Vol. 64, No. 8

JOURNAL OF VIROLOGY, Aug. 1990, p. 3760-3769

0022-538X/90/083760-10$02.00/0 Copyright C 1990, American Society for Microbiology

Simian Virus 40 DNA Replication Correlates with Expression of a Particular Subclass of T Antigen in a Human Glial Cell Line CAROL A. DEMINIE' AND LEONARD C. NORKIN12* Department of Microbiology2 and Graduate Program in Molecular and Cellular Biology,' University of Massachusetts, Amherst, Massachusetts 01003 Received 30 January 1990/Accepted 19 April 1990

Immunocytochemistry and in situ hybridization were used to identify simian virus 40 (SV40) large T-antigen expression and viral DNA replication in individual cells of infected semipermissive human cell lines. SV40 infection aborts before T-antigen expression in many cells of each of the human cell lines examined. In all but one of the human cell lines, most of the T-antigen-producing cells replicated viral DNA. However, in the A172 line of human glial cells only a small percentage of the T-antigen-expressing cells replicated viral DNA. Since different structural and functional classes of T antigen can be recognized with anti-T monoclonal antibodies, we examined infected A172 cells with a panel of 10 anti-T monoclonal antibodies to determine whether viral DNA replication might correlate with the expression of a particular epitope of T antigen. One anti-T monoclonal antibody, PAb 100, did specifically recognize that subset of A172 cells which replicated SV40 DNA. The percentage of PAb 100-reactive A172 cells was dramatically increased by the DNA synthesis inhibitors hydroxyurea and aphidicolin. Removal of the hydroxyurea was followed by an increase in the percentage of cells replicating viral DNA corresponding to the increased percentage reactive with PAb 100. The pattern of SV40 infection in A172 cells was not altered by infection with viable viral mutants containing lesions in the small t protein, the agnoprotein, or the enhancer region. Finally, in situ hybridization was used to show that the percentage of human cells expressing T antigen was similar to the percentage transcribing early SV40 mRNA. Thus, the block to T-antigen expression in human cells is at a stage prior to transcription of early SV40 mRNA.

The outcome of infection by simian virus 40 (SV40) largely depends on the species of the host cell (see reference 36 for a review). Monkey cells are considered to be fully permissive for SV40. They support the production of relatively large viral yields, and infection results in extensive cytopathology. Rodent cells are considered to be nonpermissive. They do not produce progeny virions and are not killed, although some cells may become transformed. Human cells are referred to as semipermissive, since they produce relatively low viral yields and fewer cells show cytopathic effects. The original purpose of this study was to understand why SV40 replicates in only a small percentage of A172 human glial cells (23). As in our earlier report (23), we found a plateau in the percentage of A172 cells which express the SV40 large T antigen. In the present study, we used in situ hybridization to identify cells expressing early viral mRNA. The percentage of cells making T antigen was found to be similar to the percentage expressing early mRNA. We also used in situ hybridization to identify cells replicating viral DNA and found that only a small percentage of T-antigenexpressing (T+) cells also replicated viral DNA. This new finding is particularly intriguing, since T antigen is the only SV40 gene product required for viral DNA replication (19). Other human cell types have been identified in which SV40 T-antigen expression is restricted to a relatively low percentage of cells (1, 3, 7, 25). However, there is no precedent for restricted SV40 DNA replication in T+ human cells. Thus, we asked whether the pattern of infection seen in the A172 cells might be characteristic of other human cell *

lines as well. Whereas SV40 T-antigen expression was restricted in many cells of each of the human cell lines that we examined, in each of those lines a large percentage of the T+ cells replicated viral DNA. Thus, the A172 cells are presently unique in that only a small percentage of T+ cells also replicate viral DNA. To explain this phenomenon in A172 cells, we considered the following aspects of T antigen. SV40 T antigen is a multifunctional protein (see references 11 and 27 for reviews). Its origin-specific binding, ATPase, and helicase activities are essential for viral DNA replication. In addition, T antigen autoregulates its own synthesis, positively regulates late viral gene expression, and is widely known as a transforming protein. T antigen is highly modified after translation, and it is believed that these modifications result in different functional subclasses of the protein. We wanted to determine whether SV40 DNA replication in individual A172 cells might correlate with the expression of a particular subclass of T antigen. For this purpose, we obtained a panel of 10 T-antigen-specific monoclonal antibodies (MAbs) which have been used to identify and partially characterize structurally and functionally distinct forms of SV40 T antigen (10, 13, 28, 32, 33). We found that 9 of the 10 MAbs reacted with approximately the same percentage of infected A172 cells, always in excess of the percentage replicating viral DNA. In contrast, the exceptional anti-T MAb, PAb 100, reacted with a similar percentage of cells as reacted with the SV40 DNA probe. Simultaneous in situ hybridization with the SV40 DNA probe and immunocytochemical detection of T antigen showed that expression of the PAb 100-reactive epitope indeed correlates with viral DNA replication in individual cells. These results suggest that SV40 DNA replication might be dependent on

Corresponding author. 3760

VOL. 64, 1990

the particular form of T antigen recognized by PAb 100. Furthermore, the modification of T antigen recognized by PAb 100 occurs in only some A172 cells. We found that treating infected A172 cells with the DNA synthesis inhibitors, hydroxyurea (HU) and aphidicolin, resulted in an unexpected increase in the percentage of PAb 100-reactive cells. After removal of the HU, the percentage of cells replicating viral DNA became equivalent to the enhanced percentage reactive with PAb 100. These results are consistent with the premise that expression of the PAb 100-reactive form of T antigen is necessary for SV40 DNA replication. Finally, the pattern of infection seen in the A172 cell line was found not to depend on the action of the SV40 small t protein, the agnoprotein, or the 72-base-pair (bp) duplication of the enhancer region. MATERIALS AND METHODS Cells and virus. All cell lines were maintained in Dulbecco modified Eagle medium (GIBCO Laboratories) supplemented with 10% fetal calf serum (Cell Culture Laboratories). CV-1, A172, and T98G cells were purchased from the. American Type Culture Collection. HeLa cells were kindly provided by C. Prives, and HS27 and HS74 cells were provided by H. Ozer. SV40 wild-type (WT) strain 830 and mutant strains pm1493 (26) and d1884 (35) were provided by T. Shenk; A72 was provided by R. Frisque. Virus stocks were prepared by harvesting CV-1 cells 4 days after infection at a multiplicity of infection of 5 PFU per cell. Viral titers were determined by plaque assay on CV-1 cells. Immunocytochemistry (ICC). Hybridoma supernatants containing MAbs PAb 100, PAb 101, PAb 103, PAb 104, PAb 105, PAb 106, PAb 107, PAb 108, and PAb 109 were kindly supplied by E. Gurney (12-14, 32, 33). Hybridoma supernatant of PAb 100 was also provided by C. Prives. PAb 100 hybridoma cells were purchased from the American Type Culture Collection, and PAb 416 (16) and PAb 597 hybridoma cells were kindly provided by E. Harlow. Hybridoma cultures were grown in Dulbecco modified Eagle medium supplemented with 10% hybridmax fetal calf serum (Sigma

Chemical Co.). Cells grown on cover slips were infected with WT or mutant SV40 for 90 min at 37°C. At various times postinfection, the cells were rinsed with phosphate-buffered saline (PBS) and fixed in cold acetone for 10 min. Cells were rehydrated with PBS and incubated for 30 min at 37°C with undiluted hybridoma supernatant and then with a 1:20 dilution of biotinylated anti-mouse immunoglobulin G (Sigma). After treatment with a 1:250 dilution of streptavidin-horseradish peroxidase (hrp) (Enzo Biochem, Inc.) for 1 h at 37°C, the cover slips were washed and the hrp was reacted with a solution of 0.01% H202 and 0.5 mg of diaminobenzidine tetrahydrochloride (DAB) per ml in PBS. The brown precipitate formed indicates the presence of cells which contain viral proteins that reacted with the antibodies. The cells were counterstained with hematoxylin, and 400 to 600 cells were scored to determine the percentage positive for viral proteins. In situ hybridizations using a biotin-labeled SV40 DNA probe. Cells grown on cover slips were infected with WT or mutant SV40 for 90 min at 37°C. At various times postinfection, cells were rinsed with PBS, fixed in 4% paraformaldehyde, and dehydrated through a graded ethanol series. In situ hybridizations with a biotinylated SV40 DNA probe were done as described by Brigati et al. (6). After rehydra-

SV40 T-ANTIGEN EXPRESSION

3761

tion in PBS, the cells were pretreated with 0.02 N HCl-PBS containing 0.01% Triton X-100 and then with 0.5 mg of pronase (Calbiochem-Behring) per ml in 0.05 M Tris hydrochloride (pH 7.6)-S5 mM EDTA. After a wash in PBS containing 2 mg of glycine per ml, the cells were treated with 100 ,ug of pancreatic RNase-10 U of RNase T1 per ml. After a 5-minute postfixation in paraformaldehyde, cells were dehydrated in ethanol. Then 20 ,ul of hybridization solution containing 2 ,ug of biotinylated SV40 DNA (Enzo) per ml, 50% formamide, 10% dextran sulfate, 200 ,ug of carrier DNA (Enzo) per ml, and 2x SSC (lx SSC is 0.15 M NaCl, 0.015 M sodium citrate) (pH 7.0) was applied to each cover slip. A second cover slip was sealed on top with rubber cement, and the viral DNA and probe DNA were denatured simultaneously for 5 min in an 80°C oven. After hybridization at 37°C overnight, the cover slips were washed in 2x SSC followed by PBS containing 0.1% Triton X-100. After a 1-h incubation with streptavidin-hrp, the hrp was visualized by incubation in PBS containing 0.01% H202 and 0.5 mg of DAB per ml. Cells recognized by the probe DNA were detected by a brown precipitate. The cells were counterstained with hematoxylin and dehydrated in ethanol and xylenes. The percentage of cells positive for viral DNA replication was determined by scoring approximately 400 to 600 cells. Simultaneous in situ hybridizations and ICC. At 48 h postinfection, cells were rinsed with PBS and fixed in cold acetone for 10 min. ICC was done by using either PAb 416 or PAb 100. Cells were then pretreated for in situ hybridization by the method of Haase et al. (5, 15). Cells were incubated in 0.2 N HCl-2x SSC (pH 7.0) at 70°C, and protein was digested with 1 ,g of pronase per ml in 0.05 M Tris hydrochloride (pH 7.6)-5 mM EDTA. After being washed in PBS containing 2 mg of glycine per ml, the cells were acetylated with 0.1 M triethanolamine (pH 7.5)-0.25% acetic anhydride for 10 min. After a wash in distilled water, cells were dehydrated in ethanol and treated with 100 ,ug of pancreatic RNase-10 U of RNase T1 per ml. Cells were postfixed in 5% paraformaldehyde for 30 min and then incubated in a 65°C solution of 95% formamide, O.1x SSC for 15 min to denature the DNA. A mixture of 0.1 x SSC and ice was added, and the cells were rinsed with distilled water and dehydrated in ethanol. The 35S-labeled DNA probe was prepared by using a nick translation kit (Bethesda Research Laboratories, Inc.) and [35S]dCTP (New England Nuclear Corp.). The 1,700- and 3,053-bp SV40 fragments generated by digesting p777B2 (pBR322 with WT SV40 cloned in the BamHI site) with HhaI and BamHI were isolated from an agarose gel and used in the nick translation reaction. The hybridization solution containing (per microliter) 2 ng of 35S-labeled SV40 DNA (107 cpm/,g), 10% dextran sulfate, 50% formamide, 0.6 M NaCl, 0.1 mM EDTA, 10 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid) (pH 2.0), 0.02% Ficoll-polyvinylpyrrolidone, and bovine serum albumin plus 200 ,ug of sonicated and denatured salmon sperm DNA per ml was denatured at 100°C for 1 min. Dithiothreitol was added to 10 mM, and 10 ,u was applied to each cover slip. After hybridization overnight at 37°C, the cover slips were washed in HWM (50% formamide, 0.6 M NaCl, 10 mM phosphate buffer [pH 6.0], and 1 mM EDTA) and then incubated in 2 x SSC for 1 h at 55°C. Cover slips were then washed for 2 to 4 h in HWM, dehydrated in ethanol containing 0.3 M ammonium acetate and dipped in NTB-2 nuclear track emulsion (Eastman Kodak Co.). After 3 to 5 days at 4°C, the slides were developed in D-19 (Kodak) and fixed in Kodak fixer. Cells stained brown with DAB

3762

DEMINIE AND NORKIN

J. VIROL.

100

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[ /

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FIG. 1. T- and V-antigen expression and viral DNA replication in CV-1 and A172 cells. CV-1 and A172 cells were infected at inputs of 0.5 and 25 PFU per cell, respectively. Cells were fixed daily postinfection and assayed for T- and V-antigen expression by ICC and for SV40 DNA replication by in situ hybridization.

precipitate were counted as T+, and cells with autoradiographic grains above background (approximately 10 to 20 per nucleus) were scored as positive for viral DNA replication. In situ hybridizations for early SV40 mRNA. At 15 h postinfection, cells were rinsed with PBS and fixed for 15 min in 4% paraformaldehyde. Cells were then pretreated for in situ hybridization by the method of Haase et al. (15). Cells were incubated in 0.2 N HCl-2x SSC (pH 7.0) at 70°C, and protein was digested with 1 ,ug of pronase per ml in 0.05 M Tris hydrochloride (pH 7.6)-S5 mM EDTA followed by two washes in PBS containing 2 mg of glycine per ml. After a wash in distilled water, cells were dehydrated in ethanol. To probe for early mRNA, a 3,053-bp SV40 fragment containing only the early region was generated by digesting p777B2 with HhaI and BamHI. The contrul probe, pBR322 DNA, was generated by digesting p777B2 with BamHI. The fragments were isolated from an agarose gel and labeled with 35S by nick translation. The hybridization solution containing 2 ng of 35S-labeled SV40 DNA per ,ul (107 cpm/lg), 10% dextran sulfate, 50% formamide, 0.6 M NaCl, 0.1 mM EDTA, 10 mM HEPES (pH 2.0), 0.02% Ficoll-polyvinylpyrrilidone, and bovine serum albumin plus 200 p.g of sonicated, denatured salmon sperm DNA-2 ,ul of RNase inhibitor (Sigma) per ml was denatured at 100°C for 1 min. Dithiothreitol was added to .10 mM, and 10 RI1 was applied to each cover slip. After hybridization overnight at 37°C, the cover slips were washed in HWM and then incubated in 2X SSC for 1 h at 55°C. Cover slips were then washed for 2 to 4 h in HWM, dehydrated in ethanol containing 0.3 M ammonium acetate, and dipped in NTB-2 nuclear track emulsion (Kodak). After 15 days at 4°C, the slides were developed in D-19 (Kodak), fixed in Kodak fixer, and counterstained with Geimsa. RESULTS T- and V (major capsid protein VPl)-antigen expression and viral DNA replication in A172 and CV-1 cells. SV40 infections of semipermissive A172 human glioblastoma cells were compared with infections of fully permissive CV-1 monkey kidney cells by observing T- and V-antigen expression and viral DNA replication in individual cells. T and V antigens were detected by ICC, using MAbs PAb 416 and PAb 597, respectively. SV40 DNA replication was detected in parallel cultures by in situ hybridization, using a biotin-labeled SV40 DNA probe. At an input of about 0.5 PFU per cell, 60% of the CV-1 cells produced both T and V antigens and replicated viral

DNA by 3 days postinfection (Fig. 1). In contrast, at an input of 25 PFU per cell, only 40% of the A172 cells expressed T antigen. Furthermore, only 5% of the A172 cells replicated viral DNA or synthesized V antigens. This shows that SV40 replication aborts in many A172 cells at a stage before T-antigen synthesis. Also, in many cells, the infection aborts after T-antigen synthesis but before DNA replication. This latter result is intriguing, since T antigen is the only viral gene product required for viral DNA replication. ICC with MAbs that recognize different T-antigen epitopes. T antigen undergoes several modifications after translation, which are believed to result in functionally distinct forms of the protein (see reference 10 for a review). T-antigen-specific MAbs have been used to identify different forms of T antigen enriched for different activities in vitro (10, 13, 28, 32, 33). We used 10 of these MAbs (PAbs 100, 101, 103, 104, 105, 106, 107, 108, 109, and 416) to determine whether expression of a particular form of T antigen might correlate with SV40 DNA replication in A172 cells. Parallel cultures of infected A172 cells were stained by ICC with each of the MAbs and examined by in situ hybridization with the SV40 DNA probe. PAb 100 was the only MAb which reacted with a similar percentage of cells as reacted with the SV40 DNA probe. Each of the other nine MAbs reacted with many more cells than reacted with the DNA probe. The results of the ICC stains with five of the MAbs and the in situ hybridizations at 48 h are shown in Table 1. CV-1 and HeLa cells were also examined by using the five MAbs listed in Table 1. In these cells, each of the MAbs, including PAb 100, reacted with a similar percentage of cells TABLE 1. Percentage of cells reactive with different anti-T MAbs and with the SV40 DNA probe % Reactive cellsb Cell linea

CV-1 HeLa A172

416

100

MAb 101

103

105

vDNA probe

46 41 34

34 35 1.7

38 40 33

41 42 25

38 42 36

37 35 1

a CV-1, HeLa, and A172 cells were infected at 0.5, 50, and 25 PFU per cell, respectively. b Cells were fixed at 48 h postinfection. T antigen was detected by ICC, using the indicated MAbs. Parallel cultures were examined by in situ hybridization to detect cells replicating viral DNA. vDNA, Viral DNA.

SV40 T-ANTIGEN EXPRESSION

VOL. 64, 1990

(although the percentage of cells reactive with PAb 100 may be somewhat lower than the percentages reactive with the other MAbs). Furthermore, in the CV-1 and HeLa cultures, the percentage of cells reactive with each of the anti-T MAbs was similar to the percentages reactive with the SV40 DNA probe. Thus, there is no apparent block in these cells after T-antigen expression.

In the above experiments, ICC and in situ hybridizations compared by using parallel cultures. To confirm that viral DNA replication occurs in only those cells expressing the PAb 100-reactive T epitope, ICC staining and in situ hybridizations were done on the same cells. This enabled us to simultaneously identify T-antigen expression and viral DNA replication in individual cells. In these experiments, A172 cells were infected at an input of 50 PFU per cell and fixed at 48 h postinfection. Viral DNA was detected with a 35S-labeled SV40 DNA probe, and T antigen was detected by ICC, using PAb 100 or the broadly reactive PAb 416 (Fig. 2). We found that 37% of the PAb 416-reactive A172 cells were reactive with the SV40 DNA probe. In contrast, .93% of the PAb 100-reactive cells were reactive with the SV40 DNA probe. A similar experiment was done using CV-1 cells infected at an input of 0.5 PFU per cell. At 48 h postinfection, 86% of the PAb 416-reactive CV-1 cells were reactive with the SV40 DNA probe and 98% of the PAb 100-reactive cells were positive for SV40 DNA replication. These results confirm that in SV40-infected A172 cell cultures, more cells express T antigen than support viral DNA replication. They also confirm that SV40 DNA replication only occurs in that small percentage of T+ A172 cells which are reactive with PAb 100. Infections of other human cell lines. Four other human cell lines were examined to see whether they would show the same pattern of infection as that seen in the A172 cells. HeLa cells were examined since HeLa extracts have been widely used to study SV40 DNA replication in vitro. T98G cells were chosen since they, like A172 cells, are derived from human glial cells. Two human fibroblast cell lines, HS27 (newborn foreskin) and HS74 (fetal bone marrow), were chosen because they are known to differ in their abilities to support SV40 DNA replication (24). In each of the human cell lines, as well as in CV-1 cells, somewhat more cells reacted with PAb 416 than with the viral DNA probe (Fig. 3). However, the discrepancy between the percentage of PAb 416-reactive cells and viral DNA-replicating cells is much larger in cultures of A172 cells than in any of the other cell lines. Furthermore, in each of the cell lines the percentage of cells replicating viral DNA was more similar to the percentage reactive with PAb 100 than with the percentage reactive with PAb 416. A similar experiment was done with an early passage of human embryonic kidney (HEK) cells. In these cultures, there were only slightly more cells reactive with PAb 416 than with PAb 100 or with the SV40 DNA probe (data not shown). Thus, early-passage HEK cells were more like the majority of the human cell lines that we examined in that most T+ cells replicated viral DNA. HS27 cells produce more viral DNA and virus than HS74 cells (24). Our results show that a greater percentage of HS27 than HS74 cells produce T antigen when each cell type is infected at the same input multiplicity (100 PFU per cell). Also, more HS27 than HS74 cells go on to replicate SV40 DNA. Thus, the greater yields of viral DNA and PFUs in the HS27 than in the HS74 cells may largely reflect the greater numbers of HS27 cells which become infected. Effect of inhibitors of DNA replication on expression of PAb were

3763

100-reactive T. One interpretation of our results is that PAb 100 recognizes a form of T antigen required for viral DNA replication. Alternatively, expression of the PAb 100-reactive epitope might be enhanced by viral DNA replication. To test this alternative hypothesis, aphidicolin and HU were added to the culture medium of infected cells to determine what effect inhibiting viral DNA synthesis might have on expression of PAb 100-reactive T antigen. In cultures of CV-1 cells infected at an input of 0.5 PFU per cell, the drugs virtually abolished reactivity with the SV40 DNA prob-e yet somewhat increased the percentage of cells reactive with PAbs 416 and 100 (Table 2). In A172 cultures infected at an input of 25 PFU per cell, the drugs completely blocked our ability to detect viral DNA in situ. Neither drug had a large effect on the percentage of A172 cells reactive with PAb 416. However, treatment with each drug resulted in a dramatic increase in the percentage of cells reactive with PAb 100, such that about as many cells reacted with PAb 100 as with PAb 416. This unexpected increase in the percentage of PAb 100-reactive cells, caused by the DNA synthesis inhibitors, shows that viral DNA replication does not underlie expression of PAb 100-reactive T antigen in individual cells. If expression of the PAb 100-reactive form of T antigen is necessary for SV40 DNA replication, then the drug-induced increase in the percentage of A172 cells reactive with PAb 100 might be followed by a corresponding increase in the percentage of cells replicating viral DNA after removal of the drugs. This was indeed the case. Treatment with HU again resulted in an increase in the percentage of PAb 100-reactive A172 cells (Table 3). By 14 h after removal of the HU, the percentage of cells replicating viral DNA became equivalent to the enhanced percentage expressing PAb 100-reactive T antigen. Note that whereas cells replicating viral DNA were detected by 8 h after removal of the HU (not shown), viral DNA replication first became apparent in most of the positive cells between 10 and 14 h. Cellular DNA. replication, as indicated by bromodeoxyuridine incorporation, was apparent by 2 h (not shown). In the untreated A172 cultures, there were many more cells reactive with PAb 416 than with PAb 100 or the viral DNA probe throughout the experiment (Table 3). Reversal of the HU block in infected CV-1 cells was also followed by the emergence of cells replicating viral DNA, corresponding in number to those expressing PAb 100-reactive T antigen (Table 3). Infection of A172 cells with SV40 mutants. Early in this study, we were interested in how the SV40 small t protein, the agnoprotein, and the 72-bp enhancer duplication in the prototype strain might affect the pattern of infection in A172 cells. These viral factors do not have well-understood roles in SV40 replication in fully permissive monkey cells. We examined viable SV40 mutants with lesions in the small t protein (d1884), the agnoprotein (pm1493), and the enhancer region (A72). d1884 has a deletion in that portion of the early region which encodes only the small t protein (35). pm1493 has a mutation in the initiation codon for the agnoprotein (26). A72 has an exact deletion of one of the 72-bp enhancer

duplications.

A172 cells were infected with WT SV40 and with each of the mutants at an input of 100 PFU per cell. In each instance, there was a block before T-antigen synthesis in at least some cells (Fig. 4). Furthermore, in each instance many more cells produced T antigen than supported viral DNA synthesis. Therefore, the pattern of SV40 infection seen in the A172 cells did not reflect the action of any of the viral factors defined by these mutants. Transcription of early SV40 mRNA in human and simian

3764

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DEMINIE AND NORKIN

n FIG. 2. Simultaneous ICC and in situ hybridization to identify T antigen and viral DNA in the same cells. CV-1 and A172 cells were infected at inputs of 0.5 and 50 PFU per cell, respectively, and fixed at 48 h postinfection. T antigen was detected by ICC, using PAb 100 or the broadly reactive PAb 416. SV40 DNA was detected with a 35S-labeled SV40 DNA probe. (A) CV-1 cells reacted with PAb 416 and the SV40 DNA probe. (B) CV-1 cells reacted with PAb 100 and the SV40 DNA probe. (C) A172 cells reacted with PAb 416 and the SV40 DNA probe. The arrow points to a cell that reacted with both PAb 416 and with the DNA probe. Other cells are seen which reacted only with PAb 416. (D) A172 cells reacted with PAb 100 and the SV40 DNA probe. All of the cells reactive with PAb 100 are also reactive with the probe. Note that in the original color photograph, T+ nuclei were stained brown with the DAB precipitate. Cells positive for viral DNA replication contained autoradiographic grains above background.

VOL. 64, 1990

SV40 T-ANTIGEN EXPRESSION 100

3765

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80

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FIG. 3. Expression of T antigen reactive with PAbs 416 and 100 and SV40 DNA replication in five human cell lines and in CV-1 cells. A172, T98G, HS27, and HS74 cells were infected at an input of 100 PFU per cell. HeLa and CV-1 cells were infected at inputs of 50 and 0.5 PFU per cell, respectively. Cells were fixed daily and assayed for T-antigen expression by ICC and for SV40 DNA replication by in situ hybridization.

cells. Early SV40 mRNA was detected by in situ hybridization in cultures of infected HeLa, A172, and CV-1 cells. T antigen was detected in parallel cultures by ICC. We found similar percentages of cells expressing T antigen as were transcribing early mRNA in each cell type (Fig. 5, Table 4). Thus, the block to T-antigen expression in human cells is at a stage prior to transcription of early mRNA.

was previously observed in A172 cells (23) and in various other human cell lines (1, 3, 7, 25). The underlying basis for this finding is not yet known. Results reported here show that the block to T-antigen expression in individual human TABLE 2. Effect of DNA synthesis inhibitors on

T-antigen expressiona % Reactive cells

DISCUSSION Our comparison of SV40 replication in permissive and semipermissive cell lines showed at least two stages at which cellular factors determine the outcome of infection. First, in many cells of each of the semipermissive lines, the productive infection aborts before T-antigen synthesis. Second, in many cells of the A172 human glial line and in fewer cells of the other human lines, the productive infection aborts after T-antigen synthesis but before viral DNA replication. A plateau in the percentage of cells expressing T antigen

CV-1

Treatment

None HU Aphidicolin

A172

PAb 416

PAb 100

vDNA

PAb 416

PAb 100

vDNA

32 44 50

33 42 47

29 1.7 0

50 48 40

16 40 41

12 0 0

a CV-1 and A172 cells were infected at 0.5 and 50 PFU per cell, respectively. HU (1 mM) and aphidicolin (20 FM) were added to cultures at 8 and 12 h postinfection, respectively. Cells were fixed at 32 h for ICC and in situ hybridization. vDNA, Viral DNA.

3766

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DEMINIE AND NORKIN

TABLE 3. Expression of PAb 100-reactive T antigen and SV40 DNA replication after treatment with HU' % Reactive cells HU

Time (h)

CV-1

A172 PAb 416

PAb 100

vDNA

PAb 416

PAb 100

vDNA

30 14 35 34 32 20 47 35 38 12 46 46 48 16 1.8 35 30 + 0 32 35 46 31 + 37 33 39 46 46 36 a A172 and CV-1 cells were infected at 50 and 0.5 PFU per cell, respectively. HU (1 mM) was added to treated cultures from 8 to 32 h postinfection. Cells were fixed at 32 and 46 h for ICC and in situ hybridization. vDNA, Viral DNA.

cells is at a stage prior to transcription of early viral mRNA. The block probably occurs after virus adsorption, since the cell surface receptors for SV40 are present on all cells in the populations (2; W. J. Atwood, J. McDonnell, and L. C. Norkin, unpublished results). Regardless of its basis, the early block might be an important determinant of the semipermissive nature of SV40 infection of human cells (24). Restricted T-antigen expression was also seen in kidney cells derived from the rhesus macaque, the natural host of SV40 (22). Indeed, it may be that the only cells in which this block has not been observed are those derived from green monkey kidneys. Thus, we suggest that the restriction of T-antigen expression in individual cells might be a more common and important feature of SV40 infection than is generally appreciated, perhaps playing a role in long-term infection in vivo (see reference 21 for a review). More surprising to us than the restriction of T-antigen

expression was the large percentage of T+ A172 human glial cells which did not replicate SV40 DNA. The failure of viral DNA replication to occur in T+ cells is surprising because T antigen is the only viral gene product required for SV40 DNA replication (19). T antigen is known to undergo several posttranslational modifications and T-antigen-specific MAbs have been used to identify and characterize structurally and functionally distinct forms of T antigen. Those studies distinguished newly synthesized and old T antigen and identified forms active in origin binding and forms that are free or associated with replicating or mature SV40 chromosomes (10, 28, 32-34). Since different subclasses of T antigen can be identified with T-antigen-specific MAbs, we considered the possibility that viral DNA replication might correlate in individual A172 cells with the expression of a particular form or epitope of T antigen. It is inferred that this form of T antigen would be expressed in only a subset of the T+ A172 cells. From a panel of 10 anti-T MAbs, we found one, PAb 100, which indeed recognized that subset of A172 cells which replicates SV40 DNA. This suggests that the modification of T antigen recognized by PAb 100 may be required for SV40 DNA replication. Although several of the known modifications of T antigen have been studied (see reference 11 for a review), it is not clear which, if any, are directly involved in DNA replication. Also, the nature of the modifications recognized by the different MAbs is not known. Nevertheless, some of the information available on PAb 100-reactive T antigen is consistent with the premise that it represents a unique modification or conformation necessary for DNA replication. PAb 100 recognizes only a small percentage of total

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SV40 T-ANTIGEN EXPRESSION

VOL. 64, 1990

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4.4-..4.

FIG5 n stuhybdiaton etet fixe ell at eprssig arl 4A72adHLcelwreiftdatnpsof10nd5PF 40mRN per cell repectively ll cultureo were 15 h potinfection(A) Uninfeted A172 clls reactedwith the erly SV4O DA probe (B Infected A172 cells reacted with the early SV4O DNA probe (C) Infected HeLa cells reacted with pBR322 (D) Infected HeLa cells reacted~~~~~~~~~~~~~~~~~~~4 with the early SV4O DNA probe~~~~~~~~~~

immunoreactive T antigen but immunoprecipitates the ma-

jority of the origin-specific DNA-binding activity (28). PAb 100-reactive T antigen also has enhanced levels of ATPase activity and complexed p53 (33). Origin-specific DNA binding, ATPase activity, and complexed p53 are each associated with the DNA replication function of T antigen (8, 9, 11, 19, 20, 29-31, 33, 34). In addition, PAb 100 was the most effective of several anti-T MAbs at inhibiting T-antigen ATPase activity (4) and replication of SV40 chromatin in vitro (M. K. Bradley, personal communication). Only initiation of replication, not elongation, was affected by PAb 100. This might explain the finding that PAb 100 reacts poorly with T antigen bound to replicating the mature SV40 chromosomes (32) if the PAb 100-reactive epitope is expressed or exposed on chromatin-bound T antigen only at the origin. Other anti-T MAbs do react well with T antigen bound to chromatin (32).

Our in situ results, together with the in vitro properties of PAb 100-reactive T antigen are consistent with the premise that the modification of T antigen recognized by PAb 100 may be necessary for SV40 DNA replication. An alternative explanation of our findings is that viral DNA replication causes expression of, or stabilizes, the PAb 100-reactive epitope. We tested this possibility by examining the effect of the DNA synthesis inhibitors aphidicolin and HU on the numbers of cells which express PAb 100-reactive T antigen. Unexpectedly, the inhibitors actually increased the numbers of cells which expressed PAb 100-reactive T antigen. Thus, viral DNA replication does not cause expression of PAb 100-reactive T antigen. This is consistent with several other findings. First, the hybridoma cells which secrete PAb 100 were generated by using spleen cells from mice immunized with SV40-transformed mouse cells which do not replicate SV40 DNA (13). Second, PAb 100-reactive T antigen can be

3768

DEMINIE AND NORKIN

J. VIROL.

TABLE 4. Percentage of cells expressing early SV40 mRNA and T antigen % Positive cellsb

Cell

linea

Early mRNA

T antigen

CV-1 HeLa A172

13 26 18

28 14

15

a CV-1, HeLa, and A172 cells were infected at inputs of 0.5, 50, and 100 PFU per cell, respectively. b Cultures were fixed at 15 h postinfection and examined by in situ hybridization to detect cells expressing early viral mRNA. Parallel cultures were fixed at 20 h postinfection to detect T antigen by ICC, using PAb 416.

immunoprecipitated from extracts of COS-7 cells (28), in which there is also no replicating SV40 DNA. The basis for the effect of the DNA synthesis inhibitors on the number of cells expressing PAb 100-reactive T antigen is not yet known. Perhaps a cellular factor necessary for expression of PAb 100-reactive T antigen is sequestered by replicating cellular DNA. The inhibition of cellular DNA synthesis might then make that factor available to modify T antigen. It is also possible that arrest of the cells in the S phase or at the G1-S boundary promotes expression of PAb 100 reactive T antigen. This might occur if the factor that modifies T antigen is most active during this part of the cycle. Aphidicolin and HU also caused a small increase in the percentage of CV-1 cells which reacted with both PAb 416 and PAb 100. This might be related to the antagonistic effect of SV40 DNA replication on early viral transcription (17, 18). The finding that the DNA synthesis inhibitors actually increased the numbers of cells expressing PAb 100-reactive T antigen led to the prediction that after removal of the drugs there might be a similar increase in the numbers of cells which replicate viral DNA. This was indeed the case. Release from the HU block resulted in an increase in the percentage of cells replicating viral DNA, corresponding to the enhanced percentage expressing PAb 100-reactive T antigen. This further supports the premise that PAb 100 recognizes a modification of T antigen which is necessary for SV40 DNA replication. There was some variability in the percentage of viral DNA-positive A172 cells in different experiments (compare Tables 1 and 2). However, in each of our experiments the percentage of viral DNA-positive cells correlated with the percentage of PAb 100-reactive cells. There was less variability in the greater percentage of cells which reacted with PAb 416, our broadly reactive anti-T MAb. Current studies are concerned with understanding why only a subset of PAb 416-reactive A172 cells also express PAb 100reactive T antigen. Those studies may identify systematic variables which affect whether individual PAb 416-reactive cells will also express the PAb 100 epitope and replicate viral DNA. In summary, we consider the correlation between expression of PAb 100-reactive T antigen and replication of viral DNA in individual A172 cells to be the most interesting finding reported here. It will be important to determine the nature of the T-antigen modification recognized by PAb 100 and to understand why that modification occurs in only some T+ A172 cells. ACKNOWLEDGMENTS We thank Maryanne Wells for expert preparation of the manuscript. We are most grateful to M. Bradley, E. 0. Major, H. Ozer,

and C. Prives for advice and helpful discussions. We thank C. Prives, E. Gurney, and E. Harlow for kindly providing hybridomas and hybridoma supematants; R. Frisque and T. Shenk for SV40 mutant strains; and H. Ozer for cell lines HS27 and HS74 and C. Prives for HeLa cells. This investigation was supported by Public Health Service grants A114049 from the National Institute of Allergy and Infectious Diseases and CA50532 from the National Cancer Institute and by a Biomedical Research Support Grant. LITERATURE CITED 1. Aaronson, S. A., and G. J. Todaro. 1968. SV40 T antigen induction and transformation in human fibroblast cell strains. Virology 36:254-261. 2. Atwood, W. J., and L. C. Norkin. 1989. Class I major histocompatibility proteins as cell surface receptors for simian virus 40. J. Virol. 63:4474 4477. 3. Blattner, W. A., A. S. Lubiniecki, J. J. Mulvihill, P. Lalley, and J. F. Fraumeni, Jr. 1978. Genetics of SV40 T antigen expression: studies of twins, heritable syndromes, and cancer families. Int. J. Cancer 22:231-238. 4. Bradley, M. K., T. F. Smith, R. H. Lathrop, D. M. Livingston, and T. A. Webster. 1987. Consensus topography in the ATP binding site of the simian virus 40 and polyomavirus large tumor antigens. Proc. Natl. Acad. Sci. USA 84:4026-4030. 5. Brahic, M., A. T. Haase, and E. Cash. 1984. Simultaneous in situ detection of viral RNA and antigens. Proc. Natl. Acad. Sci. USA 81:5445-5448. 6. Brigati, D. J., D. Myerson, J. J. Leary, B. Spalholz, S. Z. Travis, C. K. Y. Fong, G. D. Hsiung, and D. C. Ward. 1983. Detection of viral genomes in cultured cells and paraffin-embedded tissue sections using biotin-labeled hybridization probes. Virology 126:32-50. 7. Carp, R. I., and R. V. Gilden. 1966. A comparison of the replication cycles of simian virus 40 in human diploid and African green monkey kidney cells. Virology 28:150-162. 8. Clark, R., K. Peden, J. Pipas, D. Nathans, and R. Tjian. 1983.

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