VIROLOGY

86, 344-353

Synthesis

(1978)

of the SV40 Viral Polypeptide

HARUMI

KASAMATSU

AND

Vpl During Infection

P. JOHN FLORY, JR.

Molecular Biology Institute, University of California, 405 Hilgard Avenue, Los Angeles, California 9~~4 Accepted December 15,1977 The synthesis of simian virus 40, SV40, polypeptides in permissively infected cells was studied by a very sensitive method utilizing ‘251-labeled antibodies to identify and quantitate polypepties immobilized in gels. The synthesis of Vpl begins about 24 hr postinfection, with the majority of the Vpl being found in the nucleus. Smaller quantities of several Vplrelated polypeptides are observed in highly purified vii-ions. Two of these species, with apparent molecular weights of about 117,999 and 199,090, are also found in infected cells. They appear long after Vpl synthesis has begun, at about 54 hr postinfection in nuclei and later, at about 95 br postinfection, in the cytoplasm.

having different molecular weights but at least some of the same antigenic determinants, as in mutant or precursor polypeptides. We have used this technique to study the biosynthesis of the major structural polypeptide, Vpl. In addition, we have detected two polypeptides which are related to Vpl and have apparent molecular weights of 117,000 and 100,000. They are predominantly located in the nuclei of infected cells.

INTRODUCTION

Seven polypeptides have been identified previously as protein components of simian virus 40, SV40, virions. Vpl, Vp2, and Vp3 have molecular weights between 45,090and 28,000. These polypeptides map between 0.76 and 0.175 units in the late region of the viral genome (Khoury et al., 1975; Rosenblatt et al., 1976). Vp4, Vp5, Vp6, and Vp7 have molecular weights between 20,000 and 10,000and are cellular histones (Lake et al, 1973). The synthesis of Vpl, Vp2, and Vp3 in infected cells has been studied by poly acrylamide gel electrophoresis of extracts from pulse-labeled cells (Anderson and Gesteland, 1972; Walter et aZ., 1972; Tegtmeyer et al, 1974). The synthesis of virusinduced polypepties with the same molecular weights as Vpl-3 begins 15 to 21 hr postinfection (Anderson and Gesteland, 1972). To study further the time-course of synthesis of the structural components Vpl-3 and the intracellular distribution of the different polypeptides, we have developed a very sensitive immunological staining technique utilizing ‘251-labeled antibodies against specific viral polypeptides to stain polypeptides immobilized in polyacrylamide gels. This method can be used both to quantitate a specific polypeptide in the presence of other unrelated polypeptides of similar molecular weight, as in a crude extract, and to detect related polypeptides

METKODS

A. Cells and virus. The TC7 clone of CV-1 cells (Robb and Huebner, 1973) was used in all experiments described here. Cells were grown in Eagle’s basal medium containing 10% fetal calf serum. They were infected at confluence with 10 PFU/cell, using plaque-purified sp12, a small plaqueforming stain. of iodoacetamideB. Preparation treated “C-labeled viral polypeptides. Twenty-four hours after infection, cells were washed with Tris buffer (140 mM NaCl, 25 mM Tris, 5 mM KCl, 1 milf CaCl2, 0.7 mikf Na2HP04, 0.5 m&f MgCl2 (pH 7.5)) and 0.5 PCi of 14C-labeled protein hydrolysate (Amersham) in 5 ml of medium containing one-tenth the normal concentrations of amino acids was added to each plate. After 60 hr, 4 ml of complete medium was added, and virus was harvested about 6 days postinfection (Kasamatsu and Wu, 344

0042-6822/78/0862-0344$02.00/O Copyright 0 1978 by Academic Press, Inc. AII rights of reproduction in any form reserved.

SYNTHESIS

197613).14C-Labeledviral polypeptides were prepared by treating this virus with 5% sodium dodecyl sulfate (SDS), 10 mM Tris (pH 7.5), 5 mM EDTA, and 5 m&f DTT and then sedimenting the lysate through a 15 to 30% linear sucrose gradient. The polypeptides were pooled and dialyzed into 0.4% SDS, 10 mM Tris (pH 8.0), and 1 n&f EDTA. DTI’ was added to 10 mM, the polypeptides were incubated at 45’ for 30 min, iodoacetamide (Sigma Chemical Co.) was added to 14 mM, and they were incubated in the dark for 30 min at room temperature. The polypeptides were dialyzed against 0.5% Triton X-100,10 mi!f Tris (pH 7.9), and 1 mM EDTA overnight. C. Preparation of Vpl-specific antisera. Antiserum to SV40 viral polypeptide Vpl was obtained from New Zealand White rabbits immunized by intradermal injections of electrophoretically homogeneous antigen as follows: Viral polypeptides were electrophoresed on SDS-polyacrylamide gels as described below. These gels were stained with Coomassie blue, the Vpl bands were excised, and Vpl was electrophoreticahy eluted. Each rabbit received 200 c(g of Vpl in 0.5 ml of saline mixed with 0.5 ml of Freund’s complete adjuvant, followed at 6week intervals by two booster injections of half of this amount. Sera were taken 2 weeks after the second booster injection. The antisera obtained in this way selectively immunoprecipitated Vpl out of 14Clabeled total viral polypeptides (Fig. 1). D. Preparation of ‘251-labeled IgG from anti-Vpl sera. The IgG fractions of these antisera were isolated by ammonium sulfate precipitation (43%saturation), dialyzed into 0.01 M potassium phosphate buffer (pH 7.5), purified by DEAE-cellulose chromatography, and labeled with 125Iby the lactoperoxidase method (Marchalonis, 1969). In a typical labeling, a 0.2~ml reaction mixture contained 4 mg of protein, 0.02 M potassium phosphate buffer (pH 7.0), 0.3 mg/ml of lactoperoxidase (Sigma Chemical Co.), and 0.25 to 0.5 mCi of Na1251.The iodination was initiated by adding 4 ~1 of 0.13 M HzOz, repeated after 5 min at room temperature. After a further 5 min, unincorporated iodine was removed on Sephadex G-25 by either a microcentrifuge-desalting technique (Neal and Florini, 1973)

345

OF SV40 Vpl vpq vp2 44

VP3

ee lmnt

4

i

6 I

!

FIG. 1. Gel electrophoresis of viral polypeptides precipitated by anti-Vpl serum. About 10 H of iodoacetamide-treated “C-labeled total viral polypeptides (Methods) in 40 4 of 0.5% Triton X-100,10 m&f Tris (pH 7.9), 1 mMEDTA, and0.15MNaCl wasincubated with 5 d of anti-VP1 serum for 90 min at room temperature and then with 40 4 of goat anti-rabbit IgG fraction (Miles) for 90 min at room temperature. The precipitate was washed with the above buffer three times, and a portion was analyzed on an SDS-7.5% polyacrylamide cylindrical gel, 0.6 x 12 cm (Fairbanks et al., 1971). After electrophoresis at 7 mA/gel for 4 hr, the gel was sliced into 2-mm slices. Each slice was incubated with 0.1 ml of 30% Hz02 at 37” overnight, suspended in 8 ml of Aquasol, and counted in a Beckman IS-250 liquid scintillation counter. The positions of Vpl, Vp2, and Vp3 in a parallel gel are indicated.

or column chromatography in 0.01 M potassium phosphate buffer, pH 7.0. The specific activity of ‘251-labeled IgG thus prepared was 1 to 5 X lo5 cpm/pg of protein. E. Nuclear and cytoplaamic extracts from SV40-infected cells. Confluent plates of TC7 cells were washed with Tris buffer, incubated for 90 min with 10 PFU of plaque-purified SV40 per cell, thoroughly rinsed with Tris buffer, and overlaid with fresh medium. At intervals after the initiation of infection, the plates were rinsed with ice-cold Tris buffer, and nuclei and cytoplasm were separated by the method of Walter et al. (1972) as modified by Tegtmeyer et al. (1974). Each plate was treated for 10 min at 0” with 1 ml of 0.5% Nonidet P-40 detergent in Tris buffer containing 0.3

346

KASAMATSU

mg/ml of phenyhnethylsulfonylfluoride. The lysed cells were scraped off the plates and centrifuged at 2000 g for 5 min at 0’ to separate cytoplasm from the nuclei, which remained intact. The nuclei were washed once, and then both nuclei and cytoplasm were made to 2% SDS, 11 mM dithiothreitol and boiled for 4 min. They were precipitated with acetone, dried, resuspended in 0.5 ml per plate of 4% SDS and 50 mM Tris (pH 6.8), and stored at -25”. Protein concentrations were determined by the Lowry method (Lowry et aZ., 1951). Control extracts were prepared by the same method from parallel cultures of uninfected cells. Each g-cm control plate yielded 1.1 mg of protein in the cytoplasmic and 0.26 mg in the nuclear extract. F. Gel Electrophoresis and ‘251-labeled IgG staining. Samples in 50 ~1 of 4% SDS, 50 m&f Tris.HCl (pH 6.8), 12 mM dithiothreitol, 10% glycerol, and 0.001% bromophenol blue were boiled for 4 min, applied to 14 x 14-cm x 1.2-mm SDS-7.5% polyacrylamide gels prepared according to Laemmli (1970), and electrophoresed at 15 to 20 mA for 4 hr. The gels were treated with 20% trichloracetic acid (TCA) at 2” without shaking for 5 to 12 hr, with 50% methanol for 30 min, with 0.1% glutaraldehyde (Polysciences) in 0.1 M sodium phosphate buffer (pH 7.0) for 1.5 hr, with 20 pg/ml of NaBH4 in BBS buffer (0.1 M H3B03,0.025 M Na2B407, 0.15 M NaCl, pH 8.5) for at least 6 hr with three changes, and finally with BBS buffer alone for at least 2 hr with one change. They were next incubated for 15 to 24 hr with 1 to 2 mg of 1251-labeled antibodies in 30 ml of BBS buffer containing 0.03% NaN3 and 1 mg/ml of bovine serum albumin, and unbound antibodies were removed by rinsing the gels in 10% BBS, 0.15 M NaCl, 0.03% NaN3 (and, in some cases, 0.5% Triton X-100) for approximately 72 hr, with changes every 6 to 12 hr. The gels were then stained for 30 to 60 min in 0.5% Coomassie brilliant blue in 10% methanol-lo% acetic acid, destained with 10% methanol-10% acetic acid for about 6 hr, treated for 1 hr with 7.5%acetic acid-l% glycerol, and dried. All gel treatments were performed at room temperature in the dark with gentle shaking except as noted, and the gels were rinsed in distilled water for 2

AND FLORY

min at each transfer. Autoradiograms were obtained by placing the dried gels in contact with Kodak No-Screen X-ray film for 2 to 3 days at room temperature. Tracings of these films were used to locate ‘251-stained bands in the dried gels. These bands were excised, diced, rehydrated by shaking 12 hr in 1 ml of 0.1% SDS at room temperature, suspended in 9 ml of Biofluor (Beckman), and scintillation counted, using the 3H + 14Cisoset. Alternatively, staining intensities were measured by densitometry of autoradiagrams, using a Joyce-Loebbel densitometer. Care was taken to ensure that the measurements fell within the linear response range of the film. RESULTS

A. Staining of Vpl in Lysates of Virions Briefly, the method described here consists of electrophoresing polypeptides through SDS-polyacrylamide gels, immobilizing them by fixation with glutaraldehyde, and staining them with 1251-labeled antibodies. The sensitivity of this method of SV40 Vpl was measured by determining the minimum amount of Vpl which could be detected in lysates of SV40 virions (Fig. 2). Different dilutions of lysates were electrophoresed through a gel which was fixed and then stained with ‘251-labeledanti-Vpl. The autoradiogram shows that the Vpl from 1 ng of viral polypeptides is clearly detectible by this method. Since Vpl constitutes roughly 75% of total viral polypeptides (Tooxe, 1973), this amount corresponds to about 0.75 ng of Vpl. The antibody preparation does not crossreact appreciably with Vp2 or Vp3 as previously shown in Fig. 1, since little staining is seen at these positions (Fig. 2). The apparent darkening of regions near Vpl seen in the l- and 1Oqg slots is probably due to overexposure during the long exposure time necessary to visualize the Vpl in the 1-ng slot. The relationship between the amount of Vpl in the gel and the amount of ‘251-labeled anti-Vpl bound was determined by excising and counting the Vpl regions from the different slots of several gels containing known amounts of Vpl. As shown in Fig. 3, a tenfold increase in the amount of Vpl applied results in about a twofold increase

SYNTHESIS

117KloOK-

-116K -94K -68K

Vpl-

-43K

\I’P2{ , vp3FIG. 2. Staining of SV40 viral polypeptides with 1251-labeled anti-Vpl. Different dilutions of lysates of SV40 virus were electrophoresed through an SDS-polyacrylamide gel, which was treated as described under Methods. The gel was stained with 1.2 mg of ‘S-labeled anti-VP1 (2.7 x 106 cpm/pg) and rinsed in BBS buffer containing 0.5% Tuition X-100. Autoradiography was for 72 hr. Amounts of total SV40 viral polypeptides applied to each slot: (1) 10 gg; (2) 1 pg; (3) 100 ng; (4) 10 ng; (5) 1 ng. The positions of Vpl, Vp2, Vp3, and the 100,000 (lOOK) and 117,ooO (117K) dalton species are indicated at the left and the positions of marker polypeptides in another slot of the same gel are indicated at the right: 116K w-galactosidase (a kind gift from Dr. I. Zabin)], 94K [phosphorylase a (Sigma)], 66K (bovine serum albumin), and 43K (ovalbumiu). The 66,000 and 93,000 dalton species were also stained but are not indicated because they are only faintly visible.

in the amount of ‘251-labeled anti-Vpl bound to the gel within the range examined. The amount of Vpl in an unknown sample may therefore be measured from the amount of ‘251-labeled anti-VP1 bound within this range. This nonlinear relationship may indicate that the diffusion of 1251labeled IgG in the gel is hindered either because of the gel concentration or because of the crosslinking of the polypeptides. In either case,the bands would be labeled only near the surface. The minimum amount of 1251-labeled

347

OF SV40 Vpl

anti-VP1 required for staining a gel was also determined. Identical virus samples each containing 1 pg of total viral polypeptides were lysed, applied to every slot of a gel, and electrophoresed. The gel was sliced longitudinally into equal pieces which were stained with different amounts of ‘251-labeled anti-Vpl (data not shown). Definite staining of Vpl was apparent even with the lowest amount of antibody used, corresponding to 190 c(gof antibody per gel in 30 ml of staining solution (Methods). In later experiments, each gel was treated with 1 to 2 mg of antibody to ensure adequate labeling. Unbound lz51-labeled antibody must be completely removed from the gel before autoradiography if high backgrounds are to be avoided. The minimum time required to elute unbound antibody was measured both in the presence and absence of Triton X100, a nonionic detergent which should reduce nonspecific binding of antibody without affecting antibody-antigen interactions. A blank gel was treated as above and then cut into disks. The disks were stained with labeled rabbit IgG and rinsed in the presence or absence of 0.5% Triton X-100 (Fig. 4). The elution of the unbound labeled IgG in the presence of the detergent is both more rapid and more complete and reaches

Amount of Vroi Polypeptlde

Vpl (pg)

FIG. 3. Relationship between ‘261-labeled anti-VP1 staining and amount of Vpl. The Vpl region of each slot of the gel shown in Fig. 2 was excised and counted as described under Methods. These results are combined with those from two identical gels which had been rinsed in BBS buffer without Triton X-100, since there was no significant difference. The number of lz51labeled anti-VP1 counts per minute bound to each Vpl band is plotted versus the amount of Vpl applied to that slot of the gel.

348

KASAMATSU

AND FLCRY

fected cells in order to study the timecourse of Vpl synthesis during infection and to determine whether other polypeptides antigenically related to Vpl can be detected in infected cells. At different times after infection, cytoplasmic and nuclear extracts were prepared as described under methods. The polypeptides were separated on gels which were then stained with 1251labeled anti-Vpl. The results are shown in Figs. 5 and 6. The amounts of total polypeptides applied to each slot of these gels were not the same. Several controls were . -included in the gels shown. The control --o-4 t I I I I slots “V” of total viral polypeptides show 0.11 20 40 60 80 100 no 1251staining at the Vp2 and Vp3 posiTime (hours) tions, confirming that the antibody is speFIG. 4. Rate of rinsing of unbound rfiI-labeled IgC cific for Vpl determinants as previously from gels. Two blank gels were treated as under Metbshown. The Vpl bands in these slots also ods, and a punch was used to cut them into identical function as standards for the intensities of 4-cm disks. These disks were stained with 1.7 mg of 1251staining. The artificial mixtures “C + 1261-labeledIgC (4.4 x 106cpm/pg) from an untreated V” of total viral polypeptides, 1 I-18,plus rabbit and rinsed in 10% BBS-O.15 M NaCl-O.03% extracts from control cells, 29 pg of cytoNaN3 (-O-O-O-) or without (-@-O-8-) 0.5% Triton X-100. At intervals, the destaining solution was plasmic or 18 pg of nuclear extract, show changed and a disk was removed, diced, and counted intensities of 1251staining identical to the in 9 ml of Biofluor. izsI counts in the disks are plotted same amount of viral polypeptides alone, as a percentage of the zero time point. After 80 to 95 slots V, demonstrating that the presence of hr in the presence of Triton X-100,430 cpm of i2’I/cm2 cellular proteins does not affect the staining of gel remained, in its absence 990 cpm of ‘Vcm2 of the viral polypeptides. remained. The intensities of 1251staining of Vpl in these extracts show several features. First, a background value half that obtained in its a large increase in the intensities of both absence. A comparison of identical 1251-la1251and Coomassie blue staining at the Vpl beled anti-Vpl-stained gels containing SV40 viral polypeptides and rinsed in the position is apparent late in infection in both presence and absence of the detergent nuclear and cytoplasmic extracts. The largshows that it does not significantly reduce est increase in nuclear Vpl occurs between the amount of antibody bound (Figs. 2 and 12 and 27 hr postinfection, to about 250 times the amount found at 2 hr postinfec3 and data not shown). Several modifications of the above 1251- tion. The increase in cytoplasmic Vpl is staining procedure were also tried. When later, between 27 and 54 hr, to about 500 SV40 virion polypeptides run on 7 to 15% times the 2-hr value. These estimates have polyacrylamide gradient gels were stained been corrected for differences in the with ‘251-labeled anti-Vpl, the staining in- amounts of polypeptides applied to the gels. tensity depended on the local gel concen- This observation indicates that synthesis of Vpl begins between 12 and 27 hr postinfectration, so that quantitation was difficult. When NaBJ& was omitted from the rinse tion. Second, the amount of Vpl per cell in the 54-hr nuclear extract is about four times buffer after glutaraldehyde fixation, counts the amount in the corresponding cytoplaswere bound over the entire gel, probably due to residual glutaraldehyde in the gel. mic extract. This esimate is based on the intensities of 1251staining relative to that of Vpl in the V slots and is also corrected for B. Synthesis of Vpl in SV40-Infected Cells the different amounts of polypeptides apWe have applied this antibody staining plied to the gels. Finally, small amounts of system to polypeptides isolated from in- Vpl are found in both nuclear and cyto-

SYNTHESIS

349

OF SV40 Vpl (b)

(a) V

C

2

6

12275495C+VV

V

CI

2 /

6 /

12 /

27 I

54 1

95 C+V 1 I

V

FIG. 5. Staining of cytoplasmic extracts from SV40-infected cells. Cytoplasmic extracts were prepared from SV40-infected cells, and ahquota were electrophoresed on a gel and stained with ‘%Iabe1ed anti-Vpl. The yields of total protein per dish at each time point after infection were: 2 hr, 0.75 mg; 6 hr, 0.88 mg; 12 hr, 0.83 mg 27 hr, 1.25 mg; 54 hr, 1.88 mg; 95 br, 0.93 mg. The loading of the gel was as follows: Total viral polypeptides (1 pg) were applied to the extreme left and right slots (“V”) of the gel. The second through eight slots from the left contained ahquota of extracts from control cells. “C,” (29 HP); and from infected ceIIs at 2 hr (20 c(g), 6 hr (23 a+$, 12 hr (22 n), 27 hr (33 pg), 54 hr (49 /.&, and 95 hr (25 pg) postinfection (slots marked “2” through “95”). The ninth slot from the left (“C + V”) received an artificial mixture of 29 pg of control extract plus 1 M of total viral polypeptides. The gel was stained with 0.8 mg of 1261-labeledanti-VP1 (2.8 x 106cpm/ag). (a) Photograph of a gel showing Coomassie blue staining. (b) Autoradiogram (65 hr-exposure). The positions of Vpl, Vp2, Vp3, and the 117,000(117K), 100,000(lOOK), 93,000 (93K), and 88,000 (88K) dalton species are indicated.

FIG. 6. Staining of nuclear extracts from SV40-infected cells. Identical to Fig. 5, except that nuclear extracts from SV40-infected cells were analyzed. Yields of total protein per dish for these extracts were: 2 hr, 0.15 mg; 6 hr, 0.10 mg; 12 hr, 0.26 mg; 27 br, 0.35 mg; 54 hr, 0.93 mg; and 95 hr, 0.50 mg. The arrangement of samples in the gel was identical to that of Plate 2, with the following amounts of each extract applied: control (18 ag), 2 hr (11 ag), 6 hr (7 ccg),12 hr (18 ag), 27 hr (25 &, 54 hr (65 MB),and 95 hr (35 pg). In this case, the ninth slot from the left (C + V) received a mixture of 18 clg of control extract plus 1 pg of total viral polypeptides.

plasmic extracts between 2 and 12 hr and may represent Vpl from infecting virus. C. Vpl-Related Polypeptides in Virions and their Synthesis in Infected Cells We have observed a number of polypeptides which are contained in small quanti-

ties in extracts from highly purified virus (Fig. 7). The molecular weights range from 21,000 to 117,000. The species appearing most persistently have molecular weights of about 21,000, 31,000, 88,000, 93,000, 100,000, and 112,000. When viral polypeptides in a gel are reacted with ‘251-labeled

350

KASAMATSU

anti-Vpl, only the 88,000, 93,000, 100,000, and 117,000 species are stained (Figs. 2, 5, and 6). Assuming that each polypeptide contains one set of Vpl determinants, the amount of each per virion can be calculated by comparing the intensities of 1251staining with that of Vpl from a known amount of virus. Staining intensities were measured both by densitometry of the autoradiograms and by counting slices of gels. The amount of ‘251-labeled anti-VP1 staining of the 117,000 and 100,000 dalton species by both methods corresponds to approximately one to four copies of each per virion. These Vpl-related polypeptides have been detected in lysates of infected cells. Both species frrst appear long after Vpl synthesis has begun. They were detected sooner in nuclear extracts, at 54 hr, than in cytoplasmic extracts, at 95 hr. This observed has been confirmed on gels containing equal amounts of total nuclear or cytoplasmic polypeptides in each slot (not shown).

FIG. 7. Staining of SV49 viral polypeptides with Coomassie blue. Lysates of SV40 virus were electrophoresed through a 3%cm long SDS-polyacrylamide gradient gel, to 15%, which was stained with Coomassie blue as described under Methods. All three slogs shown contain total viral polypeptides: 125 pg in each of slots 1 and 2, and 12.5 pg in slot 3. The positions of Vpl and Vp3 are indicated at the left. The positions of marker polypepties in an identical gel are shown at right: 94K (phosphorylase a, (Sigma)); and 43k, 27K, and 14K (trimers, dimers, and monomers of a 14,399 dalton polypeptide, purchased from BDH Chemical Co.).

AND

FLORY DISCUSSION

We have used the immunological staining method described above to study further the synthesis of both SV40 Vpl and Vplrelated viral polypeptides in infected cells. Previous pulse-labeling experiments have shown that the synthesis of virus-induced polypeptides of the same molecular weight as Vpl begins approximately 24 hr postinfection (Ozer and Tegtmeyer, 1972; Anderson and Gesteland, 1972). At 52 hr postinfection, about 85% of the 45,000 dalton pulse-labeled polypeptides are in the nucleus (Walter et al., 1972). The antibody staining method provides evidence that at least some of this material is Vpl and enables the amounts of Vpl present at different stages of infection to be measured. We also first detect a large increase in the amount of Vpl in nuclei between 12 and 27 hr postinfection. The amount of Vpl in cytoplasmic extracts did not rise abruptly until much later, between 27 and 54 hr postinfection, suggesting a rapid transport of newly synthesized Vpl from the cytoplasm to the nucleus at earlier times. We find approximately four times more Vpl in the nucleus than in the cytoplasm at 54 hr postinfection. The small, nearly constant amounts of Vpl observed prior to the burst of Vpl synthesis at 24 hr postinfection probably come from the virus used for infection. The multiplicity of infection used corresponds to about 1000 virus particles per cell, of which about 500 should be firmly adsorbed to the cell (Barbanti-Brodano et al., 1970). The amounts of Vpl in the 2 to 12-hr infected cell extracts were therefore measured to estimate the number of virions adsorbed. The intensities of 125Istaining in the different Vpl bands were measured by densitometry of the autoradiograms, care being taken to ensure that all measurements fell within the linear response range of the fihn. The Vpl bands in the V slots containing known amounts of virus were used to convert the Vpl in the intracellular extracts into virion equivalents. These values were divided by the number of cells used to make the aliquots of each extract applied to the gel. The result is that each cell contains Vpl polypeptides equivalent

SYNTHESIS

to about 420 virions, 160 in the cytoplasm, and 260 in the nucleus. This value is consistent with the interpretation that the Vpl seen between 2 and 12 hr is derived from virions bound or absorbed during the infection step. The general concept of immunologically identifying proteins separated on gels by electrophoresis has been applied in several different ways (Williamson, 1971; Keck et al., 1973; Stumph et al., 1974; Burridge, 1976). The method described here incorporates several important improvements: the precipitation of proteins in the gel with TCA, glutaraldehyde fixation, and use of a single antibody for staining. Precipitation of the protein in the gel improves the efficiency of crosslinking by glutaraldehyde (Keck et al, 1973). TCA was chosen since virtually all polypeptides are insoluble in 20% TCA. The glutaraldehyde fixation should ensure that polypeptides do not diffuse out of the gel during the long staining and rinsing treatments. Direct staining with a single antibody against the polypeptide of interest greatly shortens the time required, since the lengthy stain and rinse periods for a second antibody are avoided. We have also shown that the background can be substantially reduced and the rinse times shortened by including 0.5% Triton X-106 in the rinse buffer. The immunological staining method described here is very sensitive, with a detection limit of less than 0.02 pmol, or 1 ng for SV40 Vpl, molecular weight 45,000. The same molar limit should theoretically apply to any polypeptide in the gel, so that even smaller masses of lower molecular weight polypeptides should be detected. The staining is specific, since the viral polypeptide bands Vp2 and Vp3, which are not related to Vpl, show only a very low background. The amount of antibody bound is related to the amount of polypeptide in the band, so that the method can be used for quantitation within the range examined. Finally, only a small amount, 406 pg, of the total IgG fraction is required to stain each gel of the size used here. Antibody staining of polypeptides in gels can be used in at least two ways. First, a specific polypeptide can be located and quantitated even in the presence of other

OF SV40 Vpl

351

unrelated polypeptides of the same molecular weight, as in a crude extract. Second, related polypeptides of different weights but carrying at least some of the same antigenic determinants can be located in a gel. The latter application is useful in defining precursor-product relationships among polypeptides. lz51-labeled antibody staining can also be used to detect altered polypeptides in viral mutants. A deletion in the region of the gene coding for the final product should reduce the molecular weights of both precursor and processed polypeptide. A deletion in the region coding for the portion of the precursor removed during processing would have a smaller precursor but a normal product if the deletion did not block processing. Mutants affecting processing, such a temperature-sensitive mutants, could show either no final product or one of altered molecular weight. In addition to Vpl, two polypeptides with apparent molecular weights of 117,000 and 100,006 are detected by ‘%I-labeled anti Vpl staining of extracts of infected cells. They were also consistently found in virions by ‘251-labeled anti Vpl staining (Fig. 2), Coomassie blue staining (Fig. 7), and labeling with 14C-amino acids (data not shown). The amounts in virus corresponded to about one to four copies of each per virion by all three methods. These low amounts are difficult to detect unless the gels are overloaded with viral polypeptides or the staining method described here is used. In infected cells, they were detected first in nuclear extracts very late in infection, about 27 br after the burst of Vpl synthesis. They were not detected until much later in cytoplasm, about 66 hr after the burst of Vpl synthesis. As noted above, the cytoplasmic extracts at this late time may contain polypeptides from newly assembled vii-ions. Somewhat larger amounts of the high molecular weight polypeptides relative to Vpl seem to be present in nuclear extracts than in cytoplasmic ones. Even larger amounts of these polypeptides are found in vii-ions. These differences in amount in relation to Vpl suggest that they are not simply multimers of Vpl formed as an artifact of the extraction procedure. The concentration of DTT used in the extraction, 12 n&i, should have ensured complete dissociation of in-

352

KASAMATSU

termolecular disulfide bonds (Walter and Deppert, 1974). High molecular weight polypeptides with tryptic digest patterns identical to Vpl have been observed in virions of other papovaviruses (Hewick et aZ., 1975; Wright and di Mayorca, 1976). It cannot be excluded that these high molecular weight polypeptides are the products of an unknown specific crosslinking which is resistant to D’M’. However, their late appearance during infection and specific localization in the nucleus despite greater amounts of Vpl in the cytoplasm suggests that they may have a function in packaging. It is unlikely that they are precursors to Vpl since the specific 16 S messenger RNA for Vpl is large enough to code for only about 45,090 daltons of protein. Their existence suggests that there may be small quantities of large messages coding for these polypeptides. ACKNOWLEDGMENTS We are grateful to Drs. N. Davidson, G. Famed, and P. Sharp for critical comments on the manuscript. We are also grateful for the skilled technical assistance of Deneille Stephens. P. J. F. was supported by Grant NISRA-CA-09056. This investigation was supported by Grant NP-223 from the American Cancer Society, by Grant CA 21766from the National Cancer Institute, DHEW, a grant from the California Institute of Cancer Research, and a grant from the Committee on Research, Academic Senate, Los Angeles Division, University of California. REFERENCES ANDERSON, C. W., and GESTELAND, R. F. (1972).

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