Viral transcription in human keratinocyte cell lines immortalized by human papillomavirus type-16

VIROLOGY

183, 331-342

(1991)

Viral Transcription

in Human Keratinocyte by Human Papillomavirus

M. ROHLFS, S. WINKENBACH, lnstitut ftir Virusforschung,

Deutsches

Cell Lines immortalized Type-l 6

S. MEYER,’ T. RUPP, AND M. DURST’

Krebsforschungszentrum,

Im Neuenheimer

Fe/d 280, 6900 Heidelberg,

Germany

Received January 23, 199 1; accepted April 1I 199 1 Human papillomavirus type-l 6 (HPV-16) transcription in two human keratinocyte cell lines (HPK) immortalized by transfection of viral DNA in vitro was analyzed by nucleotide sequencing of cDNA clones, and in addition by primer extension analysis and Sl nuclease and exonuclease VII digestion of poly(A)+ RNA. A novel mRNA species which probably initiates in the E7 ORF and in which the 5’-part of the El ORF (splice donor at position (pos.) 880) is joined to an exon comprising the entire E2 ORF (splice acceptor at pos. 2708) was found in both cell lines. This mRNA has the potential to encode a full-length E2 protein, which is known to function as a repressor of transcription initiated at P97. cDNAs derived from the late region of the viral genome and the use of a late polyadenylation signal at pos. 7320-7325 are described. In agreement with RNA data published by others the major promoter for HPV-16 transcription is located at pos. 97. mRNA species encoding full-length or truncated forms of the E6 protein, and species characterized by an Q 1ss1 EliAE4 splice junction (which provided the E4 open reading frame (ORF) with an ATG triplet) were identified. Academic

Press,

Inc

In cervical cancer biopsies HPV DNA is frequently integrated into the host chromosomal DNA, but the transcriptional activity of the viral E6 and E7 ORFs appears to be selectively retained which is also in support of the above experimental evidence suggesting that these genes play an essential role in transformation or in maintenance of the tumorigenic phenotype or both (for review, see: zur Hausen, 1989). In the HPV-16 positive cervical carcinoma derived cell lines CaSki and SiHa and in two primary cervical cancer biopsies three different mRNA species spanning the E6/E7 region have been identified. One species is characterized by a full-length E6 and an E7 ORF, while the other two species consist of a truncated E6* ORF (E6*I or E6*ll; generated by splicing) and an E7 ORF (Smotkin and Wettstein, 1986; Smotkin et al., 1989). The E7 protein is probably expressed by the E6*I/E7 mRNA species (Smotkin et a/., 1989). The E6 and E7 proteins of HPV16 and -18 were shown to bind in vitro to the tumor suppressor proteins p53 and pl05-RB, respectively (Dyson et a/., 1989; Milnger et a/., 1989b; Werness et a/., 1990). This protein-protein interaction may interfere with the regulation of normal cell growth and may offer one explanation on how papillomavirus infection leads to abnormal cell proliferation. We have established a number of human keratinocyte cell lines (HPKIA, HPKIB, and HPKII) by transfection of human primary foreskin keratinocytes with cloned HPV-16 DNA (Durst et al., 1987). In analogy to the tumorigenic CaSki and SiHa cells, the viral genome

INTRODUCTION Papillomaviruses cause hyperproliferations of cutaneous or mucosal epithelium at different sites of the body of both man and animals. To date some sixty different human papillomaviruses are known (for review, see: De Villiers, 1989). A subset of these viruses is found in the anogenital tract. HPV-6 and -1 1 are the etiological agents of condylomata acuminata while others such as HPV types 16, 18, 31, 33, 35, 39, and 52 are associated with precancerous lesions (squamous intraepithelial neoplasia) and squamous cell carcinomas (Dllrst eta/., 1983; Boshat-t eta/., 1984; Beaudenon et a/., 1986, 1987; Lorincz et al., 1986, 1987; zur Hausen and Schneider, 1987; Shimoda et al., 1988; Yajima et a/., 1988). Recently it was shown that the DNA of cervical cancer associated HPV types can immortalize human primary keratinocytes in culture (Durst et al., 1987; Pirisi et a/., 1987; Kaur et al,, 1989). This immortalizing activity was mapped to the E6 and E7 ORFs of the viral genome (Barbosa et al., 1989; Hawley-Nelson et a/., 1989; Munger ef a/., 1989a). This property is not shared by HPV-6 and -11 (Schlegel et al., 1988; Pecoraro et a/., 1989; Woodworth et al., 1989).

’ Present address: lnserm Unit& 186, Oncologic Molkulaire, Institut Pasteur de Lille, 1 Rue Calmette, F-5901 9 Lille Ckdex, France * To whom correspondence and reprint requests should be addressed. 331

0042-6822191

$3.00

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction m any form reserved.

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ROHLFS ET AL.

in HPK cells persists in a stably integrated form and is transcriptionally active. However, HPK cells are not tumorigenic in nude mice (DOrst et a/., 1987, 1991). It is speculated that an altered pattern of HPV-16 transcription could influence the growth characteristics of the host cell. In CaSki and SiHa cells viral DNA integration has uncoupled the 3’-part of the early region from the viral promoter P97 upstream of the E6 and E7 ORFs (Smotkin and Wettstein, 1986; Baker et a/., 1987). This 3’-part of the early region also encodes the viral E2 protein which is known to suppress the transcriptional activity of the homologous promoter P97 (Romanczuk et a/., 1990). In contrast, in HPK cell lines and a cell line derived from a human premalignant genital lesion (WI 2; Stanley et al., 1989; Doorbar et a/., 1990) the entire early region is transcribed. A negative regulatory mechanism which controls E6/E7 gene expression would therefore be conceivable in these cells. We have performed a detailed analysis of the viral transcription of HPV-16 immortalized cell lines in order to characterize transcription of the entire early region of the viral genome. A detailed knowledge of viral transcription of HPV-16 immortalized cell lines and of cell lines derived from premalignant and malignant cervical lesions will not only provide the basis for understanding HPV gene regulation, but may also show up differences in the transcriptional pattern which could correlate with a benign or a malignant phenotype of HPV-infected cells. MATERIALS

AND METHODS

Cell lines HPKIA, HPKIB, and HPKII cell lines were established by transfection of human primary foreskin keratinocyte cultures with the entire HPV-16 genome and subsequent selection for unlimited growth in vitro (Diirst et a/., 1987). For transfection, the prototype DNA clone of HPV-16 was used, which has a characteristic frame shift mutation in the El ORF at pos. 1 137 (Dllrst et a/., 1983; Seedorf et a/., 1985). This frameshift mutation was shown to be unique for the prototype HPV-16 DNA (Matsukura et al., 1986; Baker eta/., 1987). The HPKIA and HPKIB cell lines harbor HPV-16 DNA in form of head-to-tail genomic repeats integrated at a single cellular site (almost three complete copies and less than two copies of the HPV-16 genome percell in the HPKIA and HPKIB cell line, respectively). Both cell lines were derived from the same transformed cell, since they have one virus-cell junction in common. Partial deletion of the integrated viral genome must have taken place at a very early stage during development of the HPKIB cell line. HPKII cells harbor about 10 copies of HPV-16 DNA per cell integrated at two different sites of

the host genome. The HPK cell lines are nontumorigenie in nude mice giving rise to transitory nodules only (Dllrst et al., 1987, 1991). CaSki, a cervical cancer derived cell line containing about 500 copies of HPV-16 DNA integrated at several chromosomal locations (Mincheva et al., 1987) was obtained from the American Type Culture Collection. The HaCaT cell line was derived from a spontaneously immortalized human keratinocyte culture and is devoid of any known HPV DNA (Boukamp et a/., 1988). Early passages of HPK cells were grown in medium enriched with various growth supplements (see DCjrst eta/., 1987). CaSki and HaCaT cells as well as HPK cells at late passages were grown in Dulbecco’s modified Eagle’s medium supplemented with 5% FKS in a 37” incubator with 5% CO*.

Construction of cDNA libraries For cDNA synthesis poly(A)+ RNA was selected from total cytoplasmic RNA of HPKIB and HPKII cells (for RNA extraction, see Maniatis, 1989) with Hybond messenger affinity paper (Orgenics). First-strand synthesis was performed using 500 mg poly(A)+RNA, AMV reverse transcriptase (Boehringer-Mannheim) and oligo (dT,,-,J (Pharmacia) as primer as described by Maniatis (1989). The second DNA strands were synthesized as described by Gubler and Hoffman (I 983). Doublestranded cDNAs were treated with Mung bean nuclease (Pharmacia) and EcoRl linkers (p(dGGAAllCC), Biolabs) were ligated to the blunt ends. Possible internal EcoRl restriction sites were methylated with EcoRl methylase (Biolabs) prior to the linker addition. After EcoRl digestion and size-fractionation on a Sepharose CL-4B column (Pharmacia) cDNA molecules of the first two fractions were ligated into the EcoRl site of vector X NM1 149 (Murray, 1983). Packaging of recombinant viral DNA (Maniatis, 1989) gave rise to cDNA libraries with cloning efficiencies of 5 X 1O6 recombinant plaques/pg cDNA. Recombinant phages were selected for by plating on Escherichia co/i POP 13b, a derivative of strain POP 101 (Murray, 1983). HPV-16 positive phages were identified by plaque-hybridization (see Benton and Davis, 1977) using the complete 32Plabeled HPV-16 genome as probe.

Pstl subfragments of HPV-16 DNA used as hybridization probes for the classification of cDNA clones (1) PstlB (pos. 7004-875); (2) PstlA (1.83-kb subfragment) (pas. 875-27 13; Pstl-Avall); (3) PstlA (0.97-kb subfragment) (pos. 2713-3692; Avall-Pstl); (4) PstlC (pos. 3692-4756); (5) PstlC, (0.41 -kb subfragment) (pos. 3692-4108; Pstl-Rsal); (6) PstlF (pos. 47565239); (7) PstlD (pos. 5239-6151; Pstl-BarnHI); (8)

HPV-16 TRANSCRIPTION

PstlE (pos. 6151-6788; 6788-7004). cDNA sequence

BarnHI-WI);

(9) /WIG (pos.

analysis

cDNA clones representing different RNA species were subcloned in Ml 3mpl9 or Ml 3mpl8 (YanischPerron et al., 1985). The nucleotide sequence of 5’and 3’-ends, splice junctions, and virus-cell fusion sites were determined by the dideoxy chain termination method (Sanger et al,, 1977) using Sequenase enzyme (U.S. Biochemical Corp.) and Ml 3 universal primer (17-mer primer, U.S. Biochemical Corp.) as well as HPV-16-specific synthetic 20-mer oligonucleotides as internal primers. HPV-16 specific oligonucleotides were synthesized using an automated DNA synthesizer (Applied Biosystems, Inc. 380A) and purified with the aid of Oligonucleotide Purification Cartridges (Applied Biosystems, Inc.). Sequence data were analyzed by a BSA computer program developed at the German Cancer Research Center and compared with the HPV16 genomic sequence published by Seedorf et a/. (1985) (see also: Halbert et al., 1988). The nucleotide sequence libraries EMBL (Release 22.0, February 1990) and GenBank (Release 63.0, March 1990) as well as the NBRF protein sequence library (Release 24.0, March 1990) were screened for homologies to sequence data derived from the cellular part of the virus-cell fusion clones. Primer extension

analysis

A 15-pmol sample of a synthetic 2 1-mer oligonucleotide (- 100 ng; complementary to the coding strand of HPV-16, pos. 139-l 59) were 5’-end-labeled using T4 polynucleotide kinase (2-4 U/PI; Boehringer Mannheim) and 30 pmol [T-~~P]ATP (5000 Ci/mmol, Amersham). Annealing and primer extension reactions were performed as described by Phelps and Howley (1987) using 4 ng labeled oligonucleotide and 15 pg cytoplasmic RNA, except for the following modifications: RNA bound primers were extended with 30 U AMV reverse transcriptase (Boehringer Mannheim) in the presence of 40 U RNasin (Promega) and 0.2 mM dNTPs (each). Actinomycin D was not added. Primer extension products were analyzed on a 6% polyacrylamide-urea sequencing gel. A nucleotide sequence reaction primed with the same oligonucleotide as that used for the primer extension reaction was employed as length marker. RNA mapping with Sl nuclease and exonuclease

VII

A 5’-end-labeled double-stranded DNA fragment of 982 nucleotides (nt) in length covering the HPV-16 genome from pos. 7779 to 867 was used as probe (see

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IN HPK

Fig. 3C). Sl nuclease mapping was performed as described for double-stranded DNA probes in Maniatis (1989) (see also Berk and Sharp, 1977). DNA and RNA were hybridized at 48” followed by digestion with 50 U Sl nuclease (400 U/PI; Boehringer Mannheim) at 37” for 30 min. For exonuclease VII mapping the same hybridization procedure as for the St analysis was used. Unprotected DNA was digested after addition of 250 ~1 67 rnn/l potassium phosphate buffer, pH 7.9, 8.3 mM EDTA, 10 mM fi-mercaptoethanol in the presence of 3 U exonuclease VII (2 U/PI; Bethesda Research Laboratories) in a final volume of 270 ~1 for 30 min at 37”. After phenol/chloroform-isoamyl alcohol extraction undissolved phosphate salts were removed by gel filtration through Sephadex G-50 and the products were precipitated with ethanol. The protected DNA-RNA hybrids were then denatured at 90” for 3 min and sepagel using the 32Prated on a 69/o polyacrylamide-urea labeled /-/inpI fragments of Bluescribe (Stratagene) (restriction fragment sizes: 503,412, 393, 337, 332, 270, 174, 109, 103, 100,93,67,65,33,28,26,22, 13,9,8, and 7 nucleotides) and a nucleotide sequence reaction of cDNA clone HPKII 29 primed with a 2 1-mer oligonucleotide (complementary to HPV-16, pos. 965-985) as length markers. RESULTS Isolation of HPV-16 positive sequence analysis

cDNA clones for DNA

For each cell line, HPKIB and HPKII, which were established by transfection of primary human foreskin keratinocytes with the prototype HPV-16 DNA (D&-St et al., 1987) a cDNA library was constructed. The prototype HPV-16 DNA is characterized by a frameshift mutation in the El ORF (Seedorf et a/., 1985; see also Fig. 1). By plating approximately 1O6 recombinant clones of each cDNA library and screening with a 32P-labeled complete genomic HPV-16 DNA probe 60 and 63 HPV-16 positive cDNA clones were identified for HPKIB and HPKII cells, respectively. The HPV-16 positive cDNAs ranged in size from 0.6 to 7.7 kb for the HPKIB cell line and from 0.6 to 6.6 kb for the HPKII cell line. The preferred size classes for HPKII were 0.8, 1.4 to 1.6, 1.8, and 2 kb, and in case of HPKIB 1.6 to 1.8 kb only. The cDNA clones were then subdivided into groups on the basis of the plaque hybridisation pattern obtained by using cloned subgenomic HPV-16 fragments spanning the entire genome as probes (see Materials and Methods). Of each group several representatives were analyzed further by reverse hybridization. Briefly, cloned Pstl subfragments of HPV-16 covering the entire genome were digested with different restriction en-

334

ROHLFS ET AL. Em RI 6619

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FIG. 1. Schematic representation of HPKII and HPKIB cDNA clones. The genetic organization of the HPV-16 genome depicted by ORFs (open boxes), utilized polyadenylation signals, and EcoRl restriction sites are shown on top. An additional thymidine residue at pos. 3903 generating an E5 ORF with an ATG triplet and an E5* ORF as described by Halbert et al. (1988) could be confirmed by sequence analysis of cDNA clones in this study. The nucleotide positions of 5’- and ?-ends and splice donor and acceptor sites are given for each cDNA clone. Because of the genomic organization of the HPV-16 DNA as shown at the top of the figure, HPKIB clone 83 had to be drawn in two parts (5’- and 3’-ends are indicated). (A) indicates spliced out sequences; open boxes refer to genes possibly encoded by these clones; zig-zag lines depict cellular sequences.

zymes thus creating small, but still distinguishable, overlapping fragments of 50-500 nt in length. These fragments were separated on a 1.2% agarose gel, transferred to a nylon membrane and hybridized with a nick-translated cDNA clone. Analysis of the hybridization pattern obtained for each of the 32P-labeled cDNA permitted a preliminary localisation of 5’- and 3’-ends and exon boundaries within a range of 50 to 300 nucleotides. Based on these results 1 1 cDNA clones representing different mRNA species were subcloned in M 13 and their nucleotide sequences were determined. HPV-16 messenger

RNA splicing

The organization of the cDNA clones as deduced from their nucleotide sequences are presented in Fig. 1. Two splice junctions were identified for cDNA clone 6 of HPKII cells. The first with a splice donor at pos. 226 and a splice acceptor at 409 yielded an E6*I ORF

which potentially encodes a truncated protein of 43 amino acids as described previously (Smotkin and Wettstein, 1986; Smotkin et a/., 1989; Tanaka et al., 1989; Schneider-Maunoury et al., 1990). The second splice junction (splice donor, pos. 880; splice acceptor, pos. 3357) fused the first 22 nucleotides of the El a ORF including its ATG triplet to the ninth codon of the E4 ORF, which itself possesses no methionine residue. The cDNA clone HPKII 6 terminated without a poly(A) sequence at pos. 4235 that is 16 nucleotides downstream of the early region polyadenylation signal. A corresponding mRNA has thus a coding potential for an E6*I, E7, and El,AE4 fusion protein as well as an E5 protein (for E 1iAE4 nomenclature, see Palermo-Dilts et a/., 1990). This particular mRNA species was also detected with high frequency in a cDNA library of a HPV16 positive primary squamous cervical carcinoma (Vormwald-Dogan, personal communication). cDNAs analogous to clone HPKII 6 (E6*I, E7, El,AE4, and E5

HPV-16 TRANSCRIPTION

IN HPK

335

Elb

TCTACC-ATCGCTGATCCTGCAGGACGTGGTCCACATTM GTl'TGC *et E2skt ACGAGG ACGAGGACAAGG AAAACG-ATGGAGACTClTTGCCAACGT

FIG. 2. Sequence analysis and structure of the splice junction identified for cDNA clone HPKII 59 with a coding capacity for a full-length E2 protein. On the left-hand side, the autoradiogram of a 6% polyacrylamide-urea sequencing gel spanning the splice junction (splice donor, pos. 880; splice acceptor, pos. 2708) is shown. The arrowhead refers to the splice junction. The structure of the mRNA as deduced from analysis of cDNA clone HPKII 59 is given beneath the ORFs of the early region of HPV-16. Nucleotide sequences around the intron-exon junctions are depicted additionally. The start codons of the Ela and E2 ORF are underlined, asterisks mark the stop codon, and the arrowhead points to the beginning of the E2 ORF.

coding potential) were shown to have transforming potential in mouse NIH 3T3 cells and rat fibroblast 3Yl cells (Tanaka et a/., 1989). A mRNA with a full-length E6 ORF is represented by cDNA clone HPKII 29, which showed a 5’-end at pos. 55 upstream of the cap site at pos. 97 mapped previously by Smotkin and Wettstein (1986) for CaSki cells. The 3’-end of this clone as well as that of clone HPKII 36 is probably artificial due to unspecific priming with oligo(dT) during cDNA synthesis at the adenosinrich region around pos. 21 17 in the corresponding mRNA. The number of cDNA clones with a full-length E6 ORF was determined by plaque hybridization using oligonucleotides derived from known exon and intron regions of the E6 ORF (Smotkin and Wettstein, 1986; Smotkin et a/., 1989; Doorbar et a/., 1990). The first one (HPV-16, pos. 274-293) originated from the intron region of all spliced ORFs (E6*I, E6*ll, and E6*lll) and the second one (HPV-16, pos. 151-171) from the exon region of E6 ORF. Of the 65 cDNAsfrom both cell lines, which encoded the E6 and E7 ORFs, only six possessed a full-length E6 ORF (see also Fig. 1, HPKII clone 29 and HPKIB clone 83), the rest of the cDNAs contained spliced E6* ORFs (data not shown). The cDNA clones HPKII 59 and HPKIB 27 each contained a splice junction that joined the splice donor at pos. 880 to an alternative splice acceptor at pos. 2708 that is 47 nt upstream of the E2 translation initiation codon (see Fig. 2). This ATG triplet is preceded by the

in frame start codon of the El a ORF at pos. 865 and an in frame terminator codon located at pos. 2722. Thus the first exon of these clones consisted of a truncated E7 ORF and a small 5’ portion of the El a ORF whereas the second exon encompassed the entire E2 ORF. For termination the early region polyadenylation signal (pos. 4214) was utilized (polyadenylation site: HPKII clone 59 at pos. 4236; HPKIB clone 27 at pos. 4231). The corresponding mRNA potentially encodes a fulllength E2 protein. The same splice event was shown for clone HPKIB 25. However, the 5’-end of this clone mapped to the E6 ORF not to the E7 ORF as for the other two cDNAs described above and the E2 ORF was interrupted by cellular sequences. cDNAs mapping to the late region of HPV-16 Three of the 123 HPV-16 positive cDNA clones mapped to the late region of the viral genome. The first nucleotide of clone HPKII 72 was located at pos. 4199 upstream of the potential ATG start triplet of the L2 ORF, the last nucleotide coincided with the internal fcoRl restriction site of HPV-16 (pos. 6819). This end of the clone was therefore probably due to insufficient methylation of the HPV-16-specific fcoRl restriction site during linker trimming. It is not clear whether this clone represents a mRNA which had initiated in the late region, since first-strand synthesis may have terminated prematurely. Moreover the polarity of the mRNA

336

ROHLFS ET AL.

remains unknown. Yet, similar mRNAs encompassing the L2 and Ll ORFs have been identified for HPV-1 (Chow et a/., 198713) HPV-6 and -11 (Chow et a/., 1987a) by the electron microscopic R-loop technique and for BPV-1 as a polyadenylated cDNA obtained from a productively infected fibropapilloma (Baker and Howley, 1987). In each case it was described as a very rare mRNA or cDNA. The corresponding mRNA might encode for a L2 protein. Clone HPKII 46 mapped to the Ll ORF (pos. 60287343) and terminated with a poly(A) sequence 18 nucleotides downstream of a polyadenylation signal AATAAA at pos. 7320. At the 5’-end of this cDNAclone 22 nucleotides with no apparent homology to either HPV-16 or Ml 3 were identified and probably represent cellular sequences. Clone HPKIB 83 represents a virus-cell fusion transcript of about 7.7 kb containing approximately 4.3 kb of cellular sequences (Fig. 1). The HPV-16 specific region covered part of the Ll ORF, the entire upstream regulatory region (URR), E6, E7, El a, and the 5’ part of the El b ORF ending at pos. 1594. The virus-cell junction was located at pos. 6157 in the vicinity of the HPV16 BamHl restriction site (pos. 6151). Since HPK cells were established by transfection of vector purified (BarnHI) partially religated HPV-16 DNA, it appears likely that this virus-cell junction is due to fusion of a linear viral molecule to cellular DNA. Because this cDNA clone lacked a poly(A) sequence, the orientation of the corresponding mRNA in HPKIB cells was determined by reverse transcription of HPK RNA followed by polymerase chain reaction (PCR). A fragment of predetermined size (175 bp) could be amplified by PCR only, if reverse transcription was primed with an oligonucleotide complementary to the coding strand of the HPV16 genome (oligonucleotide complementary to pos. 6241-6259; for PCR a second oligonucleotide primer taken from the flanking cellular sequence was used) (data not shown). Initiation of transcription of the corresponding mRNAs is thus directed from a cellular promoter upstream of the 5’ flanking cellular sequences. The same 175-bp fragment was amplified using HPKIB DNA indicating that the virus-cell junction of this cDNA clone represents an authentic breakpoint for viral integration (data not shown). Virus-cell

fusion transcripts

One virus-ceil fusion transcript, clone HPKIB 83, of the HPKIB cell line in which the HPV-16 DNA is integrated at a single cellular site (Dlirst et a/., 1987) has been described above. In contrast to this virus-cell fusion transcript, which was shown to initiate at a cellular promoter in the 5’-flanking cellular sequences, the initi-

ation of another fusion transcript, represented by clone HPKIB 25, was directed from the viral P97. This polyadenylated cDNA comprised the E6*I, E7, and the 5’-part of the E2 ORF, and cellular sequences at the 3’-end. The virus-cell junction was mapped at pos. 3572 in the E2 ORF and probably represents the opposite breakpoint for viral integration. For clone 50 of the HPKII cell line an E6*I and E7 ORF was identified. The El ,AE4 fusion ORF was interrupted by cellular sequences with a virus-cell junction site at pos. 3406 again in the E2/E4 ORF. This site may correlate with one breakpoint of the viral genome in this cell line (two integration sites were identified for HPKII; data not shown), since no splice donor or acceptor consensus sequence was evident in the HPV-16 genome at this position. In contrast the virus-cell junction of clone HPKII 8 most likely represents HPV-16 sequences spliced into the host genome, since the last HPV-16 specific nucleotide at pos. 880 corresponds to the common HPV-16 splice donor (see HPKII clones 6, 50, and 59 and HPKIB clones 25 and 27). The cellular origin of the DNA sequences flanking the viral sequences of clone HPKIB 83 and HPKIB 25 was confirmed by hybridization of a ?labeled subfragment of each clone, which was expected to represent unique cellular regions, to digested HPKIA, HPKIB, HPKII, and placenta DNA (data not shown). No sequence homology was found between the sequenced cellular regions of the different cDNAs. The EMBL and GenBank sequence libraries, and NBRF protein sequence library were searched for sequences related to the flanking cellular sequences, but no significant homology could be found. Utilization

of HPV-16 polyadenylation

signals

cDNA clones which mapped to the early region of HPV-16 only (clone HPKII 59 and HPKIB 27) were polyadenylated 12 and 17 nucleotides downstream of the termination signal AATAAA at pos. 42 14, respectively. This appears to be the commonly used polyadenylation signal for mRNAs of the early region (Seedorf et a/., 1985; Tanaka et al., 1989). Of the three late putative mRNA polyadenylation signals, which map at pos. 7261, 7320, and 7660, the one at pos. 7320 was used by the one and only polyadenylated cDNA clone (HPKII 46) which mapped to the late region. In this case the polyadenylation site was 18 nucleotides downstream of this 3’-terminating signal, also referred to as LP2 (Kennedy et a/., 1990). Mapping of 5’-ends of HPV-16 E6 transcripts HPKIA, HPKIB, and HPKII cells

in

The sequenced 5’-ends of the cDNA clones encompassing the E6*I ORFvaried from pos. 108 to 150, all of

HPV-16 TRANSCRIPTION

them being located downstream of the second ATG triplet of the E6 ORF (pas. 104) and the cap site at pos. 97 which has previously been described by Smotkin and Wettstein (1986) and Smotkin et al. (1989) for the HPV-16 positive cervical carcinoma cell lines CaSki and SiHa. It is very likely that the lack of information at the 5’-end of these cDNA clones is due to a premature termination of first-strand synthesis and/or to Mung bean digestion during cDNA preparation. To map the 5’-ends of the corresponding mRNAs precisely and to investigate the existence of a possible minor RNA species containing an E6*ll splice junction, primer extension as well as Sl nuclease and exonuclease VII digestions were performed. CaSki cells were used as a control since the HPV-16 E6/E7 transcripts in this cell line were mapped previously by Smotkin and Wettstein (1986). The strategy employed together with results and interpretation are presented in Fig. 3. Primer extension reaction resulted in a cluster of bands from pos. 95 to 98, at pos. 103, and at pos. 106 for HPKIB and HPKII RNA. The exact position of the primer extension products was determined by a nucleotide sequence reaction which was primed with the same oligonucleotide that was also used for primer extension. Another rather prominent signal around pos. 22 was consistently observed. Furthermore, a TATA box (TATATAA) is located 31 nt upstream of this position in the HPV-16 sequence. This may be indicative of an additional initiation site of transcription in the URR and correlates with the sequence data obtained for clone HPKII 29 which had a 5’-end upstream of P97 at pos. 55. Sl nuclease digestion of HPKIA and HPKII poly(A)+ RNA resulted in three bands of about 770, 458, and 341 nt in length in agreement with results published for CaSki cells (Smotkin and Wettstein, 1986). The band of 458 nt, which represents the second exon of mRNAs containing an E6*I splice junction, was the most prominent for HPKIA and HPKII and also for CaSki. Similarly mRNAs with an unspliced E6 ORF or with an E6*ll splice junction represent only minor species in each of the cell lines examined, giving rise to protected fragments of about 770 nt and 341 nt in length, respectively. In the exonuclease VII digestion experiments only one band of about 770 nt was protected for all three cell lines indicating the same 5’-end for all mRNAs encompassing the E6 region. The clusters of bands in the exonuclease VII protection assay located in the bottom part of the gel appeared in all lanes including HaCaT poly(A)+ RNA (negative control) and a control lane containing the digested 32P-labeled probe only. These degradation products persisted in further experiments in

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IN HPK

which higher exonuclease VII concentrations were used. On the basis of the Sl nuclease and exonuclease VII protection assays as well as the cDNA analysis it is concluded that the vast majority of HPK mRNAs spanning the E6 ORF carried an E6*I splice junction and only rare species contained either an unspliced E6 ORF or an E6*ll splice junction.

DISCUSSION Because of the role of E6 and E7 as putative transforming proteins in virus-linked carcinogenesis attention has mainly focussed on the expression of the HPV-16 and -18 E6/E7 ORFs in genital tumors and tumor derived cell lines. A detailed transcriptional analysis of these genes may reveal differences that relate to the benign or malignant phenotype of the cells or tissue in question. In the HPV-16 positive cervical carcinoma cell lines CaSki and SiHa three different transcripts spanning the E6/E7 region were mapped: a major mRNA with an E6*I ORF (splice donor, pos. 226; splice acceptor, pos. 409) a minor one with a full-length E6 ORF, and a minor mRNA with an E6*ll ORF (splice donor, pos. 226; splice acceptor, pos. 526) (Smotkin and Wettstein, 1986; Smotkin ef al., 1989). The cell lines (HPK) investigated in this study were established by transfection of human primary foreskin keratinocytes with cloned HPV-16 DNA. The viral genome in these cells is stably integrated and transcriptionally active, but the cell lines are not tumorigenic in nude mice (Durst et a/., 1987, 1991). For the HPKIB and HPKII cell lines a total of 65 cDNA clones spanning the E6/E7 region were identified. Of these, 59 individual clones showed a spliced E6* ORF and only 6 contained a full-length E6 ORF. E6*I and full-length E6 cDNAs have been identified by sequence analysis. The spliced E6*I ORF represented the major transcript of the E6 region in HPK cells as shown by Sl nuclease and exonuclease VII digestion, whereas full-length E6 and E6*ll mRNAs represented minor species. In fact the latter was only identified by Sl nuclease analysis. The same classes of E6 mRNAs have previously been described for the SK-v cell line, which has been established from a vulvar intraepithelial neoplasia (Schneider-Maunout-y eta/., 1990) and for a number of HPV-16 containing premalignant and malignant cervical lesions (examined by PCR preceded by reverse transcription; Cornelissen et al., 1990; Johnson et al., 1990). In contrast, a cell line derived from a low grade cervical lesion (W12), which is unique among HPV-16 containing cell lines, because the viral genome persists as multicopy episome, only two E6 mRNA species, E6*I and E6*lll, but no full-length E6

338

ROHLFS ET AL.

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HPV-16 TRANSCRIPTION

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FIG. 3. (A) Primer extension analysis of HPKIB and HPKII total cytoplasmic RNA. A 5’.labeled oligonucleotide (21-mer; complementary to HPV-16 pos. 139-l 59) was used as primer for reverse transcription. The primer extension products were fractionated on a 6% polyacrylamideurea sequencing gel. In the lanes designated ATGC a DNA sequencing reaction of clone HPKII 29 primed with the same oligonucleotide is shown. The nucleotide positions of the primer extension products are marked by arrowheads. (B) Sl nuclease and exonuclease VII mapping of the E6 coding exons. In the Sl nuclease panel the 32P-labeled DNA probe with a length of 982 nt, which was used for both Sl and Exo VII digestions, is shown. The control lane shows digestion of the DNA probe only. In the lanes for HPKIA, HPKII, and CaSki poly(A)+ RNA protected bands of approximately 770. 458, and 341 nt in length are visible. The position of the bands are indicated by arrowheads on the left hand side. HaCaT poly(A)+ RNAwas used as negative control for both Sl and Exo VII digestions. The Exo VII panel shows in the control lane the 3*P-labeled DNA probe digested with 3 U exonuclease VII without prior hybridization to RNA. A protected band of about 770 nt is visible for HPKIA, HPKII, and CaSki poly(A)+ RNA. The molecular size markers (lane designated marker) are 3ZP-labeled Hinpl fragments of Bluescribe DNA. Fragment sizes in nucleotides from top to bottom are 503, 412, 393, 337, 332, and 270. To estimate the length of protected fragments more precisely a DNA sequencing reaction done with a 21 -mer oligonucleotide (complementary to HPV-16, pos. 965-985) is shown. The samples were separated on a 6% polyacrylamide-urea gel. (C)At the top the E6/E7 region of HPV-16 is presented with ORFs indicated as open boxes. The position of ATG triplets in each ORF are marked by a vertical line. The v-labeled probes used for primer extension, Sl nuclease, and exonuclease VII analyses and the extended or protected bands, respectively, are indicated for each method separately. The Hphl and Ncol restriction sites of HPV-16 were utilized to generate the DNA probe for the RNA-DNA protection assays. The bottom part of the figure assigns the protected bands after Sl nuclease digestion (Indicated as thick lines) to the appropriate exon of the E6 region. (A) represents spliced out sequences.

and E6*ll spliced mRNAs were identified (Doorbar et a/., 1990). The E6*lll mRNA has the potential to encode a not yet identified E6*lll protein containing amino acids derived from an out of frame region of the EZ/E4 ORF at the C terminus. An E6*lll mRNA was not identified among the HPK cDNA clones analyzed and was not tested for by PCR. A consistent qualitative difference between mRNA species spanning the E6/E7

ORF, which correlates with either a benign, a premalignant or a malignant phenotype of the cell line or tissue in question is not evident so far. A detailed analysis of a large number of clinical biopsies by reverse transcription and PCR in combination with an analysis of the topographical distribution of the viral RNA in this very heterogeneous tissue might shed some more light on this issue.

340

ROHLFS ET AL.

A novel cDNA species spanning the E2 ORF was identified in HPKIB and HPKll cells (HPKII clone 59; HPKIB clone 27). A short leader exon composed of a truncated E7 ORF and the first 22 nucleotides of the Ela ORF is joined to a second exon which spans the entire E2 ORF (splice donor, pos. 880; splice acceptor, pos. 2708). The splice acceptor is positioned 47 nt upstream of the putative E2 translation initiation codon (pos. 2755). A corresponding mRNA has the potential to encode a full-length E2 protein. Expression of an E2 protein by this mRNA species would require internal reinitiation, since the E2 ATG triplet is preceded by a small ORF characterized by the “strong” El a initiation codon (pos. 865; Kozak et al., 1986) and an in frame terminator codon (pos. 2722). Rotenberg et a/. (1989a,b) recently identified an analogous splice junction for HPV-11. Furthermore, they showed that this class of cDNA (which also encompassed an intact E6 and E7 ORF in the first exon) is coding for a functionally active E2 protein, which transactivated the E2-dependent conditional enhancer of the HPV-11 URR. Also in the case of these HPV-1 1 cDNAs internal reinitiation would be a prerequisite for translation of an E2 protein. In the HPV-16 positive cervical carcinoma derived cell line CaSki and in the W12 cell line, which harbors HPV16 DNA as episomal plasmid, a full-length E2 transcript could not be identified (Smotkin and Wettstein, 1986; Doorbar et al., 1990). Because a full-length E2 protein could potentially be expressed in the HPKIB and HPKII cell line, it will be of interest to investigate, if it is functionally active in these cells. Moreover, the effect of E2 expression on the steady-state level of E6 and E7 transcripts in HPK cells should be investigated, since the full-length E2 protein has been shown to act as a repressor of the basal activity of the homologous promoter at pos. 97 (Romanczuk et a/., 1990). The 5’-end of both c-DNAs described above (HPKII 59 and HPKIB 27) was located at nt 624 in the E7 ORF. These 5’-ends could be artificial due to incomplete cDNA synthesis. Yet, based on RNase protection assays performed with an in vitro transcript covering the 5’-part of the E7 region (preliminary data; not shown) we assume, that internal initiation of transcription in the E7 ORF is a more likely explanation. Similar mRNA species with a 5’-end in the E7 ORF were observed for HPV-1 1, HPV-6 (Chow et a/., 1987a), and HPV-1 (Palermo-Dilts et al,, 1990). Further support for promoter activity in the E7 ORF of HPV-16 is given by primer extension analysis of RNA from W12 cells (initiation sites ranging from pos. 626 to 795; Doorbar et a/., 1990). However, in the W12 cell line mRNAs which initiated in the E7 ORF encoded an El AE~ and E5 protein, but not the full-length E2 protein.

Analysis of cDNA clones also permitted the precise mapping of viral integration sites. For the HPKIB cell line one of the breakpoints within the viral genome was located at pos. 6157 in the Ll ORF (cDNA clone HPKIB 83). This was verified by PCR on RNA and DNA level (data not shown). It is very likely that cDNA HPKIB 25 represents the opposite virus-cell junction (pos. 3572 in the E2 ORF). Since the HPKIB cells harbor approximately one and a half copies of HPV-16 at a single integration site as deduced from Southern blot analysis (data not shown), cDNA clone HPKIB 27 which encompassed the entire E2 ORF, may originate from the intact HPV-16 genome, whereas clone HPKIB 25 may originate from the incomplete part of the viral genome. In HPKII cells about ten copies of the HPV-16 DNA are integrated at two different chromosomal sites. cDNA clone HPKII 50 may originate from a virus-cell fusion transcript spanning a possible integration site at pos. 3406 again in the E2/E4 region, whereas clone HPKII 8 is most likely a product of HPV-16 RNA spliced into flanking cellular sequences. At the 5’-end of clone HPKII 46, 22 nucleotides not related to either HPV-16 or M 13 were detected by sequencing. This clone may therefore also represent a virus-cell junction. Of the three late putative polyadenylation signals with the sequence AATAAA, the signal at pos. 7320 (LP2) was utilized in this cDNA clone. This is in agreement with results obtained by Kennedy et al. (1990). They showed that for HPV-16 LP2 was utilized in vitro in HeLa cell extracts as well as in vivo after transfection of HeLa cells rather than the two other putative late polyadenylation signals (LPl , pos. 7261; LP3, pos. 7660). Primer extension analysis of HPKIB and HPKII RNA revealed heterogeneous initiation around P97 (bands at pos. 95 to 98, 103, and 106). A similar heterogeneous initiation around P97 was also published by Schneider-Maunoury et al. (1990) for Sk-v cells, a HPV16 positive cell line derived from a vulvar intraepithelial neoplasia (see also Smotkin and Wettstein, 1986). A signal around position 22 which consistently appeared, is suggestive of an additional initiation site upstream of P97 in the URR. Furthermore, a TATA box is located 31 nt upstream of this position in the HPV-16 genome. The 5’-end of cDNA clone HPKII 29 (at nt 55) would also be in support of this assumption. Schneider-Gadicke and Schwarz (1986) mentioned possible additional initiation sites 100, 1 15, 210, and 350 nucleotides upstream of the E6 ATG triplet of HPV-18 in the cervical carcinoma cell lines HeLa, C41, and SW 756. They analyzed 16 cDNAclones derived from the three different cell lines. The 5’-ends of two SW 756 clones (SW 756 7-7 and SW 756 7.23) were located at pos. 69 and 62 upstream of the major pro-

HPV-16 TRANSCRIPTION

moter at pos. 105 of HPV-18. Both cDNA clones showed an unspliced exon for the E6 ORF. This is consistent with cDNA clone HPKII 29. There may be a correlation between transcriptional initiation upstream of P97 and P105, respectively, and an unspliced E6 ORF. However, further analysis is needed in support of this interpretation, especially since no respective signal could be detected in our Sl nuclease and exonuclease VII mapping studies. ACKNOWLEDGMENTS We are grateful to Drs. H. zur Hausen and L. Gissmann for continued support and discussion and to Drs. M. Pawlita, E. Schwarz, and G. Sczakiel for critical reading of the manuscript. The patient assistance of M. MUller in computer usage is gratefully acknowledged. We thank D. Glitz for technical assistance, Dr. H. Bludau and V. Vormwald-Dogan for technical advice, and Drs. P. Boukamp and N. Fusenig for HaCaT ceils. This work was supported by the Deutsche Forschungsgemeinschaft (DO 162/l and DO 162/l -1).

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