Vol. 64, No. 2

JOURNAL OF VIROLOGY, Feb. 1990, p. 950-956

0022-538X/90/020950-07$02.00/0 Copyright C) 1990, American Society for Microbiology

Phenotypic Analysis of Bovine Papillomavirus Type 1 E2 Repressor Mutants PAUL F. LAMBERT,t* BRAD C. MONK, AND PETER M. HOWLEY Laboratory of Tumor Virus Biology, National Cancer Institute, Bethesda, Maryland 20892 Received 1 August 1989/Accepted 17 October 1989

The bovine papillomavirus type 1 (BPV-1) E2 open reading frame encodes three proteins: the E2 transcriptional transactivator, the E2 transcriptional repressor (E2-TR), and the E8/E2 fusion peptide. In this study, we describe the phenotypes of BPV-1 mutants which are disrupted in their capacity to encode either the E2 transcriptional repressor or the E8/E2 fusion peptide. We also describe experiments which demonstrate that the E8/E2 gene product functions similarly to E2-TR. In the context of the entire viral genome, disruption of E8/E2 expression had little effect on viral processes, whereas disruption of E2-TR expression resulted in a complex phenotype marked by a 10- to 20-fold increase in viral DNA plasmid copy number as well as increased transformation potential. A double mutant, defective in the expression of both E8/E2 and E2-TR proteins, had high levels of E2 transactivation activity yet had reduced plasmid replication capacity and a delayed capacity to transform rodent cells.

Bovine papillomavirus type 1 (BPV-1) transformation of mouse cells has served as a model for genetic studies of the papillomaviruses (19a). The viral genome persists as a multicopy DNA plasmid in BPV-1-transformed mouse cells, and a subset of the viral genes, the early genes, are expressed at low levels (14, 26). Viral gene expression appears to be at least partially under the control of the viral E2 transcriptional regulatory circuit, which is comprised of the E2 transactivator, the E2 repressor, the E2 transcriptional repressor (E2-TR), and the E8/E2 fusion protein (5, 22, 42, 48; this study). All three proteins are expressed in BPV-1transformed cells (18, 21). The E2 transactivator, the product of the full-length E2 open reading frame (ORF), activates viral transcription through E2-responsive elements (10, 11, 13, 41), which are cis-acting enhancer elements located predominantly within the long control region (LCR) of the viral genome. The E2 transactivator binds to a partial palindromic sequence (1, 31), multiple copies of which are located within the E2-responsive elements (10, 13, 40, 41) and elsewhere in the viral genome (27). The E2 repressor was shown to be encoded by the 3' half of the same ORF, and its translation was predicted to initiate at an internal ATG codon (22) which is located just downstream of the P3080 promoter (2). A second E2 gene product that was truncated at the N terminus was previously predicted (15, 23) to be the translation product of a viral RNA spliced from nucleotide (nt) 1234 to nt 3225 (5, 44) which fuses the upstream E8 ORF to the 3' end of the E2 ORF. Both the E2 repressor and the E8/E2 fusion protein contain the DNAbinding domain of the E2 transactivator, which maps to the C-terminal 100 amino acids of the E2 ORF (30), and all three E2 proteins share the capacity to dimerize with themselves and each other (7, 29). Both competitive DNA binding and subunit mixing have been proposed as potential mechanisms by which the repressor molecules inhibit E2 transactivation. The purpose of this study was to identify the transcriptional regulatory activity of the E8/E2 gene product and to char-

acterize the biological consequences of disrupting the transcriptional repressor activities encoded by BPV-1. An E8/E2 cDNA was constructed (Fig. 1A) and tested for its ability to function as a repressor in the two assays originally utilized in the identification of E2 repressor activity: inhibition of BPV-1 transformation and repression of E2 transactivation (22). In both assays, the E8/E2 cDNA was functionally identical to E2-TR. Cotransfection of the plasmid pE8/E2 with an equal amount of the full-length BPV-1 plasmid p142-6 resulted in a greater-than-10-fold decrease in the number of transformed foci of mouse C127 cells (Fig. 1B). In transient cotransfections of monkey CV-1 cells, the E8/E2 cDNA plasmid repressed the ability of the E2 transactivator to enhance chloramphenicol acetyltransferase (CAT) expression from the reporter plasmid by 5- to 10-fold (Fig. 1C). Not surprisingly, the E8/E2 gene product did not function as an E2 transactivator (data not shown), since it does not contain the N-terminal E2 domain which is required for transactivation (8, 12, 29). Thus, the E8/E2 gene encodes a transcriptional repressor which is functionally indistinguishable from the previously identified E2 repressor E2-TR. In agreement with our data, Choe et al. (5) have isolated a partial cDNA from BPV-1-transformed C127 cells encoding the E8/E2 gene and have also concluded that the E8/E2 gene functions to inhibit E2 transcriptional activation. To examine the role of the repressor genes E2-TR and E8/E2 in the transcriptional regulation of BPV-1, base substitution mutations predicted to disrupt these genes were engineered into p142-6, a plasmid containing the full-length viral genome cloned in the bacterial vector pML2D (36). To disrupt the E2-TR gene, a missense mutation was generated altering T to C at nt 3092, giving rise to the plasmid p1472-1 (Fig. 2). Multiple synthetic oligonucleotides were utilized to generate a double-stranded DNA fragment with the BPV-1 sequence from the FspI site at nt 3023 to the HgaI site at nt 3113 and containing the T-to-C base substitution at nt 3092. This DNA fragment was then cloned into p142-6 cut at the same restriction sites, FspI and HgaI, by partial digestion to give the clone p1472-1. The T-to-C base substitution in p1472-1 caused a Met-to-Thr amino acid change in the E2 ORF. When engineered into the E2-TR cDNA p1153 (Fig. 2), this mutation eliminated the ability of this cDNA to

* Corresponding author. t Present address: Department of Oncology, McArdle Laboratory

for Cancer Research, University of Wisconsin-Madison, 450 N. Randall Ave., Madison, WI 53706.

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5'-CCGGGTTGCTGAAAATGAAGCTAACCGTGTTCTTACGCCCCTCCAGAGATCGCCCAGACG-3'. This sequence corresponds to BPV-1 nt 1193 to 1234 and is contiguous with nt 3225 to 3238. The sequence of pE8/E2 was confirmed by direct sequence analysis. (B) Inhibition of BPV-1 transformation. Subconfluent C127 cells in 6-cm dishes were transfected by the calcium phosphate precipitation protocol, as previously described (22), with 1 ,ug of the wild-type BPV-1 plasmid p142-6 with or without 1 pg of pE8/E2 plasmid DNA. Plates were stained with methylene blue 2 weeks posttransfection. Darkly stained areas are transformed foci. (C) Thin-layer chromatography of acetylated (fast-migrating forms at top) and unacetylated (origin at bottom) forms of 14C-chloramphenicol taken from reactions in which the substrate was incubated for 30 min with 50 pug of protein extracted from CV-1 cells transiently transfected, as previously described (22), with the indicated amounts of the following plasmids: p407-1, 5 ,ug; pC59, 1 ,ug; pE8/E2, 3 ,ug; p1151 (cDNA-vector control plasmid [22]), 3 ,ug. repress E2 transactivation (data not shown), in agreement with the finding that this ATG codon is utilized for translation initiation of E2-TR (21). The capacity for this mutation to disrupt E2-TR expression is further described in the accompanying paper by Riese et al. (35). To disrupt the E8/E2 gene, a G-to-A base substitution was engineered at nt 1235, giving rise to the plasmid p1471-1 (Fig. 2). This mutation was generated by the protocol of Kunkel (19), using a single-stranded recombinant M13mpl8 clone containing the SmaI (nt 945)-to-EcoRI (nt 2113) BPV-1 subgenomic fragment and the antisense oligonucleotide primer with the sequence 5'-CTCCAGATACAGG-3'. The sequenced clone mutated at nt 1235 was used to reconstruct the full-length mutant clone p1471-1 by exchanging the XmaI (nt 943)-to-BspMII (nt 1582) fragment of the mutated M13 recombinant into p142-6. The G-to-A base substitution at nt 1235 is a silent mutation in the E8 ORF and causes a Val-to-Ile amino acid change in the El ORF. This mutation altered the 5' splice sequence from 5'-AGGT-3' to 5'AGAT-3'. The capacity of this mutation to disrupt utilization of the nt 1234 splice site was confirmed by analysis of RNA isolated from cells transfected by p1471-1 (data not shown). A double repressor mutant, p1474-1, was also generated by introducing both of the individual mutations into the fulllength BPV-1 genome (Fig. 2); the EcoRI (nt 2113)-to-KpnI (nt 3455) fragment from p1472-1 was exchanged into p1471-1. Hubbert et al. (18) identified the presence of three E2 peptides in BPV-1-transformed rodent cells, with sizes of 48, 31, and 28 kilodaltons (kDa). The E2-TR (in pl472-1) and E8/E2 (pl471-1) mutant plasmids were used in identifying

the E2 genes responsible for encoding the three E2 immunospecific peptides (21); the most abundant 31-kDa E2specific protein maps to the E2-TR gene; the less abundant 28-kDa E2-specific protein maps to the E8/E2 gene; and the largest and least abundant 48-kDa E2-specific protein maps to the E2 transactivator gene. The analysis by Lambert et al. (21) confirmed the genetic identity of the E2 immunospecific peptides and provided proof that, in addition to the E2 transactivator, both the E8/E2 and E2 repressor genes are expressed in BPV-1-transformed cells. Furthermore, it established that the repressor gene products were in greater abundance than the transactivator and that E2-TR was the predominant repressor protein (Table 1). The disruption of the E2 repressor genes was also shown (21) to alter the relative abundance of E2 transactivator to E2 repressor gene products in cells harboring the repressor mutants p1471-1, p1472-1, and p1474-1 (Table 1). We were therefore interested in measuring the transactivation potential of these mutants. Levels of E2 transactivation were measured by using the reporter plasmid p407-1, which contains the BPV-1 LCR as an enhancer cloned upstream of the simian virus 40 early promoter. Expression of the CAT gene in this plasmid is dependent upon the presence of E2 transactivator activity (42) and is proportional to the relative levels of the E2 transactivator and repressor proteins (22). CAT activity was measured in monkey CV-1 cells cotransfected with the reporter plasmid and either mutant or wildtype BPV-1 plasmids (Table 1). Disruption of the E8/E2 gene (in p1471-1) led to no observable change in levels of E2 transactivation compared with the level of wild-type BPV-1,

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pE8/E2 FIG. 2. Location of point mutations on the full-length BPV-1 genomic map and transformation potential of wild-type and mutant genomes. At the top is a map of the BPV-1 circular genome linearized at nt 7000 for ease of presentation. The numbered boxes indicate the locations of both the early and late (Li and L2) ORFs. The position and identity of the viral transcriptional promoters are given by the horizontal arrows at right angles. The stipled box indicates the position of E2-responsive element 1 within the LCR. The location of the bacterial plasmid vector pML2D sequence at the unique BamHI site is indicated (36). The vertical arrows indicate the position of nt 1234 and 3225 splice signals. The nucleotide sequences surrounding nt 1235 and 3092 are given below the line drawing, and arrows iadicate the base change introduced by the respective mutations at each site; Horizontal brackets indicate the codons in the E8 and E2 ORFs. Below the nucleotide sequences are the line drawings for the wild-type (p142-6) and the mutant plasmids, with solid boxes indicating the presence of point mutations. Transformation assay data for each plasmid are given to the right of each line drawing. The left-hand column presents the number of foci generated on a 6-cm dish of subconfluent C127 cells transfected as previously described (22) with 1 p.g each of the BPV-1 plasmids and stained at 2 weeks with methylene blue. The right-hand column presents the number of soft-agar colonies generated per microgram of plasmid DNA transfected into C127 cells, as previously described (20). Briefly, 6-cm dishes of subconfluent C127 cells were transfected, and 48 h later the cells were trypsinized, suspended in 0.3% agar, and fed for 4 weeks, at which time the number of colonies was determined. At the bottom are indicated the BPV-1 coding regions of pC59 (48), which encodes the E2 transactivator; p1153 (22), which encodes E2-TR; and pE8/E2, which is described in Fig. 1A.

measured by CAT activity. A 10-fold increase in CAT activity, however, was observed with the ATG missense mutant p1472-1, in which E2-TR is disrupted. A 30-fold increase in CAT activity was observed with the double mutant p1474-1. The transactivation potential of the mutants, therefore, appears to correlate with their coding capacity; the double mutant, which expresses only the transactivator, had the greatest capacity to activate the E2-responsive promoter. To further examine the biological effects of these mutations, mouse C127 cells were transfected with the mutant or wild-type BPV-1 genomes and analyzed for viral transcription, viral DNA replication, and cellular transformation. Levels of viral transcription and DNA replication were measured in pooled G418-resistant cell populations. These cell lines were derived by cotransfection of each viral plasmid with the plasmid pMMTneo, which encodes the

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bacterial neomycin resistance gene transcribed from the mouse metallothionein promoter (25). Cellular transformation was measured by focus formation and by soft-agar colony formation of transfected C127 cells as well as by analysis of pooled G418-resistant, cotransfected cell populations. Cellular transformation by BPV-1 involves the direct action of two independent viral genes, the ES and the E6 oncogenes (38, 47), and possibly the E7 gene (32). The E2 genes are believed to indirectly affect the transforming capacity of BPV-1 through their modulation of viral promoters which direct the expression of these oncogenes, primarily the E2-responsive promoters: P89 (11, 41) and P2443 (16, 33). A recent study (4), though, has proposed a direct involvement of the E2 genes in the immortalization of primary rodent fibroblasts. To test the transforming capacity of the E2 repressor mutants, mouse C127 cells were trans-

VOL. 64, 1990

NOTES

TABLE 1. Relative abundance of E2 proteins and levels of transactivation in BPV-1 repressor mutants Level of E2 Protein with molecular mass (kDa)a:

BPV-1 plasmid DNA

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a Relative abundance of E2 proteins present in C127 cells harboring the indicated BPV-1 plasmid DNAs. The abundance of the 48-kDa E2 species present in wild-type-transfected cells was arbitrarily set at 1.0. The data were taken from Lambert et al. (21) with the exception of that for p1474-1 (P. Lambert, N. Hubbert, J. Schiller and P. Howley, unpublished data). b Relative rates of chloramphenicol acetylation for extracts of CV-1 cells transiently transfected with 5 ,ug of p407-1 and 3 ,g of the indicated BPV-1 plasmid. The rate of acetylation in p142-6-transfected cells was arbitrarily set at 1.0. All rates were corrected for background CAT activity in cells transfected with p407-1 only.

fected with the wild-type or the repressor mutant BPV-1 DNAs, and the efficiency of focus formation or the efficiency of colony formation in soft agar was measured (Fig. 2). The plasmid p1471-1 was similar to wild-type (p142-6) BPV-1 in its ability to induce formation of foci and soft-agar colonies. The plasmid p1472-1 displayed a reproducible twofold increase in its efficiency of focus formation (Fig. 2). The foci observed with p1472-1 appeared sooner and grew larger than those resulting from wild-type BPV-1 transfection; cell populations derived from individual cloned p1472-1 foci grew to higher cell densities and characteristically turned the media acidic more quickly than did wild-type BPV-1-transformed cells (data not shown). In the soft-agar assay, p1472-1 gave rise to at least 20-fold more colonies, which appeared within 7 days posttransfection, than did p142-6 (wild-type BPV-1), whose colonies appeared in 14 to 18 days. In addition, p1472-1 colonies grew to larger diameters than did colonies appearing in wild-type-transfected cells. The double repressor mutant (pl474-1) was impaired in its transformation capacity in each of these transformation assays (Fig. 2). Some foci were observed in a modified focus assay, however, in which the cells were trypsinized 48 h posttransfection and plated onto two 6-cm dishes. The time of appearance of these foci was significantly delayed, and the efficiency of focus induction was less than 5% that observed with the wild-type plasmid in this assay. Experiments were performed to determine whether the double mutant was

cytotoxic, given the absence of transformation in the standard transformation assays. The double mutant was cotransfected with several different plasmids which provided dominant selection, such as G418 resistance by using pMMTneo and BPV-1-independent transformation by using pEJRAS and pHARAS, which encode activated forms of the RAS oncogene. There was no significant decrease in the efficiency of selection for either of these dominant markers (data not shown), indicating that the double repressor mutant was not cytotoxic. The cells generated by cotransfection of the neomycin resistance gene plasmid pMMTneo with each of the BPV-1 plasmids were analyzed for their capacity to grow as colonies in soft agar after continued cell passage (Fig. 3 [experiment performed at cell passage 17]). Cells harboring the E8/E2 mutant p1471-1 exhibited a similar capacity to grow as colonies in soft agar as did the wild-type BPV-1 transformants, whereas cells harboring the E2-TR mutant p1472-1 grew more aggressively in soft agar, with approximately 59c of the input cells forming colonies as compared with less than 0.5% for the wild-type BPV-1 transformants. Cells harboring the double mutant p1474-1 acquired a transformed phenotype after continued passage, as clearly evidenced by their capacity to efficiently form large colonies in soft agar. To determine whether the increased transformation capacity seen with the double mutant in expanded populations of G418-resistant cells was reflective of levels of viral oncoproteins, E5 immunoprecipitations were performed (Fig. 4A). Cells harboring the E8/E2 repressor mutant p1471-1 had E5 levels which were comparable to those of cells harboring the wild-type BPV-1 plasmid p142-6. Cells harboring the E2-TR mutant p1472-1 had approximately 10-fold-higher levels of E5 compared with the levels in wild-type BPV-1-transfected cells. This corresponded both to the highly transformed phenotype of these cells (Fig. 2 and 3) and to elevated expression of an E5-CAT fusion gene (35). Cells harboring the double mutant p1474-1 had E5 levels which were up to threefold higher than those of the wild type. This high level of E5 expression was reproducible in different populations of cells harboring p1474-1, including cell lines at an earlier passage (passage 5; data not shown). Levels of viral transcription were measured by Northern analysis (Fig. 4B) of total cellular RNA extracted from the G418-resistant C127 cell populations. RNA was extracted from early (passage 5) and late (passage 17) passage cells. The small difference in the hybridization pattern at passage 17 likely resulted from slight RNA degradation in these samples, which was reflected also in hybridization to the control ,-actin probe (data not shown). The levels of viral "I

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FIG. 4. Levels of E5 protein, total viral RNA, and DNA copy number in pooled G418-resistant cell populations. Cells used in these assays described in the legend to Fig. 3. (A) Cells (at passage 17) harboring the indicated BPV-1 plasmid were metabolically labeled with [35S]methionine for 3 h, and E5 was immunoprecipitated as previously described (39 [with modifications by Goldstein and Schlegel, manuscript in preparation]). The arrow indicates the position of the E5 peptide. Molecular mass markers are shown on the left. The control (c) cell line was pooled G418-resistant colonies of C127 cells transfected with pMMtneo only. (B) Northern analysis of total cellular RNA from pooled G418-resistant colonies harboring the indicated BPV-1 plasmids extracted at the indicated passage. Total cellular RNA (2.5 ,ug) was electrophoresed on a 1.4% agarose gel, transferred to a nylon membrane (GeneScreen), and hybridized according to the protocol of the manufacturer by using 32P-labeled BPV-1 DNA as the probe. Mobilities of RNA standards are indicated at left (Kb, kilobases). Control (c) RNA was from the same cells as in panel A. (C) Southern analysis of total cellular DNA extracted from pooled G418-resistant colonies harboring the indicated BPV-1 plasmids extracted at passage 17. DNA (2.5 ,ug) was sheared by passage through a 22-gauge needle five times, electrophoresed on a 0.7% agarose gel, and transferred to nitrocellulose paper. Hybridization was performed by using 32P-labeled BPV-1 DNA as the probe. The first lane contained 25 pg of p142-6 plasmid. The positions of form I and form II DNAs are indicated at left. Also included are DNA samples obtained from cells harboring either of the replication-defective BPV-1 genomes (p745-1 and p743-23) and the BPV-1 mutant altered at the 5' splice site at nt 3225 (plO39-1) (15, 34). Control (c) DNA was obtained from same control cells as in panel A.

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RNA in the cells harboring p1471-1 were equal to or less than twofold higher than those in the cells harboring the wild-type viral genome p142-6. The level of virus-specific RNA in cells harboring p1472-1 was twofold higher than that of the wild type at passage 5 but increased to eightfold higher at passage 17, which is the same passage used for the analysis of colony formation in soft agar (Fig. 3), E5 expression (Fig. 4A), and viral DNA content (Fig. 4C). The level of viral RNA seen in the double mutant p1474-1-transfected cells was at least fivefold lower than that in the wild-type-transfected cells even at the later passage, when aggressive growth in soft agar and high levels of E5 expression were measured. The BPV-1 double-stranded circular genome persists as a multicopy plasmid in virally transformed rodent cells, which may correspond to the viral replicative state in the basal epithelial cells and the dermal fibroblasts of a fibropapilloma (26). The virus encodes both cis and trans genetic elements required for plasmid replication (19a). The E2 transcriptional regulatory circuit is involved in the maintenance of the viral plasmid state, since disruption of the E2 transactivator gene product leads to the loss of plasmid replication (6, 9, 34). The E2-TR and E8/E2 mutations were therefore predicted to affect the viral plasmid DNA replicative capacity. To examine the state of the viral DNA, wild-type and mutant viral plasmids were cotransfected with plasmid pMMTneo and drug-resistant colonies were cloned or pooled; total genomic DNA was isolated and probed for BPV-1-specific DNA (Fig. 3C). Cells harboring the mutant p1471-1, which was disrupted in the expression of E8/E2, had a plasmid DNA copy number similar to that of wild-type transformant cells,

ranging from 50 to 100 copies per cell in the different pooled populations analyzed. Cells containing the E2-TR mutant p1472-1 had a plasmid copy number of 500 to 1,000, which was 10- to 20-fold higher than that of wild-type transformants. Significant rearrangement of the input p1472-1 DNA was evidenced by heterogeneity of the form I DNA. These rearrangements appeared to be confined principally to the bacterial plasmid vector sequences, since restriction digestion with BamHI, which releases the viral DNA insert, gave rise predominantly to full-length BPV-1 genomic fragments (data not shown). The high-copy-number phenotype of p1472-1 was also observed when cells were selected directly for their transformed phenotype by cloning foci (data not shown). The double mutant p1473-1 was also replication competent but had a reduced copy number of 5 to 25, in the range of 10 to 30% that of the wild-type viral plasmid. The low plasmid copy number and the lack of cytotoxicity of this double mutant indicate that this is not a runaway replicon. In this study, the E8/E2 gene product is shown to be an E2 transcriptional repressor which, like E2-TR, inhibits the activity of the E2 transactivator. Disruption of the E8/E2 gene by a mutation at the nt 1234 5' splice signal in the viral genome had no observable effect on E2 transactivation capacity, viral transcription, viral replication, or levels of virally induced cellular transformation. Perhaps this is reflective of the fact that E8/E2 protein is in low abundance compared with the alternate repressor E2-TR (20). The absence of a phenotype for p1471-1 is of added interest because the point mutation of the splice signal at nt 1235 was predicted to affect the expression of at least one other viral

VOL. 64, 1990

gene, E1-M, which is apparently required for stable plasmid replication (3, 28). The El-M gene product is a 23-kDa phosphoprotein encoded by a spliced message which utilizes the nt 1234 5' splice signal (46). Yet the mutant p1471-1, which is defective in its utilization of the nt 1234 5' splice signal, is replication competent. M. Botchan (unpublished observation) has observed that the 23-kDa El phosphoprotein is replaced by an apparent full-length El ORF phosphoprotein in cells harboring an identical nt 1235 point mutation mutant. It is possible that this larger El protein retains the El-M activity, permitting the nt 1235 mutant to remain replication competent. Disruption of the major E2 repressor gene E2-TR had a pleiotropic effect on viral processes, including increases in E2 transactivation activity, viral RNA, viral plasmid copy number, transforming potential, and abundance of the viral E5 oncoprotein. Increases in these processes were predicted, given that there was a large change in the relative abundance of the transactivator to repressor proteins resulting from the lack of E2-TR expression in this mutant (21). This change in relative abundance was significant, despite an increased level of E8/E2 expression by this mutant (Table 1). A similar study was performed on an identical ATG missense mutant at nt 3092 and arrived at a similar conclusion that disruption of E2-TR resulted in an increase in the potential of the virus to transactivate, replicate, and transform (35). It will be of interest to understand the consequences of disrupting E2-TR expression in dermal fibroblasts and in basal epithelial cells of a bovine fibropapilloma. The behavior of the double repressor mutant, disrupted in the expression of both E2-TR and E8/E2, is complex and enigmatic. As predicted, transactivation activity was 30-fold higher than that of the wild-type viral genome in CV-1 cells (Table 1). Yet the steady state level of viral RNA in stably transfected C127 cells was 5- to 10-fold lower than that of the wild-type virus; correspondingly, the mutant viral plasmid copy number was low, and there was a delay in the observance of a transformed phenotype. One possible explanation for the delayed onset of transformation is that, in the absence of E2-TR and E8/E2 expression, there is a perturbation in the temporal sequence of events occurring subsequent to the introduction of the viral genome into the recipient cell. This could cause a delay in the time needed to obtain the threshold level of viral oncoprotein expression sufficient for cellular transformation. In the context of this argument, coselection of transfected cells by using G418 could provide the necessary expansion of recipient cells, allowing them to outgrow the nonrecipient cells, which are thought to inhibit cellular proliferation (24). It remains to be understood why the absence of both E2 repressors, E2-TR and E8/E2, would retard the transformation process whereas the absence of only the major repressor, E2-TR, causes a more predictable increase in viral processes. The complex phenotype of the double mutant raises the possibility that either E8/E2 or E2-TR encodes functions in addition to the repression of E2 transactivation and that the loss of such functions is phenotypically manifested only in the complete absence of repressor activity. It is also possible that the behavior of the double mutant supports the proposal that the full-length BPV-1 E2 gene product, the E2 transactivator, can under certain circumstances repress BPV-1 transcription, as described previously for the BPV-1 P7185 promoter (43) and, similar to the capacity of the full-length E2 gene product, can repress the HPV-18 P97 promoter (45). Alternatively, it is possible that BPV-1 encodes additional transcriptional regulators which

NOTES

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might override the E2 transcriptional circuit under certain conditions, as had been proposed in the context of DNA replication-defective BPV-1 mutants disrupted in the El ORF (20, 37). This study provides evidence that BPV-1 utilizes two mechanisms to express E2 repressor activities and that these

repressor activities play important roles in modulating viral processes. It is curious that BPV-1 encodes two repressor genes. There are multiple stages in the life cycle of the papillomavirus. Perhaps E2-TR and E8/E2 play different roles in this temporal process. E2-TR also contains more of the E2 coding region than does E8/E2, raising the possibility that the two proteins may have different activities which have eluded detection with our current assays. Irrespective, the data generated in this study lead to the conclusion that the high excess in the levels of the E2 repressor gene products over the E2 transactivator gene product in mouse cells transformed by wild-type BPV-1 is required to establish and maintain the tight regulation of viral processes in these cells. The coding potential for multiple E2 gene products in some human papillomaviruses (17) suggests that this may provide a general mechanism for regulating the transactivation potential of the papillomaviruses. We acknowledge the expert technical assistance of J. Kim. We are grateful to A. Griep, B. Spalholz, and F. Thierry for critically reading the manuscript and to D. DiMaio and M. Botchan for communication of results prior to publication. LITERATURE CITED 1. Androphy, E. J., D. R. Lowy, and J. T. Schilier. 1987. Bovine papillomavirus E2 trans-acting 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 tissues. EMBO J. 6:1027-1035. 3. Berg, L., M. Lusky, A. Stenlund, and M. Botchan. 1986. Repression of bovine papillomavirus replication is mediated by a virally encoded trans-acting factor. Cell 46:753-762. 4. Cerni, C., B. Binetruy, J. T. Schiller, D. R. Lowy, G. Meneguzzi, and F. Cuzin. 1989. Successive steps in the process of immortalization identified by transfer of separate bovine papillomavirus genes into rat fibroblasts. Proc. Natl. Acad. Sci. USA 86:3266-3270. 5. 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. 6. DiMaio, D., and J. Settleman. 1988. Bovine papillomavirus mutant temperature defective for transformation, replication and transactivation. EMBO J. 7:1197-1204. 7. Dostatni, N., F. Thierry, and M. Yaniv. 1998. A dimer of BPV-1 E2 protein containing a protease resistant core interacts with its DNA target. EMBO J. 7:3807-3816. 8. Giri, I., and M. Yaniv. 1988. Study of the E2 gene product of the cottontail rabbit papillomavirus reveals a common mechanism of transactivation among the papillomaviruses. J. Virol. 62: 1573-1581. 9. Groff, D. E., and W. D. Lancaster. 1986. Genetic analysis of the 3' early transformation and replication functions of bovine papillomavirus type 1. Virology 150:221-231. 10. 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. Nucleic Acids Res. 15:10267-10284. 11. 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 type 1 by a product of the viral E2 gene. EMBO J. 6:145-152. 12. Haugen, T. H., L. P. Turek, F. M. Mercurio, T. P. Cripe, B. J. Olson, R. D. Anderson, D. Siedl, M. Karin, and J. C. Schiller.

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1988. Sequence specific and general transactivation by the BPV-1 E2 transactivator require an N-terminal amphipathic helix-containing E2 domain. EMBO J. 7:4245-4253. 13. 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. 14. Heilman, C. A., L. Engel, D. R. Lowy, and P. M. Howley. 1982.

Virus-specific transcription in bovine papillomavirus transformed mouse cells. Virology 119:22-34. 15. 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. 16. 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. 17. Hirochika, H., T. R. Broker, and L. T. Chow. 1987. Enhancers and trans-acting E2 transcriptional factors of papillomaviruses. J. Virol. 61:2599-2606. 18. Hubbert, N. L., J. T. Schiller, D. R. Lowy, and E. J. Androphy. 1988. Bovine papillomavirus transformed cells contain multiple E2 proteins. Proc. Natl. Acad. Sci. USA 85:5864-5868. 19. Kunkel, T. A. 1985. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA 82:488-492. 19a.Lambert, P. F., C. C. Baker, and P. M. Howley. 1988. The genetics of bovine papillomavirus type 1. Annu. Rev. Genet. 22:235-258. 20. 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. 21. 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. 22. 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: 69-78. 23. Lambert, P. F., B. A. Spalholz, and P. M. Howley. 1987. Evidence that bovine papillomavirus type 1 may encode a negative transcriptional regulatory factor. Cancer Cells 5:15-22. 24. Land, H., A. C. Chen, J. P. Morgenstern, L. F. Parada, and R. A. Weinberg. 1986. Behavior of myc and ras oncogenes in transformation of rat embryo fibroblasts. Mol. Cell. Biol. 6: 1917-1925. 25. Law, M. F., J. C. Byrne, and P. M. Howley. 1983. A stable bovine papillomavirus hybrid plasmid that expresses a dominant trait. Mol. Cell. Biol. 3:2110-2116. 26. 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. 27. 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. 28. Lusky, M., and M. R. Botchan. 1985. Genetic analysis of bovine papillomavirus type 1 trans-acting replication factors. J. Virol. 53:955-965. 29. McBride, A. A., J. C. Byrne, and P. M. Howley. 1989. E2 polypeptides encoded by bovine papillomavirus type I form dimers through the common carboxyl-terminal domain: transactivation is mediated by the conserved amino-terminal domain.

J. VIROL. Proc. Natl. Acad. Sci. USA 86:510-514. 30. McBride, A. A., R. Schlegel, and P. M. Howley. 1988. The carboxy terminal domain shared by the bovine papillomavirus E2 transactivator and repressor proteins contains a specific DNA binding activity. EMBO J. 7:533-539. 31. 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. 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 expression regulates expression of the viral E5 transforming gene. J. Virol. 62:3608-3613. 34. Rabson, M. S., C. Yee, Y. C. Yang, and P. M. Howley. 1986. Bovine papillomavirus type 1 3' early region transformation and plasmid maintenance functions. J. Virol. 60:626-634. 35. Riese, D. J., II, J. Settleman, K. Neary, and D. DiMaio. 1990. Bovine papillomavirus E2 repressor mutant displays a highcopy-number phenotype and enhanced transforming activity. J. Virol. 64:944-949. 36. Sarver, N., J. C. Byrne, and P. M. Howley. 1982. Transformation and replication in mouse cells of a bovine papillomaviruspML2 plasmid vector that can be rescued in bacteria. Proc. Natl. Acad. Sci. USA 79:7147-7151. 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. Schilier, J. T., W. C. Vass, and D. R. Lowy. 1984. Identification of a second transforming region in bovine papillomavirus DNA. Proc. Natl. Acad. Sci. USA 81:7880-7884. 39. Schlegel, R., M. Wade-Glass, M. S. Rabson, and Y.-C. Yang. 1986. The E5 transforming gene of bovine papillomavirus encodes synthesis of a small hydrophobic protein. Science 233: 464-466. 40. 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. 41. Spalholz, B. A., P. F. Lambert, C. L. Yee, and P. M. Howley. 1987. Bovine papillomavirus transcriptional regulation: localization of the E2 responsive elements of the long control region. J. Virol. 61:2128-2137. 42. Spalholz, B. A., Y. C. Yang, and P. M. Howley. 1985. Transactivation of a bovine papillomavirus transcriptional regulatory element by the E2 gene product. Cell 42:183-191. 43. Stenlund, A., G. Bream, and M. Botchan. 1987. A promoter with an internal regulatory domain in part of the origin of replication in BPV-1. Science 236:1666-1671. 44. Stenlund, A., J. Zabielski, H. Ahola, J. Moreno-Lopez, and U. Petterson. 1985. Messenger RNAs from the transforming region of bovine papillomavirus type 1. J. Mol. Biol. 182:541-554. 45. Thierry, F., and M. Yaniv. 1987. The BPV-1 E2 trans-acting protein can be either an activator or repressor of the HPV-18 regulatory region. EMBO J. 6:3391-3397. 46. Thorner, L., N. Bacay, J. Choe, and M. Botchan. 1988. The product of the bovine papillomavirus type 1 modulator gene is a phosphoprotein. J. Virol. 62:2474-2482. 47. Yang, Y. C., H. Okayama, and P. M. Howley. 1985. Bovine papillomavirus contains multiple transforming genes. Proc. Natl. Acad. Sci. USA 82:1030-1034. 48. Yang, Y. C., B. A. Spalholz, M. S. Rabson, and P. M. Howley. 1985. Dissociation of transforming and transactivating functions for bovine papillomavirus type 1. Nature (London) 318:575-577.