Activation of the SV40 late promoter: Direct effects of T antigen in the absence of viral DNA replication

0692.8674/84/020381-09$02.00/O

Cell,Vol. 36, 381-389, February1984, Copyright(B 1984 by MIT

Activation of the SV40 Late Promoter: Direct Effects of T Antigen in the Absence of Viral DNA Replication Janis M. Keller and James C. Alwine Department of Microbiology University of Pennsylvania School of Medicine Philadelphia, Pennsylvania 19104

Summary We have examined the activation of the SV40 late promoter by inserting the late promoter and the viral origin of replication into chloramphenicol acetyltransferase (CAT) transient expression vectors. Very little late promoter activity was detected in CV1 cells, compared with high activity in COS cells, in which replication occurs due to endogenous T antigen. Nonreplicative counterparts of these plasmids, containing a mutated origin of replication, produced significantly more late promoter activity in COS cells than any of the plasmids in CV-1 cells. When plasmids were cotransfected into CV-1 cells with a plasmid that supplies T antigen, the nonreplicative plasmid displayed 30% of the activity of the replicative plasmid. Using mutant T antigens unable to replicate viral DNA, late promoter activation occurred only with mutant T antigens that retain DNA binding activity. These results demonstrate that T antigen can substantially stimulate late promoter activity directly and independent of viral DNA replication. Introduction The lytic cycle of SV40 involves two phases of gene expression (see Tooze, 1981, for complete review). Upon final uncoating of the viral DNA in the nucleus, a cellular RNA polymerase II initiates transcription from the promoter for the early region (see Figure 1). This culminates in the synthesis of the large and small tumor antigens (T antigens). Large T antigen returns to the nucleus, where it interacts with specific sequences in the early promoter/ origin of replication region. These interactions result in the autoregulation of early gene transcription and the initiation of viral DNA synthesis (Tegtmeyer, 1972; 1974; Reed et al., 1975, 1976; Alwine et al., 1977b; Khoury and May, 1977; Tjian, 1978, 1981; Rio et al., 1980; Hansen et al., 1981). Following the initiation of viral DNA synthesis, increased transcription of the late region begins and rises rapidly. Late region RNA becomes very abundant during the late phases of infection in order to produce the large quantities of viral structural proteins (VP1 , VP2, and VP3) needed to form progeny virions (Acheson, 1981). Thus the temporal regulation of the two coding regions allows for the orchestrated developmental expression of genes needed first for establishing the viral infection and initiating replication, and then for making the structural proteins needed to encapsidate the newly synthesized viral DNA. The SV40 early transcriptional promoter and its control

have been well characterized (Reed et al., 1976; Alwine et al., 1977b: Khoury and May, 1977; Rio et al., 1980; Benoist and Chambon, 1981; Gruss et al., 1981; Moreau et al., 1981; Hansen et al., 1981; Fromm and Berg, 1982, 1983; Weiher et al., 1983; Rio and Tjian, 1983) and presently represents one of the prototypical models of an RNA polymerase II promoter. In contrast, the mechanism of temporal control of late transcription is not understood. In comparisons with the sequences of known RNA polymerase II promoters, no definable promoter-like elements are detected within a fragment of SV40 DNA that appears to contain the entire late promoter (Fromm and Berg, 1982, 1983; Contreras et al., 1982; Hansen and Sharp, 1984; J. M. Keller and J. C. Alwine, personal observation). This fragment lies between nucleotides 5171 and 270 (SV numbering, Buchman et al., 1981) spanning the origin of replication. These observations, coupled with the very heterogeneous nature of the 5’ ends of late transcripts (Ghosh et al., 1978, 1982; Piatak et al., 1981), suggest that the SV40 late promoter represents an alternate class of RNA polymerase II promoters containing different signals with different requirements for activation. For this reason, understanding the mechanism of expression of the late genes is of general interest. In considering the mechanism of activation of late transcription, it has been argued that the accumulation of late RNA, at late times of infection, is largely due to the amplification of the genome (Rio et al., 1980; Tjian, 1981). This would occur through the action of T antigen in initiating DNA synthesis and autoregulating early transcription. This argument assumes that the late promoter is relatively weak, but constitutive; thus transcripts from it would become abundant only after extensive gene amplification. Although genome amplification certainly affects the levels of late RNA, evidence exists that indicates that late RNA accumulates in the absence of genome amplification (Reed et al., 1976; Alwine and Khoury, 1980). Thus more complicated transcriptional controls are indicated. The effect of T antigen on late transcription has been questioned for many years. Previous studies have demonstrated that virally infected cultures that produce a temperature-sensitive T antigen are unable to initiate late transcription at the nonpermissive temperature. However, once initiated at the permissive temperature, shifting the culture to the nonpermissive temperature showed no effect on the maintainance of late transcription (Cowan et al., 1973; Reed et al., 1976; Alwine et al., 197713). Thus it was suggested that T antigen may influence late transcription indirectly and transiently, as a consequence of its role in DNA replication. However, other experiments suggest that T antigen, perhaps in combination with a host factor, may exert a direct effect on the maintainance of late transcription (Parker and Stark, 1979; Alwine and Khoury, 1980). In the present report we have examined the effects of T antigen and replication on the activation of the late genes. Using transient expression vectors based on the chloramphenicol acetyl transferase (CAT) plasmid (Gorman et al.,

Cell 382

1982) we have separated viral DNA replication from other T antigen effects. Although genome amplification certainly affects the levels of late gene expression, our results demonstrate that late promoter activity can be substantially stimulated by a direct effect of T antigen, which occurs in the absence of, and independent of, DNA replication or genome amplification. A stimulatory effect of T antigen on late gene expression has also been independently reported by J. Brady and G. Khoury (personal communication). Our results further indicate that this stimulatory function correlates with the ability of T antigen to bind to viral DNA.

Results Constructions The plasmids used in this study were constructed from pSVO-cat (Gorman et al., 1982) which contains no eucaryotic promoter, but has a Hind Ill site, 37 bp upstream of the CAT gene AUG, into which a promoter element can be inserted. In addition, pSVO-cat contains both the SV40 small T antigen intron and the SV40 early polyadenylation addition signal downstream of the CAT gene sequence. Thus when the CAT gene is placed under the control of a eucaryotic promoter a properly processed, translatable mRNA can be produced in a eucaryotic cell. The SV40 Hind III C fragment (SV40 nucleotide 5171 to 1046, shown as a heavy line in Figure 1) was selected for use as the late promoter fragment. This fragment contains the SV40 origin of replication, the early promoter, the putative late promoter, and about one-third of the late coding region (see Figure 3). The rationale for starting with this large fragment was to include any sequences downstream of the 5’ end of the mRNA that may have regulatory functions (Piatak et al., 1981; Alwine, 1982; Hay et al., 1982; Fradin et al., 1982). The fragment was cloned into the Hind Ill site of pSVO-cat and selected for orientation such that the CAT gene was under late promoter control.

The resulting plasmid, pll-cat, is shown in Figure 2, and in partial linear form in Figure 3, along with pertinent SV40 features of the Hind Ill C fragment. The pL2cat plasmid (Figure 3) was constructed from pL1 -cat by removing the 274 bp fragment of DNA between the Eco RV site at SV40 nucleotide 772 and the Hind III site at SV40 nucleotide 1046, and replacing this sequence with a Sal I linker. As will be discussed below, pll-cat and pL2cat can replicate when T antigen is supplied, for example in COS cells (Gluzman, 1981). Nonreplicative counterparts of these plasmids, called pLln-cat and pL2n-cat, were constructed from pL1 -cat and pL2cat, respectively. The nonreplicative plasmids differ only by having 6 bp deleted at the Bgl I site within the SV40 replication origin (see Figure 3). For these constructions we used plasmid ~6-1 (Gluzman et al., 1980) which contains an SV40 genome, with the 6 bp deletion at its origin of replication, cloned at the

Frgure 2. pL1 -cat Plasmid The darker lines on the map are SV40 sequences. The SV40 Hind III C fragment (shown in Figure 1) contains the late promoter, the SV40 origin of repltcation, the intron sequence for the 19s late mRNA, and a portion of the late coding sequence. The position of the CAT sequence under the control of the SV40 late promoter is shown. The SW0 small t antigen intron and early polyadenylation site are included in the plasmid and their locations are shown. The lighter line is pBR322 sequences that contain the @lactamase gene (Amp’) and the plasmid origin of replication.

Figure 3. Linear Map of the SV40 Hind Ill C Fragment pL1 -cat

Figure 1. Map of SW0 Showing Early and Late RNA, the Origin of Replcation. and the Regron of DNA Used in Constructing the Plasmids r Jsed rn thus Study (Sold Portion of the Genome) Arrowheads

show the Hind III sites at nucleotides

1046 and 5171

as it Appears

rn

Included are the major SV40 features of this fragment: restriction sites, late mRNA start sites, splice site and coding regions, and translational start codons (AUG) known to be used in SV40 or the CAT gene. Also shown above the map are the features of the SV40 early promoter (72 bp repeats and the 21 bp repeats), the origin of replication, and the T antigen binding sites. The sequences retained in pL2cat are shown below the map of pll-cat. The positron of the 6 bp deletion that inactivates the origin of replrcation is indicated by the triangles.

T Antigen Directly 383

Activates

the SV40 Late Promoter

Barn HI site in pMKl6 #1 The 366 bp fragment from the Kpn I site at SV40 nucleotide 294 to the Hind III site at SV40 nucleotide 5171, spanning the SV40 origin of replication, was isolated from p6-1 and used to replace the corresponding regions of pL1 -cat and pL2-cat. All plasmids were characterized by digestion with appropriate restriction enzymes.

Characterization of Late Promoter Activity from the Various pL-Cat Plasmids The ability of the putative late promoter region, cloned into pll-cat, pL2cat, pLln-cat, and pL2ncat, to express the CAT gene was tested by transfecting purified plasmid DNA into ceils and testing cell extracts, prepared 48 hr after transfection, for resultant CAT activity (see Experimental Procedures). A criticism of the use of transient expression vectors for studying promoter activity is the variability in transfection efficiencies from experiment to experiment and even from plate to plate within a given experiment. Three methods were used to standardize the influence of transfection variability on our results: 1) The experiments have been repeated a number of times with different plasmid preparations and low passage cells. 2) The plasmid DNA was extracted (Hi& 1967) from one-half of the cells used to prepare the CAT extracts and the amount of plasmid DNA was quantitated by blot analysis (Southern, 1975). The CAT activity was then standardized to DNA concentration. 3) A plasmid containing a cDNA copy of the rabbit P-globin gene, with the long terminal repeat of Rous sarcoma virus as promoter (Gorman et al., 1983) was cotransfected with the CAT plasmid. RNA was prepared from a portion of the same cells used for preparing the CAT extracts. The amount of @-globin RNA produced by this independent plasmid was quantitated by northern analysis (Alwine et al., 1977a) and used as an internal standard against which CAT activity could be compared. The results of these standardization procedures are incorporated into the overall results given in the paper and are not shown separately. CV-1 and COS-1 cells (SV40 transformed CV-1 cells that constitutively produce T antigen; Gluzman, 1981) were transfected with pL2-cat, pL2n-cat, or their parent plasmid pSVO-cat, which contains no eucaryotic promoter. The comparative results of assays, within the linear range of the assay, are shown in Table 1. The activity is expressed as milliunits (mu) CAT activity per 3 X 105 cells. In COS-1 Table 1. Comparative Cells

Activities pt2cat

cos-1 cv-1

80.0 0.8

of pLZ- and pL2ncat pL2ncat 7.5 0.3

in COS-I

and CV-I

psvo-cat 0 0

Activity is expressed as milliunits (mu) CAT activity par 3 x lo5 cells, One unit of CAT activity catalyzes the acetylation of one nanomole of chloramphenicol per minute at 30” C and pH 7.8. Units of CAT activity were calculated from standard curves generated with known amounts of purified CAT (P-L Biochemicals).

cells pL2-cat shows high activity, as expected, because the plasmid can replicate using the SV40 origin of replication This contrasts to the low activity of pL2-cat in CV-1 cells where no T antigen is present. Surprisingly, the nonreplicative plasmid, pL2ncat, showed significantly more activity in COS-1 cells than in CV-1 cells. This plasmid should not be able to replicate in COS-1 cells; thus, the results imply that T antigen may be stimulating late gene expression in the absence of replication. In similar assays using pLl- and pL1 n-cat, similar results are obtained except that the activity of pll-cat is consistently three times less than pL2-cat. Preliminary observations from this laboratory indicate that the difference is due to the removal of the translational start codon for VP-3 in pL2cat (see Figure 3) thus eliminating competition for ribosomes. Alternatively, the length of the mRNA from the cap site to the CAT start codon may affect translational efficiency. Because pL2cat showed higher activity we used it and its derivatives for most other experiments involving CAT assays. Because the overall results indicated that T antigen may be stimulating late gene expression in the absence of replication, we wanted to characterize further the pL-cat plasmids in order to confirm that the observation was not due to aberrent transcription or unexpected replication in the case of the nonreplicative plasmid.

Nuclease Sl Analysis of pL-CAT Plasmid Transcripts To determine whether the SV40 late promoter and, more specifically, the SV40 late initiation sites were being properly used in our expression vector system, RNA was extracted from COS cells 48 hr after transfection with pL2cat and compared to SV40 lytic RNA (from CV-1 cells) by Sl analysis (Berk and Sharp, 1978). The results are shown in Figure 4. The probe was the 471 bp fragment extending from the Bgl I site at SV40 nucleotide 5242 to the Hint II site at nucleotide 470 (spanning the origin of replication). Probe labeling was done in vitro with ‘*P deoxynucleotide triphosphates and T4 DNA polymerase. The results indicate that the plasmid-derived RNAs have the same 5’ ends as lytic RNA. The 5’ end assignments in Figure 4 were made from this and other Sl mapping experiments (not shown), along with reference to the extensive 5’ end characterization done by Ghosh et al. (1978). In Figure 4, bands are indicated by size and the SV40 nucleotide corresponding to their mapped 5’ ends. Where only one number is shown the band continues to the end of the probe; the bands marked with asterisks map completely within the probe, between the nucleotides shown. Only one major band (a) appears in the plasmid lane and not in the lytic RNA lane. This band has not appeared in other Sl analyses of similar plasmid-derived RNA; thus it appears to be an artifact of this analysis. The relative intensities of the bands in the plasmid lane are similar to the relative intensities of the bands in the lytic RNA lane, indicating that 5’ end utilization or 5’ end

Cdl 384

selection is approximately the same for the virus and the plasmid. Overall, the data in Figure 4, as well as other Sl analysis (not shown), indicate that no CAT-encoding mRNAs initiate at sites other than known SV40 late initiation sites. In addition, the relative utilization of these sites is very similar between the virus and the plasmid. Similar results are obtained using RNA derived from pL2ncat (not shown); however, the signal is approximately 10 times weaker than experiments with pL2cat. This is to be expected from an examination of Table 1, which shows that the CAT activity of pL2ncat is approximately 10% of pL2-cat, thus predicting 10 times less promoter activity and IO times less RNA.

5’-end Bases

4P

350,

.

120

.

168

*

192

. .

564

212. 204.

318 147,

325

*

110, Figure 4. Nuclease

1

St Analysis

Tests of the Replication Capacities of the Various pL-CAT Plasmids Previous work showed that the 6 base deletion at the Bgl I site of ~6-1, within the SV40 origin of replication, renders the origin nonfunctional for replication (Gluzman et al., 1980). Using our CAT plasmid constructions we tested to be certain that nonreplicative plasmids retained this replication defect. COS cells were transfected with 5 pg of plasmid DNA or SV40 DNA. At various times after transfection (3, 24, and 48 hr) plasmid DNA was isolated by the procedure of Hirt (1967) and analyzed by quantitative dot-blot analysis. Equal portions of extract were dot-blotted, then hybridized with 32P nick-translated pBR322 DNA, which detects only the bacterial portions of the plasmid and not the SV40 portions (see SV40 controls). In a separate experiment the SV40 transfected samples were hybridized with 32P nick-translated SV40 DNA as a positive control of replication. Figure 5 shows the results of such an experiment comparing pll-cat and pLln-cat (comparable results are obtained using pL2-cat and pL2ncat). Clearly, the replicative plasmids pL1, pL2, and pL6b (see legend to Figure 5) do replicate in the COS cells as indicated by the accumulation of DNA with time. In contrast, the nonreplicative plasmid, pLln, does not accumulate but decreases with time. It can be argued that the pLln-cat DNA may replicate, but so inefficiently that it does not equal breakdown. To rule out this possibility, the pL1 and pL1 n DNA, extracted at 48 hr after transfection, was cleaved with the restriction endonuclease Mbo I. This enzyme will not cleave plasmid DNA that has been methylated in bacteria; however, if the plasmid replicates in a eucaryotic cell the resulting nonmethylated DNA can be cleaved. The results indicated that the pll-cat DNA from

168-290*

264~373* of SV40 Lytic RNA and pL2cat

RNA

Total cellular RNA was extracted from COS cells at 48 hr after transfection with pL2cat (pL2) DNA or after mock transfection with no DNA (noninf.). SW0 RNA was harvested from CV-IP cells 48 hr after infection. The probe was SV40 DNA extending from the Hint II site at nucleotide 470 to the Bgl I site at nucleotide 5242. The probe was labeled in vitro with 9 using T4 polymerase (see Experimental Procedures) so that the labeled nucleotides spread over the length of late coding strand of the probe fragment. The probe was hybridized to IO rg of RNA from pL2-cat or mock-transfected ceils, or to 3 pg of RNA from SV40-infected cells in 80% formamide conditions at 50°C for 13 hr. The samples were electrophoresed on an 8% acrylamide:bisacrylamide (40:1), 7 M urea get. The sizes of the major bands in bases are given to the left of the gel. On the right of the gel are given the sites of the 5’ ends of each of the bands. All of these initiation sites have been reported previously (Ghosh et at., 1978). The bands marked with asterisks, running at 122 and 109 bases, utilize splice donor sites that are within the probe at SW0 nucleotides 290 and 370, respectively. The band marked “a” appears only in the pL2-cat RNA lane. Since it has not appeared in three other nuclease Si assays using similar RNAs. we conclude that it is an artifact of this particular assay.

Figure 5. Quantitative Dot-Blot Various pL-cat Plasmids

Analysis

of the Replication

Capacity

of

COS cells were transfected with 5 pg of plasmid or SV40 DNA. DNA was extracted by the procedure of Hirt (1967) at 3. 24, and 48 hr afler transfection and quantitated by dot-blot analysis on nitrocellulose. In the first six lanes pBR322 DNA (pBR) was the 3ZP-labeled hybridization probe. In the last two lanes SV40 DNA was the hybridization probe. Lanes: noneno DNA transfected; pL1 -pLl -cat DNA transfected; pL1 n-pL1 n-cat DNA transfected; pt--pL2cat DNA transfected; pLGb-pLGb-cat DNA transfected (pLGb-cat is a replicatrve construction derived from pll-cat, which has deleted the SV40 sequences between the Hind Ill site at nucleotide 1046 and the Sph 1 site at nucleotide 128); SV40-SV40 DNA transfected.

T Antigen Directly Activates 385

the SV40

Late Promoter

the 48 hr samples was completely Mbo l-sensitive, whereas the small amount of pLln-cat DNA remaining in the cells was insensitive (not shown). We conclude from the results of these two experiments that the replication defect characterized in ~6-1 (Gluzman et al., 1980) is present in our nonreplicative CAT plasmid constructions.

The Effect of T Antigen on Late Promoter Activity from the pL-CAT Plasmids The preceding experiments indicated that our initial observation, that T antigen stimulated late promoter activity in the absence of replication, was correct and not due to abnormal transcription or unexpected replication in the case of the nonreplicative plasmids. Therefore we further tested the effect of T antigen on late promoter activity by transfecting CV-IP cells with pL2-cat and pL2n-cat, either with or without a cotransfected plasmid capable of supplying T antigen. CAT activity was determined at various times after transfection. The T antigen producing plasmid was ~6-1 (Gluzman et al., 1980), which has been described in a previous section. This plasmid expresses the early genes very well but cannot replicate because it contains the same 6 bp origin deletion as our nonreplicative plasmids. By using this plasmid to supply T antigen we avoided the possibility of reconstructing a functional origin in the nonreplicative CAT plasmids due to homologous recombination during cotransfection. Figure 6 shows the results of the time-course experiment. This experiment was repeated three times with different plasmid preparations and different preparations of low-passage cells. The results were remarkably consistent. The presence of ~6-1 had a definite stimulatory effect on both pL2-cat and pL2n-cat compared to either plasmid transfected alone. The activity of pL2-cat was higher than pL2n-cat, apparently due to the ability of pL2-cat to replicate. Still, the level of activity of pL2n-cat increased to approximately 30% of the pL2cat activity. This relative percentage is higher than we noted in COS cells where pL2n was 8%-10% the activity of pL2 (see Table 1). At this point we have no explanation for this difference between cell lines. The low levels of activity produced by each plasmid alone in CV-IP cells are quantitative levels of activity, since backgrounds are very low in this assay as judged by the activity of the promoterless parent plasmid pSVO-CAT (See Table 1). This low level of late transcriptional activity turns on and reaches a plateau relatively quickly in the course of the transfection. This basal activity, as measured in CV1 cells, is consistently higher for pL2-cat than pL2n-cat, suggesting that the 6 bp deletion at the origin of replication affects it. This observation may indicate that there are two late promoters or that there is a two-stage mechanism of late promoter activation (see Discussion).

Comparison of the Activity of pL2-cat in COS, C2, C6, Cll, and CV-IP Cells The data above indicate that T antigen had a substantial stimulatory effect on late transcription in the absence of

ura

Figure 6. Time Course of the Effect of T Antigen on Late Promoter from pL2-cat or pL2n-cat in CV-IP Cells

Activity

CV-IP cells were transfected with 4 cg of pL2-cat or pL2n-cat supplemented with either 4 pg of p61 DNA (to supply T antigen) or 4 pg of sonicated calf thymus DNA. At the indicated times the cells were harvested and CAT activity measured and standardized. Spots of acetylated chloramphenicol were removed from the thin-layer plate and counted in the liquid scintillation counter. The results were converted to units of CAT activity by comparison to a standard curve generated with known amounts of purified CAT enzyme.

replication. This stimulation was also noted in COS cells that produce a wild-type T antigen. In contrast to COS cells, several SV40-transformed CV-1 cell lines (C2, C6, and Cll) have been isolated, which produce defective large T antigens unable to initiate replication of viral DNA (Gluzman et al., 1977). The DNA-binding activity of the T antigens from these cell lines has been determined (Scheller et al., 1982; Prives et al., 1983). The T antigens from C2 and Cl 1 cells bind to viral DNA while C6 T antigen shows no binding activity. Plasmids pL2-cat and pL2n-cat were transfected into cultures of COS, C2, C6, Cl 1, and CV-IP cells and harvested after 48 hr for CAT assay. The results, shown in Figure 7, indicate that the difference in levels of late transcription between the nonreplicative and replicative plasmids in COS cells was no longer present in any of the cell lines containing defective T antigens. This was apparently due to the block in replication. However, the mutant T antigens, which retain their ability to bind to the viral DNA (C2 and Cl l), still had a stimulatory effect on late transcription compared to either the nonbinder (C6) or normal CV-IP cells, which contain no endogenous T antigen. In both C2 and Cl 1 we note that the nonreplicative plasmid was stimulated more than the replicative plasmid. This is a somewhat surprising result, and may indicate a favorable interaction between a mutant T antigen and an

Cell 386

Figure 7. Comparison of the Late Promoter cat In COS, C2, C6, Cl 1, and CV-IP Cells

Activity

of pL2-cat

and pL2n

5 pg of either pL2cat (2) or pL2ncat (2n) were transfected into the various cell lines, harvested after 48 hr, and assayed as described in Experimental Procedures. The resuftant spots of acetylated chloramphenicd were removed from the thin-layer plate and counted in a liquid scintillation counter. The results were compared to a standard curve generated with known amounts of purified CAT enzyme and converted to units of CAT activity. COS cells contain a wild-type T antigen; C2 and Cl1 cells contain a T antigen that can bind to DNA but cannot initiate replication of viral DNA; C6 cells contain a T antigen that can neither initiate replication nor bind to DNA: CV-IP cells contain no endogenous T antigen.

altered origin of replication. However, since the observation does not argue against our conclusions, its explanation will await future experimentation. In order to show that the lack of stimulation by C6 cells was accurate and not due to a possible lack of T antigen in that cell line, all of the cell lines were labeled for 18 hr with %S-methionine and cell extracts (equalized to represent equivalent numbers of cells) were immunoprecipitated with hamster anti-T antigen serum. The immunoprecipitates were electrophoresed on SDS polyacrylamide gels and autoradiographed (Figure 8). Clearly C2, C6, and Cl 1 cells contain equivalent amounts of immunoprecipitable T antigen This result supports the conclusions drawn from Figure 7. In addition, the immunoprecipitation results indicate that COS cells contain considerably more T antigen than any of the other cell lines. This may account for the greater stimulation of pL2n noted in COS cells (Figure 7) compared to the stimulation of either plasmid in C2 and Cl 1 cells. Alternatively, the lower stimulatory activity of C2 and Cl 1 T antigens could be due to the fact that they are mutants and are not necessarily expected to function as well as the wild-type protein. Overall, the results confirm the conclusions that T antigen has a direct stimulatory effect on late gene expression, independent of and separate from replication or genome amplification. Furthermore, the data indicate that the stimulatory effect of T antigen correlates with the retention of DNA-binding activity. Discussion We have constructed a series of transient expression vectors in which transcription of the chloramphenicol acetyltransferase (CAT) gene is under the control of the SV40

Figure 8. lmmunoprecipitation Cl 1 Cells

of T Antigen from CV-IP, COS, C2, C6, and

Extracts from the equivalent of 4 x 1Or’ %-methionine-labeled cells were immunoprecipitated with hamster anti-T antigen serum and analyzed on SDS polyacrylamide gels (see Experimental Procedures). The T antigen band is noted (T). The lanes on the right represent 10 ~1 of the total extracts from each cell line. Lane M contains size markers with molecular weights (mw) expressed in kilodaltons.

late promoter. Using these vectors we have separated late transcriptional activity from replication by inserting nonfunctional origins of replication into the plasmids and by transfecting the plasmids into cell lines that produce mutant T antigens unable to replicate DNA. Results from both types of experiments show that T antigen exerts a direct stimulatory effect on late promoter activity independent of, and in the absence of, viral DNA synthesis. Comparing the promoter activities of plasmids that differed only by the functionality of their viral origins of replication we found that T antigen substantially stimulated late promoter activity from the replicative plasmids. In comparison, both replicative and nonreplicative plasmids produced very low activity in the absence of T antigen, This T antigen-induced stimulation cannot be attributed entirely to replication or amplification, because the nonreplicative plasmid was stimulated in the presence of T antigen to yield CAT activity at levels corresponding to 10% of the replicative plasmid activity in COS cells (Table 1) and 30% in CV-1 cells (Figure 6). The difference in the relative percentage of stimulation between the two cell lines is not understood. The 30% level of stimulation detected in the CV-1 cell experiment indicates that a very high level of late promoter activity is occurring from the few nonreplicative plasmids in the cells, compared to an increasing number of genomes in the cells transfected with the replicative plasmids. Considering this, the stimulatory effect of T antigen appears to be substantial and

;;7ntigen

Directly Activates

the SV40 Late Promoter

may account for much of the late promoter activity of the lytic infection. This does not imply that genome amplification is not involved in attaining the full expression of the late region. Certainly the data indicate the positive effects of replication; however, amplification is clearly not the only major effector. Our experiments with C2, C6, and Cl 1 cells verify the conclusions that T antigen stimulates late promoter activity independent of replication. In these studies the failure to replicate was due to a defect in the T antigen. Under these conditions we noted stimulation of the late promoter (from both the replicative and nonreplicative plasmids), provided that the mutant T antigen retained DNA-binding capabilities, This may imply that DNA binding is involved in the activation of the late promoter; however, strict interpretation of the data allows only the conclusion of a correlation between DNA binding and late promoter activation. Indeed, the activation of the late promoter may occur by mechanisms that do not involve direct or specific interactions of T antigen with DNA, suggesting that T antigen may function in coordination with host factor(s), a proposal that has been made previously (Parker and Stark, 1979; Alwine and Khoury, 1980). In this regard, understanding the mechanism of T antigen activation of the late promoter may be aided by studies of the adenovirus EIA protein. Preliminary studies in this laboratory indicate that EIA protein will directly replace T antigen in stimulating 340 late promoter activity. When the replicative and nonreplicative pL-cat plasmids were transfected into CV-IP cells (in the absence of T antigen), we detected a low but quantitative basal level of late promoter activity, which appeared and reached a plateau value relatively soon after the transfection (Figure 6). This activity could account for the low levels of late RNA reported to appear early in the lytic cycle and in nonpermissive infections (Parker and Stark, 1979; Lange et al., 1981). Interestingly, the 6 bp deletion at the origin of replication of the nonreplicative plasmids consistently lowers this basal activity (Figure 6) although it does not seem to affect the ability of T antigen to stimulate the promoter. Contreras et al. (1982) have previously reported a direct effect on late transcription by similar deletions in the origin of replication. Our data suggest that the origin deletions affect only a basal activity. The delineation of a basal activity and an inducible activity suggests the possibility of there being two overlapping late promoters or a two-stage mechanism for late promoter activation. Although other mechanisms are possible, our data suggest that SV40 gene expression in the lytic system is regulated in a developmental fashion. A strong enhancerdriven promoter is first used to initiate the infection by allowing efficient expression of the early genes. This ensures the production of T antigen, one of the early gene products, which performs multiple functions in the establishment of the infection. Ultimately, T antigen allows the progression to the later phases of the infection by interacting with the viral genome at the origin of replication/ promoter region in such a way that the efficient early

promoter is down-regulated and DNA synthesis is initiated (Tegtmeyer, 1972; Reed et al., 1976; Alwine et al.; 1977b; Khoury and May, 1977; Rio et al., 1980; ‘Hansen et al., 1981). Our data extend this scenario, showing that T antigen also up-regulates the late promoter as part of the process to produce the very high levels of structural proteins needed to encapsidate the newly synthesized viral DNA. This difference in the effect of T antigen on’the enhancer-dependent early promoter and the structurally distinct late promoter implies that the late promoter is representative of a different class of RNA polymerase II promoters, possibly a class of promoters that require specific control proteins for activation. Experimental Cells, Virusas,

Procedures Plasmids,

Bacteria,

Enzymes

and Reagents

COS ceils (Gluzman, 1981) C2. C6. and Cl1 cetls (Gluzman et al., 1977) were a gift from Y. Gluzman. COS cells were grown in DMEM supplemented with 5%-10% fetal catf serum (FCS) and containing 4.5 g glucose per tier. C2, C6, Cll, and CV-IP cells were grown in DMEM supplemented with 5%-10% FCS. Virus stocks and DNA were prepared using CV-IP cells and SV40 wild-type strain 776 (Alwine, 1982; Hirt, 1967). Plasmid pSVO-cat (Gorman et al., 1982) was a gift from C. Gorman. Plasmid ~6-1 (Gluzman et al., 1980) was a gift from Y. Gluzman. All other plasmids are described in this paper and were constructed and isolated by standard recombinant DNA techniques using E. coli strain HBlOl. Restriction enzymes, T4 DNA polymerase, and T4 DNA ligase were purchased from either Bethesda Research Laboratories or New England Biolabs and were used as directed by the manufacturer. Purified chloramphenicol acetyl transferase was purchased from P. L. Biochemicak. Inc. “C-chloramphenicol was purchased from New England Nuclear, Inc. Sal I linkers were purchased from P. L. Biochemicals, Inc.

Transfection

Procedure

The calcium phosphate precipitation procedure described by Gonan et al. (1982) was used to introduce vector DNA into cells. Briefly, 3.5 X IO5 cells were plated onto 60 mm plates 24 hr prior to transfection. Fresh media containing 10% FCS was placed on the cells 3 hr prior to transfection. The DNA for transfection (5 to 10 ag) was made 120 mM in CaC12 and was added to an equal volume of 2 X HEPES buffered saline (HBS) (2 X HBS: 0.28 M NaCI; 50 mM HEPES; 2.8 mM NazHW,, adjusted to, pH 7.1) under a stream of nitrogen. The DNA-&PO, precipitate was added djrectly to the medium on the cells, and the cells were placed in a 37°C CO, incubator for 3 to 4 hr. The medium was then removed, the cells washed with serumfree medium, and then treated for 2 min with 2 ml 15% glycerol in I X HBS. After the treatment with glycerol the cells were washed three times with serum-free medium and fed with DMEM containing 10% FCS, and incubated at 37°C until they were harvested.

CAT Assay The cells were generally harvested and assayed for CAT activity at 48 hr after transfection. This time had been previously determined to be within the linear range for CAT expression in CV-1 cells or lines of CV-1 origin. The assay method of German et al. (1982) was used. Briefly the CAT enzyme was released from the cells into 100 pl of 250 mM Tris-HCI (pH 7.8) by sonication. The enzyme assay was done in a final volume of 170 ~1 of 250 mM Tris-HCI (pH 7.8) containing 20 to 80 ~1 of the cell extract, 20 cl of 4 mM acetyi co-enzyme A, and 0.1 &i of “Cchloramphenicd (56 pCi/mmole; New England Nuclear Corp.). To quantitate CAT activity standard curves were generated using known amounts of purified CAT enzyme (P. L. Biochemicak, Inc.). The enzyme assay was run for 30 to 45 min at 37OC and stopped by adding 0.2 ml ethyl acetate. The chloramphenicol was extracted from the reaction mix by three treatments with 0.2 ml ethyl acetate. After centrifugal evaporation the chloramphenicol was dissolved in 15 ~1 ethyl acetate and spotted on silica thin-layer chromatography plates. Unreacted chloramphenicol was separated from the mono- and diacetytated forms by ascending chromatography in chloroform:methanol (95:5). Fol-

Cell 388

lowrng autoradiography the amount of chloramphenicol acetytated was quantitated by cutting the spots out and counting them in a scintillation counter or by scanning the autoradiograph with a densitometer. Dot-Blot Analysis After transfection of cells with plasmrd or viral DNA for varrous times, the DNA was extracted by the procedure of Hirt (1967). The extracted DNA was purified by phenol extraction, followed by ethanol precrpitation. The pellets of precipitated nucleic acids were suspended in 200 pl of TNE (10 mM Tris, pH 7.5, 10 mM NaCI, 2 mM EDTA). 100 pl was sonrcated to shear the DNA using a Sonicator model W-10 with a microprobe (1 min at setting 5). This results in an average fragment size of approximately 800-loo0 nucleotrdes. The sonicated samples were then digested with 10 fig RNAase A (Worthington) for 30 min at 37°C to remove RNA. After phenol extraction and ethanol precipitation the DNA was dissolved in 225 pl of TNE. Portions of each sample (50 al) were boiled for 5 min, quenched on ice, and adjusted to 10 X SSC, 1 M ammonium acetate. This was immediately dotblotted onto nitrocellulose (S&S BA85) using an S&S Minifold apparatus. The nitrocellulose had been wetted and washed with 10 X SSC. 1 M ammonium acetate prior to addition of the sample. After the addition of the sample, the sample wells were washed three times with IO x SSC, 1 M ammonrum acetate. After blotting the filter was floated onto 0.1 x SSC. 1 M ammonium acetate (to remove the salt), air-dried, and baked for 2 hr at 80°C In the vacuum oven. The filter was pretreated and hybridized using standard aqueous conditions (Southern, 1975). Kodak XAR-5 X-ray film was used for autoradiography either at room temperature or at -70°C ustng DuPont Cronex Intensifying Screens. RNA Analysis Total RNA was extracted from ceils 48 hr after transfection by the method of Villarreal (1981). The RNA preparations were freed of DNA by treatment for 45 mm at 37°C with RNAase-free DNAase that had been prepared by described methods (Tullis and Rubin, 1980). The RNA was analyzed by Northern blot analysis (Alwine et al., 1977a) or by nuclease Si analysis (Berk and Sharp, 1978). The probe for Sl analysis was labeled with “P in vitro using T4 DNA polymerase (O’Farrell et al., 1980). Briefly, 0.2 rg of SV40 DNA was cut at the single &I 1 site at the onqrn of replrcation (See Figure 1) and digested for 25 min at 37°C with 0.25 U of T4 DNA polymeraseto take advantage of the 3’ exonuclease actrvity of the enzyme. Then the solution was made 300 pM in dCTP, dATP. and dGTP and IO pCi of a-=P dTTP (at 3006 Ci/mmole. Amersham Corp.) was added. The incubation was continued for 30 min at 37°C. This amount of digestion and fill-in will label over the entire region, which will ultimately be Isolated as the probe. The resulting labeled DNA was cut with Hint II, which cuts at SV40 nucleotide 470, and the 471 bp fragment was agarosegel-isolated. Hybridization for Sl analysis was carried out In 80% formamrde hybridization conditions at 50°C for 13 hr. Each sample was then treated wrt 100 U Si (P. L. Biochemicals, Inc.) for 1 hr at 37°C. Samples were electrophoresed on an 8% acrylamrde-bisacrylamide (40.1) gel containing 7 M urea. Analysis of T Antigen CV-1, COS, C2. C6. and Cl 1 cells were grown to 80% confluence on 60 mm plates. At that time the medium on the plates was reduced to 3 ml and 50 pCi of ?S-methionine was added to each plate. After 18 hr at 37°C the cells were harvested, immunoprecipitated with hamster anti-T serum, and analyzed on SDS polyacrylamide gels as described by Alwine and Khoury (1980). At the time of harvest identical plates of unlabeled cells were trypsinrzed and counted. The amount of extract used in each immunoprecrpitate was then equalized to reflect the extract from 4 X 105 cells.

We thank Moshe Sadofsky, Susan Carswell, and Elizabeth Blankenhorn for general good nature and helpful discussions. We also thank Lori Bloom for experimental assistance and Edna Matta for technical assistance. We gratefully acknowledge Laura D. Pastore for typrng the paper. J. M. K. is a predoctoral trainee supported by Public Health Service Grant T32 CN 07229. This Investigation was supported by Public Health Servrce Grant CA 33656 awarded by the National Cancer Institute.

The costs of publrcation of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

August

10, 1983; revised October

28, 1983

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mouse

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