Proc. Nati. Acad. Sci. USA Vol. 89, pp. 3159-3163, April 1992

Microbiology

Disruption of either the El or the E2 regulatory gene of human papillomavirus type 16 increases viral immortalization capacity (human epithelial cells/immortaflization)

HELEN ROMANCZUK* AND PETER M. HOWLEY Laboratory of Tumor Virus Biology, National Cancer Institute, Bethesda, MD 20892

Communicated by Bernard Moss, December 23, 1991

region are intact but that the viral El or E2 gene region is often disrupted (14-19). This integration pattern has suggested that the loss of expression of the El and/or E2 gene might provide a selective growth advantage to the HPV-infected cell and, as such, play a role in the malignant progression of an HPVpositive lesion. The role of the HPV El and the E2 genes in viral transformation, however, has not previously been studied directly. Analyses of the HPV-16 and HPV-18 P9 and P105 promoters, which control expression of the viral E6 and E7 transforming genes, have shown that these promoters possess a basal activity in primary human keratinocytes that can be repressed by the full-length papillomavirus E2 gene products (20-22). The E2 repression ofthe HPV P,7 and P105 promoters occurs, at least in part, by the binding of E2 to its cognate ACCN6GGT motifs within the long control region (LCR) upstream of each promoter (20-23). Four E2 motifs are contained within the HPV-16 (Fig. 1) and HPV-18 LCRs, with three motifs located in close proximity to the individual promoters (20, 23). Integration in the EJ/E2 gene region, with the subsequent loss of E2 expression, could thus lead to release of the E6/E7 promoter from the negative regulation by E2. The role of the HPV El gene product is not yet known, but the frequent interruption of El in carcinomas suggests that this gene may also have a negative regulatory function with respect to viral transformation. The studies presented here were designed to examine whether or not the HPV-16 El and E2 genes have an effect on the efficiency of immortalization of primary human epithelial cells. In the context of a full-length wild-type HPV-16 genome, mutations were generated in the El or the E2 gene, or in the E2 protein-binding sites upstream of the transforming gene promoter P97. We show that the "wild-type" HPV-16 genome can immortalize primary human keratinocytes with a low efficiency and that mutations that disrupt either the El or the E2 gene increase immortalization capacity. Mutations of the E2 protein-binding sites proximal to the P9 promoter did not fully alleviate E2-mediated repression, indicating that other mechanisms must be involved in the negative regulation of viral immortalization functions by E2. These results provide genetic evidence that El and E2 encode functions that negatively regulate the efficiency of viral immortalization of human epithelial cells.

ABSTRACT The "high-risk" human papillomavirus types 16 (HPV-16) and 18 (HPV-18) have been etiologically implicated in the majority of human cervical carcinomas. In these cancers, the viral DNAs are often integrated into the host genome so that expression of the El and the E2 genes is lost, suggesting that disruption of these regulatory genes plays an important role in carcinogenic pgression. Previous studies defiming the viral genes affecting PV-16 transformation functions have used the "prototype" viral genome, which was cloned from a human cervical carcinoma and later discovered to harbor a mutation in the El gene. In this study, we have corrected this mutation and have evaluated the effect of mutations of either the El or the E2 gene on the efficiency of HPV-16 immortalization of human keratinocytes. Mutation of either the El gene or the E2 gene in the background of a "wild-type" HPV-16 genome markedly increased immortalization capacity. Mutations were also generated in the E2binding sites located upstream of the P,7 promoter, which directs synthesis of the viral E6 and E7 transforming genes. E2 negatively regulates the Pg. promoter through bindin at adjacent sites. Surprly, the mutation of these sites only partially relieved the negative effect of E2 on viral immortalization, implicating additional mechaniss in theE2 repression of viral immortalization functions. Our results provide genetic evidence that the El and E2 gene products each can repress HPV-16 immortalization and support the hypothesis that a selective growth advantage is provided by integration of the viral-genome in a manner that causes the loss of expression of either El or E2. A subgroup of human papillomaviruses (HPVs) is associated with genital intraepithelial neoplasias and cervical carcinomas. The two viruses found in most of these lesions are HPV types 16 (HPV-16) and 18 (HPV-18). HPV-16 DNA and HPV-18 DNA can immortalize primary human keratinocytes in culture (1-3) and can induce histological abnormalities in organotypic cultures similar to those seen in clinical lesions in vivo (4, 5). Previous analyses have shown that the expression of both the E6 and E7 genes of the "high-risk" HPVs is necessary for the efficient immortalization of primary human keratinocytes (5-7). Furthermore, the E6 and E7 genes are consistently expressed in the HPV-positive cervical cancers and in cell lines derived from such cancers (8-13), supporting the hypothesis that expression of E6 and E7 plays a role in cervical carcinogenesis. Analyses of the state of the viral DNA in HPV-positive lesions have generally correlated the presence of extrachromosomal viral genomes in benign preneoplastic lesions and of integrated viral DNA in cancers (8, 13-16). The integration patterns of the viral genomes in cancers and in cancer-derived cell lines indicate that the E6 and E7 genes and their upstream transcriptional regulatory

MATERIALS AND METHODS Cell Culture. Primary human keratinocytes were prepared and maintained as described (24). Cells were transfected by using lipofectin as described (20) and a total of 5 pug of DNA. Viral DNA was released from vector sequences before transAbbreviations: HPV, human papilloma virus; HPV-16 and HPV-18, HPV type 16 and type 18, respectively; BPV-1, bovine papillomavirus type 1; ORF, open reading frame; LCR, long control region; nt, nucleotide(s); TTL, translation termination linker. *To whom reprint requests should be addressed.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3159

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Microbiology: Romanczuk and Howley

LCR

(TTLs), as described (25) into specific restriction endonuclease cleavage sites described in the text. The prototype HPV-16 isolate (17) p769.1 contains the deletion of a single base at nt 1138 in the El ORE. p1318 is a control plasmid containing the human f-actin promoter and poly(A) addition site in a prokaryotic vector (6).

~~~~~~~~El

HPV-16

000

000

E4

E2

L2

E5 FIG. 1. HPV-16 genome. The HPV-16 genome is schematically represented with early-region (E) and late-region (L) ORFs. The P97 promoter (-..) and the E2-binding sites (e) within the LCR upstream of the early-region genes are shown.

fection. Transformed foci were counted 7 weeks after transfection, after formalin fixation and methylene blue staining. Recombinant DNA. The plasmid p1203 was constructed by Karl Munger, National Cancer Institute, Bethesda, MD, and contains the HPV-16 genome, with an uninterrupted El open reading frame (ORF), inserted into the BamHI site of pML2D. This plasmid was constructed by exchanging the Nco I/PflMI fragment [nucleotides (nt) 863-2926] of the "prototype" HPV-16 DNA described by Durst et al. (17) with the corresponding fragment of a SiHa (cervical carcinoma cell line)-derived DNA clone that contains an intact El ORE (16). All E2-binding site mutations and El and E2 ORF mutations were generated in the background of p1203. E2binding site mutations were generated as described (20). An EcoRI/Hpa II fragment (nt 7455-57) containing the E2binding site mutations was exchanged for the analogous fragment of p1203 to generate LCR mutations in the context of the full HPV-16 genome. El and E2 mutations were generated by the insertion of translation termination linkers

A

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RESULTS The HPV-16 El ORF Negatively Regates Viral Immortalization Efficiency. The prototype HPV-16 DNA clone, first isolated from a cervical carcinoma containing integrated viral DNA (17), contains a deletion of nt 1138, resulting in a frameshift mutation in the El gene (16, 26). Previous analyses have shown that this cloned DNA efficiently immortalizes primary human keratinocytes (1, 6, 24). In preliminary experiments with an HPV-16 DNA clone with a corrected El ORF, we noticed a reduced immortalization efficiency in comparison to the prototype DNA, suggesting that the difference in immortalization capacity between these two clones was a consequence of the integrity ofthe El ORE. To test this hypothesis, a mutation was generated in the intact El ORE of the corrected DNA clone by inserting a TTL at nt 1311, close to the location of the single base deletion in the prototype HPV-16 DNA clone (Fig. 2B). Wild-type HPV-16 DNA (p1203), prototype DNA (p769. 1), and the El TTL-mutated DNA (pl6-Ne) were assayed for their ability to immortalize primary human keratinocytes, by using a quantitative assay that selects for resistance to terminal differentiation induced by serum and calcium (24). The immortalization and altered differentiation phenotype is mediated by the E6 and E7 gene products (6) expressed from the P97 promoter (10). In three separate experiments, DNAs mutated in the El ORE (p769.1, p16-Ne) were up to 8-fold more efficient at immortalization than wild-type DNA (p1203) (Fig. 2B). Furthermore, the differentiation-resistant foci obtained with the El-mutated DNAs were routinely visible earlier than foci induced by wild-type DNA. These results show that the disruption of the HPV-16 El gene increases viral immortalization capacity. Disruption of the E2 ORF Increases HPV-16 Immortalization Efficiency. E2 can repress the P97 and P105 promoters of HPV-16 and HPV-18, respectively (20-23). In the context of the viral genome, E2 is predicted to repress expression of the E6 and E7 transforming genes, and the loss of E2 function 2128 24 2326

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p1203 I 1138

p769.1

a

1

31

47

43

1

18

17

26

1311

p1-Nt L TrL

FIG. 2. Immortalization of primary keratinocytes with DNAs mutated in the El ORF. (A) El ORF, showing internal ATGs ( ) (B) El ORFs of wild-type (p1203) and El-mutated DNAs (p769.1 and pl6-N). AG represents the deletion at nt 1138 of the prototype HPV-16 DNA (17, 26). The site of TTL insertion (nt 1311) is shown. The sites of El mutations are downstream of the E4 splice donor site (27, 28). Results are from three separate experiments. .

Microbiology: Romanczuk and Howley

Proc. Natl. Acad. Sci. USA 89 (1992)

should, therefore, increase expression of those viral oncoproteins. Previous studies could not detect any difference in immortalization efficiencies of the prototype HPV-16 genome and the prototype genome harboring E2 mutations (6). Together with the results of Fig. 2, it seemed that the El mutation in the prototype clone could have masked the effects of an E2 regulatory gene mutation. To test this hypothesis, we examined the transformation efficiency of viral DNAs with E2 mutations in the context of the wild-type viral genome plasmid p1203. E2 mutations were generated in p1203 by inserting TTLs into the coding sequences of the E2 ORF as shown in Fig. 3B. Primary human keratinocytes were transfected with wildtype or E2-mutated HPV-16 DNAs and assayed for their immortalization efficiency in three separate experiments, shown in Fig. 3B. Each plasmid with a mutated E2 gene (p16-Pt, p16-St, or pl6-PtS1) exhibited -10-fold greater immortalization capacity than wild-type HPV-16 DNA (p1203). The highest efficiency of immortalization was routinely exhibited by p16-Pt DNA, which contains a mutation in the transactivating domain of E2. Each of the plasmids with E2 ORF mutations induced foci that appeared =2 weeks earlier than the foci induced by wild-type HPV-16 DNA and =1 week earlier than the foci induced by the El-mutated DNAs. In addition, the foci induced by the E2-mutated genomes were generally larger than the foci obtained with wild-type HPV-16 DNA. These data clearly indicate that HPV-16 E2 gene disruption dramatically increases HPV-16 immortalization capacity. Combined El and E2 Mutations Are Neither Synergistic Nor Additive in Their Increased Immortalization Phenotype. To determine whether the combination of an El mutation with an E2 mutation would further increase the immortalization capacity of an HPV-16 genome, the immortalization efficiency of DNAs mutated in either the El ORF or the E2 ORF was compared with that of DNAs mutated in both ORFs. The immortalization efficiency of HPV-16 genomes mutated in both El and E2 could not be distinguished from that of DNAs with mutations in the El or the E2 ORF alone in terms of comparative numbers of immortalized colonies each induced (data not shown). These results are consistent with the previous studies of HPV-16 E2 mutants that were done by using the prototype HPV-16 genome with an El mutation (6).

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Therefore, mutation of either the El or the E2 gene is sufficient to impart a high efficiency of immortalization for HPV-16. Only Part of the Negative Regulation of Viral Immortalization Functions by E2 Is Mediated Through Promoter-Proximal E2-Binding Sites. In primary human keratinocytes, the P97 promoter of HPV-16 has a basal level of activity that depends on the integrity of cellular factor recognition sites in the upstream LCR (29-32). Modulation of this basal activity occurs through the interaction of virally encoded E2 gene products with specific sites in the LCR (20, 33). To test the importance of the promoter-proximal E2-binding sites on regulation of viral immortalization functions, mutations were generated in one, two, or three of these sites, and the resulting mutated DNAs were assayed for their immortalization capacity. Single or double-nucleotide substitutions in the LCR E2binding sites were introduced into the full-length HPV-16 DNA background as shown in Fig. 4. The specific mutations disrupt the palindromic sequences in the E2-binding sites (34) and have been shown to no longer bind the bovine papillomavirus type 1 (BPV-1) E2 protein (20). The p16 plasmid was reconstructed with wild-type nucleotide sequences and is, therefore, equivalent to p1203. Primary human foreskin keratinocytes were transfected with either wild-type HPV-16 DNA (p16) or E2-binding site-mutated DNAs (pl6-Lx, pl6-Lxx, or pl6-Lxxx) (Fig. 4B). The DNAs with mutations in the promoter-proximal E2-binding sites had slight but reproducibly increased immortalization efficiencies compared with wild-type DNA (Fig. 4A). Surprisingly, the increase in immortalization efficiency of the E2-binding site-mutated DNAs was not of the magnitude seen with the E2-ORF-mutated DNAs. Furthermore, the colonies induced by wild-type or E2-binding sitemutated DNAs appeared at -4 weeks after transfection, whereas E2-ORF-mutated DNAs appeared 2 weeks after transfection. These results supported the hypothesis that E2 does, indeed, repress the P97 promoter through an interaction with promoter-proximal E2-binding sites but also indicated that the E2 repression of the HPV-16 immortalization functions must involve additional mechanisms. It is possible that E2 negatively regulates viral immortalization by a mechanism independent of its ability to bind to viral DNA. Alternatively,

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FIG. 3. Immortalization of primary keratinocytes with HPV-16 DNA mutated in E2 ORF. (A) E2 ORF, showing internal ATGs ( I ). (B) Sites of mutations (TTL insertions) in E2 ORFs. The PflMI site (nt 2922) is downstream of the translation initiation codon for the full-length E2 transactivator. The Sty I site (nt 3438) is downstream of an ATG that could encode a carboxyl-terminal DNA-binding protein. Results are from three different experiments.

Microbiology: Romanczuk and Howley

3162 A

Proc. Natl. Acad. Sci. USA 89

(1992)

7859 ... TGTGTGCAA

CCGTTTTGGGT|TACACATTTACAAGCAACTTATATAATAATACTAAACTACAATA 50

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ATTCATGTATAAAACTAAGGGCGTAACCGAAATCGGT TGA CCGAAACCGG TAGTATAAAACA P97 GACATTTATGCACCAAAAGAGAACTGCA ...

B

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ACCGAAACCGGT ...... ACCGAAATCGGT ......... ACCGTTT-GGGT .........

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ACTGAAACCGGT ...... ACCGAAATCGGT ......... ACCGTTTTGGGT .........

p16-Lx

ACTGAAACCGGT ...... ACCGAAATCAGT ......... ACCGTTTTGGGT .........

p16-Lxx **

... ATTGTTTTGGGT ......

*

*

ACCGAAATCAGT ...... ACTGAAACCGGT

p16-Lxxx

mutations in the LCR E2-binding sites could potentially affect the basal activity of the viral promoter(s) directing E6 and E7 expression, thus dampening the negative regulatory effects of El and E2. Therefore, the immortalization capacity of DNAs with combined E2 ORF mutations and E2-binding site mutations was next tested. The wild-type LCR in the plasmids with either single or double E2 ORF mutations (p16-P1, p16-St, and p16-PtSt) was replaced by an LCR with one, two, or three E2-binding site mutations (pl6-Lx, pl6-Lxx, and pl6-Lxxx). These DNAs were then assayed for their immortalization capacity. All E2 ORF mutations increased the HPV-16 immortalization efficiency, regardless of whether the LCR E2-binding sites were intact or mutated (Table 1). Mutating the E2-binding sites did not dramatically affect basal activity of the viral promoter(s) Table 1. Mutations in E2 ORF and LCR E2-binding sites increase HPV-16 immortalization capacity ORFs Colonies per 106 E2-binding sites* cellst disrupted Plasmid disrupted 1 None None p1203 E2TA 38 None p16-Pt E2TA 17 1 pl6-LxP, 45 E2TA 1, 2 pl6-LxxPt 13 1-3 E2TA pl6-LxxxP, E2DB 23 None pl6-S, 1 24 E2DB pl6-LxSt 33 E2DB 1, 2 pl6-LxxSt 1-3 E2DB 11 pl-LxxxSt E2TA/DB 18 None p16-PtSt 1 E2TA/DB ND pl6-LxPtSt 43 E2TA/DB 1, 2 pl6-LxxPS, 29 1-3 E2TA/DB pl6-LxxxPS, 0 p1318 DB, DNA-binding domain; ND, not TA, transactivating domain; done. *Sites are numbered in proximity to promoter. tValues are per 5 ,g of DNA and at 7 weeks after transfection.

...

FIG. 4. Immortalization of human keratinocytes with HPV-16 DNAs mucolonies per 106 cells tated in their E2-binding sites. (A) Nucleotide sequence of HPV-16 upstream of the P97 promoter, showing the three E2-binding sites most proximal to the 2 1 promoter. The E2-binding sites are boxed, and the P97 promoter is denoted by an arrow. (B) Mutations in the E2binding sites proximal to the P97 promoter were generated in the context of 6 3 a full-length wild-type HPV-16 genome. A wild-type LCR was also constructed (p16) and, as such, is similar to p1203. Open boxes below consensus sites signify sites capable of binding 5 6 E2; dark boxes are sites that no longer bind E2. Sites are numbered in terms of proximity to the promoter. *, Nucleotide mutated from the original se8 6 quence. Results are from two separate experiments.

directing expression of the immortalization functions. Together with the results of Figs. 3 and 4, these results show that an E2 ORF mutation has a dominant phenotype over that of E2-binding site mutations and argues that only part of the E2 negative regulation of viral immortalization functions by E2 is mediated through the promoter-proximal E2-binding sites.

DISCUSSION In this study, we have used a quantitative assay to show that mutations in either the El or the E2 gene of HPV-16 substantially increase the efficiency of immortalization of human keratinocytes, suggesting that the El and E2 genes can negatively regulate viral immortalization functions. These studies support the concept that elimination of El and E2 gene expression through viral integration in cervical cancers could provide a selective growth advantage to the cell. The El and E2 functions involved in regulating the efficiency of immortalization appear to be through a common pathway because there is no additive or synergistic effect of double mutants. It is possible that the HPV-16 El and E2 gene products can associate in a complex, as recently shown for the analogous BPV-1 proteins (35, 36). Similarities in the organizations of the papillomavirus genomes and in the El and E2 proteins (37, 38) suggest that these protein functions are probably conserved between BPV-1 and genital tractassociated HPVs. Mechanism of E2 Repression. The E2-mediated regulation of the HPV-16 P97 promoter and the analogous HPV-18 P105 promoter appears to be a complex mechanism dependent, in part, on the interaction of E2 with its cognate binding sites in the proximity of the promoter. It has been proposed that E2 binding near the promoter may interfere with assembly of the transcription initiation complex necessary for promoter activity (20-23). Mutation of the E2-binding sites proximal to the P97 promoter would be predicted to relieve E2-mediated promoter repression. In the context of the full-length HPV-16 viral genome, the relief of P97 promoter repression would be

Microbiology: Romanczuk and Howley expected to cause increased transcription of the E6 and E7 transforming genes. Although an increase in the immortalization capacity of the viral genome was seen with the E2-binding site mutants, the magnitude of the increase was modest compared with E2 ORF mutants. These results suggest that the regulation of viral immortalization capacity by E2 must involve a mechanism additional to repression of the P97 promoter through the promoter-proximal E2-binding

sites. Our knowledge of the biochemistry of E2 that might help explain this additional negative regulatory role in viral immortalization is limited. E2 may interact with other cis elements in the viral genome to modulate transcriptional efficiency. Alternatively, E2 may effect viral transcriptional efficiency through an indirect mechanism. Recent studies have shown an association between BPV-1 E2 and the general transcription factor SP1 (39) and the viral replication factor El (35, 36). If the analogous HPV E2 has these properties, then it is probably necessary for both the maintenance of viral replication and the regulation of viral transcription. An absence of E2 would then preclude a tight regulation of replication and transcription processes, resulting in the integration of viral DNA and derepression of viral gene expression. Another possibility is that the effect of E2 is not transcriptional but occurs through an interaction with cellular proteins directly involved in the immortalization process. Mechanism of El Repression. Little is known about the HPV El gene products, so much of our understanding of the mechanism of El regulation is by analogy with BPV-1. Recent studies have shown that the BPV-1 El and E2 gene products are essential for viral DNA replication (40) and that El ORF mutations affect increased viral transcription and transformation (41, 42). If the analogous HPV gene products function in a related manner, then the mutation of these genes may disrupt DNA replication and cause integration of the viral genome, altering patterns of viral transcription contributing to immortalization. Those cells with increased levels of transforming gene products would have a selective growth advantage. Additionally or alternatively, El may have a more direct role in the immortalization process through its interaction with E2 or with cellular factors involved in the immortalization process. Regulation of the HPV-16 immortalization functions is complex, involving cellular factors and, as shown in this study, viral factors. This regulation can only partly be explained at the transcriptional level by the binding of E2 protein to its cognate sites immediately upstream of the P97 promoter. Other mechanisms of regulation may also be transcriptional or may involve other El and E2 activities. This study provides genetic evidence that the elimination of the negative regulatory effects of El and E2 through integration, as observed in a high percentage of HPV-positive cervical cancers, increases the immortalization capacity of the viral genome. This action could be one of the early genetic events that occurs during carcinogenic progression. We thank C. Yee for primary human keratinocytes and J. Byrne for oligonucleotide synthesis. We are grateful to Drs. J. Benson, F. DelVecchio, A. McBride, K. Munger, and S. Van de Pol for helpful comments concerning the manuscript. H.R. was supported by the Leukemia Society of America.

Durst, M., Dzarlieva-Petrusevska, R. T., Boukamp, P., Fusenig, N. E. & Gissmann, L. (1987) Oncogene 1, 251-256. 2. Pirisi, L., Yasumoto, S., Feller, M., Doniger, J. & DiPaolo, 1.

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Disruption of either the E1 or the E2 regulatory gene of human papillomavirus type 16 increases viral immortalization capacity.

The "high-risk" human papillomavirus types 16 (HPV-16) and 18 (HPV-18) have been etiologically implicated in the majority of human cervical carcinomas...
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