Vol. 28, No. 1
JOURNAL OF VIROLOGY, Oct. 1978, p. 314-323 0022-538X/78/0028-0314$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Evidence for an Adenovirus Type 2-Coded Early Glycoprotein YUN-HUA JENG, WILLIAM S. M. WOLD,* AND MAURICE GREEN Institute for Molecular Virology, St. Louis University School of Medicine, St. Louis, Missouri 63110
Received for publication 28 February 1978
We have identified an adenovirus type 2 (Ad2)-induced early glycopolypeptide with an apparent molecular weight of 20,000 to 21,000 (20/21K), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 20/21K polypeptide could be labeled in vivo with [3H]glucosamine. [35S]methionine- and [3H]glucosamine-labeled 20/21K polypeptides bound to concanavalin A-Sepharose columns and were eluted with 0.2 M methyl-a-D-mannoside. The pulse-labeled polypeptide appeared as a sharp band with an apparent molecular weight of 21K, but after a chase it converted to multiple bands with an average molecular weight of 20K. This variability in electrophoretic mobility is consistent with glycosylation or deglycosylation of the 20/21K polypeptide. Analysis of the pulse and pulsechase-labeled forms by using partial proteolysis indicated that the polypeptides were highly related chemically, but not identical. Most of the 20/21K polypeptide is localized in the cytoplasm fraction of infected cells lysed by Nonidet P-40. The 20/21K polypeptide and a 44K polypeptide, labeled with [35S]methionine or [3H]glucosamine in Ad2-infected human cells, were precipitated by a rat antiserum against an Ad2-transformed rat cell line (T2C4), but not by antisera against three other Ad2-transformed rat cell lines, or by serum from nonimmune rats. The partial proteolysis patterns of the 20/21K and the 44K polypeptides were indistinguishable, indicating that the two polypeptides are highly related, and suggesting that the 44K polypeptide might be a dimer of the 20/21K polypeptide. The 20/21K polypeptide was also induced in Ad2-early infected monkey and hamster cells. These results imply that the 20/21K polypeptide is synthesized in Ad2-infected human, monkey, and hamster cells, and in one but not all Ad2transformed rat cells. Thus, the 20/21K polypeptide is probably viral coded rather than cell coded and viral induced.
Polypeptides induced in cells during early stages (i.e., before viral DNA replication) of infection by human adenovirus type 2 (Ad2) are of great interest because they may regulate viral DNA replication, transcription, and cell transformation (34). We have attempted to identify Ad2-induced early polypeptides by labeling infected and mock-infected cells with [35S]methionine, subjecting the protein extracts to electrophoresis through sodium dodecyl sulfate-polyacrylamide slab gels (SDS-PAGE), and comparing the infected and mock-infected polypeptide bands seen in autoradiograms. We have also identified Ad2 early polypeptides by immunoprecipitation, using antisera against Ad2-transformed rodent cells. Prominent early polypeptide bands with the following approximate apparent molecular weights have been detected: 73,000 (73K), 53K, 21K, 19K, 15K, 11.5K, and 1lK (8, 10, 14). Additional minor polypeptide bands of about 17.5K, 15.5K, 13.5K, 13K, 12K, 8.8K, and 8.3K can be observed in some gels, or can be immunoprecipitated by antisera against certain lines of Ad2-transformed rat cells (Wold
and Green, unpublished data). Similar in vivolabeled polypeptides have been identified by other laboratories (12, 23). Similar polypeptides have also been observed by cell-free translation of polyribosomal RNA from Ad2-early infected cells and by immunoprecipitation, using a rabbit antiserum against early infected HeLa cells (23). Six polypeptides (72K, 44 to 50K, 19K, 15.5K, 15K, and 11K) have been mapped by cell-free translation of early mRNA purified by hybridization to restriction endonuclease DNA fragments (18). Relatively little is known about the chemistry of these polypeptides or their biological functions. The 73K polypeptide is a phosphoprotein (14, 17, 19, 22) that is viral coded (9, 18, 30), that binds to single-stranded DNA (24, 27, 29), and that apparently functions in viral DNA replication (28, 31). This protein has been purified to homogeneity and partially characterized (19, 27). The 53K (8, 16) and the 15K polypeptides (8) are candidate transformation proteins because they are immunoprecipitated by antisera against Ad2- or Ad5-transformed rodent cells. 314
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Genes coding polypeptides of 44 to 50K and 15K polypeptides have been mapped in the transforming region of the Ad2 genome (18). The 11K polypeptide is localized mainly in the nuclear fraction (3, 23; our unpublished data). The 73K, 21K, 15K, 11K, and possibly 8.3K polypeptides are present in a soluble complex that synthesizes Ad2 DNA, suggesting a possible role for these in viral DNA replication (21). Ishibashi and Maizel (13) reported that an Ad2-induced early 19K polypeptide could be labeled in vivo with [3H]glucosamine. In this report, we present evidence that our 21K polypeptide is an Ad2-coded early glycopolypeptide, in that it can be labeled with [3H]glucosamine, it binds to concanavalin A (ConA)-Sepharose, its mobility changes in pulse-chase experiments, it is immunoprecipitated by antisera to Ad2transformed cells (T2C4), and it is synthesized in Ad2-infected monkey and hamster cells. MATERIALS AND METHODS Chemicals. L-['S]methionine (Met) (400 Ci/ mmol) and [3H]glucosamine (20 Ci/mmol) were purchased from New England Nuclear Corp.; 1-/i-Darabinofuranosylcytosine (ara-C), ConA-Sepharose, and methyl-a-D-mannoside were purchased from Sigma Chemical Co.; acrylamide was purchased from Eastman Kodak Co.; Nonidet P-40 was purchased from Shell Chemical Corp.; N,N-methlenebisacrylamide, N,N,N',N'-tetramethyladenediamine, and ammonium persulfate were purchased from Bio-Rad Laboratories; and Staphylococcus aureus V8 protease was purchased from Miles Laboratories, Inc. Cell culture and virus infection. Suspension cultures of human KB cells were grown in Eagle minimal essential medium (MEM) containing 5% horse serum. Cells at a concentration of 6 x 106/ml were infected with 100 PFU of Ad2 (strain 38-2) per cell in medium without horse serum. After 1 h of adsorption, cells were suspended at a concentration of 3.5 x 105/ml in medium with 5% horse serum. Cyclohexamide (CH; 25 ,Lg/ml) was added to some cultures at 1 h postinfection (p.i.). Ara-C (20 ,ug/ml) was added at 4 h p.i. At 9 h p.i., cells were washed and suspended either in warm Met-free medium or medium with 10% the normal glucose concentration; both media contained 5% horse serum and 20 ,ug of ara-C per ml. Cells, at a concentration of 3.5 x 105/ml, were labeled with [3S]Met (10 t,Ci/ml) or [3H]glucosamine (10 liCi/ml) for various times. At the end of the labeling period, cells were centrifuged and washed twice with cold phosphatebuffered saline lacking Ca2' and Mg2e. Mock-infected cells were similarly labeled, except that virus was not added. CV-1 (monkey) cell monolayers were maintained in 75-cm2 plastic flasks containing MEM with 5% calf serum, and in an atmosphere of 5% CO2. Nearly confluent monolayers were infected (or mock infected) with Ad2 in MEM without serum. After 2 h of adsorbtion, MEM with 5% calf serum (with or without 25 yg of CH per ml) was added to 25 ml, and incubation continued. Ara-C (20 ,ug/ml) was added at 4 h p.i. Cells
Ad2 EARLY GLYCOPROTEIN
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were labeled from 9 to 20 h p.i. with [3S]Met in Metfree MEM containing 20 jig of ara-C per ml. Cell fractionation. Infected or mock-infected cells labeled with [3S]Met were washed and then suspended in isotonic high pH buffer (33). All steps were at 0 to 4°C, and all buffers contained 1 to 2 mM phenylmethylsulfonyl fluoride. Cells were lysed by using 0.5% Nonidet P-40 for 5 min. Lysis was monitored by phase-contrast microscopy until 99% complete. Nuclei were pelleted by centrifugation at 200 x g for 3 min. The cytoplasmic supernatant was obtained by centrifugation at 12,000 x g for 20 min to remove mitochondria. Isolated nuclei were resuspended in phosphate-buffered saline, and treated with 0.86% Tween 40 and 0.43% sodium deoxycholate to remove outer and inner nuclear membranes (11). The nucleardetergent suspension was vigorously mixed in a Vortex mixer for 30 s, and the nuclei were collected by centrifugation at 500 x g for 3 min. The pellet was resuspended in cold phosphate-buffered saline with the addition of 1% Triton X-100 and disrupted by sonic treatment for 10 min in a Raytheon sonic oscillator. The nucleoplasm supernatant and nuclear pellet were separated by centrifugation at 12,000 x g for 20 min. Portions of subfractionated samples were precipitated with 10% cold trichloroacetic acid for analysis of labeled polypeptides. ConA-Sepharose column chromatography. Affinity chromatography of glycoproteins on ConASepharose columns was carried out as described by Stohlman et al. (26), with the following modifications. All steps were at 4°C. The column (1.5 x 25 cm) was first equilibrated with 10 bed volumes of 50 mM Trishydrochloride (pH 7.5), 50 mM NaCl, 1 mM MnCl2, and 2 mM phenylmethylsulfonyl fluoride (buffer A) containing 0.2 M methyl a-D-mannoside, followed by buffer A containing 0.2% Nonidet P-40 and 0.2% sodium deoxycholate (buffer A-detergent). The cytoplasmic protein fraction (no CH pretreatment) was diluted 25-fold with buffer A, and sodium deoxycholate was added to a final concentration of 0.2%. The sample was disrupted by sonic treatment (see above), centrifuged at 12,000 x g for 20 min, dialyzed extensively against buffer A-detergent, and loaded onto the ConASepharose column. The column was washed with buffer A-detergent until no significant radioactive material was eluted. The adsorbed proteins were eluted with buffer A containing 0.2 M methyl-a-D-mannoside. The column flow-through, wash, and methyl-a-D-mannoside-eluted fractions were concentrated by precipitation with cold 10% trichloroacetic acid and washed with acetone to remove trichloroacetic acid and detergent. Radioimmunoprecipitation of proteins. T2C4 cells, from an Ad2-transformed rat cell line (6), were obtained from P. H. Gallimore. Antiserum was prepared in rats against extracts of T2C4 cells, and the immunoglobulin G (IgG) fraction was purified (8). Infected and mock-infected labeled cell extracts were assayed for T2C4-specific polypeptides by the doubleantibody immunoprecipitation procedure, as described earlier (8). Portions containing equal counts per minute from infected and mock-infected samples were incubated with T2C4 or nonimmune rat IgG at 4°C for 18 h. Then goat serum anti-rat IgG was added, and the sample was incubated for 2 h at 37°C. Precipitates
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were collected by centrifugation for 90 s at 23,000 x g in a Beckman 152 microfuge, washed three times with phosphate-buffered saline containing 0.5 M urea, 0.5% Nonidet P-40, and 1% sodium deoxycholate, suspended in an equal volume of gel electrophoresis sample buffer without 2-mercapthoethanol, and analyzed by SDSPAGE.
SDS-PAGE and autoradiography. SDS-PAGE was carried out as described elsewhere (14). Unless stated otherwise, 8 to 21% gradient acrylamide 10-cm slab gels were used. Radiofluorography as described by Bonner and Laskey (1) was used to detect [1H]labeled polypeptides. The absorbance of bands in autoradiograms was monitored by using a Joyce-Loebel densitometer. The area under the absorbance tracing was calculated as the quantitative estimation of the intensity of bands. Partial proteolysis. The relationship between [;35S]Met-labeled 44K and 20/21K polypeptides immunopreciptated by T2C4 antiserum was investigated, using the partial proteolysis procedure essentially as described by Cleveland and colleagues (4). The polypeptides immunoprecipitated by T2C4 antiserum were resolved by SDS-PAGE, the gels were dried, and the fluorographs were developed. The 44K and 20/21K bands were cut from the gel, and the gel slices were inserted into wells (4 mm wide, 0.75 mm thick, containing soaking buffer which consisted of: 0.125 M Tris-hydrochloride [pH 6.8], 0.1% SDS, and 1 mm EDTA) of a second slab gel, and hydrated by soaking for 30 min. The proteolysis gel consisted of a 4-cm stacking gel of 9% acrylamide (pH 6.8) and a 5-cm running gel of 17% acrylamide (pH 8.8). S. aureus V8 protease was diluted to 2 mg/ml in soaking buffer containing 10% glycerol and 0.025% bromophenol blue, and 10 Al was added to wells containing the 44K and 20/21K gel slices. Electrophoresis was carried out with the gel apparatus connected to a circulating water bath at 20°C. The sample was subjected to electrophoresis (25 mA per gel) until the bromophenol blue band had almost reached the bottom of the stacking gel. The power was shut off for 30 min to allow the protease to digest the [K5S]Met-labeled polypeptides that had migrated from the gel slices. Electrophoresis was then continued until the dye reached the bottom of the running gel, and the gels were dried and fluorographed.
RESULTS Identification of Ad2-induced early polypeptides and demonstration of electrophoretic mobility changes in a 20/21K polypeptide in pulse-chase experiments. The electropherograph in Fig. 1 illustrates [35S]Metlabeled Ad2-infected and mock-infected cell polypeptides. In this and all other experiments, ara-C (20 ,ig/ml) was added at 4 h p.i. to inhibit viral DNA replication. Therefore, cells were in early stages of infection. Lanes A to H in Fig. 1 show polypeptides from cells incubated with CH before labeling, a procedure that enhances the synthesis of Ad2-specific early polypeptides relative to host polypeptides (10). Six Ad2-induced
J. VIROL.
polypeptides are clearly visible: DBP (the singlestranded DNA binding protein of about 73K daltons), 20/21K, 19K, 15K, 11.5K, and 11K. Lanes A and B and lanes C and D show infected and mock-infected polypeptides labeled 9 to 10 and 9 to 12 h p.i., respectively. In these lanes (A and C), the 20/21K polypeptide appeared as a fairly sharp band of about 21K daltons. However, when cells were labeled for long periods, e.g., 9 to 24 h p.i. (lanes E and F), or 9 to 12 h p.i. followed by a 12-h chase in complete MEM (lanes G and H), the intensity of the 21K band decreased, and new bands of roughly 20K appeared. Lanes I to L illustrate polypeptides from cells labeled without preincubation with CH (only DBP and the 20/21K bands are visible). Again, a distinct 21K band was apparent in cell extracts labeled 9 to 12 h p.i. (lanes I and J), but this band became reduced in intensity and seemed to increase in mobility in cells labeled for 9 to 24 h p.i. (lanes K and L). These results provide initial evidence that the 20/21K polypeptide was modified after translation. Since glycosylation of polypeptides affects their SDSPAGE mobility, we concentrated on the 20/21K polypeptide as a candidate glycopolypeptide. Figure 2 illustrates the analysis of the pulse (30 min) 21K polypeptide and the pulse-chase (30 min followed by a 15-h chase) 19K to 20K forms of this polypeptide by the partial proteolysis procedure. Lane A shows the pulse-labeled 21K polypeptide before chromatography on ConA-Sepharose, and lanes B to J show pulseand pulse-chase-labeled forms (purified on ConA-Sepharose). In this experiment, the chased polypeptide formed a broad band ranging from apparent molecular weight of 19K to 20K. Lanes C, F, and I represent the 20K region, and lanes D, G, and J represent the 19K region. Most of the partial proteolysis polypeptides of the pulse and pulse-chase forms clearly coincide, confirming that the 21K and 20K are highly related. However, the polypeptides are not identical, because the chased forms, especially 19K, contained at least one band (e.g., second from bottom in lanes D and G) not found among the proteolysis products of the pulse form. Note that in lanes A to G some of the polypeptide has spontaneously "polymerized" to 44K. This was a reproducible phenomenon. Cellular localization of the 20/21K polypeptide. To further analyze the 20/21K polypeptide, we established which cellular fraction contained the majority of the polypeptide. Ad2infected and mock-infected cells were labeled with [35S]Met from 9 to 12 h p.i., lysed with 0.5% Nonidet P-40, and then fractionated into nuclear pellet, nuclear membrane, nucleoplasm, and cytoplasm. The vast majority of 20/21K poly-
Ad2 EARLY GLYCOPROTEIN
VOL. 28, 1978
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IIK FIG. 1. Identification of Ad2-induced early polypeptides with and without CH pretreatment, and pulsechase experiments demonstrating mobility changes in a 20/21Kpolypeptide. Ad2-infected and mock-infected cells were prepared. CH (25 pg/ml) was added to some cultures at 1 h p.i. (lanes A to H). Ara-C (20 pg/ml) was added to all cultures at 4 h p.i.. At 9 h p.i., all cultures were washed and resuspended in warm Met-free medium containing ara-C and then labeled with [3S]Met for various time periods. Protein extracts were prepared and were analyzed by SDS-PAGE. An autoradiograph of a dried 17-cm gel is shown. (A) Infected, labeled 9 to 10 h p.i.; (B) mock infected, labeled 9 to 10 h p.i.; (C, I) infected, labeled 9 to 12 h p.i.; (D, J) mock infected, labeled 9 to 12 h p.i.; (E, K) infected, labeled 9 to 24 h p.i.; (F, L) mock infected, labeled 9 to 24 h p.i.; (G) infected, labeled 9 to 12 h p.i., and chased for 12 h; (H) mock infected, labeled 9 to 12 h p.i., and chased for 12 h.
318
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FIG. 2. Partial proteolysis (S. aureus V8 protease) of pulsed 21K and pulse-chased 35S-labeled 19 to 20K forms of the 20/21K polypeptide. Infected cells were pulse-labeled with [35S]Met for 30 min or for 30 min followed by a 15-h chase. Cytoplasm extracts were prepared and were purified by chromatography on ConASepharose followed by SDS-PAGE. The 21K pulse and 20K or 19K chase polypeptide bands were cut from the gel and analyzed by partial proteolysis as described in the text, using 0.5, 5, or 50 yg of protease per lane. (A) Pulse-labeled 21K polypeptide before chromatography on ConA-Sepharose; (B, E, H) pulse-labeled 21K polypeptide purified on ConA-Sepharose; (C, F, I) pulse-chase-labeled 20K after ConA-Sepharose; (D, G, J) pulse-chase labeled 19Kpolypeptide after ConA-Sepharose. peptide was present in the cytoplasm fraction TABLE 1. Distribution of radioactivity and the 20/21Kpolypeptide in subcellular fractions (Table 1). Labeling of the 20/21K polypeptide with totl 20/21K 20/21K Mock-infected Infected cells 2021
[3H]glucosamine, and immunoprecipitation with antiserum directed against an Ad2transformed rat cell line (T2C4). Infected and mock-infected cells were labeled with [3H]glucosamine to test whether the label was incorporated into the 20/21K polypeptide. As shown in Fig. 3, the only Ad2-specific polypeptide labeled with [3H]glucosamine had about the same electrophoretic mobility as the 20/21K polypeptide (lane B). Antiserum against T2C4 cells precipitated the 'H-labeled 20/21K polypeptides, as well as a second polypeptide of about 44K (lane D). The 20/21K and 44K polypeptides were not precipitated from infected cell extracts by nonimmune rat sera (lane E), or from mockinfected cell extracts by either T2C4 or nonimmune rat sera (lanes F and G). The T2C4 antiserum also immunoprecipitated viral specific early [35S]Met-labeled polypeptides of 53K, 44K, 20/21K, 19K, 18K, 15K, 14.5K, 13.5K, 12K, and 11.5K (Wold and Green, unpublished data). The 20/21K and 44K polypeptides precipitated by the T2C4 antiserum were assayed by the partial proteolysis procedure (4) to test whether they were chemically related. [35S]Metlabeled polypeptides were assayed instead of
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' Total ¢'S counts per minute in the infected whole-cell preparation was 278 x 10', and in the mock-infected preparation was 280 x 10. Infected and mock-infected proteins were resolved by SDS-PAGE, and autoradiograms were developed. The autoradiograms were scanned by using a Joyce-Loebel densitom-
eter to obtain the total area represented by each fraction. These values indicate the percentage of the total scan area represented by the 20/21K polypeptide, minus contribution
from mock-infected polypeptides. ' Calculations are based on a value of 100% for the estimated 20/21K counts per minute in the whole-cell fraction.
["H]glucosamine-labeled polypeptides, because larger amounts of radioactivity could be obtained. [35S]Met-labeled polypeptides immunoprecipitated by the T2C4 antiserum were resolved by SDS-PAGE, and fluorograms were developed. The 20/21K and 44K bands were cut
Ad2 EARLY GLYCOPROTEIN
VOL. 28, 1978
A
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FIG. 3. Identification of polypeptides labeled in vivo with [3H]glucosamine and immunoprecipitated by antiserum against 72C4 cells. Ad2-infected and mock-infected cells were labeled with [3H]glucosamine. Total cell extracts were prepared and immunoprecipitated by using T2C4 IgG and nonimmune rat IgG. Equal volumes of each precipitate were analyzed by SDS-PAGE. (A) Marker polypeptides: [3S]Met-labeled, early infected polypeptides prepared by the CH enhancement procedure. (B-G), [3H]glucosamine-labeled extracts. (B) Infected, before immunoprecipitation; (C) mock infected, before immunoprecipitation; (D) infected extract versus T2C4 IgG; (E) infected extract versus nonimmune rat IgG; (F) mock-infected extract versus 72C4 IgG; (G) mock-infected extract versus nonimmune rat IgG.
from the dried gel and analyzed by partial proteolysis as described above. The partial proteolysis slab gel is shown in Fig. 4. The same polypeptide bands were generated by protease digestion of the 20/21K and 44K polypeptides, indicating that they are highly related chemically. Proteolysis of the 20/21K and 44K polypeptides by using other protease concentrations also indicated that they are highly related. Binding of the 20/21K polypeptide to ConA-Sepharose and its elution by methyla-D-mannoside. To obtain further evidence that the 20/21K polypeptide is a glycopolypeptide, 35S-labeled protein extracts were subjected to affinity chromatography on columns of ConASepharose. Only certain carbohydrate moieties (a-D-glucopyranosides, and a-N-acetyl-D-glucosaminides) bind strongly to ConA (20), so that if the 20/21K polypeptide contains these sugars it should bind to the ConA-Sepharose column. Infected and mock-infected cells were labeled with [35S]Met for 15 h, and cell cytoplasms were prepared and then loaded onto ConA-Sepharose
columns. The columns were washed and then eluted with 0.2 M methyl-a-D-mannoside. The column flow-through, wash, and eluted fractions were analyzed by SDS-PAGE. Only the mannoside-eluted infected cell fraction (lane F) contained detectable 20/21K polypeptide (Fig. 5). These results suggest that the 20/21K polypeptide contains sugar residues specific for ConA. The 20/21K polypeptide is synthesized in Ad2-infected monkey cells. The immunoprecipitation of both [3H]glucosamine and [35S]Met-labeled 20/21K polypeptide by the T2C4 antiserum suggests that the 21K polypeptide is viral coded rather than cell coded and viral induced. The 20/21K polypeptide (and the DBP, 11K, 8.8K, 8.3K polypeptides) was synthesized in Ad2-early infected monkey (CV-1) cells (Fig. 6). The 21K, DBP, and 11K were also synthesized in Ad2-early infected hamster cells (not shown). These results provide further evidence that the 21K, DBP, and 11K polypeptides are viral coded.
320
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DISCUSSION Our results provide strong evidence that the Ad2-induced 20/21K polypeptide is a glycopolypeptide. The polypeptide can be labeled with [3H]glucosamine, and it binds to ConA-Sepharose columns and is eluted with methyl-a-Dmannoside. When labeled with [35S]Met for short periods (30 min to 3 h), it appears as a single, relatively sharp band with an apparent molecular weight of about 21K. However, if labeled for long periods, or if pulse-labeled and then chased, the polypeptide appears as multiple bands with apparent molecular weights of 20K to 21K. Partial proteolysis data indicate that the pulse-labeled 21K and the pulse-chase-labeled 20K polypeptides are highly related, although not identical. Both pulse (30 min) and pulse (30 min)-chase (12 h in complete MEM) forms of [35S]-labeled 20/21K polypeptide bind to ConASepharose, and therefore apparently are glycosylated. The multiple bands of the chased
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20/21K polypeptide may indicate heterogeneity in sugar content, because glycosylation affects (usually decreases) the SDS-PAGE mobility of polypeptides. We cannot exclude the possibility that there is another distinct polypeptide of
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FIG. 5. ConA-Sepharose column chromatography: identification of [3S]Met-labeled Ad2-infected and mock-infected polypeptides in the flow-through, wash, and methyl-a-D-mannoside eluted fractions. Ad2infected and mock-infected cells were labeled with [3S]Met from 9 to 24 h p.i. The cytoplasm fraction was prepared and chromatographed on ConA-Sepharose. Appropriate column fractions were pooled and analyzed by SDS-PAGE. (A) Marker polypeptides: Ad2-infected KB cells extracts labeled with [3SJMet by the CH enhancement procedure; (B) infected, flow-through fraction; (C) mock infected, flow-through fraction; (D) infected, wash fraction; (E) mock infected, wash fraction; (F) infected, methyl a-D-mannoside eluate; (G) mock infected, methyl-a-D-mannoside eluate.
VOL. 28, 1978
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about 20K to 21K that cannot be detected in these single-dimension gels. Our results also strongly suggest that the 20/21K polypeptide is coded by an early Ad2 gene, and is not a cell coded polypeptide induced by Ad2. Both [3H]glucosamine and [35S]Metlabeled 20/21K polypeptide were immunoprecipitated by rat antiserum against T2C4 cells. T2C4 is a line of Ad2-transformed rat cells (6) that contains all four of the Ad2 early gene blocks (7) and synthesizes RNA derived from all four blocks (5). The polypeptide was not precipitated by nonimmune rat serum, or by rat antisera against three other Ad2-transformed rat cells (not shown). Therefore, it is probable that the Ad2 sequences present in T2C4 cells (but not in the other 3 Ad2-transformed rat lines) synthesize the 20/21K polypeptide. A 20/21K polypeptide was also induced in Ad2-early infected monkey and hamster cells. These results suggest that the 20/21K polypeptide is viral coded, because it is improbable (but not impossible) that a cell-coded 20/21K polypeptide would be synthesized by Ad2-infected human, monkey, and hamster cells, and by one but not all Ad2-transformed rat cells. We are currently attempting to purify the 20/21K polypeptide for further chemical, physical, and biological characterization. The protein seems to exist in multiple charge forms, because it is present in fractions eluted from ion-exchange resins by salt concentrations of from 10 mM to 0.4 M. Also, it seems to exist in multiplesize forms, as reflected by its behavior on gel filtration columns. An example of this is shown in Fig. 3, where the 20/21K polypeptide spontaneously polymerized to 44K, the 44K spontaneously converted to 21K, and both the 44K and the 20/21K polypeptide polymerized to species of high molecular weight. This type of apparent size and charge heterogeneity is typical of many glycoproteins. We have not attempted to reduce and alkylate the 20/21K polypeptide to determine whether sulfhydral groups are involved in the polymerization of the polypeptide. Chin and Maizel (2) reported that a 35S-labeled polypeptide (E2) was a component of the plasma membrane. E2, apparently, is the same polypeptide that Ishibashi and Maizel (13) reported to be labeled with [3H]glucosamine. The 20/21K polypeptide probably corresponds to E2, because we have seen no indication that any other early protein is glycosylated. Our finding FIG. 6. [35S]Met-labeled polypeptides induced in Ad2-early infected monkey (CV-1) cells, with and without CH (25 pg/ml) pretreatment. (A) Infected, no CH; (B) infected, with CH; (C) mock infected, no CH; (D) mock infected, with CH.
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that the 20/21K polypeptide is located mainly in the cytoplasm of Nonidet-P40-lysed cells would be consistent with the possibility that this polypeptide is a membrane component. However, we have also found the 20/21K polypeptide in a complex purified from lysed nuclei that synthesizes Ad2 DNA (21). The 20/21K polypeptide can also be immunoprecipitated by T2C4 antiserum from the nucleoplasm of cells. Therefore, a nuclear role for this polypeptide is not excluded. Glycoproteins are widely distributed in nature and serve a variety of structural, lubricating, enzymatic, hormonal, and plasma membrane-associated functions (15, 25, 32). The finding that an Ad2-early gene product is a glycoprotein is of interest, especially if it is found to serve a regulatory function. Studies on the 20/21K polypeptide should be of interest not only regarding Ad2 replication, but also the general role of glycosylation in protein function. ACKNOWLEDGMENTS We thank H. Thornton for assistance in cell culture and in preparation of the T2C4 antiserum, and C. Devine for technical assistance. We are grateful to P. H. Gallimore for a gift of the T2C4 cells. This work was supported by Public Health Service grants Al 01725-19 from the National Institute of Allergy and Infectious Diseases and CA 21824-01 from the National Cancer Institute, and by contract NOI CP 43359 from the Virus Cancer Program within the National Cancer Institute. W.S.M.W. was partially supported by a fellowship from the Medical Research Council of Canada. M.G. is the recipient of a Research Career Award (5 K06 AI 04739) from the National Institute of Allergy and Infectious Diseases.
1.
2.
3. 4.
5.
6.
7.
8.
LITERATURE CITED Bonner, W. M., and R. A. Laskey. 1974. A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83-88. Chin, W. W., and J. V. Maizel, Jr. 1976. Polypeptides of adenovirus. VII. Further studies of early polypeptides in vivo and localization of E, and E2A to the cell plasma membrane. Virology 71:518-530. Chin, W. W., and J. V. Maizel, Jr. 1976. The polypeptides of adenovirus. VIII. The enrichment of E3 (11,000) in the nuclear matrix fraction. Virology 76:79-89. Cleveland, D. W., S. G. Fischer, M. W. Kirschner, and U. K. Laemmli. 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252:1102-1106. Flint, S. J., P. H. Gallimore, and P. A. Sharp. 1975. Comparison of viral RNA sequences in adenovirus 2transformed and lytically infected cells. J. Mol. Biol. 96:47-68. Gallimore, P. H. 1974. Interactions of adenovirus type 2 with rat embryo cells. Permissiveness, transformation and in vitro characteristics of adenovirus transformed rat embryo cells. J. Gen. Virol. 25:263-273. Gallimore, P. H., P. A. Sharp, and J. Sambrook. 1974. Viral DNA in transformed cells. II. A study of the sequences of adenovirus 2 DNA in nine lines of transformed rat cells using specific fragments of the viral genome. J. Mol. Biol. 89:49-72. Gilead, Z., Y.-H. Jeng, W. S. M. Wold, K. Sugawara, H. M. Rho, M. L. Harter, and M. Green. 1976. Im-
J. VIROL. munological identification of two adenovirus 2-induced early proteins possibly involved in cell transformation. Nature (London) 264:263-266. 9. Grodzicker, T., C. Anderson, J. Sambrook, and M. B. Mathews. 1977. The phvsical locations of structural genes in adenovirus DNA. Virology 80:111-126. 10. Harter, M. L., G. Shanmugam, W. S. M. Wold, and M. Green. 1976. Detection of adenovirus type 2-induced early polypeptides using cycloheximide pretreatment to enhance viral protein synthesis. J. Virol. 19:232-242. 11. Holtzman, E., I. Smith, and S. Penman. 1966. Electron microscopic studies of detergent treated HeLa cell nuclei. J. Mol. Biol. 17:131-135. 12. Ishibashi, M., W. W. Chin, and J. V. Maizel, Jr. 1977. The polypeptides of adenovirus. IX. Partial purification of early proteins and observations on late proteins of type 5 adenovirus. Virology 83:1-15. 13. Ishibashi, M., and J. V. Maizel, Jr. 1974. The polypeptides of adenovirus. VI. Early and late glycopolypeptides. Virology 58:345-361. 14. Jeng, Y.-H., W. S. M. Wold, K. Sugawara, Z. Gilead, and M. Green. 1977. Adenovirus type 2 coded singlestranded DNA binding protein: in vivo phosphorylation and modification. J. Virol. 22:402-411. 15. Kornfeld, R., and S. Kornfeld. 1976. Comparative aspects of glycoprotein structure. Annu. Rev. Biochem. 45:217-237. 16. Levinson, A. D., and A. J. Levine. 1977. The group C adenovirus tumor antigens: identification in infected and transformed cells and a peptide map analysis. Cell 11:871-879. 17. Levinson, A. D., E. H. Postel, and A. J. Levine. 1977. In tito and in vitro phosphorylation of the adenovirus type 5 single-stranded-specific DNA-binding protein. Virology 79:144-159. 18. Lewis, J. B., J. F. Atkins, P. R. Baum, R. Solem, R. F. Gesteland, and C. W. Anderson. 1976. Location and identification of the genes for adenovirus type 2 early polypeptides. Cell 7:141-151. 19. Linne, T., H. Jornvall, and L. Philipson. 1977. Purification and characterization of the phosphorylated DNA-binding protein from adenovirus-type-2-infected cells. Eur. J. Biochem. 76:481-490. 20. Poretz, R. D., and I. J. Goldstein. 1970. An examination of the topography of the saccharide binding sites of concanavalin A and the forces involved in complexation. Biochemistry 9:2890-2896. 21. Rho, H. M., Y.-H. Jeng, W. S. M. Wold, and M. Green. 1977. Association of adenovirus type 2 early proteins with a soluble complex that synthesizes adenovirus DNA in vitro. Biochem. Biophys. Res. Commun. 79:422-428. 22. Russell, W. C., and G. E. Blair. 1977. Polypeptide phosphorylation in adenovirus-infected cells. J. Gen. Virol. 34:19-35. 23. Saborio, J. L., and B. Oberg. 1976. In vivo and in vitro synthesis of adenovirus type 2 early proteins. J. Virol. 17:865-875. 24. Shanmugam, G., S. Bhaduri, M. Arens, and M. Green. 1975. DNA binding proteins in the cytoplasm and in a nuclear membrane complex isolated from uninfected and adenovirus 2 infected cells. Biochemistry 14:332-337. 25. Spiro, R. G. 1973. Glycoproteins, p. 349-467. In C. B. Anfinson, J. T. Edsall, and F. M. Richards (ed.), Advances in protein chemistry, vol. 27. Academic Press Inc., New York. 26. Stohlman, S. A., 0. R. Eylar, and C. L. Wissman, Jr. 1976. Isolation of the dengue virus envelope glycoprotein from membranes of infected cells by concanavalin A affinity chromatography. J. Virol. 18:132-140. 27. Sugawara, K., Z. Gilead, and M. Green. 1977. Purification and molecular characterization of adenovirus
VOL. 28, 1978 type 2 DNA-binding protein. J. Virol. 21:338-346. 28. Sugawara, K., Z. Gilead, W. S. M. Wold, and M. Green. 1977. Immunofluorescence study of the adenovirus type 2 single-stranded DNA binding protein in infected and transformed cells. J. Virol. 22:527-539. 29. van der Vliet, P. C., and A. J. Levine. 1973. DNA binding proteins specific for cells infected by adenovirus. Nature (London) 246:170-174. 30. van der Vliet, P. C., A. J. Levine, M. J. Ensinger, and H. S. Ginsberg. 1975. Thermolabile DNA binding proteins from cells infected with a temperature-sensitive mutant of adenovirus defective in viral DNA synthesis. J. Virol. 15:348-354. 31. van der Vliet, P. C., J. Zandberg, and H. S. Jansz. 1977. Evidence for a function of the adenovirus DNA-
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binding protein in initiation of DNA synthesis as well as elongation of nascent DNA chains. Virology 80:98-110. 32. Waechter, C. J., and W. J. Lennarz. 1976. The role of polyprenol-linked sugars in glycoprotein synthesis. Annu. Rev. Biochem. 45:95-112. 33. Wall, R., J. Weber, Z. Gage, and J. E. Darnell. 1973. Production of viral mRNA in adenovirus-transformed cells by the post-transcriptional processing of heterogeneous nuclear RNA containing viral and cell sequences. J. Virol. 11:953-960. 34. Wold, W. S. M., M. Green, and W. Buttner. 1978. Adenoviruses, p. 673-768. In D. P. Nayak (ed.), The molecular biology of animal viruses, vol. 2. Marcel Dekker. Inc., New York.
JOURNAL OF VIROLOGY, Oct. 1978, p. 314-323 0022-538X/78/0028-0314$02.00/0 Copyright © 1978 American Society for Microbiology
Printed in U.S.A.
Evidence for an Adenovirus Type 2-Coded Early Glycoprotein YUN-HUA JENG, WILLIAM S. M. WOLD,* AND MAURICE GREEN Institute for Molecular Virology, St. Louis University School of Medicine, St. Louis, Missouri 63110
Received for publication 28 February 1978
We have identified an adenovirus type 2 (Ad2)-induced early glycopolypeptide with an apparent molecular weight of 20,000 to 21,000 (20/21K), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 20/21K polypeptide could be labeled in vivo with [3H]glucosamine. [35S]methionine- and [3H]glucosamine-labeled 20/21K polypeptides bound to concanavalin A-Sepharose columns and were eluted with 0.2 M methyl-a-D-mannoside. The pulse-labeled polypeptide appeared as a sharp band with an apparent molecular weight of 21K, but after a chase it converted to multiple bands with an average molecular weight of 20K. This variability in electrophoretic mobility is consistent with glycosylation or deglycosylation of the 20/21K polypeptide. Analysis of the pulse and pulsechase-labeled forms by using partial proteolysis indicated that the polypeptides were highly related chemically, but not identical. Most of the 20/21K polypeptide is localized in the cytoplasm fraction of infected cells lysed by Nonidet P-40. The 20/21K polypeptide and a 44K polypeptide, labeled with [35S]methionine or [3H]glucosamine in Ad2-infected human cells, were precipitated by a rat antiserum against an Ad2-transformed rat cell line (T2C4), but not by antisera against three other Ad2-transformed rat cell lines, or by serum from nonimmune rats. The partial proteolysis patterns of the 20/21K and the 44K polypeptides were indistinguishable, indicating that the two polypeptides are highly related, and suggesting that the 44K polypeptide might be a dimer of the 20/21K polypeptide. The 20/21K polypeptide was also induced in Ad2-early infected monkey and hamster cells. These results imply that the 20/21K polypeptide is synthesized in Ad2-infected human, monkey, and hamster cells, and in one but not all Ad2transformed rat cells. Thus, the 20/21K polypeptide is probably viral coded rather than cell coded and viral induced.
Polypeptides induced in cells during early stages (i.e., before viral DNA replication) of infection by human adenovirus type 2 (Ad2) are of great interest because they may regulate viral DNA replication, transcription, and cell transformation (34). We have attempted to identify Ad2-induced early polypeptides by labeling infected and mock-infected cells with [35S]methionine, subjecting the protein extracts to electrophoresis through sodium dodecyl sulfate-polyacrylamide slab gels (SDS-PAGE), and comparing the infected and mock-infected polypeptide bands seen in autoradiograms. We have also identified Ad2 early polypeptides by immunoprecipitation, using antisera against Ad2-transformed rodent cells. Prominent early polypeptide bands with the following approximate apparent molecular weights have been detected: 73,000 (73K), 53K, 21K, 19K, 15K, 11.5K, and 1lK (8, 10, 14). Additional minor polypeptide bands of about 17.5K, 15.5K, 13.5K, 13K, 12K, 8.8K, and 8.3K can be observed in some gels, or can be immunoprecipitated by antisera against certain lines of Ad2-transformed rat cells (Wold
and Green, unpublished data). Similar in vivolabeled polypeptides have been identified by other laboratories (12, 23). Similar polypeptides have also been observed by cell-free translation of polyribosomal RNA from Ad2-early infected cells and by immunoprecipitation, using a rabbit antiserum against early infected HeLa cells (23). Six polypeptides (72K, 44 to 50K, 19K, 15.5K, 15K, and 11K) have been mapped by cell-free translation of early mRNA purified by hybridization to restriction endonuclease DNA fragments (18). Relatively little is known about the chemistry of these polypeptides or their biological functions. The 73K polypeptide is a phosphoprotein (14, 17, 19, 22) that is viral coded (9, 18, 30), that binds to single-stranded DNA (24, 27, 29), and that apparently functions in viral DNA replication (28, 31). This protein has been purified to homogeneity and partially characterized (19, 27). The 53K (8, 16) and the 15K polypeptides (8) are candidate transformation proteins because they are immunoprecipitated by antisera against Ad2- or Ad5-transformed rodent cells. 314
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Genes coding polypeptides of 44 to 50K and 15K polypeptides have been mapped in the transforming region of the Ad2 genome (18). The 11K polypeptide is localized mainly in the nuclear fraction (3, 23; our unpublished data). The 73K, 21K, 15K, 11K, and possibly 8.3K polypeptides are present in a soluble complex that synthesizes Ad2 DNA, suggesting a possible role for these in viral DNA replication (21). Ishibashi and Maizel (13) reported that an Ad2-induced early 19K polypeptide could be labeled in vivo with [3H]glucosamine. In this report, we present evidence that our 21K polypeptide is an Ad2-coded early glycopolypeptide, in that it can be labeled with [3H]glucosamine, it binds to concanavalin A (ConA)-Sepharose, its mobility changes in pulse-chase experiments, it is immunoprecipitated by antisera to Ad2transformed cells (T2C4), and it is synthesized in Ad2-infected monkey and hamster cells. MATERIALS AND METHODS Chemicals. L-['S]methionine (Met) (400 Ci/ mmol) and [3H]glucosamine (20 Ci/mmol) were purchased from New England Nuclear Corp.; 1-/i-Darabinofuranosylcytosine (ara-C), ConA-Sepharose, and methyl-a-D-mannoside were purchased from Sigma Chemical Co.; acrylamide was purchased from Eastman Kodak Co.; Nonidet P-40 was purchased from Shell Chemical Corp.; N,N-methlenebisacrylamide, N,N,N',N'-tetramethyladenediamine, and ammonium persulfate were purchased from Bio-Rad Laboratories; and Staphylococcus aureus V8 protease was purchased from Miles Laboratories, Inc. Cell culture and virus infection. Suspension cultures of human KB cells were grown in Eagle minimal essential medium (MEM) containing 5% horse serum. Cells at a concentration of 6 x 106/ml were infected with 100 PFU of Ad2 (strain 38-2) per cell in medium without horse serum. After 1 h of adsorption, cells were suspended at a concentration of 3.5 x 105/ml in medium with 5% horse serum. Cyclohexamide (CH; 25 ,Lg/ml) was added to some cultures at 1 h postinfection (p.i.). Ara-C (20 ,ug/ml) was added at 4 h p.i. At 9 h p.i., cells were washed and suspended either in warm Met-free medium or medium with 10% the normal glucose concentration; both media contained 5% horse serum and 20 ,ug of ara-C per ml. Cells, at a concentration of 3.5 x 105/ml, were labeled with [3S]Met (10 t,Ci/ml) or [3H]glucosamine (10 liCi/ml) for various times. At the end of the labeling period, cells were centrifuged and washed twice with cold phosphatebuffered saline lacking Ca2' and Mg2e. Mock-infected cells were similarly labeled, except that virus was not added. CV-1 (monkey) cell monolayers were maintained in 75-cm2 plastic flasks containing MEM with 5% calf serum, and in an atmosphere of 5% CO2. Nearly confluent monolayers were infected (or mock infected) with Ad2 in MEM without serum. After 2 h of adsorbtion, MEM with 5% calf serum (with or without 25 yg of CH per ml) was added to 25 ml, and incubation continued. Ara-C (20 ,ug/ml) was added at 4 h p.i. Cells
Ad2 EARLY GLYCOPROTEIN
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were labeled from 9 to 20 h p.i. with [3S]Met in Metfree MEM containing 20 jig of ara-C per ml. Cell fractionation. Infected or mock-infected cells labeled with [3S]Met were washed and then suspended in isotonic high pH buffer (33). All steps were at 0 to 4°C, and all buffers contained 1 to 2 mM phenylmethylsulfonyl fluoride. Cells were lysed by using 0.5% Nonidet P-40 for 5 min. Lysis was monitored by phase-contrast microscopy until 99% complete. Nuclei were pelleted by centrifugation at 200 x g for 3 min. The cytoplasmic supernatant was obtained by centrifugation at 12,000 x g for 20 min to remove mitochondria. Isolated nuclei were resuspended in phosphate-buffered saline, and treated with 0.86% Tween 40 and 0.43% sodium deoxycholate to remove outer and inner nuclear membranes (11). The nucleardetergent suspension was vigorously mixed in a Vortex mixer for 30 s, and the nuclei were collected by centrifugation at 500 x g for 3 min. The pellet was resuspended in cold phosphate-buffered saline with the addition of 1% Triton X-100 and disrupted by sonic treatment for 10 min in a Raytheon sonic oscillator. The nucleoplasm supernatant and nuclear pellet were separated by centrifugation at 12,000 x g for 20 min. Portions of subfractionated samples were precipitated with 10% cold trichloroacetic acid for analysis of labeled polypeptides. ConA-Sepharose column chromatography. Affinity chromatography of glycoproteins on ConASepharose columns was carried out as described by Stohlman et al. (26), with the following modifications. All steps were at 4°C. The column (1.5 x 25 cm) was first equilibrated with 10 bed volumes of 50 mM Trishydrochloride (pH 7.5), 50 mM NaCl, 1 mM MnCl2, and 2 mM phenylmethylsulfonyl fluoride (buffer A) containing 0.2 M methyl a-D-mannoside, followed by buffer A containing 0.2% Nonidet P-40 and 0.2% sodium deoxycholate (buffer A-detergent). The cytoplasmic protein fraction (no CH pretreatment) was diluted 25-fold with buffer A, and sodium deoxycholate was added to a final concentration of 0.2%. The sample was disrupted by sonic treatment (see above), centrifuged at 12,000 x g for 20 min, dialyzed extensively against buffer A-detergent, and loaded onto the ConASepharose column. The column was washed with buffer A-detergent until no significant radioactive material was eluted. The adsorbed proteins were eluted with buffer A containing 0.2 M methyl-a-D-mannoside. The column flow-through, wash, and methyl-a-D-mannoside-eluted fractions were concentrated by precipitation with cold 10% trichloroacetic acid and washed with acetone to remove trichloroacetic acid and detergent. Radioimmunoprecipitation of proteins. T2C4 cells, from an Ad2-transformed rat cell line (6), were obtained from P. H. Gallimore. Antiserum was prepared in rats against extracts of T2C4 cells, and the immunoglobulin G (IgG) fraction was purified (8). Infected and mock-infected labeled cell extracts were assayed for T2C4-specific polypeptides by the doubleantibody immunoprecipitation procedure, as described earlier (8). Portions containing equal counts per minute from infected and mock-infected samples were incubated with T2C4 or nonimmune rat IgG at 4°C for 18 h. Then goat serum anti-rat IgG was added, and the sample was incubated for 2 h at 37°C. Precipitates
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were collected by centrifugation for 90 s at 23,000 x g in a Beckman 152 microfuge, washed three times with phosphate-buffered saline containing 0.5 M urea, 0.5% Nonidet P-40, and 1% sodium deoxycholate, suspended in an equal volume of gel electrophoresis sample buffer without 2-mercapthoethanol, and analyzed by SDSPAGE.
SDS-PAGE and autoradiography. SDS-PAGE was carried out as described elsewhere (14). Unless stated otherwise, 8 to 21% gradient acrylamide 10-cm slab gels were used. Radiofluorography as described by Bonner and Laskey (1) was used to detect [1H]labeled polypeptides. The absorbance of bands in autoradiograms was monitored by using a Joyce-Loebel densitometer. The area under the absorbance tracing was calculated as the quantitative estimation of the intensity of bands. Partial proteolysis. The relationship between [;35S]Met-labeled 44K and 20/21K polypeptides immunopreciptated by T2C4 antiserum was investigated, using the partial proteolysis procedure essentially as described by Cleveland and colleagues (4). The polypeptides immunoprecipitated by T2C4 antiserum were resolved by SDS-PAGE, the gels were dried, and the fluorographs were developed. The 44K and 20/21K bands were cut from the gel, and the gel slices were inserted into wells (4 mm wide, 0.75 mm thick, containing soaking buffer which consisted of: 0.125 M Tris-hydrochloride [pH 6.8], 0.1% SDS, and 1 mm EDTA) of a second slab gel, and hydrated by soaking for 30 min. The proteolysis gel consisted of a 4-cm stacking gel of 9% acrylamide (pH 6.8) and a 5-cm running gel of 17% acrylamide (pH 8.8). S. aureus V8 protease was diluted to 2 mg/ml in soaking buffer containing 10% glycerol and 0.025% bromophenol blue, and 10 Al was added to wells containing the 44K and 20/21K gel slices. Electrophoresis was carried out with the gel apparatus connected to a circulating water bath at 20°C. The sample was subjected to electrophoresis (25 mA per gel) until the bromophenol blue band had almost reached the bottom of the stacking gel. The power was shut off for 30 min to allow the protease to digest the [K5S]Met-labeled polypeptides that had migrated from the gel slices. Electrophoresis was then continued until the dye reached the bottom of the running gel, and the gels were dried and fluorographed.
RESULTS Identification of Ad2-induced early polypeptides and demonstration of electrophoretic mobility changes in a 20/21K polypeptide in pulse-chase experiments. The electropherograph in Fig. 1 illustrates [35S]Metlabeled Ad2-infected and mock-infected cell polypeptides. In this and all other experiments, ara-C (20 ,ig/ml) was added at 4 h p.i. to inhibit viral DNA replication. Therefore, cells were in early stages of infection. Lanes A to H in Fig. 1 show polypeptides from cells incubated with CH before labeling, a procedure that enhances the synthesis of Ad2-specific early polypeptides relative to host polypeptides (10). Six Ad2-induced
J. VIROL.
polypeptides are clearly visible: DBP (the singlestranded DNA binding protein of about 73K daltons), 20/21K, 19K, 15K, 11.5K, and 11K. Lanes A and B and lanes C and D show infected and mock-infected polypeptides labeled 9 to 10 and 9 to 12 h p.i., respectively. In these lanes (A and C), the 20/21K polypeptide appeared as a fairly sharp band of about 21K daltons. However, when cells were labeled for long periods, e.g., 9 to 24 h p.i. (lanes E and F), or 9 to 12 h p.i. followed by a 12-h chase in complete MEM (lanes G and H), the intensity of the 21K band decreased, and new bands of roughly 20K appeared. Lanes I to L illustrate polypeptides from cells labeled without preincubation with CH (only DBP and the 20/21K bands are visible). Again, a distinct 21K band was apparent in cell extracts labeled 9 to 12 h p.i. (lanes I and J), but this band became reduced in intensity and seemed to increase in mobility in cells labeled for 9 to 24 h p.i. (lanes K and L). These results provide initial evidence that the 20/21K polypeptide was modified after translation. Since glycosylation of polypeptides affects their SDSPAGE mobility, we concentrated on the 20/21K polypeptide as a candidate glycopolypeptide. Figure 2 illustrates the analysis of the pulse (30 min) 21K polypeptide and the pulse-chase (30 min followed by a 15-h chase) 19K to 20K forms of this polypeptide by the partial proteolysis procedure. Lane A shows the pulse-labeled 21K polypeptide before chromatography on ConA-Sepharose, and lanes B to J show pulseand pulse-chase-labeled forms (purified on ConA-Sepharose). In this experiment, the chased polypeptide formed a broad band ranging from apparent molecular weight of 19K to 20K. Lanes C, F, and I represent the 20K region, and lanes D, G, and J represent the 19K region. Most of the partial proteolysis polypeptides of the pulse and pulse-chase forms clearly coincide, confirming that the 21K and 20K are highly related. However, the polypeptides are not identical, because the chased forms, especially 19K, contained at least one band (e.g., second from bottom in lanes D and G) not found among the proteolysis products of the pulse form. Note that in lanes A to G some of the polypeptide has spontaneously "polymerized" to 44K. This was a reproducible phenomenon. Cellular localization of the 20/21K polypeptide. To further analyze the 20/21K polypeptide, we established which cellular fraction contained the majority of the polypeptide. Ad2infected and mock-infected cells were labeled with [35S]Met from 9 to 12 h p.i., lysed with 0.5% Nonidet P-40, and then fractionated into nuclear pellet, nuclear membrane, nucleoplasm, and cytoplasm. The vast majority of 20/21K poly-
Ad2 EARLY GLYCOPROTEIN
VOL. 28, 1978
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IIK FIG. 1. Identification of Ad2-induced early polypeptides with and without CH pretreatment, and pulsechase experiments demonstrating mobility changes in a 20/21Kpolypeptide. Ad2-infected and mock-infected cells were prepared. CH (25 pg/ml) was added to some cultures at 1 h p.i. (lanes A to H). Ara-C (20 pg/ml) was added to all cultures at 4 h p.i.. At 9 h p.i., all cultures were washed and resuspended in warm Met-free medium containing ara-C and then labeled with [3S]Met for various time periods. Protein extracts were prepared and were analyzed by SDS-PAGE. An autoradiograph of a dried 17-cm gel is shown. (A) Infected, labeled 9 to 10 h p.i.; (B) mock infected, labeled 9 to 10 h p.i.; (C, I) infected, labeled 9 to 12 h p.i.; (D, J) mock infected, labeled 9 to 12 h p.i.; (E, K) infected, labeled 9 to 24 h p.i.; (F, L) mock infected, labeled 9 to 24 h p.i.; (G) infected, labeled 9 to 12 h p.i., and chased for 12 h; (H) mock infected, labeled 9 to 12 h p.i., and chased for 12 h.
318
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FIG. 2. Partial proteolysis (S. aureus V8 protease) of pulsed 21K and pulse-chased 35S-labeled 19 to 20K forms of the 20/21K polypeptide. Infected cells were pulse-labeled with [35S]Met for 30 min or for 30 min followed by a 15-h chase. Cytoplasm extracts were prepared and were purified by chromatography on ConASepharose followed by SDS-PAGE. The 21K pulse and 20K or 19K chase polypeptide bands were cut from the gel and analyzed by partial proteolysis as described in the text, using 0.5, 5, or 50 yg of protease per lane. (A) Pulse-labeled 21K polypeptide before chromatography on ConA-Sepharose; (B, E, H) pulse-labeled 21K polypeptide purified on ConA-Sepharose; (C, F, I) pulse-chase-labeled 20K after ConA-Sepharose; (D, G, J) pulse-chase labeled 19Kpolypeptide after ConA-Sepharose. peptide was present in the cytoplasm fraction TABLE 1. Distribution of radioactivity and the 20/21Kpolypeptide in subcellular fractions (Table 1). Labeling of the 20/21K polypeptide with totl 20/21K 20/21K Mock-infected Infected cells 2021
[3H]glucosamine, and immunoprecipitation with antiserum directed against an Ad2transformed rat cell line (T2C4). Infected and mock-infected cells were labeled with [3H]glucosamine to test whether the label was incorporated into the 20/21K polypeptide. As shown in Fig. 3, the only Ad2-specific polypeptide labeled with [3H]glucosamine had about the same electrophoretic mobility as the 20/21K polypeptide (lane B). Antiserum against T2C4 cells precipitated the 'H-labeled 20/21K polypeptides, as well as a second polypeptide of about 44K (lane D). The 20/21K and 44K polypeptides were not precipitated from infected cell extracts by nonimmune rat sera (lane E), or from mockinfected cell extracts by either T2C4 or nonimmune rat sera (lanes F and G). The T2C4 antiserum also immunoprecipitated viral specific early [35S]Met-labeled polypeptides of 53K, 44K, 20/21K, 19K, 18K, 15K, 14.5K, 13.5K, 12K, and 11.5K (Wold and Green, unpublished data). The 20/21K and 44K polypeptides precipitated by the T2C4 antiserum were assayed by the partial proteolysis procedure (4) to test whether they were chemically related. [35S]Metlabeled polypeptides were assayed instead of
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' Total ¢'S counts per minute in the infected whole-cell preparation was 278 x 10', and in the mock-infected preparation was 280 x 10. Infected and mock-infected proteins were resolved by SDS-PAGE, and autoradiograms were developed. The autoradiograms were scanned by using a Joyce-Loebel densitom-
eter to obtain the total area represented by each fraction. These values indicate the percentage of the total scan area represented by the 20/21K polypeptide, minus contribution
from mock-infected polypeptides. ' Calculations are based on a value of 100% for the estimated 20/21K counts per minute in the whole-cell fraction.
["H]glucosamine-labeled polypeptides, because larger amounts of radioactivity could be obtained. [35S]Met-labeled polypeptides immunoprecipitated by the T2C4 antiserum were resolved by SDS-PAGE, and fluorograms were developed. The 20/21K and 44K bands were cut
Ad2 EARLY GLYCOPROTEIN
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11.5K
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FIG. 3. Identification of polypeptides labeled in vivo with [3H]glucosamine and immunoprecipitated by antiserum against 72C4 cells. Ad2-infected and mock-infected cells were labeled with [3H]glucosamine. Total cell extracts were prepared and immunoprecipitated by using T2C4 IgG and nonimmune rat IgG. Equal volumes of each precipitate were analyzed by SDS-PAGE. (A) Marker polypeptides: [3S]Met-labeled, early infected polypeptides prepared by the CH enhancement procedure. (B-G), [3H]glucosamine-labeled extracts. (B) Infected, before immunoprecipitation; (C) mock infected, before immunoprecipitation; (D) infected extract versus T2C4 IgG; (E) infected extract versus nonimmune rat IgG; (F) mock-infected extract versus 72C4 IgG; (G) mock-infected extract versus nonimmune rat IgG.
from the dried gel and analyzed by partial proteolysis as described above. The partial proteolysis slab gel is shown in Fig. 4. The same polypeptide bands were generated by protease digestion of the 20/21K and 44K polypeptides, indicating that they are highly related chemically. Proteolysis of the 20/21K and 44K polypeptides by using other protease concentrations also indicated that they are highly related. Binding of the 20/21K polypeptide to ConA-Sepharose and its elution by methyla-D-mannoside. To obtain further evidence that the 20/21K polypeptide is a glycopolypeptide, 35S-labeled protein extracts were subjected to affinity chromatography on columns of ConASepharose. Only certain carbohydrate moieties (a-D-glucopyranosides, and a-N-acetyl-D-glucosaminides) bind strongly to ConA (20), so that if the 20/21K polypeptide contains these sugars it should bind to the ConA-Sepharose column. Infected and mock-infected cells were labeled with [35S]Met for 15 h, and cell cytoplasms were prepared and then loaded onto ConA-Sepharose
columns. The columns were washed and then eluted with 0.2 M methyl-a-D-mannoside. The column flow-through, wash, and eluted fractions were analyzed by SDS-PAGE. Only the mannoside-eluted infected cell fraction (lane F) contained detectable 20/21K polypeptide (Fig. 5). These results suggest that the 20/21K polypeptide contains sugar residues specific for ConA. The 20/21K polypeptide is synthesized in Ad2-infected monkey cells. The immunoprecipitation of both [3H]glucosamine and [35S]Met-labeled 20/21K polypeptide by the T2C4 antiserum suggests that the 21K polypeptide is viral coded rather than cell coded and viral induced. The 20/21K polypeptide (and the DBP, 11K, 8.8K, 8.3K polypeptides) was synthesized in Ad2-early infected monkey (CV-1) cells (Fig. 6). The 21K, DBP, and 11K were also synthesized in Ad2-early infected hamster cells (not shown). These results provide further evidence that the 21K, DBP, and 11K polypeptides are viral coded.
320
J. VIROL.
JENG, WOLD, AND GREEN
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44
DISCUSSION Our results provide strong evidence that the Ad2-induced 20/21K polypeptide is a glycopolypeptide. The polypeptide can be labeled with [3H]glucosamine, and it binds to ConA-Sepharose columns and is eluted with methyl-a-Dmannoside. When labeled with [35S]Met for short periods (30 min to 3 h), it appears as a single, relatively sharp band with an apparent molecular weight of about 21K. However, if labeled for long periods, or if pulse-labeled and then chased, the polypeptide appears as multiple bands with apparent molecular weights of 20K to 21K. Partial proteolysis data indicate that the pulse-labeled 21K and the pulse-chase-labeled 20K polypeptides are highly related, although not identical. Both pulse (30 min) and pulse (30 min)-chase (12 h in complete MEM) forms of [35S]-labeled 20/21K polypeptide bind to ConASepharose, and therefore apparently are glycosylated. The multiple bands of the chased
nP
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I
FIG. 4. Partial proteolysis (S. aureus V8protease) of the 44K and 20/21K [35S]Met-labeled polypeptides immunoprecipitated by T2C4 antiserum (A) 44K, (B) 21K. Both polypeptides were treated with 5 pLg of protease.
20/21K polypeptide may indicate heterogeneity in sugar content, because glycosylation affects (usually decreases) the SDS-PAGE mobility of polypeptides. We cannot exclude the possibility that there is another distinct polypeptide of
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)
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.....
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-1
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-
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I;
FIG. 5. ConA-Sepharose column chromatography: identification of [3S]Met-labeled Ad2-infected and mock-infected polypeptides in the flow-through, wash, and methyl-a-D-mannoside eluted fractions. Ad2infected and mock-infected cells were labeled with [3S]Met from 9 to 24 h p.i. The cytoplasm fraction was prepared and chromatographed on ConA-Sepharose. Appropriate column fractions were pooled and analyzed by SDS-PAGE. (A) Marker polypeptides: Ad2-infected KB cells extracts labeled with [3SJMet by the CH enhancement procedure; (B) infected, flow-through fraction; (C) mock infected, flow-through fraction; (D) infected, wash fraction; (E) mock infected, wash fraction; (F) infected, methyl a-D-mannoside eluate; (G) mock infected, methyl-a-D-mannoside eluate.
VOL. 28, 1978
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321
about 20K to 21K that cannot be detected in these single-dimension gels. Our results also strongly suggest that the 20/21K polypeptide is coded by an early Ad2 gene, and is not a cell coded polypeptide induced by Ad2. Both [3H]glucosamine and [35S]Metlabeled 20/21K polypeptide were immunoprecipitated by rat antiserum against T2C4 cells. T2C4 is a line of Ad2-transformed rat cells (6) that contains all four of the Ad2 early gene blocks (7) and synthesizes RNA derived from all four blocks (5). The polypeptide was not precipitated by nonimmune rat serum, or by rat antisera against three other Ad2-transformed rat cells (not shown). Therefore, it is probable that the Ad2 sequences present in T2C4 cells (but not in the other 3 Ad2-transformed rat lines) synthesize the 20/21K polypeptide. A 20/21K polypeptide was also induced in Ad2-early infected monkey and hamster cells. These results suggest that the 20/21K polypeptide is viral coded, because it is improbable (but not impossible) that a cell-coded 20/21K polypeptide would be synthesized by Ad2-infected human, monkey, and hamster cells, and by one but not all Ad2-transformed rat cells. We are currently attempting to purify the 20/21K polypeptide for further chemical, physical, and biological characterization. The protein seems to exist in multiple charge forms, because it is present in fractions eluted from ion-exchange resins by salt concentrations of from 10 mM to 0.4 M. Also, it seems to exist in multiplesize forms, as reflected by its behavior on gel filtration columns. An example of this is shown in Fig. 3, where the 20/21K polypeptide spontaneously polymerized to 44K, the 44K spontaneously converted to 21K, and both the 44K and the 20/21K polypeptide polymerized to species of high molecular weight. This type of apparent size and charge heterogeneity is typical of many glycoproteins. We have not attempted to reduce and alkylate the 20/21K polypeptide to determine whether sulfhydral groups are involved in the polymerization of the polypeptide. Chin and Maizel (2) reported that a 35S-labeled polypeptide (E2) was a component of the plasma membrane. E2, apparently, is the same polypeptide that Ishibashi and Maizel (13) reported to be labeled with [3H]glucosamine. The 20/21K polypeptide probably corresponds to E2, because we have seen no indication that any other early protein is glycosylated. Our finding FIG. 6. [35S]Met-labeled polypeptides induced in Ad2-early infected monkey (CV-1) cells, with and without CH (25 pg/ml) pretreatment. (A) Infected, no CH; (B) infected, with CH; (C) mock infected, no CH; (D) mock infected, with CH.
322
JENG, WOLD, AND GREEN
that the 20/21K polypeptide is located mainly in the cytoplasm of Nonidet-P40-lysed cells would be consistent with the possibility that this polypeptide is a membrane component. However, we have also found the 20/21K polypeptide in a complex purified from lysed nuclei that synthesizes Ad2 DNA (21). The 20/21K polypeptide can also be immunoprecipitated by T2C4 antiserum from the nucleoplasm of cells. Therefore, a nuclear role for this polypeptide is not excluded. Glycoproteins are widely distributed in nature and serve a variety of structural, lubricating, enzymatic, hormonal, and plasma membrane-associated functions (15, 25, 32). The finding that an Ad2-early gene product is a glycoprotein is of interest, especially if it is found to serve a regulatory function. Studies on the 20/21K polypeptide should be of interest not only regarding Ad2 replication, but also the general role of glycosylation in protein function. ACKNOWLEDGMENTS We thank H. Thornton for assistance in cell culture and in preparation of the T2C4 antiserum, and C. Devine for technical assistance. We are grateful to P. H. Gallimore for a gift of the T2C4 cells. This work was supported by Public Health Service grants Al 01725-19 from the National Institute of Allergy and Infectious Diseases and CA 21824-01 from the National Cancer Institute, and by contract NOI CP 43359 from the Virus Cancer Program within the National Cancer Institute. W.S.M.W. was partially supported by a fellowship from the Medical Research Council of Canada. M.G. is the recipient of a Research Career Award (5 K06 AI 04739) from the National Institute of Allergy and Infectious Diseases.
1.
2.
3. 4.
5.
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LITERATURE CITED Bonner, W. M., and R. A. Laskey. 1974. A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur. J. Biochem. 46:83-88. Chin, W. W., and J. V. Maizel, Jr. 1976. Polypeptides of adenovirus. VII. Further studies of early polypeptides in vivo and localization of E, and E2A to the cell plasma membrane. Virology 71:518-530. Chin, W. W., and J. V. Maizel, Jr. 1976. The polypeptides of adenovirus. VIII. The enrichment of E3 (11,000) in the nuclear matrix fraction. Virology 76:79-89. Cleveland, D. W., S. G. Fischer, M. W. Kirschner, and U. K. Laemmli. 1977. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J. Biol. Chem. 252:1102-1106. Flint, S. J., P. H. Gallimore, and P. A. Sharp. 1975. Comparison of viral RNA sequences in adenovirus 2transformed and lytically infected cells. J. Mol. Biol. 96:47-68. Gallimore, P. H. 1974. Interactions of adenovirus type 2 with rat embryo cells. Permissiveness, transformation and in vitro characteristics of adenovirus transformed rat embryo cells. J. Gen. Virol. 25:263-273. Gallimore, P. H., P. A. Sharp, and J. Sambrook. 1974. Viral DNA in transformed cells. II. A study of the sequences of adenovirus 2 DNA in nine lines of transformed rat cells using specific fragments of the viral genome. J. Mol. Biol. 89:49-72. Gilead, Z., Y.-H. Jeng, W. S. M. Wold, K. Sugawara, H. M. Rho, M. L. Harter, and M. Green. 1976. Im-
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VOL. 28, 1978 type 2 DNA-binding protein. J. Virol. 21:338-346. 28. Sugawara, K., Z. Gilead, W. S. M. Wold, and M. Green. 1977. Immunofluorescence study of the adenovirus type 2 single-stranded DNA binding protein in infected and transformed cells. J. Virol. 22:527-539. 29. van der Vliet, P. C., and A. J. Levine. 1973. DNA binding proteins specific for cells infected by adenovirus. Nature (London) 246:170-174. 30. van der Vliet, P. C., A. J. Levine, M. J. Ensinger, and H. S. Ginsberg. 1975. Thermolabile DNA binding proteins from cells infected with a temperature-sensitive mutant of adenovirus defective in viral DNA synthesis. J. Virol. 15:348-354. 31. van der Vliet, P. C., J. Zandberg, and H. S. Jansz. 1977. Evidence for a function of the adenovirus DNA-
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binding protein in initiation of DNA synthesis as well as elongation of nascent DNA chains. Virology 80:98-110. 32. Waechter, C. J., and W. J. Lennarz. 1976. The role of polyprenol-linked sugars in glycoprotein synthesis. Annu. Rev. Biochem. 45:95-112. 33. Wall, R., J. Weber, Z. Gage, and J. E. Darnell. 1973. Production of viral mRNA in adenovirus-transformed cells by the post-transcriptional processing of heterogeneous nuclear RNA containing viral and cell sequences. J. Virol. 11:953-960. 34. Wold, W. S. M., M. Green, and W. Buttner. 1978. Adenoviruses, p. 673-768. In D. P. Nayak (ed.), The molecular biology of animal viruses, vol. 2. Marcel Dekker. Inc., New York.
