Ability of Nonpermissive Mouse Cells to Express a Simian

Transcription

1 JOURNAL OF VIROLOGY, June 1981, p X/81/ $02.00/0 Vol. 38, No.3 Ability of Nonpermissive Mouse Cells to Express a Simian Virus 40 Late Function(s) MICHELE LANGE, EVELYNE MAY, AND PIERRE MAY* Institut de Recherches Scientifiques sur le Cancer, Villejuif, France Received 11 December 1980/Accepted 10 March 1981 Mouse cells are fully nonpermissive for simian virus 40 (SV40). Infection does not lead to detectable virus replication. In this report, it was shown, first, that spliced 16S and 19S SV40 late mrna were present in cytoplasmic and polysomal polyadenylated acid+ RNA preparations from SV40-infected baby mouse kidney cells. The 16S and 19S SV40 late mrna's produced in infected baby mouse kidney cells were identical to or similar to the 16S and 19S SV40 late mrna's produced in permissive monkey cells as judged by their Si mapping patterns performed with the late strand of HpaII-BamHI fragment B and by their sedimentation patterns in a sucrose gradient. It was also shown that the 16S late mrna from infected baby mouse kidney cells could be translated into a polypeptide which was identical to or similar to virion protein VP1 in every aspect examined, including the pattern of peptide mapping by limited proteolysis. Second, we reported that mouse kidney cells produced detectable, although low, levels of SV40 virion protein VP1, as shown by the sodium dodecyl sulfatepolyacrylamide gel autoradiogram of [35S]methionine-labeled proteins immunoprecipitated by a rabbit antiserum directed against SV40 virion proteins. Third, it was reported that infected baby mouse kidney cells produced late mrna's either (i) when the infection was done at a restrictive temperature with the nonleaky tsa58 mutant or (ii) in cells treated with 100 yg of cycloheximide per ml, in which large T antigen synthesis was inhibited by more than 99.9%. This suggested that large T antigen was not required for the synthesis of late mrna in mouse cells. 940 During the lytic cycle of simian virus 40 (SV40), SV40 mrna is synthesized in two phases. During the early phase, which lasts until the beginning of viral DNA replication, there is a synthesis of two early (19S) mrna's coding for the SV40 small t and large T antigens, respectively. In the late phase, beginning with the commencement of viral DNA replication, two late mrna's (19S and 16S) are synthesized; the late 16S mrna codes for VP1 protein, and the late 19S mrna codes for VP2 and VP3 proteins. Until recently, it was usually assumed that only "early" mrna was produced early in productively infected monkey cells and in abortively infected mouse cells where SV40 DNA replication could not be detected. Nevertheless, experimental evidence is accumulating that SV40 (or polyoma) late transcription and viral DNA replication are not as closely linked as previously believed (for reviews, see references 1 and 2). A study of the SV40 transcription during productive infection of BSC-1 monkey cells with early temperature-sensitive mutants of SV40 grown at 410C (a nonpermissive temperature) indicated the presence of a small but reproducible fraction of late virus-specific cytoplasmic RNA (22). A late viral transcription was shown to be initiated in the lytic cycle in the absence of viral DNA replication, by characterizing the SV40 transcriptional complexes isolated by the Sarkosyl extraction procedure (6, 12). The cytoplasm of CV1 monkey cells at early periods of productive infection with SV40 was shown to contain late mrna's (16). In addition, the presence of late mrna's was observed in the cytoplasm of CVI infected with an SV40 tsa mutant which had been maintained at 41 C and continuously cultured in the presence of cycloheximide, suggesting that the late transcription did not require the A gene product (16). In the case of polyoma virus, it was shown that viral transcription during productive infection of mouse 3T6 cells revealed the appearance of RNA complementary to both DNA strands in the nuclei and the cytoplasm of the cells before viral DNA synthesis was detectable (31). The question of whether a late transcription occurs during an abortive infection with SV40 was also investigated by Khoury et al. (20, 21); these authors consistently found the appearance

2 VOL. 38, 1981 of late transcripts in nonpermissive mouse cells infected with this virus. Whether the late transcripts detected in infected (nonpermissive) mouse cells were destined to be processed and to become functional mrna(s) remained an open question. In this report, we show that SV40-infected mouse cells do produce spliced and functional late mrna(s) and that they synthesize a small amount of SV40 virion protein VP1. Moreover, we report that SV40 late mrna's are produced by infected mouse cells either when infection is performed at a nonpermissive temperature with the nonleaky tsa58 mutant or when large T antigen synthesis is inhibited more than 99.9% by cycloheximide (100,ug/rml). MATERLALS AND METHODS Cels and virus. Primary mouse kidney (BMK) cell cultures were prepared from 10-day old Swiss albino CR1 mice. The BMK cells were grown in 10- cm-diameter plastic petri dishes in Dulbecco modified Eagle medium (DMEM) supplemented with 10% calf serum (GIBCO Laboratories) and were used for infection 3 days after they had reached confluence; they were infected at a multiplicity of 50 PFU/cell. After 1 h of adsorption (at 37 C, unless specified otherwise), the BMK cells were refed with DMEM without added serum. The established monkey cell line CV1 was cultivated in Eagle minimum essential medium (MEM) plus 10% calf serum. Confluent monolayers of CV1 cells were infected at a multiplicity of 25 PFU/ cell; after 1 h of adsorption at 37 C, cells were refed with MEM plus 2% calf serum. SV40 virus strains were the wild-type SVi (also designated SVLP) (44), and the temperature-sensitive mutant tsa58 (41). Cell fractionation. Cells in cm-diameter petri dishes were washed twice with 5 ml per petri dish of buffer I (10 mm triethanolamine (ph 8)-25 mm NaCl-5 mm MgCl2-250 mm sucrose). After the medium was removed from the cells, 2.5 ml per 10 petri dishes of cold buffer I plus 1% Nonidet P-40 (Shell Chemical Co.) was added; where necessary (in preparation of polysomes), 2 mg of yeast RNA per ml (type XI; Sigma Chemical Co.), which had been repurified by phenol extraction, was added as a carrier. The mixture was left for 5 min, and the lysate was then carefully scraped off the surface and collected into a glass centrifuge tube (Sorvall HB4 rotor). Nuclear and cytoplasmic fractions were separated by centrifugation at 2,500 rpm for 5 min at 4 C and recovered in the pellet and the supernatant, respectively. The cytoplasmic fraction either was used for extracting RNA or was further fractionated to prepare polyribosomes. Preparation of polyribosomes. The cytoplasmic fraction (from 20 petri dishes) was centrifuged at 10,000 rpm (Sorvall SS 34 rotor) for 20 min. The resulting post-mitochondrial supernatant was placed into a tube containing 0.25 nil of 10% sodium deoxycholate (final concentration of sodium deoxycholate was 0.5%). The mixture was centrifuged through a 0.5 M/2 M sucrose double layer (3 ml of 0.5 M sucrose MOUSE CELLS EXPRESS SV40 LATE FUNCTION(S) 941 over 2.5 ml of 2 M sucrose) in a Spinco fixed-angle 65 rotor at 38,000 rpm for 2.5 h at 4 C. The pellet was then suspended carefully in 0.75 ml of buffer I. (As judged from an analytical sucrose gradient centrifugation, more than 80% of the suspension consisted of polyribosomes). We added to the suspension 0.1 ml of 0.1 M EDTA, thus obtaining the polyribosomal preparation to be used for extracting RNA (see below). Preparation of cytoplasmic and polysomal RNAs. The fluids designated as the cytoplasmic or the polysomal fractions, respectively (see above), were diluted with 10 volumes of 0.01 M triethanolamine buffer (ph 7.4) (4 C) containing 50 mm NaCl, 6 mm EDTA, and 1.1% sodium dodecyl sulfate (35). An equal volume of a mixture of phenol-chloroform-isoamyl alcohol in the ratio 50:50:1 was added, the extraction mixture was blended in a Vortex mixer at room temperature for 5 min and then centrifuged at 8,500 rpm for 5 min in the Sorvall HB4 rotor. The aqueous phase and the interphase were reextracted with an equal volume of phenol-chloroform-isoamyl alcohol mixture and recentrifuged. After this, the aqueous phase and the interphase were extracted with an equal volume of chloroform at room temperature for 5 min and centrifuged as described above, and the RNA was precipitated from the aqueous phase with 2 volumes of ethanol after addition of sodium acetate (ph 5) to a final concentration of 0.15 M. Preparation of cytoplasmic (or polysomal) poly(a)+ RNA. The cytoplasmic (or polysomal) RNA was passed through an oligodeoxythymidylic acid-cellulose column to recover the cytoplasmic (or polysomal) polyadenylated [poly(a)+] RNA and then precipitated with ethanol (35). Preparation of early and late strands of SV40 DNA HpaH-BamHI restriction fragments A and B. Closed circular SV40 DNA labeled in vivo with 32P04 (specific activity: 5 x 105 to 5 x 106 cpm/,ug) was digested to completion with single-site restriction endonucleases HpaII (0.725 map units) and BamHI (0.14 map units). These enzymes cleave SV40 DNA near the early and late SV40 junctions (0.66 and 0.17 map units) (22). The individual vidual strands of the fragments were separated by the method of Hayward (17). The separated strands were purified as described by Alwine and Khoury (3). The resulting purified separated strands were dialyzed against 0.1x SSC. RNA mapping by gel electrophoresis of endonuclease Si-resistant RNA-DNA hybrid. The S1 mapping method used was that of Berk and Sharp (5), slightly modified according to May et al. (26). For the hybridization reaction cytoplasmic or polysomal poly(a)+ RNA was annealed with 10- to 20-fold molar excess of 'P[DNA] probe (20 to 30 ng) consisting of the early strand (AE) of HpaII-BamHI fragment A or the late strand (BL) of HpaII-BamHI fragment B. The conditions of DNA excess were established by titration of different samples. RNA amounts used in hybridization mixtures were as indicated in the legends to the figures. Antiserum. Antiserum directed against the SV40 virion proteins (predominantly VP1) was prepared by intravenous injection of purified SV40 virion proteins into rabbits, as described by Tevethia (42). This antiserum was designated as anti-vp serum. Normal rab-

3 942 LANGE, MAY, AND MAY bit serum was used as a control. Immunoprecipitation and electrophoresis of proteins extracted from infected cells. SV40-infected BMK and SV40-infected CV1 cells were labeled at the times and for the periods indicated below with 100,iCi of L-[35S]methionine (750 to 930 Ci/mmol; The Radiochemical Centre, Amersham, England) in methionine-free medium. After labeling, the proteins were extracted from the cells as described by Kress et al. (23). The soluble extract (from 107 CV1 cells or 108 BMK cells) was incubated with 2 1l of rabbit anti-vp serum or normal rabbit serum. The immune complexes were isolated by the method of Schwyzer et al. (37) slightly modified by Kress et al. (23) with the new modification introduced by Ito (18) to reduce the background in final autoradiograms. Immune complexes were desorbed from a protein A-Sepharose column with 100 pd of 7 M urea in TBS buffer (25 mm Tris-hydrochloride [ph 9]-137 mm NaCl-5 mm KCl-1 mm CaC mm MgCl2-0.7 mm Na2HPO4-10% [wt/vol] glycerol). The eluted samples were diluted 10 times with TBS buffer and reincubated ovemight with 20,ld of settled protein A-Sepharose. The immune complexes were then reeluted from the protein A-Sepharose beads with 50,Al of electrophoresis buffer containing 0.08 M Tris-hydrochloride (ph 6.8), 2% sodium dodecyl sulfate (SDS), 5% 2-mercaptoethanol, 15% (wt/vol) glycerol, and 0.001% bromophenol blue and incubated at 100 C for 10 min before analysis by SDS-polyacrylamide gel electrophoresis (PAGE) (24). Conditions for SDS-PAGE have been previously described (23). Translation of gradient-fractionated mrna. The cytoplasmic poly(a)+ RNA sample from infected BMK cells (250 Ag of RNA) in 400 Al of a buffer containing 10 mm triethanolamine (ph 7.4), 50 mm NaCl, and 1 mm EDTA (used for preparing sucrose gradients) was centrifuged in 16 ml of 15 to 30% (wt/ vol) sucrose gradients in a Spinco SW27.1 rotor at 23,000 rpm for 22 h at 20 C. Fractions (0.4 ml) were collected from the top of the tubes with an ISCO 640 density gradient fractionator, and absorbance at 254 nm was monitored simultaneously with an UA-4 absorbance monitor. Each fraction was precipitated with 2 volumes of ethanol after addition of sodium acetate (ph 5) to a final concentration of 0.15 M. After ethanol precipitation, the RNA from each fraction was pelleted, dissolved in 30 1,l of water, and stored at -20 C (this RNA solution was also used in Si mapping analyses). The nuclease-treated rabbit reticulocyte lysate system (30) was programmed with 20 pi of the RNA solution from each fraction (200,l of reaction mixture containing 60,Ci of L-[nS]methionine, 750 to 930 Ci/mmol; the Radiochemical Centre). Incubation was for 1 h at 30 C. The reaction was stopped by adding Nonidet P-40 to a final concentration of 2%. The translation products were immunoprecipitated with control and anti-vp sera (1,lA of serum per 100,lI of lysate). The immune complexes were isolated by the method of Schwyzer et al. (37), and analysis was carried out by gel electrophoresis, as described above. Peptide mapping. Peptide mapping by limited proteolysis was performed by the digestion procedure for proteins in gel slices as described by Cleveland et al. (9). The protease used was Staphylococcus aureus V8 protease (Miles Laboratories, Inc.). RESULTS J. VIROL. Production of early and late SV40 mrna's in (nonpermissive) BMK cells. We first wanted to determine whether the cytoplasm of BMK cells abortively infected with SV40 contained spliced late mrna's. Poly(A)+ cytoplasmic RNA extracted at various times after SV40 infection from BMK cells was analyzed by Si gel mapping. The 32P-labeled SV40 DNA probes used in the present study were the early strand (AE) of HpaII-BamHI fragment A and the late strand (BL) of HpaII-BamHI-B. Fragment B contains essentially all of the late gene region except for the terminal portions located between and map units and between 0.66 and map units, respectively. Fragment A contains all of the early region plus those terminal portions of the late region lacking in fragment B. Figure 1A shows the nuclease S1 analysis of poly(a)+ cytoplasmic RNA samples from BMK cells at 12 h and from CV1 at 48 h after SV40 infection, respectively. The analysis of RNA from infected BMK cells with the AE singlestrand probe shows the predicted bands for the mrna's which encode large T and small t antigens (3, 5, 11, 27, 32). We observed a band migrating at 2,050 nucleotides corresponding to the 3'-exon (RNA segment distal to the single splice) which is common to both early mrna's; a 600-nucleotide band of 5'-exon of small t antigen mrna (the RNA segment proximal to the single splice); and a 300-nucleotide band of 5'- exon of large T antigen mrna. Using the same DNA probe to examine the early RNA in the poly(a)+ cytoplasmic RNA from CV1 infected for 48 h, we observed the same bands, except that the 2,050-nucleotide band was replaced by a 1,945-nucleotide band. Alwine and Khoury (3) have demonstrated that this 1,945-nucleotide band is colinear with the 2,050-nucleotide band, but is shortened at the 3' end. The shortening of this band could be due to RNA-RNA hybridization between late and early SV40 RNA, which could displace the last 100 bases of early RNA from the labeled probe. The analysis of RNA from infected CV1 cells with the BL single-strand probe (Fig. 1A) shows the predicted bands for the 19S and 16S late SV40 mrna's. Two bands migrating at 1,100 nucleotides and 2,000 nucleotides correspond to the 3'-exon of 16S and 19S late mrna's, respectively. The band of 180 nucleotides (Fig. 1) corresponds to the 5'-exon of 16S (and possibly of 19S) late mrna, since Si mapping analysis with

4 VOL. 38, 1981 MOUSE CELLS EXPRESS SV40 LATE FUNCTION(S) 943 A _- _ oc Y mco V) V) I I (A r''l I --~~l 48h 12h AE BL AE BL B I 6h 9h AE BL AE BL - lr 0n 12h 21h AE BL AE BL il; * ~ ~ ~ ~ XD!-110 SOW;- 0;.;;XS, ; 18 FIG. 1. Nuclease Sl analysis of SV40 cytoplasmic poly(a)+ RNAs Cytoplasmic poly(a)+ RNA from CVJ or from BMK cells infected for the indicated time with SV40 was hybridized to the early strand (AE~) of the larger fragment (A) of 32P-labeled SV40 DNA generated by digestion with the restriction endonucleases HpaII and BamHI (0.144 to map unit), or to the late strand (BL) of the smaller (B) HpaII-BamHI fragment. Samples were hybridized, nuclease Si treated, electrophoresed, and autoradiographed as described in the text. Input RNA amounts were as follows: 2.5 and 0.3 pg for hybridizing CVI cell RNA to AE and BL probes, respectively; 5 and 10 pg for hybridizing BMK cell RNA to AE and BL probes, respectively. Kodirex X-ray films were exposed to gels for 2 days (A) or 8 days (B). Numbers at the left of the gels are marker sizes in nucleotides. HpaII-BamHI BL probe essentially allows the detection of that leader sequence (5'-exon) which is common to 16S and 19S mrna species and maps between nucleotides 243 and 444 (13). This leader sequence indeed generates a 180- nucleotide band corresponding to a DNA segment mapping between nucleotides 267 and 444, since the point of cleavage of SV40 late DNA strand by HpaII restriction enzyme is between nucleotides 266 and 267. It should be noted that both the bodies (3'-exons) of 16S and 19S late mrna's, respectively, may be attached to several other leader sequences (13, 15) which are probably not detectable by this S1 mapping. Using the same DNA probe to examine the late RNA in poly(a)+ cytoplasmic RNA from infected BMK cells (Fig. 1), it is possible to detect the 1,100 nucleotide- and 180-nucleotide bands characteristic of 16S late mrna. The 2,000-nucleotide band characteristic of 19S late mrna 3'-exon is also detectable, though rather faint. (Fig. 1B, 9-h and 12-h RNAs; Fig. 2, cytoplasmic RNA). The same band appears much darker in the S1 pattern of RNA from infected BMK cells treated with 100,ig of cycloheximide per ml, due to drug-induced overproduction of late mrna's (see Fig. 8). When the same S1 analysis was done with polysomal poly(a)+ RNA extracted from SV40- infected BMK cells (Fig. 2), the 1,100-nucleotide band, corresponding to the 3'-exon of late 16S mrna, was found. The amount of 16S mrna used in this analysis is probably too low to permit the detection of the corresponding 5'- exon. These results indicate that mouse kidney cells produce detectable amounts of spliced late 16S and 19S mrna's. As in the lytic cycle, the 16S mrna appears to be the major species of late mrna. The intensity of the bands in the autoradi-

5 944 LANGE, MAY, AND MAY * (A CL E% o 0 _1 ' OC r U.. AE BL AE BL ti:r: :0 0'.E': DE.' 0: X- 0-00; a0es?,00 t, ;.?, E; C:: <: --0-0:f- 10-j-t : SX :;: :: CE :aje: -0:0 -t ;:: ::;. :: :: fffisx i;0:0:: :: i:-: -0-: X f. 00 FIG. 2. Nuclease Si analysis of SV40 polysomal poly(a)+ RNAs. Polysomalpoly(A)+ RNA from BMK cells infected for 12 h with SV40 was hybridized to HpaII-BamHI AE and BL probes. Samples were hybridized, S1 nuclease treated, electrophoresed, and autoradiographed as described in the text. For comparison, cytoplasmic poly(a)+ RNA extracted from SV40-infected BMK cell cultures after 12 h of infection was hybridized in parallel to the same probes, S1 nuclease treated, and electrophoresed on the same gel. Input RNA amounts were 2.5 and 5 pg for hybridizations to AE and BL probes, respectively. Kodirex X-ray films were exposed to gels for 10 days. Numbers at the left of the gels are marker sizes in nucleotides. J. VIROL. ograms can be considered as an approximate measure of the relative amounts of the various RNA species, since the hybridizations with the labeled DNA probes were performed under conditions of DNA excess. Therefore, the experiment in Fig. 1B allows us to compare the time courses of appearance of spliced early versus late SV40 mrna's in infected BMK cells and, in fact, both time courses appear to be virtually parallel. Considering the intensities of the major bands observed in the Si mapping patterns obtained with AE and BL DNA probes, i.e., the 2,050-nucleotide band and the 1,100-nucleotide band, respectively, we can see (i) that both these bands are detectable as early as 6 h after infection; (ii) that their intensities become maximum at around 9 to 12 h after infection; and (iii) that they virtually disappear at 21 h after infection. This time course is similar to that of the synthesis of hybridizable SV40 RNA in the same system, as reported by May et al. (28). The fact that both time courses of SV40 mrna syntheses are parallel suggests that the same SV40 DNA molecules are available as templates for the synthesis of late and early transcripts. Cell-free translation product of the late 16S mrna produced in mouse cells. The next experiment shows that the cytoplasmic fraction isolated from SV40-infected BMK cells contained late mrna(s) functionally competent in directing the synthesis of late protein(s). Poly(A)+ cytoplasmic RNA extracted at 12 h postinfection from SV40-infected cells was fractionated through a sucrose density gradient. The fractionated RNAs were translated in the reticulocyte system, and the translation products were reacted with normal rabbit serum and rabbit anti-vp serum. The immunoprecipitates were analyzed by SDS-PAGE followed by autoradiography. For comparison, the cytoplasmic poly(a)+ RNA extracted at 48 h postinfection from SV40-infected CV1 cells was translated in the reticulocyte system, the translation products were immunoprecipitated with rabbit anti-vp serum, and the immunoprecipitate was analyzed by SDS-PAGE in the identical gel. The results (Fig. 3) show that the poly(a)+ cytoplasmic RNA contains a class of RNA encoding a protein which comigrates with the major virion protein VP1, whose synthesis is directed by the predominant late SV40 mrna from CV1 cells. As expected, the sedimentation profile of late messenger translational activity detected in BMK cells (Fig. 3) is very similar to that of the 16S late mrna observed by S1 mapping of the fractionated RNAs along the gradient (Fig. 4). Moreover, the protein whose in vitro synthesis is directed by the predominant late mrna from BMK cells appears to be identical to VP1, as judged by peptide mapping analysis of both proteins, performed according to Cleveland et al. (9) (Fig. 5). Production of the major virion protein

6 VOL. 38, 1981 MOUSE CELLS EXPRESS SV40 LATE FUNCTION(S) 945 SV40 inf. BMK cells liu 1s P- VP V V N V N V N V N V N V N V N V V FIG. 3. PAGE of proteins made in response to gradient-fractionated cytoplasmic mrna's from SV40- infected BMK cells. Cytoplasmic poly(a)+ RNA from SV40-infected BMK cells was fractionated on a 16-ml linear (15 to 30% [wt/vol]) sucrose gradient by centrifugation in a Spinco 27.1 rotor at 23,000 rpm for 22 h at 20 C. Forty 0.4-ml fractions were collected. The RNA from each fraction was precipitated with 2 volumes of ethanol after addition of sodium acetate (ph 5) to a final concentration of 0.15 M, and it was then redissolved in 30,ul of water. The reticulocyte lysate was programmed with 20 p1 of the RNA solution from each fraction, and the translation products were immunoprecipitated with rabbit anti- VP serum (V) and with normal rabbit serum (N) and analyzed by SDS-PAGE. Sedimentation was from right to left. Fractions 15 and 25 correspond to positions of28s and 18S rrna's, respectively, sedimented in a parallel gradient. The fraction numbers are indicated above each pair of tracks. For comparison, cytoplasmic poly(a)+ RNA was extracted at 48 h postinfection from SV40-infected CVJ cells and then translated in the reticulocyte system. The translation products were immunoprecipitated with rabbit anti-vp serum, and the immunoprecipitate was analyzed by SDS-PAGE in the identical gel (7.5% polyacrylamide). The numbers on the left indicate the positions of the molecular-weight markers (Mr x 10-3). Kodak Kodirex film was exposed to the gel for 4 days. VP1 by SV40-infected BMK cells. The presence in infected BMK cells of translatable 16S late mrna, at least part of which is apparently polyribosome-associated, prompted us to investigate whether these cells produce detectable levels of virion protein VP1. BMK cells (15 plates) were labeled with 100 t,ci of [35S]methionine per ml from 14 to 15 h after SV40 infection. After the labeling period, soluble extracts were prepared from the cells and were reacted with normal rabbit serum and anti-vp serum. The immune complexes were isolated by the method of Schwyzer et al. (37) modified by Ito (18) as described above, and they were analyzed by SDS-PAGE followed by autoradiography. For comparison, parallel extracts of SV40-infected CV1 cells labeled at 47 to 48 h postinfection were analyzed in the identical SDS-polyacrylamide gel for the presence of protein(s) precipitable with anti-vp serum. The SDS- PAGE pattern of immunoprecipitable proteins from infected BMK cells reveals the presence of a band corresponding to a molecular weight of approximately 45,000 and comigrating with the major SV40 virion protein VP1 from SV40-infected CV1 cells (Fig. 6). From this experiment, we infer that infected BMK cells do contain detectable levels of newly synthesized SV40 virion protein VP1. However, these levels must be very low since we were unable to detect the presence of virion protein VP1 in these cells by the indirect immunofluorescence technique using the same anti-vp serum (data not shown). Thus, the question of whether the synthesis of protein VP1 occurs in all the infected BMK cell population or only in a fraction of this population remains open. Production of early and late SV40 mrna's in BMK cells in the absence of A gene activity. We next examined whether the synthesis of SV40 late mrna's occurring in

7 946 LANGE, MAY, AND MAY 18S FIG. 4. Sl mapping of the sucrose gradient-fractionated cytoplasmic RNAs from SV40-infected BMK cells by hybridization to the late strand of HpaII- BamHI fragment B. A 10-,ul portion of each fractionated mrna of the sucrose gradient represented in Fig. 3 was hybridized to the late strand (BJ) of 32plabeled HpaII-BamHI fragment B. The hybrid molecules were nuclease Sl treated, electrophoresed, and autoradiographed as described in the text. The fraction number is indicated below each track. Numbers on the left are marker sizes in nucleotides. nonpermissive cells required the synthesis of a functional large T antigen. BMK cells were first infected with the nonleaky SV40 temperature-sensitive mutant tsa58. It should be noted that tsa mutants are defective in the production of a functional large T antigen required for viral DNA synthesis (36, 38, 40). tsa58 mutants fail to produce any detectable progeny virus at temperatures above 390C (39). Moreover, we have verified that no newly synthesized radioactive viral DNA is detectable in the Hirt supernatant from CV1 cells infected at 410C with this mutant. After a 1-h adsorption period at 33 or 410C, respectively, the cells were incubated at 330C for 17 h or at 410C for 12 h, respectively. At this point, the cytoplasmic poly(a)+ RNA was isolated from the cells and analyzed by S1 mapping performed with the HpaII-BamHI BL probe. Figure 7 shows that the BMK cells infected at 410C (nonpermissive temperature) contained a detectable level of spliced 16S late mrna, as judged by the presence, in the Si mapping pattern, of the typical J. VIROL. 1,100-nucleotide band corresponding to the 3'- exon of 16S late mrna. We then studied the effects of a cycloheximide treatment of infected BMK cells on the synthesis of early and late SV40 mrna's. Cycloheximide (0, 15, 50, or 100,ug/ml) was added to infected BMK cells immediately after the 1-h period of virus adsorption. Cytoplasmic poly(a)+ RNA was isolated from infected cells at 14 h postinfection and analyzed by Si gel mapping performed with HpaII-BamHI AE and BL probes. The Si mapping patterns (Fig. 8) reveal that cycloheximide treatment results in an overproduction of SV40 early RNAs and that late mrna's are produced in cycloheximidetreated cells and that the latter are, in fact, overproduced in the presence of 100,ug of cycloheximnide per ml. The overproduction of early SV40 mrna's in infected BMK cells treated with cycloheximide is similar to that observed in SV40-infected CV1 monkey cells exposed to the same drug (16). This overproduction probably results from two distinct effects. First, an increased rate of early transcription can be due to A I CVI 40---ii 29 - BMK B_ o N en o - Li CD pg protease FIG. 5. Digestion during re-electrophoresis of the translation products observed in the gel shown in Fig. 3. The band of VP1 (from infected CVI cells) and the bands comigrating with it and corresponding to gradient fractions were cut from the SDS gel (Fig. 3). The band of VPI was divided into five samples, which were placed respectively in the five sample wells labeled CVI (A). The gel slices corresponding to fractions 24 to 28 were placed from left to right, respectively, in the five sample wells labeled BMK (B). Each slice was overlaid with S. aureus V8 protease, according to Cleveland et al. (9). The numbers on the left indicate the molecular weights (Mr x lo-) of the markers. The protease concentrations are given above each track. The gel (15% polyacrylamide) was fluorographed using the autoradiography enhancer EN3HANCE (New England Nuclear Corp.). Kodak SB5 film was exposed to the gel at -70 C for 21 days.

8 VOL. 38, 1981 MOUSE CELLS EXPRESS SV40 LATE FUNCTION(S) 947 the decreased production of large T antigen, on the basis of the autoregulation model of Tegtmeyer (33, 41). Secondly, cycloheximide seems to produce a rather generalized increase in mrna content, with the drug probably acting m > ax _ "c C_. CD 0 (n) U1 48h 18h.66-VPI N V N V FIG. 6. Virion protein VP1 in SV40-infected BMK cells was labeled with 100,pCi of[3s]methionine per ml from 14 to 15 h. After labeling, soluble extract from the cells was immunoreacted with rabbit anti- VP serum (1< and normal rabbit serum (N). The immune complexes were isolated and electrophoresed in a 7.5% SDS-polyacrylamide gel. For comparison, SV40-infected CV) cells were labeled with 100,uCi of [36S]methionine per ml from 47 to 48 h after infection. After labeling, soluble extract from these cells was immunoprecipitated with anti- VP serum, and the immunoprecipitate was analyzed by SDS-PAGE in the identical gel, revealing essentially the band of virion protein VP1, as expected. The gel was fluorographed with the autoradiography enhancer EN3HANCE (New England Nuclear Corp.). Kodak SB5 film was exposed to the gel at -70 C for 21 days C 410C FIG. 7. SI gel analysis of cytoplasmic RNAs isolated from tsa-infected cells. BMK cells were infected with tsa58 mutant either at 33 C for 17 h or at 41 C for 12 h. At this point, the cytoplasmic poly(a)+ RNA was isolated from the cells and analyzed by S1 mapping performed with HpaII-BamHI BL probe. Input RNA amounts were 25 pg in each hybridization mixture. Kodirex X-ray film was exposed to the gel for 10 days. Numbers on the left are marker sizes in nucleotides. by promoting the stability of mrna's (19, 25, 46). The latter effect could also account for the overproduction of late mrna's in the presence of 100,ug of cycloheximide per ml. In a parallel experiment designed to determine the level of cycloheximide-induced inhibition of T antigen synthesis, infected BMK cells were exposed to cycloheximide with the same drug concentration and for the same period. From 12 to 13 h postinfection, the cells were incubated in methionine-free MEM and then labeled from 13 to 14 h postinfection with 100,uCi of [3S]methionine per ml added in methionine-free MEM. Cycloheximide was present in methionine-free MEM and in the medium used for labeling. Treatment with 15, 50, and 100,ug of cycloheximide per ml resulted in a 95, 98.3, and 99.3% inhibition of protein synthesis, respectively, as

9 948 LANGE, MAY, AND MAY I AE BL E _- In _ S I AE BL AE BL AE BL than 73, 97.8, and 99.9% after treatment with this antibiotic at concentrations of 15, 50, and 100 jg/ml, respectively. The fact that we observe a production of SV40 -E. c) E EE J. VIROL u.. u I + fl) o z o- o u FIG. 8. Sl gel analysis ofthe cytoplasmicpoly(a)+ RNAs isolated at 14 h after infection from BMK cells treated with various concentrations of cycloheximide (CH). BMK cells were infected with SV40 and treated with CH at the indicated dose from 1 to 14 h after infection. At 14 h postinfection, cytoplasmic poly(a)+ RNA was extracted from the cells. These RNAs were hybridized to 32P-labeled HpaII-BamHI AE or BL probes (as indicated), nuclease S1 treated, and electrophoresed as described in the text. Input RNA amounts were 2.5 and 10 jlg for hybridizing RNA to AE and BL probes, respectively. The gel was exposed to Kodirex X-ray film for 5 days. Numbers on the left are marker sizes in nucleotides. measured by [35S]methionine incorporation into trichloroacetic acid-precipitable material. Soluble extracts from the cells were immunoprecipitated with hamster anti-sv40 tumor serum, and the immunoprecipitates were analyzed by SDS- PAGE followed by autoradiography, as described by Kress et al. (23). A relatively long exposure period was used to enhance detection of large T antigen from drug-treated cells, resulting in an overexposition of the large T antigen band from untreated cells. As judged by a comparison of band intensities made by densitometry of the autoradiogram (Fig. 9), the synthesis of large T antigen was inhibited by more tu N tu N t:u N tu FIG. 9. SDS-polyacrylamide gel autoradiography of labeled T antigens from extracts of SV40-infected BMK cells treated with cycloheximide (CH). SV40- infected BMK cells were treated with the indicated dose of CH from 1 to 14 h postinfection and labeled with [355]methionine (100 jici/ml) from 13 to 14 h after infection. After labeling, soluble extract from the cells was reacted with hamster anti-sv40 tumor serum (tu) and with normal hamster serum (N). The immunoprecipitates were analyzed by SDS-PAGE and autoradiographed. The gel was at 12.5% polyacrylamide. Kodirex X-ray film was exposed to the gel for 8 days. Numbers on the left indicate the molecular weights (Mr x 10-3) of the markers. Bands T and t correspond to large and small T antigens, respectively.

10 VOL. 38, 1981 late mrna's in (i) infected BMK cells after treatment with the higher dose (100,ug/ml) of cycloheximide resulting in a 99.9% inhibition of large T antigen synthesis and (ii) BMK cells infected with the mutant tsa58 at 410C (restrictive temperature) suggests that SV40 late transcription in BMK cells does not require a functional A gene activity. MOUSE CELLS EXPRESS SV40 LATE FUNCTION(S) 949 DISCUSSION Mouse cells are considered as fully nonpermissive for SV40. Infection does not lead to detectable virus replication (2, 43). Up to now it has been felt that SV40-infected mouse cells did not produce either functional late mrna or SV40 virion protein(s). In the present study, we first found that spliced 16S and 19S SV40 late mrna's were present in cytoplasmic and polysomal poly(a)+ RNA preparations from SV40-infected (nonpermissive) BMK cells. The 16S and 19S SV40 late mrna's produced in infected BMK cells indeed turned out to be identical to or similar to the 16S and 19S SV40 late mrna's produced in permissive monkey cells, respectively, as judged by their Si mapping patterns performed with the late strand of HpaII-BamHI fragment B (Fig. 1, 2, and 4), and by their sedimentation patterns (Fig. 3 and 4). Moreover, we showed that the 16S mrna from infected BMK cells could be translated in the rabbit reticulocyte lysate system into a polypeptide which was identical to or similar to virion protein VP1 in every aspect we examined, including the pattern of peptide mapping by limited proteolysis (Fig. 3 and 5). It is noteworthy that the ratio of the amount of late mrna to that of early mrna is of the same order in infected BMK cells (5 to 10%, as determined by hybridization of RNA with cloned region-specific viral DNA fragments immobilized on filters [M. Lange, unpublished data]) as in CV1 monkey cells at the early phase of lytic infection (1 to 5% according to Parker and Stark [29]). Secondly, we have reported evidence that infected BMK cells did produce detectable, although low, levels of virion protein VP1, as shown by the SDS-polyacrylamide gel autoradiogram of [3S]methionine-labeled proteins immunoprecipitated from the cells (Fig. 6). This confirms that at least a part of the late mrna's produced by the cells was polyribosome-associated functional mrna. Our results have implications for the nature of the factors determining the abortive responses of mouse cells to SV40 infection. Previous studies on the regulatory mechanism of SV40 late gene expression in mouse cells led Graessmann and Graessmann (14) to hypothesize that a cellular repressing-type factor prevents the expression of the late SV40 genes in these cells. Our results do not fit well with this hypothesis. On the other hand, Watkins (45) suggested that cells which are nonpermissive for SV40 might lack factor(s) specifically required for the translation of SV40 late mrna's. From our results, it may be inferred that if such specific factors are necessary for SV40 late mrna translation, they are present in nonpermissive mouse cells, although we cannot exclude the possibility that the translation efficiency of late mrna's is lower in mouse cells than in monkey cells. Other interesting observations reported here are that infected BMK cells did produce late mrna's either (i) when the infection was performed at 410C with the nonleaky tsa58 mutant or (ii) in cells continuously treated after the virus adsorption period with 100,ug of cycloheximide per ml, in which large T antigen synthesis was inhibited by more than 99.9%. In the latter case, a drug-induced overproduction of late mrna's was, in fact, noted. These observations are similar to those reported by Handa and Sharp (16) concerning the synthesis of SV40 late mrna during the early phase of lytic infection of monkey cells. Our observations suggest that late mrna synthesis in mouse cells does not require large T antigen, although we cannot completely rule out the alternative possibility that late mrna synthesis requires large T antigen but at an exceedingly low concentration (29). Our suggestion that large T antigen is not required for late mrna synthesis fits well with the hypothesis that the positive regulatory effect of large T antigen exerted on late mrna synthesis in lytic infection of monkey cells (4, 7, 10, 22, 29, 33) is not due to a direct interaction of large T antigen with the late promoter(s) but might be indirectly mediated by another effect of large T antigen, such as increasing the number of SV40 DNA molecules transcribed (7) through its role in initiating viral DNA replication (8, 38). This latter hypothesis is also consistent with the recent findings of Rio et al. (34), who, using a cell-free RNA synthesizing system, showed that the D2 protein (biologically equivalent to SV40 large T antigen) inhibited SV40 early transcription, but had no effect on transcription of SV40 late sequences, and that efficiency of late transcription was enhanced relative to that of early transcription as the concentrations of both early and late promoters increased. ACKNOWLEDGMENTS We thank M. Kress for helpful discussions and for performing the peptide mapping of proteins by limited proteolysis, J. Borde and C. Breugnot for their excellent assistance, C. Bois-

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