Vol. 32, No. 2

JOURNAL OF VIROLOGY, Nov. 1979, p. 648-660

0022-538X/79/11-0648/13$02.00/0

Intracellular Localization of Viral Polypeptides During Simian Virus 40 Infection HARUMI KASAMATSU* AND ANGELA NEHORAYAN Department of Biology and Molecular Biology Institute, University of California at Los Angeles, Los Angeles, California 90024

Received for publication 2 March 1979

African green monkey kidney cells infected by simian virus 40 were analyzed by immunofluorescence techniques for the nature and the time course of the appearance of viral polypeptides during infection. Reagents used in the study were anti-Vpl sera and affinity-purified anti-Vpl immunoglobulin G, anti-Vp3 sera, antivirus (anti-V) sera, and anti-tumor antigen sera. The results are summarized as follows. (i) Three types of staining, nuclear, perinuclear, and perinuclear accompanied by cytoplasmic staining, were observed in infected cells in reaction with anti-Vpl antibody. In addition, a highly structured staining was observed at the periphery of nuclei of infected cells late in infection. (ii) In reaction with anti-Vp3 serum, the staining was confined within nuclei of cells throughout infection. (iii) Vpl and Vp3 antigens seem to occupy different spacial regions of the nuclear area in cells. (iv) Vpl and Vp3 antigens were expressed simultaneously during infection. (v) Centriolar staining observed early in infection paralleled the appearance of tumor (T-) antigen until 24 h after infection, after which time the frequency of positive centriolar staining decreased as infection progressed. (vi) T-antigen was first expressed at about 8 h after infection, and Vpl and Vp3 antigens were first expressed at about 20 h after infection.

The genome of simian virus 40 (SV40) has a defmed molecular structure and a number of genes. After virus infection of permissive cells, each gene is expressed during a particular time period. In the early phase of infection, a portion of the viral genome is transcribed, and T-antigen, U-antigen, and tumor-specific transplantation antigen are expressed. Subsequently, certain enzyme activities and host DNA synthesis are induced. About 15 h after infection, progeny viral DNA is replicated, and at the same time, a new species of viral DNA-specific RNAs appears in the cytoplasm and is translated into viral structural polypeptides. These polypeptides and newly made viral DNA, together with host histones, are assembled into mature virus particles (19). In particular, the structural polypeptides of SV40, Vpl, Vp2, and Vp3, have been shown to be synthesized at a similar rate in the late phase of a productive infection, and these polypeptides are metabolically stable (1, 5, 10, 14, 20). To study further the time course of synthesis of the structural viral polypeptides and the intracellular distribution of the different polypeptides, we prepared monospecific sera against purified viral polypeptides Vpl and Vp3 and used them in the present study. MATERIALS AND METHODS Cells and virus. TC7 monkey cells were cultured

in plastic dishes, using Dulbecco modified Eagle medium supplemented with 0.3 mg of arginine-hydrochloride per ml, 1.5 mg of glutamine per ml, 20 ,ug of histidine-hydrochloride per ml, and 1.5 mg of glucose per ml, containing 10% (vol/vol) fetal bovine serum. For immunofluorescence studies, cells were seeded on glass cover slips (22 by 22 mm; Gold Seal, Clay Adams) in petri dishes. When infected with virus, cells were cultured in medium containing 3% fetal bovine serum and 125 ,tg of streptomycin sulfate per ml, 31.25 ,ig of kanamycin per ml, 250 ,ug of n-butyl-p-hydroxybenzoate per ml, and 625 U of penicillin G per ml. Wildtype SV40, plaque-purified strain 776, was used to infect subconfluent cells at a multiplicity of 10. Preparation and characterization of antisera. Antisera to SV40 viral polypeptide Vpl or Vp3 were obtained from New Zealand white rabbits immunized by intradermal and scapular injections of electrophoretically homogeneous antigens. Total viral polypeptides were electrophoresed on 7 to 15% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gels (15 by 30 by 0.15 cm). After Coomassie brilliant blue staining, the Vpl and Vp3 bands were excised, lyophilized, and ground as finely as possible with mortar and pestle. The powdered sample was suspended in 1 ml of phosphate-buffered saline (PBS; pH 7.5), emulsified with an equal volume of complete Freund adjuvant, and injected into rabbits. About 100 Mg of antigen was used per injection. Injections were repeated with incomplete adjuvant at 4-week intervals. Sera were tested for anti-Vpl or anti-Vp3 activity by immunodiffusion against SDS-dissociated total viral polypeptides. Sera were taken from the positively reacting 48

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VIRAL POLYPEPTIDES DURING SV40 INFECTION

animals. Antisera used in the study were obtained from bleedings obtained 3 weeks after the third booster injection. The specificity of the anti-Vpl serum and of the anti-Vp3 serum were checked by the methods described by Burridge (2). Anti-Vpl immunoglobulin G (IgG) reacted specifically with Vpl polypeptide, and anti-Vp3 serum reacted specificially with Vp3 polypeptide (Fig. 1). There seems to be very little cross-reactivity to Vp3 polypeptide in the anti-Vpl IgG and to Vpl polypeptide in the anti-Vp3 serum. Although the amount of Vp2 polypeptide contained in virus was similar to that of Vp3 polypeptide, no obvious "25I-labeled band was detectable in the gels which had been reacted with either anti-Vpl or anti-Vp3 serum. Both antisera recognized virus particles, and about 1.3 x 1010 virus particles were precipitated with about 1 1A of anti-Vpl serum or 0.2 ,l1 of anti-Vp3 serum. Anti-SV40 virion (anti-V) serum was obtained from a rabbit hyperimmunized with intact SV40 virus, a gift from K. Takemoto, National Institutes of Health. It was used at a 1:20 dilution. The sera contained antiT activity and reacted with purified Vpl and Vp3 polypeptides. Anti-T serum, also a gift from K. Takemoto, was used at a 1:25 dilution. Antisera to rabbit IgG were obtained from a goat immunized with 1 mg of rabbit IgG in 1 ml of PBS and 1 ml of Freund adjuvant at 3-week intervals. Antisera used in these studies were from the bleeding obtained after the third booster injection. Preparation of fluorescein IgG. Fluorescein-conjugated goat anti-rabbit IgG was prepared by a method described previously (18). A crude goat IgG fraction was obtained by ammonium sulfate (48%) precipitation of goat anti-rabbit IgG sera at 4°C and dialyzed against PBS. 1.8 ml of dialyzed sample, containing 125 mg of IgG, was mixed with 0.2 ml of 0.5 M sodiumcarbonate buffer (pH 9.5) and 3.1 mg of fluorescein isothiocyanate (Sigma Chemical Co.). The mixture was incubated for 5 min at room temperature, centrifuged at 3,000 rpm for 10 min, and fractionated on a Sephadex G-25 column to remove unbound fluorescein. Fluorescein-conjugated IgG was further purified on a DEAE-cellulose column. The fraction eluted by 0.125 M NaCl in 17.5 mM phosphate buffer (pH 6.3) was used throughout the study. Preparation of anti-Vpl IgG by affinity chromatography. Viral polypeptides were isolated from virus by sedimentation through a 15 to 30% sucrose gradient in 0.01 M Tris, 1 mM EDTA, and 0.5% SDS as described previously (11). After mixing with a tracer amount of '25I-labeled viral polypeptides prepared by the method described by Greenwood et al. (9), the viral polypeptides were then dialyzed extensively against 0.1 M sodium-borate buffer (pH 7.5) containing 0.1% SDS. Specific activity of the viral polypeptide was 2.2 x 103 cpm/pg of protein. About 2.74 mg of protein in 10 ml of buffer was added to 0.5 g of solid Affi-gel 10 (Bio-Rad Laboratories). The mixture was left at 4°C for 2 h, with occasional shaking, then placed on a shaker bath for 24 h at 4°C for complete coupling. This mixture was packed into a column (1 by 20 cm) and washed with 15 ml of 0.1 M sodium-borate buffer containing 0.1% SDS, 20 ml of 0.1 M sodium-borate buffer containing 2% SDS, and finally 30 ml of 0.1 M sodium-borate buffer containing 0.1% SDS to remove

anti Vp3

anti

649

Vpi

Vp3

FIG. 1. Autoradiograms of gels stained with antisera. Virus (2 jig in the anti- Vpl lane and 10 pg in the anti-Vp3 lane) in 50 pl of 2% SDS, 50 mM Tris (pH 6.8), 10 mM dithiothreitol, 10% glycerol, and 0.001% bromophenol blue was boiled for 4 min and applied either to an SDS-7.5% polyacrylamide gel (anti-Vpl) or an SDS-10% polyacrylamide gel (anti-Vp3) prepared by the method of Laemmli (12). After electrophoresis, the gels were treated with 46% methanol8% acetic acid for 1 h and equilibrated with buffer A (50 mM Tris, pH 7.4, 0.15 M NaCl, 5 mM EDTA, and 0.02% NaN3) for at least 20 h with four changes. For the study of anti-Vpl specificity, the gel was incubated for 20 h with 200 pg of 125I-labeled IgG fraction of anti- Vpl serum (5 x 105 cpm/g) in 10 ml of buffer A containing 1 mg of bovine serum albumin per ml. Unbound antibodies were removed by rinsing the gel in buffer A. For the study of anti- Vp3 specificity, the gel was reacted with 200 pl of anti-Vp3 serum in 30 ml of buffer A containing 2.5 mg gelatin per ml for 20 h, rinsed thoroughly with buffer A, and incubated with 3 pg of 1251-labeled protein A (3 x 106 cpm/pg) in 30 ml of buffer A with 2.5 mg ofgelatin per ml for 20 h. The above treated gels were stained with Coomassie brilliant blue, destained, treated with 1% glycerol in buffer A for 1 h, dried, and autoradiogrammed with X-ray films (Cronex 4; DuPont) with an intensifying screen (Cronex Quanta II; DuPont). Anti- Vpl IgG or protein A (Pharmacia) was labeled with 1251 by the chloramine T method (9) or a modified chloramine T method, respectively. In the modified chloramine T method, the reaction was quenched with tyrosine (0.4 mg/ml) instead of sodium metabisulfite. The positions of Vpl and Vp3 polypeptides in the gels, indicated in the photograph, were identified from the Coomassie brilliant blue staining of the viral polypeptides and of marker polypeptides (bovine serum albumin, ovalbumin, trypsinogen, and /3-lactoglobulin).

650

KASAMATSU AND NEHORAYAN

uncoupled viral polypeptides from the beads. Each eluate was checked for radioactivity; it was found that approximately 1 mg of polypeptide was bound to 0.5 g of beads. The column was then washed sequentially with: 50 ml of 10 mM Tris (pH 8.5)-10 mM dithiothreitol-0.5% SDS; 100 ml of 10 mM Tris (pH 8.5); 20 ml of 10 mM Tris (pH 7.9)-0.15 M NaCl-1 mM EDTA-0.5% NP-40; 10 ml of the same buffer containing 5 mg of bovine serum albumin per ml; 70 ml of the same buffer without bovine serum albumin; and 70 ml of 10 mM Tris (pH 8.5)-i mM EDTA-0.15 M NaCl. About 1.5 ml of anti-Vpl serum was then loaded slowly onto the column. The flow-through fraction was reloaded several times; the column was then washed extensively with 10 mM Tris (pH 8.5)-i mM EDTA0.15 M NaCl, until no absorbance could be measured in the wash at 280 nm. Flow rate of the column was about 1 ml/min. Adsorbed antibody was eluted by the application of 0.2 M glycine-hydrochloride saline buffer (pH 2.4), and fractions were collected. Peak fractions were detected by absorbance at 280 nm and combined. The solution was adjusted to neutrality with NaOH and dialyzed overnight against 10 mM Tris (pH 7.5)-i mM EDTA-0.15 M NaCl. Gammaglobulin was concentrated by precipitation with an equal volume of saturated ammonium sulfate suspended in water and dialyzed against 10 mM Tris (pH 7.5)-i mM EDTA-0.15 M NaCl. Approximately 0.80 mg of specific IgG was obtained from 1.5 ml of antiVpl serum. For the immunofluorescence studies, the specific IgG was diluted to 0.5 mg/ml in PBS containing 10 mg of bovine serum albumin per ml. Fixation, staining, and analysis of samples. At various time points after infection, cover slips were removed from the culture, rinsed well in PBS, fixed in a 1:1 mixture of acetone and methanol for 3 min, left another 3 min at -20°C, and air dried. These cover slips were stored at -20°C for later use. Uninfected cells were prepared in a similar manner. Other fixation methods, such as 0.24% glutaraldehyde fixation followed by 0.5% Triton X-100 treatment in PBS, were used in earlier studies for comparison with acetonemethanol fixation. No change in the staining patterns was observed. The preparations were overlaid with 25 t,l of appropriate dilutions of antiserum (1:5 for antiVpl and anti-Vp3, 1:20 for anti-V, and 1:25 for anti-T) or specific anti-Vpl IgG fraction for 60 min at 37°C, rinsed well in PBS, and then stained with 25 1L of fluorescein-conjugated goat anti-rabbit IgG (0.5 mg/ ml) or goat anti-hamster IgG at a 1:100 dilution. After a further thorough washing, the cover slip was inverted onto a drop of Elvanol (DuPont Co.). The cells were observed with a Leitz Orthoplan microscope equipped with a Ploemopak for incident illumination, using optics composed of a 63x objective and Leitz filter H and K530 barrier filter. Specimens were photographed with an Orthomat camera, using Kodak Tri-X or Plus-X film. After microscopic observations, the number of positively stained cells of a total of about 300 cells was counted. Types of positively stained cells were classified from a randomly picked sample of 100 positively stained cells. Samples were photographed, and the negatives were used for the area measurements. The Olympus micrometer was used to obtain the magnification factor.

J. VIROL.

Measurement of area. The majority of the cells possessed circular or ellipsoid nuclei exhibiting either uniform or perinuclear staining. Based on this, an estimated cross-sectional area enclosed by the surface stained with antibodies was computed as S = (ab/4)7T, expressed as square micrometers, from the measured lengths of the shorter (a) and the longer (b) diameters. Measurements of the distances a and b were made to the outermost part of the stained nuclear region. Although cells with irregularly shaped nuclei also exist, they were omitted from the computation. Measurements for about 25 nuclei were taken for each time point.

RESULTS Localization of viral polypeptide Vpl during productive infection. Antiserum against Vpl was used to detect the localization of the antigen in the cells throughout infection. Infected cultures were harvested at various time points after infection, fixed in acetone-methanol, reacted with Vpl antisera, stained with fluorescein-conjugated goat anti-rabbit gammaglobulin (IgG), and examined for the localization of the antigen in the cells. Several different types of fluorescent stainings-perinuclear, nuclear, and cytoplasmic-were observed. Uninfected cells did not exhibit any detectable fluorescent staining. Anti-Vpl IgG purified by affinity chromatography was also tested to check the Vpl specificity of these stainings. Identical results were obtained. The remaining studies were carried out with anti-Vpl IgG. No Vpl staining was observed within 16 h after infection (Fig. 2a). At about 20 h after infection, 1 to 2 of 1,000 cells showed nuclear or perinuclear staining. Diffuse cytoplasmic staining appeared in some of the stained cells. At about 24 h after infection, three distinct types of staining were observed: (i) uniform nuclear staining (Fig. 2b), (ii) perinuclear staining (Fig. 2c), and (iii) perinuclear staining accompanied by cytoplasmic staining (Fig. 2c and d and 3a). These three types of stained cells were also observed at approximately 37, 48, and 60 h after infection. The frequency of each type is presented in Table 1. The intensity of cytoplasmic staining increased late in infection, whereas the fraction of stained cells with staining confined to the nucleus or peri-nucleus decreased. In general, photographs were taken at the sharpest focal point of the perinuclear and cytoplasmic staining (Fig. 3a and b and 2c). This tends to make the finer structures of the nuclear region out of focus, since a cell possesses a threedimensional structure. In fact, a highly structured network of staining was observed in the nuclei at different focal planes (Fig. 3). This suggests that the antigens at the periphery of nuclei possess an ordered structure.

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VIRAL POLYPEPTIDES DURING SV40 INFECTION

651

FIG. 2. Fluorescence micrographs of cells stained with anti- Vpl IgG. Staining pattern observed at 16 h (a) and 24 h (b, c, and d) after infection. Bar represents 50 pm.

652

J. VIROL.

KASAMATSU AND NEHORAYAN TABLE 1. Results of staining with anti-Vpl

gammaglobulina Type of staining (%)

TimePeiu Perinu-

after in- Positive fection cells (%) Nuclear

(h)

Perinu-

~~~~clear

and

ando

Anti-Vpl area

0im')

plasmic 12

0

-

-

16 20 24

0 0 9

-

-

-

-

-

-

-

-

19

50

32

370.5 ± 51.6

37

37

3

8

89

490.1+± 77.4

48

55

6

8

86

60

76

0

3

97

506.5 ± 119.6 607.4 ± 110.2

--

Values were computed as described in the text.

Actual diameters of nuclei, defined as nuclei visualized after the staining, appeared to be different as infection progressed (Fig. 2 and 3). To obtain quantitative differences, the cross-sectional area enclosed by the surface stained with antibody to Vpl was computed as described in the text. The average area measured is presented in Table 1. Between 24 and 60 h after infection, there was an increase in area of approximately 60%. Assuming that these regions confined by the nuclear staining are circular in form, one can convert the area to the corresponding diameter. The average diameter of stained nuclei was 22 ,um at 24 h after infection and 28 ,im at 60 h after infection. Thus, a difference of about 6 ,um in diameter was observed in this time period. Centriolar staining. The time course of appearance of viral polypeptide Vp3 during infection was studied in a similar fashion with antiVp3 sera. Anti-Vp3 sera were used throughout the study, since isolation of pure anti-Vp3 IgG fraction was not possible at this stage. Two types of response were observed: (i) Staining at the centriolar region of cells, which we call centriolar staining (Fig. 4a-d and 5a-d) and (ii) nuclear staining (Fig. 4c-f ). The nuclear staining will be discussed later. The centriolar staining was sometimes accompanied by fibrous staining radiating from the centriolar region into the cytoplasm (Fig. 4a and b) and across the nucleus (Fig. 4b and a). Uninfected control cells did not exhibit any detectable fluorescent staining, except in the centriolar region. A faint fluorescent centriolar staining is detectable in about 10 to 15% of the uninfected cells. At 24 h after infection, about 82% of cells exhibited the centriolar

staining (Fig. 5a) and 5% of cells exhibited nuclear staining (Fig. 5b and Table 2). To see whether the centriolar staining is related to the presence of either Vp3 polypeptide or Vp3-related polypeptide, anti-Vp3 sera were preadsorbed to virus before use in the staining of infected cells. The frequency of the centriolar staining was not affected by the preadsorption of the sera to virus (Fig. 5c and 5d); however, nuclear staining was completely abolished. Very rarely (less than 1%) cells with intense centriolar staining and very faint nuclear staining (Fig. 5d) were observed. Perhaps anti-Vp3 activity was not completely removed by the preadsorption. The above preadsorption experiment indicates that the observed centriolar staining is not specific to Vp3 but that nuclear staining is specific to Vp3. Centriolar staining appeared very early in infection (Table 2). The frequency of strong centriolar staining increased rapidly, i.e., 82% of the cells showed positive centriolar staining by 24 h after infection; however, the frequency decreased as the infection progressed. This decrease in the frequency of centriolar staining might be related in some way to the function of the centriole in infection or may be due to lack of detection of centriolar staining during the late stages of infection, since the strong nuclear staining, the appearance of which coincides with the decrease of detectable centriolar staining, might be masking the clarity of the centriole. Localization of viral polypeptide Vp3 during productive infection. As mentioned in the previous section, nuclear staining, which is specific to Vp3 antigen, was observed during infection. The percentage of cells with nuclear staining increased from about 5% at 24 h after infection to 62% (48 h after infection) and 83% (60 h after infection) (Table 2). The intensity of the nuclear staining was typically greater later in infection (cf. Fig. 4c and f). Essentially the same results were obtained with antisera at 1:2, 1:5, 1:10, and 1:50 dilutions; no significant cytoplasmic staining was seen even with the most concentrated antiserum (that is, greater than the faint, diffuse stain seen early in infection with specific sera or seen at any time with control sera). Although nuclear staining is still uniform, a higher intensity of fluorescence was observable at the periphery of the nuclear area in

approximately 25% of the stained cells late in infection (Table 2). The diameter of stained nuclei was calculated from the cross-sectional area enclosed by the surface stained with antibody to Vp3 for the respective time points (Table 2). A gradual increase in the area of stained nuclei was observed

VOL. 32, 1979

VIRAL POLYPEPTIDES DURING SV40 INFECTION

653

I FIG. 3. Fluorescence micrographs of cells stained with anti-Vpl IgG. The micrographs were taken at different focal planes of the same cells: a and d, b and e, or c and f. Staining patterns observed at 24 h (a and d) and 60 h (b, c, e, and f) after infection. Bar represents 50 um.

throughout infection. Between 24 and 60 h after infection, an increase of approximately 40% in the area of stained nuclei, which corresponds to a difference of 4 ,um in diameter, was observed. The nuclear area stained with anti-Vp3 sera appeared to be smaller than the total nuclear or

perinuclear area stained by anti-Vpl. Localization of T and V antigens during infection. The time course of appearance of T and V antigen was also studied to correlate this with that of Vpl and Vp3 antigens. The staining with anti-T sera gave results similar to those of

654

KASAMATSU AND NEHORAYAN

J. VIROL.

d

r---"

FIG. 4. Fluorescence micrographs of cells stained with anti- Vp3 sera. Staining patterns observed at 12 h (a and b), 24 h (c), 37 h (d), 48 h (e), and 60 h (f) after infection. Bar represents 50 ,M.

previous investigators (6, 16); therefore micrographs are omitted. The fluorescence of cells reacted with anti-T sera, observable at 12 h after infection, was confined to the nucleus. The fre-

quency of nuclear staining increased with the progress of the infection. In addition, the average area of the nuclear region stained increased with time (Table 3), similar to that of cells reacted

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VIRAL POLYPEPTIDES DURING SV40 INFECTION

FIG. 5. Fluorescence micrographs of cells stained with anti- Vp3 sera and virus-preadsorbed Anti- Vp3 sera. (a and b) Staining patterns observed with antiVp3 at 24 h after infection; (c and d) stainingpatterns observed with the sera preadsorbed to virus particles as described in the text. Bar represents 50 pm.

with anti-Vp3, but less than that for anti-Vpl. Reaction with anti-V sera, which contained some anti-T activity, revealed the following. Up to 20 h after infection, stained cells predominantly exhibited nuclear staining identical to that observed in anti-T staining, as shown in Fig. 6a-c. The nuclear staining observed at 20 h after infection remained unchanged after preadsorption of sera to virus particles. Perinuclear staining resembling that of anti-Vpl appeared at about 20 h after infection (Fig. 6d-e). Sometimes both nuclear and peri-nuclear stainings could be observed separately in one cell (Fig. 6d-f). Cytoplasmic staining appeared at about 24 h after infection in 18% of stained cells (Fig. 7a-d) and its frequency continued to increase with time. Fine structures at different focal planes analogous to those described for anti-Vpl stainings were also observed in anti-V stainings late in infection (Fig. 7). The average area of stained nuclear region increased in a similar manner to cells reacted with anti-Vpl (Table 3).

655

Expression and localization of late gene products during productive infection. Percent positive staining obtained in different antigen-antibody reactions (Tables 1-3) are expressed as a function of time in Fig. 8. Reaction to anti-T sera exhibits a biphasic curve. The number of cells positively stained with anti-T sera increases linearly at a rate of 4.4%/h until approximately 20 h after infection, at which time the rate decreases. Extrapolating this curve to 0% positive staining indicates the time at which the T-antigen is first expressed under these conditions. Thus, T-antigen is produced starting at about 8 h after infection. Centriolar staining observed in anti-Vp3 reactions somewhat parallels the expression of the T-antigen until 24 h after infection, after which time the frequency of positive centriolar staining decreases as infection progresses. A linear increase in percent positive staining was observed for cells reacted with anti-Vpl. Extrapolation indicates 20 h after infection to be the time at which Vpl first appears. Identical results are obtained for anti-Vp3 reactions, excluding centriolar staining. There is thus a lag of approximately 12 h between the expression of the early gene product, T-antigen, and that of the late gene products Vpl and Vp3. It may be concluded that the expression of early and late gene products is controlled differently. The above results agree with the results obtained by analyses of pulse-labeled polypeptides on SDS-polyacrylamide gels after virus infection (1, 5, 14, 20), and by analyses of total cellular polypeptide of infected cells with radiolabeled anti-Vpl IgG (10). TABLE 2. Results of staining with anti-Vp3 seruma

Type of staining (% Time Centrioafter in- lar- Positive At- 3 cells Nuclear area fection stained (4.tm) and (%)b (% Nuclear peri(h) cells nuclear'

12 16 20 24

48 57 73 82

0 0 0 5

100

0

265.0±+

37

54

37

100

0

340.0°±

48

34

62

77

23

14

83

321.3 ± 65.7 379.9 ±

-

_

-

-

-

-

129.0

72.7 60

II

25

75

I

I

1

~~~~~112.6

a Values were computed as described in the text. b Nuclear-stained cells independent of centriolar

staining. 'Uniform nuclear staining accompanied by intense

perinuclear staining.

656

KASAMATSU AND NEHORAYAN

J. VIROL.

FIG. 6. Fluorescence micrographs of cells stained with anti- V sera. Staining patterns were observed 16 h (a and b) and 20 h (c, d, e, and f) after infection. Bar represents 50 pm.

We observed the diameter difference of stained nuclei increased as infection proceeded, stained nuclei through the course of infection in as shown in (Tables 1-3). (ii) The diameter of two ways. Diameters were computed from the anti-Vpl-stained nuclei was consistently larger cross-sectional area enclosed by the surface than that of anti-Vp3-stained nuclei. The area stained by antibodies. (i) The diameter of of stained nuclei (Tables 1-3) is plotted as a

VOL. 32, 1979

VIRAL POLYPEPTIDES DURING SV40 INFECTION

657

.. --..._I ..sE'd' FIG. 7. Fluorescence micrographs of cells stained with anti-V sera. The micrographs were taken at two different focal planes (a and b; c and d) of the same cells. Staining patterns observed at 37 h (a and b) and 60 h (c and d) after infection. Bar represents 50 pm. TABLE 3. Results of staining with anti-T or anti- V seraa

TimeTpoi after tive inec(h)

cels

A

-o-

Anti-T area

tive

Anti-V area

(mi2)

cels

(1=2)

12 16 20 24 37 48 60

22 25 42 181.9 ± 51.6 35 248.6 ± 53.9 66 227.5 ± 42.2 56 248.6 ± 53.9 74 243.9 ± 84.4 78 349.4 ± 46.9 76 321.3 ± 63.3 86 326.0 ± 65.7 86 398.7 ± 168.8 92 565.1 ± 75.0 94 415.1 ± 72.7 100 45.0.1 ± 119.6 a Values were computed as described in the text.

function of time in Fig. 9. The results indicate that, indeed, the cross-sectional area enclosed by the surface which is stained with antibody to Vpl is consistently larger than that of nuclei stained with antibody to Vp3. For example, at 37 h after infection, 340 and 490 ,m2 are the respective average areas of stained nuclei of Vp3and Vpl-positive cells. Assuming that these nuclear regions are circular in shape, the corresponding diameters are 20.8 and 25.0,um, respectively. Thus, there is a diameter difference of approximnately 4 ,um late in infection. This suggests that Vpl and Vp3 antigens occupy different spacial regions of the nuclear area.

0/

0~~~~~~~~

50) 0)

a.

\0 0

0

20

40

20 40 Hours After Infection

60

60

FIG. 8. Expression of T antigen, Vpl antigen, Vp3 antigen, and centriolar staining during infection. Frequency of stained cells observed in reaction with anti- Vpl IgG (Table 1), anti- Vp3 (Table 2), and antiT (Table 3) and of centriolar staining is plotted as a function of time after infection. Symbols: [-F, T antigen; Em- - -El, centriolar staining; O-O, Vpl antigen; A A, Vp3 antigen.

DISCUSSION The use of monospecific antisera to Vpl or to Vp3 has permitted us to determine the cellular localization of these antigens at various time points during infection, relative to that of T and

658

KASAMATSU AND NEHORAYAN 750-

0)

0

500-

z

-

,/

C

.0 U) 0

250-

a)

_,

(n

-

I

0

I

20 40 Hours After Infection

I

60

FIG. 9. Comparison of the nuclear area observed between anti- Vpl-stained nuclei and anti- Vp3stained nuclei. The average area of stained nuclei, obtained from Tables 1 and 2, is plotted (together with a standard deviation) as a function of time after infection. Symbols: A--- -A, area of anti- Vpl -stained nuclei; O-0, area of anti- Vp3-stained nuclei.

V antigens in general. The detection of an antidepends on the local concentration of the antigen, on the titer of each antiserum, and on the availability of exposed antigenic determinant sites. If an antigen is synthesized on cytoplasmic polyribosomes and transported very rapidly to the nucleus, its staining will not be observed in the cytoplasm. A change in the rates of release from polysomes and transport may result in an observable change in intracellular localization. In each antibody reaction, about 105 cells per cm2 were stained with 5 yl of serum. If all of the 105 cells are infected and produce 103 virus particles per cell, 108 virus particles will be contained in the cells late in infection. We have determined that about 1.3 x 1010 isolated virus particles are immunoprecipitated with either 1 IL of anti-Vpl serum or 0.2 ,ul of anti-Vp3 serum. Therefore, the 5 ,ul of serum applied was more than sufficient to interact with all of the assembled virus as well as the unassembled polypeptides. Note that neither antiserum would distinguish the physical nature of the antigens studied. Our results show that Vpl and Vp3 antigens appear at the same time, about 20 h after infection (12 h after the first expression of T-antigen), that the localization of the two antigens is spacially different, and that the frequency of centriolar staining increases with the appearance of gen by immunofluorescence

J. VIROL.

T-positive cells during the early phase of infection. Staining with anti-V serum showed two patterns in infected cells: a nuclear staining similar to that of anti-T staining, and perinuclear and cytoplasmic staining similar to that of anti-Vpl staining. Since anti-V serum also reacts with isolated Vpl and Vp3 polypeptides, the staining observed late in infection which is similar to that of the anti-Vpl reaction (Fig. 3 and 7) is probably due to reaction with Vpl polypeptides in the cytoplasm and at the nuclear periphery rather than to mature virus particles in the cytoplasm. At this stage of infection, mature virus particles have been observed only in nuclei (8). Since this serum also contains some anti-T activity, it is difficult to differentiate the nature of the nuclear staining observed late in infection. Graessmann et al. (7) measured the concentration of intranuclear T-antigen and intranuclear V-antigen in permissive CV1 monkey cells by immunofluorescence and cytofluorometry. Accumulation of V-antigen was detected only in cells which attained a certain concentration of T-antigen. We have so far failed to detect any obvious presence of Vp3 in the cytoplasm of infected cells. The very diffuse cytoplasmic staining observed was similar to that in uninfected cells. In comparison, in polyoma-infected cells, V3 polypeptide, analogous to Vp3 of SV40, is first detected in the cytoplasm (13). Perhaps rapid transport of newly synthesized Vp3 polypeptides from cytoplasm to the nuclei occurs, and further assembly into virus particles may result in the apparent absence of the antigen in the cytoplasm. For example, the fluorescent staining with anti-T sera detected the presence of antigen in nuclei, even though polyadenylic acid-containing mRNA which directs the synthesis of Tantigen has been isolated from the cytoplasm of virus-infected cells (17). The two different types of nuclear staining, uniform nuclear staining, and perinuclear staining, were observed in reaction with anti-Vpl IgG. The relationship between these two is not clear. They may represent transient forms to one another or may have resulted separately from some unknown processes. The intense cytoplasmic staining observed with anti-Vpl IgG late in infection may result from the change in the rate of virus assembly of newly synthesized polypeptides. Labeled major capsid proteins were shown to be incorporated into virus particles soon after their synthesis; however, at a later stage of infection the major portion of labeled capsid protein was not incorporated immediately into viral particles (15). On the other hand, newly synthesized Vp3 polypeptides seem

VOL. 32, 1979

VIRAL POLYPEPTIDES DURING SV40 INFECTION

to be effectively transported into the nuclei for virus assembly throughout infection. Centriolar staining observed in reaction with anti-Vp3 was not specific to Vp3. When infected cells, at 15 h postinfection, were reacted with antisera against tubulin, the staining pattern was completely different from that of anti-Vp3 sera. The centriolar region, which was positively stained with anti-Vp3 sera, was devoid of the staining although microtubule fibers radiating from centriolar region in the cells were stained (unpublished data). Therefore, observed centriolar staining is independent of the presence of tubulin. Of three anti-Vp3 sera tested, two samples gave very strong centriolar staining and one gave very weak centriolar staining (unpublished data). Some control sera also showed intense centriolar staining in infected cells (unpublished data). Therefore, the observed centriolar staining is likely to be a result of cross-reacting activity in the sera (3). About 10 to 15% of uninfected cells could be distinguished as having positive centriolar staining with the anti-Vp3 serum. Thus, distinct centriolar staining is less frequently encountered in uninfected cells, whereas distinct and enlarged centriolar stainings appeared in infected cells at high frequency. Although centriolar staining is not Vp3 specific, the distinct increase in frequency observed very early in infection, accompanied by T-positive staining and followed by a decrease suggests that the centriole may have a function in the early course of viral infection. The observation that Vpl and Vp3 antigens occupy different spacial regions of the nuclear area is quite intriguing. With the observation techniques used here, it was not possible to determine the position of the nuclear membrane relative to the staining by anti-Vpl and by antiVp3. However, we propose that late in infection the Vp3 antigen is found primarily within the nucleus, whereas Vpl lies in a shell about 2 ,Lm in depth just outside the nuclear membrane. The ordered structures observed at the periphery of nuclei in the reaction with anti-Vpl IgG may suggest the specific association of Vpl antigen to some subcellular organelle, i.e., the Vpl synthesis may take place at membrane-bound ribosomes of endoplasmic reticulum. Detailed study of the localization of antigens at the level of electron microscopy is necessary. Although a number of investigators have studied the expression of SV40 gene products by other methods, the present study is the first application of immunofluorescence to observe the time course and the subcellular localization of viral polypeptides after infection with monospecific antisera to Vpl or Vp3. Similar studies with temperature-

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sensitive mutants may shed further light on the function of the several viral genes in virus assembly. ACKNOWLEDGMENTS We thank N. Davidson and T. Maniatis for their interest and advice, and also G. Fareed and J. Jordan for the use of the fluorescence microscope, E. McNeil for the T-antigen assay, and D. Stephens for technical help. This research was supported by American Cancer Society grant NP-223 and Public Health Service grant CA21768 from the National Cancer Institute, and by the California Institute of Cancer Research.

LITERATURE CITED 1. Anderson, C. W., and R. F. Gesteland. 1972. Pattern of protein synthesis in monkey cells infected by simian virus 40. J. Virol. 9:758-765. 2. Burridge, K. 1976. Changes in cellular proteins after transformation: identification of specific glycoproteins and antigens in sodium dodecyl sulfate gels. Proc. Natl. Acad. Sci. 73:4457-4461. 3. Connolly, J. A., and V. I. Kalnins. 1978. Visualization of centrioles and basal bodies by fluorescent staining with nonimmune rabbit sera. J. Cell Biol. 79:526-532. 4. Fey, G., and B. Hirt. 1974. Fingerprints of polyoma virus proteins and mouse histones. Cold Spring Harbor Symp. Quant. Biol. 39:235-241. 5. Fischer, H., and G. Sauer. 1972. Identification of virusinduced proteins in cells productively infected with simian virus 40. J. Virol. 9:1-9. 6. Gilden, R. V., R. L. Carp, F. Taguchi, and V. Defendi. 1965. The nature and localization of the SV40-induced complement-fixing antigen. Proc. Natl. Acad. Sci. U.S.A. 53:684-692. 7. Graessmann, A., M. Graessmann, E. Guhl, and C. Mueller. 1978. Quantitative correlation between simian virus 40 T-antigen synthesis and late viral gene expression in permissive and nonpermissive cells. J. Cell Biol. 77:R1-R8. 8. Granboulan, N., P. Tournier, R. Wicker, and W. Bernhard. 1963. An electron microscope study of the development of SV40 virus. J. Cell Biol. 17:423-441. 9. Greenwood, F. C., W. M. Hunter, and J. S. Glover. 1963. The preparation of 13'I-labeled human growth hormone of high specific radioactivity. Biochem. J. 89: 114-123. 10. Kasamatsu, H., and P. J. Flory. 1978. Synthesis of SV40 viral polypeptide Vpl during infection. Virology 86:344-353. 11. Kasamatsu, H., and M. Wu. 1976. Protein-SV40 DNA complex stable in high salt and sodium dodecyl sulfate. Biochem. Biophys. Res. Commun. 68:927-936. 12. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:680-685. 13. McMillen, J., and R. A. Consigli. 1977. Immunological reactivity of antisera to sodium dodecyl sulfate-derived polypeptides of polyoma virions. J. Virol. 21:1113-1120. 14. Ozer, H. L. 1972. Synthesis and assembly of SV40. I. Differential synthesis of intact virions and empty shells. J. Virol. 9:41-51. 15. Ozer, H. L., and P. Tegtmeyer. 1972. Synthesis and assembly of SV40. II. Synthesis of the major capsid protein and its incorporation into viral particles. J. Virol. 9:52-60. 16. Rapp, F., T. Kitahara, J. S. Butel, and J. L. Melnick. 1964. Synthesis of SV40 tumor antigen during replication of Simian Papovavirus (SV40). Proc. Natl. Acad. Sci. U.S.A. 52:1138-1142. 17. Smith, A. E., S. T. Bayley, T. Wheeler, and W. F.

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Mangel. 1975. Cell-free synthesis of polyoma and SV40 viral proteins, p. 331-338. In A. L. Haenni and G. Beaud, (ed.), In vitro transcription and translation of viral genomes. INSERM, Paris. 18. Sternberger, L. A. 1974. Immunocytochemistry. Prentice-Hall, Inc., Englewood Cliffs, N.J.

J. VIROL. 19. Tooze, J. 1973. The molecular biology of tumor viruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 20. Walter, G., R. Roblin, and R. Dulbecco. 1972. Protein synthesis in SV40-infected monkey cells. Proc. Natl. Acad. Sci. 69:921-924.