Vol. 65, No. 11

JOURNAL OF VIROLOGY, Nov. 1991, p. 5710-5720 0022-538X/91/115710-11$02.00/0 Copyright © 1991, American Society for Microbiology

Regulation of Early Gene Expression from the Bovine Papillomavirus Genome in Transiently Transfected C127 Cells PAUL SZYMANSKI AND ARNE STENLUND* Cold Spring Harbor Laboratory, P.O. Box 100, I Bungtown Road, Cold Spring Harbor, New York 11724-2206 Received 24 April 1991/Accepted 21 July 1991

Expression of bovine papillomavirus (BPV) early gene products is required for viral DNA replication and establishment of the transformed phenotype. By the use of a highly efficient electroporation system, we have examined for the first time the transcriptional activity of BPV promoters in their natural genomic context in a replication-permissive cell line. We have determined that a qualitatively distinct stage of transcription is not detectable prior to DNA replication in transiently transfected cells. This suggests that the transcriptional activity of the BPV genome in stably transformed cells represents the early stage of BPV gene expression. Quantitative differences in promoter activity between transiently transfected and stably transformed cells suggest that subtle changes in gene expression may control progression of the viral life cycle. Deletion analysis demonstrated that the E2 transactivator protein stimulates all of the early promoters through sequences located in the upstream regulatory region. This E2-dependent enhancer was found to be highly redundant, and particular E2 binding sites did not display a preference for particular promoters. Despite this dependence on a common cis-acting sequence, the various promoters displayed different sensitivities to the E2 transactivator. The findings that E2 regulates all promoters and, with the exception of the E2 repressors, that no other known viral gene product appears to affect transcription indicate that the E2 system functions as the master regulator of BPV early gene expression.

formation of nonfunctional transcription complexes might also occur, as suggested by the finding that the E8/E2 protein can repress the basal activity of the P2 (P89) promoter (4). The E2 proteins are expressed from several different viral promoters. The E2C and E8/E2 repressors are expressed from the PS (P3080) and P3 (P89) promoters, respectively (4, 21), and the E2 transactivator is expressed from the P2, P3, and P4(P2443) promoters (12, 33, 41, 49). Since at least the P2 and P4 promoters have been shown to be regulated by the E2 activator and repressor proteins in a URR-dependent manner (8, 9, 12, 33, 40, 41), it is possible that an autoregulatory circuit based on the E2 proteins is crucial for control of BPV gene expression. Indeed, it appears that a precise regulation of gene expression is necessary for control of BPV copy number, since mutations in the E2C repressor result in a high-copy-number phenotype in stably transformed cell lines (20, 34). In addition, repressor mutants replicate to higher levels than the wild type in transient replication assays (47). To explore the regulatory circuit that controls the BPV life cycle, it is necessary to examine transcriptional regulation in the environment in which BPV is capable of replication, establishment and maintenance of an episomal state, and transformation. Mouse C127 cells have been the cell line of choice for the genetic analysis of BPV because transformed cells are easily recognizable by their morphology, thereby permitting quantitative focus assays (7). The transcripts present in stably transformed cells have been examined in detail (2, 43, 51), but the study of transcriptional regulation with this system is complicated by the indirect effects of engineered mutations on the viral life cycle. This limitation can be overcome by the use of transient expression experiments, in which mutations can be assayed for their direct effects on transcription. In a previous report, we described the development of a highly efficient electroporation system and its use in a transient replication assay (46). In this study we use the

The papillomaviruses are small DNA viruses that cause benign epithelial tumors (warts) in a variety of animal species and are sometimes associated with malignant carcinomas. Virus production is limited to terminally differentiated keratinocytes, and it has not yet been possible to propagate any of the papillomaviruses in a cell culture system (17). Bovine papillomavirus (BPV) has been chosen as the prototype for the molecular and genetic analysis of the papillomaviruses because it transforms certain rodent cells in culture and persists as a multicopy nuclear episome (14, 22), a state believed to be analogous to the latent infection of the basal epithelial cells and dermal fibroblasts of a fibropapilloma (17). Gene expression is limited to the early region of the virus and results in low levels of viral mRNAs and proteins (17). One major transcription-regulatory system of BPV involves the interaction of the viral E2 gene products with enhancer elements in the upstream regulatory region (URR), also referred to as the long control region (9, 42). The product of the E2 open reading frame (ORF) is a transcriptional activator that binds as a dimer (6, 27) to the sequence ACCN6GG/TT (1, 10, 23, 30, 31), where N is any nucleotide. There are 17 such sites in the BPV genome, 11 of which are located in the URR (23). In addition to the transactivator protein, the E2 ORF encodes two repressor proteins: E2C (also called E2TR), which contains only the carboxy-terminal DNA-binding and dimerization domains of E2 (21); and E8/E2, which consists of the first 11 amino acids encoded by the E8 ORF fused to the carboxy-terminal domain of E2 (4, 11, 19). The repressors are believed to function by competing with the transactivator homodimers for binding to DNA or by associating with the transactivator to form inactive heterodimers (21). Displacement of cellular factors or the *

Corresponding author. 5710

REGULATION OF BPV EARLY GENE EXPRESSION

VOL. 65, 1991

electroporation method in conjunction with a sensitive RNase protection assay, which allows us to examine the transcriptional activity of BPV promoters in the intact viral genome in C127 cells. The ultimate goal was to unify the examination of transcriptional regulation with the previous findings concerning replication and transformation. Our results suggest that the latent stage of the BPV life cycle, as represented in stably transformed cells, reflects the early stage of viral gene expression. We also found that the P2, P3, P4, and P5 promoters are all stimulated by the E2 transactivator protein through common sequences in the URR and that no other known viral gene product (except the E2 repressor proteins) appears to affect promoter activity. The differential sensitivity of the four promoters to the E2 transactivator suggests that other regulatory elements located outside of the URR may act to determine the degree of E2 responsiveness. MATERIALS AND METHODS Plasmid constructions. (i) Test plasmids. pMLBPV

con-

tains the entire BPV genome linearized at the unique BamHI site and inserted into pML (25). pE2TTL is identical to pMLBPV except for an XbaI translation termination linker inserted into the E2 ORF at BPV nucleotide (nt) 3351. Linker deletion mutants of the BPV HindIII-HpaI fragment were derived from linker insertion mutants described previously (26) and were inserted in place of the wild-type HindIII-HpaI fragment of pE2TTL to generate the URR mutants. pSitel5Al and pSitelA5S were constructed by digesting pE2TTL with BstEII, treating with mung bean

nuclease, and religating. The resulting mutations

are:

for

pSitelSAl, from ACCCCTCCTG£TAACCA to ACCCCTC CTGTAACCA; for pSitelSA5, from ACCCCTCCTGGTA

5711

imately 2.0 x 106 to 2.5 x 106 per 10-cm dish were trypsinized 16 to 20 h after seeding and pelleted by centrifugation. The cell pellet was resuspended in DMEM-10% FBS-5 mM BES [N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, pH 7.2] at a density of 3 x 107 to 4 x 107 cells per ml and stored on ice. Cell suspension (0.25 ml) was mixed with plasmid DNA plus 50 ,ug of denatured salmon sperm DNA in a disposable cuvette and electroporated at 240 V and 960 p,F with a Bio-Rad Gene Pulser equipped with a capacitance extender. The cells were then transferred to a 15-ml centrifuge tube, left on ice for 15 to 20 min, resuspended in 10 ml of DMEM-10% FBS-5 mM BES, and pelleted by centrifugation. The cell pellet was resuspended in 10 ml of DMEM-10% FBS and plated onto a 10-cm dish. RNase protection analysis of RNA. Cytoplasmic RNA was prepared from cells 30 h posttransfection by the Nonidet P-40 method and treated with DNase I to remove plasmid DNA (36). Uniformly labeled riboprobes were prepared with SP6 RNA polymerase by the method of Melton et al. (28). RNA was dissolved in 20 RI of 40 mM PIPES [piperazineN,N'-bis(2-ethanesulfonic acid), pH 6.7]-0.4 M NaCl-1 mM EDTA containing 105 cpm (Cerenkov) of probe, denatured at 85°C for 5 min, and hybridized at 60°C overnight. A digestion mix (0.3 ml) of 10 mM Tris (pH 7.5)-0.3 M NaCl-5 mM EDTA-40 jig of RNase A per ml-2 ,ug of RNase T1 per ml was added and incubated at 16°C for 45 min. Digestion was terminated by addition of 20 RI of 10% sodium dodecyl sulfate and 5 ,ul of 10-mglml proteinase K, followed by incubation at 37°C for 30 min. Samples were phenol-chloroform extracted, ethanol precipitated, and electrophoresed on 5% polyacrylamide-8 M urea gels. Quantitation was performed by exposing the dried gels to storage phosphor screens and analyzing with a Molecular Dynamics PhosphorImager equipped with ImageQuant software.

ACCA to ACCCCTCCTGCA. pSitel7 contains a point mutation in the first half-site of E2 binding site 17 at nt 3090 (ACC to ACA). pSitel6/17 (gift of Rong Li) contains the same mutation in site 17 plus a point mutation in the second half-site of site 16 at nt 2931 (G£LT to GCT). pElSmaTTL contains an XbaI translation termination linker inserted into the SmaI site of pMLBPV. pElABgl/ Bcl was constructed by digestion of a cloned BPV SmaIEcoRI fragment with BglII and BclI, religation, excision of the SmaI-EcoRI fragment, and insertion into SmaI- and EcoRI-digested pMLBPV. (ii) Expression vectors. The E2 expression vector pHSE2 is identical to pCGE2 (46) except that the cytomegalovirus promoter-enhancer was replaced with the heat shock protein 70 promoter. The El expression vector pCGEag contains BPV nt 619 to 2766 and 3881 to 4450 and expresses the full-length El protein, as described previously (46). ppA36 contains the human P-globin gene driven by its own promoter truncated to immediately upstream of the TATA box (45). (iii) SP6 polymerase templates. The BPV promoter-specific templates pSPP2, pSPP3, pSPP4, and pSPP5 contain BPV nt 7945 to 239 (HpaI-RsaI), 796 to 1008 (DraI-HincII), 2113 to 2499 (EcoRI-Hinfl), and 3023 to 3175 (FspI-DpnI), respectively, inserted into pSP64 or pSP65 (28). The 3-globin template pSP6,350 was described previously (13). To produce run-off transcripts, plasmids were linearized in the vector polylinker sequences, except for pSPP4, which was linearized at the BPV BstEII site (nt 2405). Cell culture and transfections. C127 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS). Cells at approx-

RESULTS BPV-specific messages expressed from viral promoters in their natural context can be detected in transiently transfected C127 cells. To examine the regulation of BPV promoters in their natural genomic context, we used a highly efficient electroporation system to transfect C127 cells with the complete BPV genome cloned into a plasmid. The development of this transfection procedure has been described previously (46). To assay the activity of each promoter individually, we produced uniformly labeled RNA probes for use in RNase protection assays, which are sensitive enough to detect very low levels of mRNA. The experiment shown in Fig. 1 demonstrates the feasibility of this approach. C127 cells were electroporated with 10 jig of pMLBPV, which contains the entire wild-type BPV genome cloned into a plasmid vector (25). Cytoplasmic RNA was extracted 30 h posttransfection and divided into four aliquots so that we could determine the activity of all the promoters in the same population of transfected cells. RNase protections were performed with the BPV promoterspecific probes mixed with a probe to detect mRNA from a cotransfected P-globin expression vector serving as an internal standard. Lanes 8, 9, 10, and 11 of Fig. 1A show BPV messages initiating at P2, P3, P4, and P5, respectively. Compared with RNA extracted from V1216 cells, a mouse cell line stably transformed by BPV (lanes 12 to 15), one can see that the positions of the initiation sites of the four promoters were the same as in the transiently transfected cells. The possibility remains, however, that additional promoters may be active in transiently transfected cells that

J. VIROL.

SZYMANSKI AND STENLUND

5712

A. 36

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are not detected by probes. Transcripts that initiate at the promoter spanned by a given probe protect only a portion of the probe from RNase digestion and thus produce a fragment smaller than the undigested probe. In contrast, RNA which initiates at upstream BPV promoters and cryptic start sites in the plasmid sequences ("readthrough" transcripts) will protect the entire length of a downstream probe. These products are indicated by RT in Fig. 1B. The BPV-specific probes were purposely designed to be quite short (Fig. 1B) so as to avoid detection of splice donors and acceptors and minimize the number of bands on the gel. However, the P3 probe detects a splice donor at BPV nt 864, resulting in a protected fragment of 68 nt. The 3-globin probe protects a large region of its cognate message and produces three bands, representing correctly initiated P-globin RNA plus RNA initiated within the upstream vector sequences and spliced at cryptic sites (45). To determine the relative abundance of mRNAs initiating

are our

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FIG. 1. Detection of BPV-specific messages in transiently transfected C127 cells. (A) RNase protection analysis. Cytoplasmic RNA was extracted from untransfected C127 cells (lanes 1 and 2), from C127 cells transfected with 1 ,ug of ppA6 (lanes 3 and 4), 2 pLg of pHSE2 plus 1 ,ug of pp36 (lanes 5 to 7), 10 pLg of pMLBPV plus 1 ,ug of pp36 (lanes 8 to 11) or from V1216 cells (lanes 12 to 15). RNAs were hybridized to a P-globin probe (lanes 1 to 15) plus probes spanning the initiation sites of P2 (lanes 8 and 12), P3 (lanes 6, 9, and 13), P4 (lanes 10 and 14), P5 (lanes 7, 11, and 15), P2 plus P4 (lanes 1, 3, and 5), or P3 plus P5 (lanes 2 and 4). Transfection mixes for lanes 3 to 11 were supplemented with pUC19 to equal 20 ,ug of total plasmid DNA plus 50 ,ug of salmon sperm DNA as the carrier. Lanes 16 and 17, undigested probes. Lane M, Mspl-digested pSP64 as size markers (in nt). RT, readthrough transcripts. (B) Diagram of the early region of BPV. The five early promoters (P1 to P5) are represented by bent arrows positioned at the major initiation sites, with probes shown as narrow horizontal lines above P2 to P5. Open bars labeled El to E8 represent ORFs. Solid boxes numbered 1 to 17 signify E2 binding sites (23).

at the viral promoters, the intensity of the bands was quantitated with a Molecular Dynamics Phosphorlmager. The value obtained was normalized to that for the P-globin signal and then to the number of cytosine residues (the labeled nucleotide in the probe) in the protected fragment. We found that the pattern of relative message abundance was quantitatively different between the cells transiently transfected with pMLBPV and the stably transformed (VI216) cells (Table 1). The P2 promoter expressed the most mRNA in VI216 cells but not in the transfected cells. The level of P4-specific message was equal to that of P2 in the transfection but was lower than that of P2 in the stable transformants. Most strikingly, the level of mRNA expressed from the P3 and P5 promoters relative to that expressed from P2 was four- to sixfold lower in V1216 cells than in transiently transfected cells. It is possible that the

VOL. 65,

1991

REGULATION OF BPV EARLY GENE EXPRESSION

TABLE 1. Relative abundance of BPV promoter-specific messages in transiently transfected and stably transformed cellsa Promoter

P2 P3 P4 P5

Relative mRNA abundance pMLBPV

V1216

1.00 0.28 1.08 2.57

1.00 0.05 0.65 0.59

a Values represent the average of five experiments arbitrarily standardized to that of the P2 promoter.

viral regulatory system has not yet reached equilibrium by 30 h posttransfection and that the VI216 pattern will be achieved over time, or that the change in relative message abundance is an indirect consequence of cellular transformation. The 30 h time point was chosen because transcription can be detected but replication has not yet begun in earnest, and therefore replicated DNA constitutes only a small fraction of the total transfected DNA present in the cells (44, 46). The finding that replication-defective El mutants (46) display wild-type transcriptional activity (see Fig. 5) confirms that replication has a negligible effect on mRNA levels at 30 h posttransfection. Preliminary experiments suggest that no dramatic changes in the expression pattern occur from 2 through 5 days after the transfection (44), throughout which time DNA replication is proceeding (44, 46). Therefore, the situation in stably transformed cells appears to represent the early stage of BPV gene expression, and a qualitatively distinct stage of transcription is not detectable prior to DNA replication in transiently transfected cells. BPV early promoters display differential sensitivities to the E2 transactivator. To examine the effect of the E2 transactivator protein on expression from the intact genome, we cotransfected C127 cells with 10 jig of pMLBPV plus 2 ,ug of pHSE2, which expresses the full-length E2 protein from the heat shock protein 70 promoter. This vector has been shown by metabolic labeling and immunoprecipitation to express only full-length E2 (47). The RNA initiating at the heat shock promoter of pHSE2 protects the entire P5 probe, but the expression from the P5 promoter within the E2 ORF is below the limit of detection (Fig. 1B, lane 7). In addition, pHSE2 is incapable of expressing the E2C repressor due to a point mutation which changes the initiation codon for E2C from ATG to ATC. Expression of the E2 protein resulted in a small increase in the level of RNA initiating at all the promoters (data not shown). Since pMLBPV carries an intact E2 ORF, we suspected that the viral promoters might already have been stimulated by E2 activator expressed from the plasmid itself and therefore would not display a true basal level. In addition, expression of the repressor forms of E2 from pMLBPV might be increased in response to the E2 activator expressed from pHSE2, thus buffering its effect. Therefore, to reduce the complexity of the system, we introduced a translation termination linker into the E2 ORF of pMLBPV to generate the plasmid pE2TTL, which is unable to express either the activator or repressor forms of E2. To quantitatively investigate the effect of the E2 protein on this template, we performed a titration with increasing amounts of pHSE2. When C127 cells were transfected with pE2TTL (Fig. 2), the presence of the E2 expression vector resulted in a much greater fold induction of RNA levels than when pMLBPV

5713

was used as the test plasmid. This was mostly due to reduced activity in the absence of pHSE2, indicating that pMLBPV expresses a significant amount of the E2 activator from its own genome. The increase in P2 and P4 promoter activity obtained with pE2TTL was of about the same magnitude as reported by others with chloramphenicol acetyltransferase (CAT) gene-containing plasmids (8, 9, 12, 33, 40, 41). The responses of all the promoters to increasing amounts of E2 expression vector are described by saturation curves, but the levels of RNA from different BPV promoters were increased to different extents, as seen most easily with a linear plot of RNA level versus amount of pHSE2 (Fig. 2B). To illustrate differences in the basal level of expression, it was most revealing to plot the same data on a full log scale (Fig. 2C), thus expanding the area of the graph near the origin. From this we see that in the absence of E2, the level of P5-specific messages was more than 10-fold greater than the level of mRNA expressed from any other promoter, suggesting that P5 may be the most productive promoter upon initial viral infection. The P5 promoter was the least responsive to E2, however, since cotransfection with 10 jig of pHSE2 resulted in only a fourfold increase in the level of RNA. The basal levels of messages from the P2 and P4 promoters were equal, but P4 was more responsive than P2 (60-fold versus 20-fold at 10 jig of pHSE2) and, at high levels of pHSE2, expressed even more mRNA than the P5 promoter (Fig. 2B). The P3 promoter displayed the lowest basal level, but its response to E2, as described by the slope of the line on the full log plot (Fig. 2C), was about equal to that of P2. This results in the level of P2-specific messages being about two- to threefold greater than the level of P3-specific messages throughout the course of the titration. Deletion analysis of the E2-dependent enhancer. It is known that the E2 binding sites located in the URR mediate the activation of the P2 and P4 promoters by the E2 protein (17). Specifically, two groups of sites, termed E2RE1 (sites 7 to 10) and E2RE2 (sites 1 and 2), were shown by CAT assays in CV-1 cells to be most important for E2-dependent enhancer activity (9, 39-41). No deletion analysis of the activation of the P3 and P5 promoters has been performed. We therefore sought to determine whether the E2-dependent enhancer affects all the early promoters in their natural context in C127 cells and, if so, which sequences in the URR were responsible for the effect. We wished to determine whether all or only a subset of the E2 binding sites contribute to the induction and whether certain E2 binding sites might be selective for different promoters. Figure 3 shows a series of deletion mutations in the URR which were cloned into the pE2TTL background. The undeleted URR in pE2TTL is referred to as wild type in this experiment. No small deletion had a severe effect on expression from any promoter in either the presence or absence of E2 (Fig. 3B), indicating that no essential basal regulatory elements are disrupted by these deletions and that no individual E2 binding site or pair of sites are absolutely required for the promoters to respond to E2. Some minor effects were observed, however. The basal level of P2-specific messages was decreased about two- to threefold by the 36/221 deletion, whereas the fold induction by E2 was the same as that of the wild type. This deletion did not decrease the basal or induced level of P3-, P4-, or P5-specific messages, suggesting that a basal element specific to the P2 promoter is located within nt 7835 to 7893. The 121/36 deletion decreased the induction of P2- and P4-specific messages about twofold, suggesting that E2 binding sites 9 and 10 make a major contribution to the E2 responsiveness of these promoters. In

5714

J. VIROL.

SZYMANSKI AND STENLUND

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FIG. 2. E2 titration. (A) RNase protection. C127 cells were transfected with 10 p.g of pE2TTL plus increasing amounts of pHSE2 plus 1 of ppM6 plus pUC19 to 21 p.g of total plasmid DNA plus carrier. Cytoplasmic RNA was extracted at 30 h posttransfection, divided into aliquots, and hybridized to the P-globin probe plus probes specific for P2 plus P4, P3, or P5, as indicated. (B and C) Linear (B) and full log (C) plots of RNA (arbitrary units) versus micrograms of E2 expression vector. Values plotted are the average of four trials; error bars represent one standard deviation. The value plotted at 0.01 ptg of pHSE2 is actually the value obtained in the absence of expression vector (lanes 1, 9, and 17). p.g

contrast, the 134/121 deletion, which affects only site 9, had no significant effect on any promoter. This suggests that site

10, which has the highest relative equilibrium binding

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stant of all the E2 binding sites (23), may make the largest contribution to the overall E2 response.

Since the experiment in Fig. 3B indicated that no small region of the URR is absolutely required for basal or induced transcription, we constructed a series of plasmids unidirectionally deleted from the 5' end of the URR. We observed a stepwise loss of activity with increasing deletion size for all promoters (Fig. 3C). The 234/Sca deletion decreased the

induced level of all promoters by about 50%. Most of this effect was due to a reduction of the basal activity, however, with only a small decrease in the E2 responsiveness. The basal levels of P2 and P4 are not seen on this exposure but are clearly visible on long exposures and are quantifiable by the Phosphorlmager. The basal level of P3 is quite difficult to detect on film, but reproducible values can be obtained by use of the Phosphorlmager. When only sites 7 to 11 were present (234/11, lane 8), only 20 to 30% of the induced wild-type activity remained (note high internal standard for P2, P3, and P4). Further deletion

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Deletion analysis of the URR. (A) Linker deletion mutants. The URR is depicted as a line, with solid boxes numbered 1 to 11 representing E2 binding sites. Lines beneath the URR correspond to sequences present in constructs, with nucleotide endpoints indicated. WT, wild type. (B) Small internal linker deletions. The absence (-) or presence (+) of the E2 expression vector is indicated. (C) Fixed 5' endpoint 3.

pE2TJTL

deletions. (D) Fixed 3' endpoint deletions. C127 cells

were transfected with 10 ~±g of wild-type or URR deletion mutants as indicated plus or minus 2 ~Lg of pHSE2 plus pUC19 to 20 iLg of total plasmid DNA plus carrier. Cytoplasmic RNA was extracted at 30 h posttransfection, divided into aliquots, and hybridized to P-globin probe plus probes specific for P2 plus P4, P3, or P5, as indicated. Basal levels of P2, P3, and P4 are visible on long exposures and are quantifiable by the Phosphorlmager. Values in panel B represent the amount of RNA in the protected fragment as determined by Phosphorlmager analysis and were standardized to the wild-type basal level for each promoter.

plus 1 jig of

ppA6

5715

5716

J. VIROL.

SZYMANSKI AND STENLUND

of sites 7 and 8 (234/134, lane 10) reduced P2 and P4 messages to about 5% of the induced wild-type level and made P3 undetectable. With larger deletions, P2 and P4 messages were undetectable even in the presence of E2. The P5 promoter was still significantly active when nearly the entire URR was deleted but was no longer stimulated by E2 (234/221, lanes 15 and 16). In Fig. 3D we assayed deletions with a fixed endpoint near the 3' end of the URR. The basal level of P2-specific messages was reduced two- to threefold by the 36/221 deletion, as it was in Fig. 3B. Successive deletions did not further reduce the P2 basal level except when the entire URR was deleted (Hin/Hpa), at which point RNA was not detectable. Deletion up to Nar/221 had no effect on the P3 or P4 basal level, but all activity was lost upon deletion of the entire URR. The P5 promoter behaved similarly, except that about 35% of the wild-type basal activity remained even after deletion of the entire URR. We conclude that a P2 promoter element is located between nt 7835 and 7894, as defined by the 36/221 deletion, and that basal elements that contribute to the activity of all promoters are located between nt 6958 and 7274, as indicated by comparison of the Hin/Hpa and Nar/221 deletions. It is likely that this region contains more than one element, since the 234/Sca deletion (nt 7187 to 7388) decreased the basal RNA level only about 50%. In the presence of E2, the constructs in Fig. 3D expressed a fairly constant amount of mRNA up to and including the Cla/221 deletion (lane 12), followed by a complete loss of E2 inducibility by the Nar/221 deletion (lanes 13 and 14). This dramatic decrease in activity was in contrast to the generally steady decrease of activity observed in Fig. 3C. Deletion of the entire URR eliminated expression from P2, P3, and P4 even in the presence of E2, whereas P5 retained some activity but was no longer E2 inducible (lanes 15 and 16). Thus, it seems that all the promoters respond to E2 through the URR and are affected similarly by deletions in this region, with the exception that P5 is not completely dependent upon the URR for basal-level expression. We conclude that specific E2 binding sites contribute to the activity of all promoters similarly and do not display a preference for certain promoters and that elements controlling the basal level of transcription are also located in the URR. Moreover, the E2-dependent enhancer is redundant, since small deletions have little effect and larger deletions generally show a stepwise loss of activity. Since cooperation between E2 molecules has been demonstrated by physical methods (16, 29) and suggested by functional analysis (39), perhaps E2 binding sites 1 to 4 were sufficient for full response in Fig. 3D as a result of being moved closer to sites 11 and 12 by removal of the intervening DNA. E2 binding sites near P4 and P5 are not required for E2 activation. As alluded to above, E2 dimers bound to distal sites on the same DNA molecule can interact through DNA looping to form multimeric complexes that can be visualized by electron microscopy (16). In addition, E2 has been functionally and physically shown to interact with DNAbound Spl to activate transcription from P4 and P5 (24, 41). This raises the possibility that the E2 binding sites located near P4 (site 15) and P5 (sites 16 and 17) may act to direct the activity of E2 bound at the URR to these distal promoters. We therefore chose to investigate the role of E2 binding sites 15, 16, and 17 in the context of the viral genome by mutating these sites and assaying expression from P4 and P5. In Fig. 4, the E2 binding-site mutants were assayed in the absence and presence of the E2 expression vector. In the

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FIG. 4. Effect of mutations in E2 binding sites proximal to P4 and P5. C127 cells were transfected with 10 ,ug of wild-type (WT) pE2TTL or E2 binding-site mutants as indicated plus 1 pLg of pIA36 plus or minus 2 ,ug of pHSE2 plus pUC19 to 20 ,ug of total plasmid DNA plus carrier. Cytoplasmic RNA was extracted at 30 h posttransfection, divided into aliquots, and hybridized to P-globin probe plus probes specific for P2 plus P4 or P5, as indicated. Basal levels of P2 and P4 are visible on long exposures and are quantifiable by the Phosphorlmager.

presence of E2, the level of P4 messages was reduced slightly by a 1-bp deletion (pSitelSAl) and severalfold by a 5-bp deletion (pSitel5A5). The Al and A5 mutations change the sequence of the second half of the palindromic binding site from GGT to GTA and GCA, respectively. The levels of P2 and P5 messages were wild type, indicating that the effect of the site 15 mutations is specific to the P4 promoter. The major effect, however, was a reduction in the basal levels of the mutants, which was detectable on long exposures. The site 15 mutants are still highly responsive to E2, albeit to a lower level than the wild type. We conclude that although binding site 15 is not absolutely required for E2 activation of the P4 promoter, it appears that this site, or more likely the sequences adjacent to it, plays at least some role in the response. Similar results were obtained upon mutation of the E2 binding sites near the P5 promoter. Although the site 17 mutation had no effect on the basal or induced activity of P5 or any other promoter, the site 16/17 double mutant reduced P5 basal expression by about 50%. The fold induction by E2 remained at wild-type levels, however, indicating that binding sites 16 and 17 are not involved in the response of the P5 promoter to the E2 protein and that the point mutation within E2 binding site 16 most likely affects a basal promoter element. The El, E6, and E6/7 gene products do not function as transcriptional regulators. Since pE2TTL is capable of expressing all documented BPV early gene products except the E2 proteins, it was necessary to determine whether the stimulatory effect of E2 on all promoters is direct or is mediated by a viral gene product that is expressed in response to E2. The investigation of El function is of particular interest, since it has been observed that El mutants display elevated P2 activity in stably transformed cell lines (18, 37). It was suggested that the El protein might repress transcription from the P2 promoter or that the increased P2 activity might be an indirect effect of the state of the viral DNA in cells transformed with wild-type BPV or

VOL. 65, 1991

WT

REGULATION OF BPV EARLY GENE EXPRESSION

P2 + P4 Sr. agl

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assayed in the pE2TTL background and were found to have no effect on the ability of any promoter to respond to E2 (data not shown).

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FIG. 5. El mutation and complementation. C127 cells were transfected with 10 ,ug of pMLBPV (WT) or El ORF mutants as indicated plus 1 ,ug of ppA36 plus or minus 1 Fg of pCGEag (El) plus pUC19 to 20 ,g of total plasmid DNA plus carrier (lanes 1 to 6). The transfection mix for lane 7 was the same except that it contained no BPV test plasmid. Cytoplasmic RNA was extracted at 30 h posttransfection, divided into aliquots, and hybridized to P-globin probe plus probes specific for P2 plus P4, P3, or P5, as indicated.

El mutants, which are present in episomal and integrated states, respectively (18, 37). We decided to test this hypothesis directly by assaying the transcriptional activity of El mutants. We mutated the El ORF either by placing a translation termination linker at the SmaI site (SmaTTL) or by deleting the 296 bp between the BgIII and BclI sites (ABgl/Bcl), which also creates a frameshift mutation. By comparing RNA from C127 cells transfected with wild-type and El mutant constructs, we found that the level of RNA expressed from the P2 promoter was unaffected by either mutation of the El ORF (Fig. 5, lanes 1, 3, and 5). Expression from the P3, P4, and P5 promoters was also unaffected by the El mutations, suggesting that the El gene product does not play a role in transcriptional regulation. Since it is possible that insufficient El protein is expressed from pMLBPV to produce a detectable effect in a transient transfection, we thought it important to employ an expression vector to achieve much higher intracellular levels of El. The plasmid pCGEag has been shown to express full-length El protein and is able to complement an El mutation in a transient replication assay (46). We cotransfected the wildtype and El mutant plasmids with pCGEag and found that the El expression vector had no effect on expression from the P2 and P5 promoters (Fig. 5). The effect of pCGEag on P3 and P4 was difficult to determine because of the large amount of mRNA from the expression vector. Therefore, the El protein does not appear to possess a transcriptional regulatory function, and in particular, it does not repress the activity of the P2 promoter. It is therefore more likely that the effect observed in previous studies (18, 37) was due to an indirect effect of the inability of the El mutants to replicate episomally. We also assayed the transcriptional activity of pMLBPV constructs which contained mutations in the E6 or E7 ORF. The 775 (E6) and 576 (E6/7) mutants, which were described previously (3), displayed wild-type mRNA levels, consistent with previous experiments with stably transformed cell lines (32). Cotransfection with plasmids which express the E6 or E6/7 protein similarly had no effect (data not shown). Lastly, the mutations in the El, E6, and E6/7 ORFs were also

DISCUSSION There is a large body of evidence suggesting that a precise regulation of gene expression is necessary for BPV to establish and maintain itself as a multicopy nuclear plasmid in transformed cells. We believed that to examine this potentially very complex regulatory circuit properly, it was desirable to study transcriptional regulation of the virus in cells in which the early part of the viral life cycle is reproduced, such as C127 cells. Previous transient expression experiments have avoided the difficulties of poor transfection efficiency of C127 cells and low activity of BPV promoters by the use of other cell lines and subgenomic fragments of BPV linked to the CAT gene. A problem inherent in such approaches is that the activity of BPV promoters may be influenced by their genomic context. In addition, since BPV contains multiple promoters and a complex splicing pattern, it is not always possible to ascribe all CAT activity to a particular promoter. Lastly, primate cell lines such as CV-1 cells are not replication permissive for BPV (35), suggesting either that the host replication machinery is not compatible with BPV or that the El and E2 proteins are not expressed from the viral genome in a manner which can support viral DNA replication. We have recently found that a plasmid carrying the BPV origin will replicate in primate cell lines when cotransfected with El and E2 expression vectors (48). This indicates that BPV host range is restricted by limitations in gene expression rather than by a block to replication per se, and therefore that primate cells do not provide the proper system for the examination of replication-permissive BPV gene expression. For these reasons, we assayed the intact viral genome in C127 cells, in which BPV is capable of expressing the factors required for replication from its own genome. Our comparison of the abundance of messages expressed from wild-type BPV shows that the transcription pattern which exists in transient transfections is qualitatively similar to that observed in stably transformed cells. The subtle quantitative differences between the transiently transfected and stably transformed cells and the results of the E2 titration raise the possibility of a gradual change in transcriptional activity and suggest ways in which the BPV regulatory program might work. The P5 promoter expresses the most RNA in the wild-type transfection and also when the E2 mutant (pE2TTL) is assayed in the absence of the E2 expression vector. If this latter situation represents the "ground state" of the virus, it appears likely that the E2C repressor is the most abundant BPV protein expressed early after viral infection. Perhaps to establish latency and maintain a stable copy number, BPV must control its replication by beginning the infection with a low level of gene expression. This is in contrast to viruses such as simian virus 40, which express a transcriptional transactivator as an early protein and replicate in a runaway fashion. This high level of P5-specific messages in the short-term assays is consistent with the abundance of transcripts from this promoter in transformed cells (43, 51). The P5 promoter appears to express only the E2C protein. The P4 promoter expresses at least as much RNA as P5 in VI216 cells, but about 90% of the transcripts from P4 are spliced to encode the E5 protein (2, 51). This means that only about 10% of the P4-specific messages could encode full-length E2, which fits

5718

SZYMANSKI AND STENLUND

fairly well with the finding that the E2C protein is 10- to 20-fold more abundant than the E2 protein in transformed cells (15, 19). Although the level of the E2C repressor is presumably initially high upon viral infection, the P2 and P4 promoters have a low but significant basal activity. At least a portion of the messages that initiate at these promoters code for the E2 transactivator (12, 33, 41, 49). That some transactivator is indeed expressed early after infection is suggested by the higher basal activity of pMLBPV than of pE2TTL. Based on the E2 titration, the level of E2 activity in the wild-type transfection is roughly equivalent to that produced by about 100 to 200 ng of the E2 expression vector. Expression of this small amount of E2 early after infection would specifically activate the P2 and P4 promoters. Since the P5 promoter would not be concomitantly stimulated, synthesis of the E2C repressor would not keep pace with that of the E2 activator. This would result in a strong positive autoregulation of E2 transactivator expression. Autoregulation of E2 expression from the P4 promoter has also been suggested by Hermonat et al. (12). In response to the rising E2 activator levels, expression of the E5 protein from the P4 promoter (2, 11, 33, 41) would increase dramatically, resulting in cellular transformation. Production of the E6 protein, which is also involved in transformation and is believed to be expressed from the P2 promoter (17), would also be increased. A significantly lower level of messages is expressed from the P3 promoter in transformed cells than in the short-term assay, suggesting that P3 may be specifically downregulated during the course of the viral infection. It is unclear what the role of the P3 promoter might be, however, since P3 is the weakest promoter under any circumstance and appears to express both the E2 activator (49) and the E8/E2 repressor (4). Perhaps this promoter supplies some sort of back-up system, as suggested by the finding that an E8/E2 mutant is phenotypically normal, whereas mutations that inactivate E8/E2 and E2C have a more severe effect than a mutation that affects E2C only (20). The E2 titration, while informative, clearly represents only part of the BPV gene expression program. The situation in stably transformed cells or in an actual viral infection is more complicated, since the repressors are also present. Perhaps BPV promoters also display differential sensitivities to the repressors to arrive at the pattern of promoter activity that is seen in transformed cells. This could explain why the level of P3-specific messages relative to P2-specific messages is considerably lower in transformed cells than in the shortterm assay, even though the P2 and P3 promoters are equally sensitive to the E2 activator. It is important to note that the differential sensitivities of the promoters to the E2 transactivator are not due simply to distance effects or a gradient of enhancer activity, since P4 responds to a greater degree than P2 and P3, which are much closer to the E2 enhancer. Therefore, the question arises as to what does determine the degree of sensitivity to the E2 transactivator. It has been recently shown that transcription factor Spl is important for the basal activity of the P4 and P5 promoters (24, 41) and physically interacts with the E2 transactivator to mediate their induction (24). Perhaps E2 can also interact with other cellular factors so that the number, arrangement, and type of factors that are bound at a given BPV promoter determine how well it responds to E2. The effect of the binding site 15 Al and A5 mutations suggests that in addition to the Spl site, other sequence elements upstream from the P4 promoter may be involved in the response to E2. These mutations delete only the down-

J. VIROL.

stream end of site 15 and therefore do not affect the Spl site. It is possible that the deletions affect some other promoter element which overlaps the downstream end of site 15, resulting in a decrease in activity. The mutations might also bring binding sites for cellular factors closer together, causing steric hindrance. This could explain the more severe effect of the larger A5 deletion than of the Al deletion. The deletion analysis presented in Fig. 3 showed that the E2-dependent enhancer of the URR activates transcription from all promoters and that all the E2 binding sites contribute to the effect. Previous reports have stated that sites 7 to 10 (referred to as E2RE1) were sufficient for full induced activity of P2 and P4 (9, 41). In our system, however, the mutant containing these sites plus site 11 (234/11) retained only 15 to 30% of the wild-type activity. Similarly, we found that E2 binding sites 1 and 2 (referred to as E2RE2) (40) were no more important than any other pair of binding sites. The URR also contains several basal transcription elements. The 36/221 deletion defines an element specific for P2 promoter activity. Basal elements that regulate all promoters are located between nt 6958 and 7388, a region that contains the constitutive enhancers described by two other groups (38, 50). The first group defined an enhancer within nt 7215 to 7351 that weakly activated the simian virus 40 promoter in C127 cells (38). The second group defined an enhancer within nt 7162 to 7275 that strongly activated transcription from the simian virus 40 early promoter but not from P2 in primary bovine fibroblasts (50). Since the former enhancer does not contain the minimal functional element of the latter enhancer, the two were thought to be different (50). The effect of the 234/Sca deletion (nt 7187 to 7389) is more in line with weak effects of the nt 7215 to 7351 enhancer than the strong effects of the nt 7162 to 7275 enhancer. This could be due to cell type specificity, since the latter enhancer displayed weak activity in C127 cells (50). It is of interest to determine how the progression of the viral life cycle is regulated from infection by a single virion to stable maintenance of about 100 copies of the viral DNA per cell. Based on our results, changes in BPV gene expression during the establishment of stable copy number are most likely quantitative and subtle. In addition to transcriptional control, regulation of RNA splicing, transport, and stability, translational control, and posttranslational modifications may also play a role in the BPV life cycle. We find it remarkable that BPV can establish and maintain itself in cultured cells with only subtle alterations in its gene expression pattern. The findings that El and E2 are required for short-term BPV replication (46) and that E2 is continuously required for viral DNA replication in stably transformed cells (5) suggest that viral gene expression controls DNA replication under all circumstances. Therefore, it seems probable that maintenance of BPV as a multicopy episome over numerous generations requires a finely tuned regulatory system. It has been shown that mutation of one form of E2 affects the level of the other forms and that E2 repressor mutants display a high-copy-number phenotype (19, 20, 34). These findings, together with our results that all promoters respond to the E2 activator through the URR and that no other BPV gene products are involved, suggest that viral homeostasis could be maintained by a delicate balance between the E2 activator and repressor proteins. ACKNOWLEDGMENTS We thank Mart Ustav for helpful discussions; Masafumi Tanaka and Rong Li for gifts of plasmids; Mart Ustav, Jacek Skowronski, and Masafumi Tanaka for critical review of the manuscript; and

VOL. 65, 1991

Rong Li and Michael Botchan for communication of results prior to publication. This work was supported by Public Health Service grant CA 13106-19. P.S. was supported by an NIH postdoctoral fellowship. REFERENCES 1. Androphy, E. J., D. R. Lowy, and J. T. Schiller. 1987. Bovine papillomavirus E2 trans-activating gene product binds to specific sites in papillomavirus DNA. Nature (London) 325:70-73. 2. Baker, C. C., and P. M. Howley. 1987. Differential promoter utilization by the bovine papillomavirus in transformed cells and productively infected wart tissue. EMBO J. 6:1027-1035. 3. Berg, L., M. Lusky, A. Stenlund, and M. R. Botchan. 1986. Repression of bovine papilloma virus replication is mediated by a virally encoded trans-acting factor. Cell 46:753-762. 4. Choe, J., P. Vaillancourt, A. Stenlund, and M. Botchan. 1989. Bovine papillomavirus type 1 encodes two forms of a transcriptional repressor: structural and functional analysis of new viral cDNAs. J. Virol. 63:1743-1755. 5. DiMaio, D., and J. Settleman. 1988. Bovine papillomavirus mutant temperature sensitive for transformation, replication, and transactivation. EMBO J. 4:1197-1204. 6. Dostatni, N., F. Thierry, and M. Yaniv. 1988. A dimer of BPV-1 E2 protein containing a protease resistant core interacts with its DNA target. EMBO J. 7:3807-3816. 7. Dvoretzky, I., R. Shober, S. K. Chattopadhyay, and D. R. Lowy. 1980. A quantitative in vitro focus assay for bovine papillomavirus. Virology 103:369-375. 8. Harrison, S. M., K. L. Gearing, S.-Y. Kim, A. J. Kingsman, and S. M. Kingsman. 1987. Multiple cis-active elements in the long control region of bovine papillomavirus type 1 (BPV-1). Nucleic Acids Res. 15:10267-10284. 9. Haugen, T. H., T. P. Cripe, G. D. Ginder, M. Karin, and L. P. Turek. 1987. Trans-activation of an upstream early gene promoter of bovine papillomavirus-1 by a product of the viral E2 gene. EMBO J. 6:145-152. 10. Hawley-Nelson, P., E. J. Androphy, D. R. Lowy, and J. T. Schiller. 1988. The specific DNA recognition sequence of the bovine papillomavirus E2 protein is an E2-dependent enhancer. EMBO J. 7:525-531. 11. Hermonat, P. L., and P. M. Howley. 1987. Mutational analysis of the 3' open reading frames and the splice junction at nucleotide 3225 of bovine papillomavirus type 1. J. Virol. 61:38893895. 12. Hermonat, P. L., B. A. Spalholz, and P. M. Howley. 1988. The bovine papillomavirus P2443 promoter is E2 trans-responsive: evidence for E2 autoregulation. EMBO J. 7:2815-2822. 13. Herr, W., and Y. Gluzman. 1985. Duplications of a mutated simian virus 40 enhancer restore its activity. Nature (London) 313:711-714. 14. Howley, P. M., and R. Schlegel. 1987. Papillomavirus transformation, p. 141-163. In N. P. Salzman and P. M. Howley (ed.), The papovaviridae, vol. 2: the papillomaviruses. Plenum Publishing Corp., New York. 15. Hubbert, N. L., J. T. Schilier, D. R. Lowy, and E. J. Androphy. 1988. Bovine papilloma virus-transformed cells contain multiple E2 proteins. Proc. Natl. Acad. Sci. USA 85:5864-5868. 16. Knight, J., R. Li, and M. R. Botchan. 1991. The activation domain of the bovine papillomavirus E2 protein mediates association of DNA-bound dimers to form DNA loops. Proc. Natl. Acad. Sci. USA 88:3204-3208. 17. Lambert, P. F., C. C. Baker, and P. M. Howley. 1988. The genetics of bovine papillomavirus type 1. Annu. Rev. Genet. 22:235-258. 18. Lambert, P. F., and P. M. Howley. 1988. Bovine papillomavirus type 1 El replication-defective mutants are altered in their transcriptional regulation. J. Virol. 62:4009-4015. 19. Lambert, P. F., N. L. Hubbert, P. M. Howley, and J. T. Schiller. 1989. Genetic assignment of multiple E2 gene products in bovine papillomavirus-transformed cells. J. Virol. 63:31513154. 20. Lambert, P. F., B. C. Monk, and P. M. Howley. 1990. Phenotypic analysis of bovine papillomavirus type 1 E2 repressor

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mutants. J. Virol. 64:950-956. 21. Lambert, P. F., B. A. Spalholz, and P. M. Howley. 1987. A transcriptional repressor encoded by BPV-1 shares a common carboxy-terminal domain with the E2 transactivator. Cell 50:6978. 22. Law, M.-F., D. R. Lowy, I. Dvoretzky, and P. M. Howley. 1981. Mouse cells transformed by bovine papillomavirus contain only extrachromosomal viral DNA sequences. Proc. Natl. Acad. Sci. USA 78:2727-2731. 23. Li, R., J. Knight, G. Bream, A. Stenlund, and M. Botchan. 1989. Specific recognition nucleotides and their DNA context determine the affinity of E2 protein for 17 binding sites in the BPV-1 genome. Genes Dev. 3:510-526. 24. Li, R., J. D. Knight, S. P. Jackson, R. Tjian, and M. R. Botchan. 1991. Direct interaction between Spl and the BPV enhancer E2 protein mediates synergistic activation of transcription. Cell 65:493-505. 25. Lusky, M., and M. R. Botchan. 1985. Genetic analysis of bovine papillomavirus type 1 trans-acting replication factors. J. Virol. 53:955-965. 26. Lusky, M., and M. R. Botchan. 1986. Transient replication of bovine papillomavirus type 1 plasmids: cis and trans requirements. Proc. Natl. Acad. Sci. USA 83:3609-3613. 27. McBride, A. A., R. Schlegel, and P. M. Howley. 1989. E2 polypeptides encoded by bovine papillomavirus 1 form dimers through the carboxy-terminal DNA-binding domain: transactivation is mediated through the amino-terminal domain. Proc. Natl. Acad. Sci. USA 86:510-514. 28. Melton, D. A., P. A. Krieg, M. R. Rebagliati, T. Maniatis, K. Zinn, and M. R. Green. 1989. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12:7035-7056. 29. Monini, P., S. R. Grossman, B. Pepinsky, E. J. Androphy, and L. A. Laimins. 1991. Cooperative binding of the E2 protein of bovine papillomavirus to adjacent E2-responsive sequences. J. Virol. 65:2124-2130. 30. Moskaluk, C., and D. Bastia. 1987. The E2 "gene" of bovine papillomavirus encodes an enhancer-binding protein. Proc. Natl. Acad. Sci. USA 84:1215-1218. 31. Moskaluk, C., and D. Bastia. 1988. Interaction of the bovine papillomavirus type 1 transcriptional control protein with the viral enhancer: purification of the DNA-binding domain and analysis of its contact points with DNA. J. Virol. 62:1925-1931. 32. Neary, K., and D. DiMaio. 1989. Open reading frames E6 and E7 of bovine papillomavirus type 1 are both required for full transformation of mouse C127 cells. J. Virol. 63:259-266. 33. Prakash, S. S., B. H. Horwitz, T. Zibello, J. Settleman, and D. DiMaio. 1988. Bovine papillomavirus E2 gene regulates expression of the viral E5 transforming gene. J. Virol. 62:3608-3613. 34. Riese, D. J., II, J. Settleman, K. Neary, and D. DiMaio. 1990. Bovine papillomavirus E2 repressor mutant displays a highcopy phenotype and enhanced transforming activity. J. Virol. 64:944-949. 35. Roberts, J., and H. Weintraub. 1986. Negative control of DNA replication in composite SV40-bovine papillomavirus plasmids. Cell 46:741-752. 36. Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 37. Schiller, J. T., E. Kleiner, E. J. Androphy, D. R. Lowy, and H. Pfister. 1989. Identification of bovine papillomavirus El mutants with increased transforming and transcriptional activity. J. Virol. 63:1775-1782. 38. Sowden, M., S. Harrison, R. Ashfield, A. J. Kingsman, and S. M. Kingsman. 1989. Multiple cooperative interactions constrain BPV-1 E2 dependent activation of transcription. Nucleic Acids Res. 17:2959-2972. 39. Spalholz, B. A., J. C. Byrne, and P. M. Howley. 1988. Evidence for cooperativity between E2 binding sites in E2 trans-regulation of bovine papillomavirus type 1. J. Virol. 62:3143-3150. 40. Spalholz, B. A., P. F. Lambert, C. L. Yee, and P. M. Howley. 1987. Bovine papillomavirus transcriptional regulation: localiza-

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42. 43. 44. 45.

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