described elsewhere. All stocks of Ad2+ ND4, however, were contaminated with adeno-associated virus. Plaque titrations were routinely performed on HeLa and CVl cells. In one experiment, primary AGMK cells were used to assay Ad2, Ad2+ ND1, Ad2+ ND2, and Ad2+ ND4. The assays were performed as described by Williams (41). The overlay medium for HeLa cells contained 0.6% agar (Difco) and 0.9% agar for CV, and AGMK cells. After incubation for 3 to 4
1238
J. VIROL.
WALTER AND MARTIN
days, 2 ml of additional overlay medium was added. Overlay medium containing neutral red and 0.9% agar was added 6 to 7 days after infection of HeLa cells, and 8 to 10 days after infection of CV, and AGMK cells. MgCl2 was omitted from the overlay medium when plaque assays were performed on CVl and AGMK cells. The plaque titers of virus stocks prepared in HeLa or CV, cells are showh in Table 1. Plaque formation in CV, cells was less efficient than in AGMK cells. Whereas Ad2+ ND1 and Ad2+ ND4 formed plaques with the same efficiency on HeLa and AGMK cells, Ad2+ ND2 formed 10-fold less plaques on AGMK cells than on HeLa cells. Ad2 showed at least a 1,000-fold difference in plaque efficiency between HeLa and AGMK cells (Table 1). The high titer stocks of Ad2+ ND3 and Ad2+ ND5 up to dilutions of 10-3 produced strong cytopathic effect (CPE) in the entire CV, monolayer, preventing the detection of plaques. Neither CPE nor plaques were observed at dilutions of 10-'. Ad2 also produced total CPE on CV1 cells up to a dilution of 10-', but a small number of plaques at a dilution of 10'. This observed CPE is presumably caused by a high concentration of free pentons in these high titer stocks and can be eliminated by trypsin treatment (9). Since in our experiments trypsin treatment of the high titer stocks was not performed, the titers of 10' PFU/ml of Ad2+ ND3 and Ad2+ ND5 on CV, cells only represent maximum estimates. A similar CPE was produced by the high titer Ad2 stock when assayed on AGMK cells. In this case, CPE was observed up to a dilution of 10-'. It is likely, therefore, that the Ad2 titer of 106 PFU/ml on AGMJK cells represents an overestimation. The titers of Ad2+ ND3 and Ad2+ ND5 stocks were not determined on AGMK. Infection and labeling of cells. For labeling experiments, 2 x 108 HeLa S3 cells were seeded on 3-cm plastic petri dishes (NUNC, Denmark) and infected 24 h later with 0.2 ml of virus. After an adsorption period of 1 h, 2 ml of growth medium, DMEM containing 10% calf serum, was added per dish. For labeling cells, the culture medium was changed to labeling medium containing 2.5% of the normal level of amino acids, 5% calf serum, and 20 ACi of "4C-labeled L-amino acid mixture per ml. A 0.6-ml amount of labeling medium was added per dish. After labeling, the cell layer was washed three times with DMEM. The cells were lysed on the dish with 0.2 ml of electrophoresis sample buffer (0.0625 M Tris, pH 6.8; 3% sodium dodecyl sulfate [SDS]; 5% 2-mercaptoetha-
nol; 10% glycerol; 0.001% bromphenol blue; 2 mM phenylmethylsulfonyl fluoride). Glycerol and bromphenol blue were omitted until after boiling. The lysates were sonically treated for 10 s with a Branson sonifier at position 4 and heated for 2 min in a boiling water bath. Polyacrylamide gel electrophoresis and scintillation autography (fluorography). The polyacrylamide gel system of Laemmli and Maizel as described by Laemmli (19) was employed, except that the gels were formed as slabs 1 mm thick and 9.5 cm long between glass plates. The general apparatus and methodology for gel electrophoresis have been described elsewhere (29). The ratio of acrylamide to bisacrylamide was 30 to 0.8. A 10-ul volume of cell lysate in electrophoresis sample buffer, containing 5 to 10 ,g of protein and approximately 20,000 to 100,000 counts/ min, were loaded per slot. Electrophoresis was performed at a constant current of 10 mA. After electrophoresis, the gels were prepared for fluorography, which is 10 times more sensitive than conventional autoradiography, as described by Bonner and Laskey (2). Briefly, the gels were dehydrated in dimethyl sulfoxide, soaked in a solution of 2,5-diphenyloxazole in dimethyl sulfoxide, immersed in water to remove dimethyl sulfoxide, dried and exposed to medical X-ray film (Kodak, RP Royal X-omat, RP/R54) at -70 C. The purity of the chemicals used for gel electrophoresis can influence the relative mobility of proteins and the quality of separations of complex mixtures of proteins (36). Therefore, we list the source of the reagents used in this study: acrylamide (Eastman 5521) was recrystallized from chloroform; bisacrylamide (Eastman 8383); N,N,N',N'-tetramethylethylenediamine (Eastman 8178); glycine (Eastman 445); SDS (BDH, specially pure, 30176); Tris (Trizma base, reagent grade, T-1503); ammonium persulfate (Baker 0762); and 2-mercaptoethanol (Eastman 4196). The molecular weight of proteins was determined by comparing their mobility on SDS polyacrylamide gels with proteins of known molecular weight. The following marker proteins were used: Ad2 proteins as characterized by Maizel et al. (30), Ishibashi and Maizel (14), and Everitt et al. (7); ,B-galactosidase of Escherichia coli (135,000 daltons); bovine serum albumin (68,000 daltons); ovalbumin (43,000 daltons); chymotrypsinogen (25,700 daltons); and cytochrome c from horse heart (12,270 daltons). Labeling and purification of virions. HeLa S3
TABLE 1. Plaque-forming ability of Ad2-SV40 hybrid viruses
Infectivity (PFU/ml)
Stock prepared reae
Virus HeLa
Ad2+ ND1 Ad2+ ND2 Ad2+ND3 Ad2+ ND4 Ad2+ND5 Ad2
aND, Not done.
9.2 2.4 4.5 6.9 1.7 3.5
x x x x x x
106 10' 109 106
1010 109
CV,
AGMK
7.0 x 10' 1.2 x 102
JOURNAL OF VIROLOGY, Nov. 1975, p. 1236-1247 Copyright © 1975 American Society for Microbiology
Simian Virus 40-Specific Proteins in HeLa Cells Infected with Nondefective Adenovirus 2-Simian Virus 40 Hybrid Viruses GERNOT WALTER* AND HAWLEY MARTIN
Tumor Virology Laboratory, The Salk Institute, Post Office Box 1809, San Diego, California 92112 Received for publication 17 June 1975
The synthesis of simian virus 40 (SV40)-specific proteins in HeLa cells infected with the nondefective adenovirus 2 (Ad2)-SV40 hybrid viruses, Ad2+ ND2, Ad2+ ND3, Ad2+ ND4, and Ad2+ ND5, was investigated. Infected-cell proteins were labeled with radioactive amino acids late after infection, when host protein synthesis was shut off, and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. All polypeptides normally seen in Ad2infected cells were found in cells infected by the hybrid viruses. In addition to the Ad2-specific proteins, cells infected with Ad2+ ND2 contain two SV40-specific proteins with apparent molecular weights of 42,000 and 56,000, cells infected with Ad2+ ND4 contain one protein with an apparent molecular weight of 56,000, and cells infected with Ad2+ ND5 contain one protein with an apparent molecular weight of 42,000. Cells infected with Ad2+ ND3 do not contain detectable amounts of proteins not seen during Ad2 infection. Pulse-chase experiments demonstrate that the SV40-specific proteins induced by Ad2+ ND2, Ad2+ ND4, and Ad2+ ND5 are metabolically unstable. These proteins are not present in purified virions. Two nonstructural Ad2-specific proteins have been demonstrated in Ad2 and hybrid virus-infected cells which have a smaller apparent molecular weight after a short pulse than after a pulse followed by a chase. The molecular weight increase during the chase may be caused by the addition of carbohydrate to a polypeptide backbone.
The expression of the simian virus 40 (SV40) genome during productive infection occurs in two phases. The early phase is characterized by the induction of the SV40-specific antigens, T (13), tumor-specific transplantation antigen (TSTA) (8), and U (26), and by the induction of cellular DNA synthesis (10, 40). The late phase begins with the synthesis of viral DNA and structural proteins. Little is known about the chemical and biological properties of the early antigens, T, TSTA, and U. They could be either proteins coded by the SV40 genome (early proteins) or, alternatively, cellular products induced or modified by the early viral proteins. The major obstacle to the detection and characterization of early SV40 proteins and early antigens is the continuation of host protein synthesis in SV40infected cells. At present, no methods are available for selectively inhibiting host protein synthesis and making the clear detection of early proteins possible. The nondefective adenovirus 2 (Ad2)-SV40 hybrid viruses, isolated by Lewis and co-workers, are a useful tool for the study of early SV40 proteins. They have the following properties:
each of the five nondefective hybrid viruses, Ad2+ ND1 (24), Ad2+ ND2, Ad2+ ND3, Ad2+ ND4, and Ad2+ ND5 (25), contains a single continuous segment of SV40 DNA covalently inserted at the same site in the Ad2 genome (3, 12, 16, 21, 32). The SV40 DNA segments differ in size: Ad2+ ND1 contains 17 to 20%; Ad2+ ND2, 32 to 36.5%; Ad2+ ND3, 7%; Ad2+ ND4, 43 to 48%; and Ad2+ ND5, 28% of the SV40 genome (16, 20, 31, 32). The segments are completely overlapping and colinear with SV40 DNA (16, 20) (Fig. 1). They have a common end point which is located in the Haemophilus influenzae restriction endonuclease fragment G of SV40 DNA and is 0.11 map units of the SV40 genome away from the cleavage site of EcoRl restriction endonuclease (16, 20, 31, 32). All SV40 DNA segments contain at their common end point a major fraction of the Hin G fragment (20) which is located in the late region of the SV40 genome. During productive infection with the nondefective hybrid viruses, the SV40 DNA segment contained in each hybrid virus is fully transcribed (18, 23). The transcripts are complementary to the minus (early) strand of SV40 DNA and consist of both early and
1236
VOL. 16, 1975
SV40-SPECIFIC PROTEINS
Ad2+ND
U U
Ad2+ ND2
1237
.
TST A
Ad2+ND3 U
TSTA
T
Ad2 ND5
Hin F
J
G
B
|
I
Late Regi
0
0.1
Restriction Endonuclease Cleavage Map C HI A D
Early Region
0.2
0.3
E
I
I
K F
Late Region
0.4 0.5 0.6 0.7 0.8 FRACTIONAL LENGTH OF SV40 DNA
0.9
1.0
FIG. 1. SV40 DNA segments in nondefective Ad2-SV40 hybrid viruses in relation to the Haemophilus influenza restriction endonuclease cleavage map of SV40. The position of the early and late gene regions in SV40 DNA is derived from the results of Khoury et al. (17) and Dhar et al. (5). Arrows indicate the direction of transcription. The dotted lines indicate the degree of variation in the length of the SV40 DNA segments as determined by different investigators (16, 20, 31, 32). The Haemophilus influenza cleavage map is derived from Danna et al. (4).
antilate sequences (18). Ad2+ ND4, containing the largest segment of the SV40 genome, induces the synthesis of three SV40-specific antigens: T, TSTA, and U (25, 27). Ad2+ ND2 induces TSTA and U, and Ad2+ ND1 induces only U antigen (25-27). The SV40 DNA regions in these hybrid viruses, which determine the expression of the early SV40 antigens, have been mapped on the SV40 genome (16, 20). The expression of the SV40 information in the nondefective hybrid viruses is controlled by the Ad2 genome and takes place at a late time during productive infection, that is, after hybrid viral DNA synthesis has been initiated (23). It is this property of the hybrid viruses which facilitates the identification and characterization of early SV40 proteins, since late during infection host protein synthesis is shut off by the adenovirus genome. As a consequence, it may be possible to detect early SV40 proteins or SV40-specific antigens in hybrid virus-infected cells by labeling with radioactive amino acids late in the infection cycle and then analyzing the labeled proteins by gel electrophoresis and autoradiography. It has recently been shown that HeLa cells infected with Ad2+ ND1 synthesize a polypeptide of molecular weight 28,000 which is not present in Ad2-infected cells (9, 28). In this report, we demonstrate the presence of specific proteins in cells infected by Ad2+ ND2, Ad2+ ND4, and Ad2+ ND5. We have provisionally designated these proteins as "SV40 specific," since their expression is caused by the SV40 information contained in the hybrid virus genomes.
MATERIALS AND METHODS Cells. HeLa S3 cells were grown in suspension in Eagle minimum essential medium supplemented with 5% calf serum or in monolayer in Dulbecco's modification of Eagle medium (DMEM) with 10% calf serum. HeLa S3 cells were only used for labeling experiments. For adenovirus plaque assays, a different type of HeLa cell with a flatter morphology was used. These cells were obtained from the Cold Spring Harbor Laboratory and grown in DMEM supplemented with 10% calf serum. CV1 cells, a line of African green monkey kidney cells (AGMK), were obtained from the American Type Culture Collection and grown in DMEM supplemented with 10% fetal calf serum. Primary AGMK cells were purchased from Microbiological Associates, Inc., and grown in DMEM with 10% fetal calf serum. Viruses. Seed stocks of nondefective Ad2-SV40 hybrid viruses Ad2+ ND1, Ad2+ ND2, Ad2+ ND3, Ad2+ ND4, and Ad2+ ND5 were obtained from A. M. Lewis, Jr. Stocks of Ad2+ ND1, Ad2+ ND2, and Ad2+ ND4 were prepared in CV, and HeLa S3 cells. Stocks of Ad2, Ad2+ ND3, and Ad2+ ND5 were prepared in HeLa S3 cells. Hybrid stocks of Ad2+ ND1, Ad2+ ND2, Ad2+ ND3, and Ad2+ ND5 were shown to be free of adeno-associated viruses by cesium chloride density centrifugation, high multiplicity infections, and heat sensitivity assays which will be described elsewhere. All stocks of Ad2+ ND4, however, were contaminated with adeno-associated virus. Plaque titrations were routinely performed on HeLa and CVl cells. In one experiment, primary AGMK cells were used to assay Ad2, Ad2+ ND1, Ad2+ ND2, and Ad2+ ND4. The assays were performed as described by Williams (41). The overlay medium for HeLa cells contained 0.6% agar (Difco) and 0.9% agar for CV, and AGMK cells. After incubation for 3 to 4
1238
J. VIROL.
WALTER AND MARTIN
days, 2 ml of additional overlay medium was added. Overlay medium containing neutral red and 0.9% agar was added 6 to 7 days after infection of HeLa cells, and 8 to 10 days after infection of CV, and AGMK cells. MgCl2 was omitted from the overlay medium when plaque assays were performed on CVl and AGMK cells. The plaque titers of virus stocks prepared in HeLa or CV, cells are showh in Table 1. Plaque formation in CV, cells was less efficient than in AGMK cells. Whereas Ad2+ ND1 and Ad2+ ND4 formed plaques with the same efficiency on HeLa and AGMK cells, Ad2+ ND2 formed 10-fold less plaques on AGMK cells than on HeLa cells. Ad2 showed at least a 1,000-fold difference in plaque efficiency between HeLa and AGMK cells (Table 1). The high titer stocks of Ad2+ ND3 and Ad2+ ND5 up to dilutions of 10-3 produced strong cytopathic effect (CPE) in the entire CV, monolayer, preventing the detection of plaques. Neither CPE nor plaques were observed at dilutions of 10-'. Ad2 also produced total CPE on CV1 cells up to a dilution of 10-', but a small number of plaques at a dilution of 10'. This observed CPE is presumably caused by a high concentration of free pentons in these high titer stocks and can be eliminated by trypsin treatment (9). Since in our experiments trypsin treatment of the high titer stocks was not performed, the titers of 10' PFU/ml of Ad2+ ND3 and Ad2+ ND5 on CV, cells only represent maximum estimates. A similar CPE was produced by the high titer Ad2 stock when assayed on AGMK cells. In this case, CPE was observed up to a dilution of 10-'. It is likely, therefore, that the Ad2 titer of 106 PFU/ml on AGMJK cells represents an overestimation. The titers of Ad2+ ND3 and Ad2+ ND5 stocks were not determined on AGMK. Infection and labeling of cells. For labeling experiments, 2 x 108 HeLa S3 cells were seeded on 3-cm plastic petri dishes (NUNC, Denmark) and infected 24 h later with 0.2 ml of virus. After an adsorption period of 1 h, 2 ml of growth medium, DMEM containing 10% calf serum, was added per dish. For labeling cells, the culture medium was changed to labeling medium containing 2.5% of the normal level of amino acids, 5% calf serum, and 20 ACi of "4C-labeled L-amino acid mixture per ml. A 0.6-ml amount of labeling medium was added per dish. After labeling, the cell layer was washed three times with DMEM. The cells were lysed on the dish with 0.2 ml of electrophoresis sample buffer (0.0625 M Tris, pH 6.8; 3% sodium dodecyl sulfate [SDS]; 5% 2-mercaptoetha-
nol; 10% glycerol; 0.001% bromphenol blue; 2 mM phenylmethylsulfonyl fluoride). Glycerol and bromphenol blue were omitted until after boiling. The lysates were sonically treated for 10 s with a Branson sonifier at position 4 and heated for 2 min in a boiling water bath. Polyacrylamide gel electrophoresis and scintillation autography (fluorography). The polyacrylamide gel system of Laemmli and Maizel as described by Laemmli (19) was employed, except that the gels were formed as slabs 1 mm thick and 9.5 cm long between glass plates. The general apparatus and methodology for gel electrophoresis have been described elsewhere (29). The ratio of acrylamide to bisacrylamide was 30 to 0.8. A 10-ul volume of cell lysate in electrophoresis sample buffer, containing 5 to 10 ,g of protein and approximately 20,000 to 100,000 counts/ min, were loaded per slot. Electrophoresis was performed at a constant current of 10 mA. After electrophoresis, the gels were prepared for fluorography, which is 10 times more sensitive than conventional autoradiography, as described by Bonner and Laskey (2). Briefly, the gels were dehydrated in dimethyl sulfoxide, soaked in a solution of 2,5-diphenyloxazole in dimethyl sulfoxide, immersed in water to remove dimethyl sulfoxide, dried and exposed to medical X-ray film (Kodak, RP Royal X-omat, RP/R54) at -70 C. The purity of the chemicals used for gel electrophoresis can influence the relative mobility of proteins and the quality of separations of complex mixtures of proteins (36). Therefore, we list the source of the reagents used in this study: acrylamide (Eastman 5521) was recrystallized from chloroform; bisacrylamide (Eastman 8383); N,N,N',N'-tetramethylethylenediamine (Eastman 8178); glycine (Eastman 445); SDS (BDH, specially pure, 30176); Tris (Trizma base, reagent grade, T-1503); ammonium persulfate (Baker 0762); and 2-mercaptoethanol (Eastman 4196). The molecular weight of proteins was determined by comparing their mobility on SDS polyacrylamide gels with proteins of known molecular weight. The following marker proteins were used: Ad2 proteins as characterized by Maizel et al. (30), Ishibashi and Maizel (14), and Everitt et al. (7); ,B-galactosidase of Escherichia coli (135,000 daltons); bovine serum albumin (68,000 daltons); ovalbumin (43,000 daltons); chymotrypsinogen (25,700 daltons); and cytochrome c from horse heart (12,270 daltons). Labeling and purification of virions. HeLa S3
TABLE 1. Plaque-forming ability of Ad2-SV40 hybrid viruses
Infectivity (PFU/ml)
Stock prepared reae
Virus HeLa
Ad2+ ND1 Ad2+ ND2 Ad2+ND3 Ad2+ ND4 Ad2+ND5 Ad2
aND, Not done.
9.2 2.4 4.5 6.9 1.7 3.5
x x x x x x
106 10' 109 106
1010 109
CV,
AGMK
7.0 x 10' 1.2 x 102
