Identification of a new promoter in the early region of the human papillomavirus type 16 genome

T. H. Braunstein1, B. S. Madsen1, B. Gavnholt1, M. W. Rosenstierne1, C. Koefoed Johnsen1 and B. Norrild1

Institute of Molecular Pathology, The Protein Laboratory, University of Copenhagen, Panum Institute, Bldg 6.2, Blegdamsvej 3C, DK-2200 Copenhagen N, Denmark1

Author for correspondence: Bodil Norrild. Fax +45 3536 0116. e-mail bono{at}biobase.dk


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
Transcription of the human papillomavirus type 16 (HPV-16) genome is controlled by several promoters; the P97 promoter is considered to be the main one. An additional promoter has been identified within the E7 ORF as well as an antisense promoter just upstream of the L2 ORF. The significance of these promoters for early and late gene expression and their activity related to cell differentiation is not known in detail. Identification of two new, previously undescribed transcription start sites at nt 542 just upstream of the E7 ORF and at nt 611 within the E7 ORF is reported. The promoter responsible for the start site at nt 542 (P542) was active in SiHa, HeLa and C33A cells. Very low promoter activity was found upstream of the nt 611 start site. The E7 protein has previously been shown to be synthesized from a polycistronic mRNA encoding both the E6 and E7 proteins under the control of the P97 promoter. The data reported in the present paper suggest that promoter P542 may control synthesis of the E7 oncoprotein from a monocistronic mRNA.


   Introduction
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Abstract
Introduction
Methods
Results
Discussion
References
 
Human papillomaviruses (HPVs) are epitheliotropic DNA viruses with a double-stranded circular DNA genome of approximately 8 kbp. The coding strand of the genome encodes six or seven early and two late proteins, and has a long control region (LCR) of approximately 750 bp (for review, see zur Hausen, 1996 ; Vousden, 1993 ). More than 80 different HPV types have been identified and new types are currently being isolated (de Villiers, 1994 ). The HPV types are divided into malignant and benign types; HPV-16 is considered to be the prototype among the malignant HPVs and HPV-11 is considered to be the benign prototype (Vousden, 1993 ). The early ORFs E1 and E2 encode the proteins necessary for viral DNA replication, episomal maintenance and transcriptional regulation, the E1{wedge}E4 ORF encodes a protein which binds to the intermediate filaments (Roberts et al., 1997 ), and the E5, E6 and E7 ORFs encode oncoproteins (Hawley-Nelson et al., 1989 ; Crook et al., 1989 ; Münger et al., 1989 ; Leptak et al., 1991 ; Conrad et al., 1993 ). The late ORFs encode the two capsid proteins L1 and L2 (for review, see zur Hausen, 1996 ).

Regulation of gene expression has been studied in several of the HPV types and the locations of the different promoter regions identified for HPV-16, -18, -31b, -11 and -6b are similar. The viruses have a major promoter in front of the E6 ORF [P97 in HPV-16 (Smotkin & Wettstein, 1986 ), P105 in HPV-18 (Thierry et al., 1987 ), P97 in HPV-31b (Hummel et al., 1992 )]. In HPV-11, the major promoter is in the E7 ORF (Chow et al., 1987 ). There are, however, promoters at nt 99 in HPV-11 and at nt 80 in HPV-6b which are responsible for E6 protein expression (Zhao et al., 1997 ; Karlen et al., 1996 ). Several additional promoters have been identified in the early region of the genome, i.e. in the E7 ORF of HPV-16 (Higgins et al., 1992 ; Doorbar et al., 1990 ; Grassmann et al., 1996 ), HPV-31b (Hummel et al., 1995 ; Ozbun & Meyers, 1997 ), HPV-11 (Nasseri et al., 1987 ; DiLorenzo & Steinberg, 1995 ) and HPV-6 (Chow et al., 1987 ; Karlen et al., 1996 ). The E7-localized promoters of HPV-16, -31b and -11 are differentiation-dependent (Cheng et al., 1995 ; DiLorenzo & Steinberg, 1995 ; Grassmann et al., 1996 ; Ozbun & Meyers, 1997 , 1998 ). A promoter is present in the E6 ORF of HPV-6b as observed by Karlen et al. (1996) and Smotkin et al. (1989) . This promoter is thought to be responsible for E7 mRNA in benign HPV types. Promoters have been identified just upstream of or within the E2 ORF such as P2598 and P3036 of HPV-18 (Karlen & Beard, 1993 ) and P3270 of HPV-6b (Karlen et al., 1996 ). Late promoters were identified within the E4 ORF of HPV-31b (P3320) (Ozbun & Meyers, 1998 ), just downstream of the E5 ORF of HPV-16 (Geisen & Kahn, 1996 ; Maki et al., 1996 ), and between the L2 and L1 genes in HPV-31b (P5600) (Karlen & Beard, 1993 ). Late mRNAs might also be initiated from promoters upstream of the P97 of both HPV-16 and HPV-31b where transcription start sites have been identified at position nt 22 of HPV-16 (Rohlfs et al., 1991 ) and nt ~7375, nt 7783, nt 7850, nt 49 and nt 77 of HPV-31b (Ozbun & Meyers, 1997 , 1998 , 1999 ). An antisense promoter in the nt 4030–4230 region of the HPV-16 genome producing E7 antisense RNA was identified recently (Belaguli et al., 1997 ). The location of promoters in HPVs resembles that in bovine papillomavirus type 1 as promoters are identified in the upstream regulatory region and in the E6, E1 and E2 ORFs (Sousa et al., 1990 ).

Based on our previous study, in which we demonstrated translation of the HPV-16 E5 protein from a polycistronic mRNA encoding the entire E2 and E5 ORFs (Johnsen et al., 1995 ), we initiated a search for promoters in the early region of the genome applying a new method for the identification of transcription start sites. We report the identification of two new transcription start sites at nt 542 and nt 611 just upstream of and in the E7 gene, respectively. The promoter region P542 was characterized and shown to be active in both the HPV-positive cell lines HeLa and SiHa, and in the HPV-negative cell line C33A. The potential specificity of this new promoter for E7 is discussed.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Cell culture.
The HPV-negative cervical carcinoma cell line C33A, the HPV-18-positive cervical cell line HeLa and the HPV-16-positive cervical cell line SiHa were maintained in Dulbecco’s modified Eagle’s 1885 medium (DMEM) supplemented with 10% foetal calf serum (FCS), 1% l-glutamine, and 1% penicillin and streptomycin at 37 °C and 5% CO2.

{blacksquare} Cell transfections.
The day before transfection, 2x105 cells were seeded in 3·5 cm Petri (6-well) dishes (NUNC). Just before transfection, the medium was replaced with serum-free DMEM containing 1% l-glutamine, and 1% penicillin and streptomycin. The cells were transfected using Lipofectamine Plus (Gibco BRL) according to the procedure recommended by the manufacturer. Each dish was transfected with 0·5 pmol plasmid DNA, adjusting for differences in the size of the constructs. Three hours after transfection, the medium was replaced with DMEM containing 10% FCS, 1% l-glutamine, and 1% penicillin and streptomycin.

{blacksquare} Cloning in the pGL3 vectors.
The vectors contain the firefly luciferase reporter gene. To increase the signal of the 97LCR construct relative to the empty vector (pGL3-Basic; Promega), particularly in C33A cells, the pGL3-Basic vector was modified by insertion of a 103 bp HPV-16 fragment (nt 2428–2530) in the KpnI site in front of the DNA fragment of interest. The inserted segment contains seven ATG codons, rendering translation of the luciferase gene from messengers originating from the vector very unlikely. The segment was PCR-amplified with Pfu polymerase (Stratagene) using specific HPV-16 primers containing restriction sites for KpnI and stop codons in both directions in all three reading frames (see Table 1). HPV-16 fragments to be tested for promoter activity were PCR-amplified with Pfu polymerase and specific primers containing restriction enzyme sequences for KpnI (sense) and NheI (antisense). The fragments were cloned into the KpnI and NheI sites in the MCS of the pGL3-Basic vector, ensuring that the first downstream ATG codon was the initiation codon for the luciferase gene (Fig. 4).


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Table 1. The names, coordinates and sequences of the primers used for PCR of HPV-16 DNA fragments and for CapFinder PCR

 


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Fig. 4. The activity of the LCR constructs and the L constructs. The map location of the potential promoters relative to the E6 and E7 ORFs are shown in the top left. DNA fragments cloned into the modified pGL3-Basic vector are shown below the map on the left; those with the same initiation site are filled in with the same pattern. Luciferase activity is shown on the right. Luciferase activity was measured relative to the activity of the 97LCR. The modified pGL3-Basic vector is shown as a negative control. Filled-in bars, SiHa cells; open bars, HeLa cells; hatched bars, C33A cells. Each column represents the mean value of at least 12 measurements; SD is marked for each column.

 
 Cloning of the selected short DNA fragments into the KpnI and NheI sites of the unmodified pGL3-Enhancer vector (Promega) was also done by PCR amplification as described above. All the DNA fragments were designed without any ATG codons between the putative cap site and the 3'-end of the fragment (Fig. 5). The mutations were designed based on a transcription factor search using Tess-String-Based Search: Basic Query Results Transfac v.3.2 and MatInspector v.2.2 on the HPV-16 sequence. The program listed transcription factors predicted to bind in the HPV-16 genome. All constructs were sequenced to verify that both the 3'- and 5'-ends were correctly inserted into the vector. The point mutations introduced in the three mutated nt 489–556 fragments were also verified by sequencing.



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Fig. 5. The activity of constructs in which fragments with only one or two transcription start sites were inserted in the pGL3-Enhancer vector. The map location of the potential promoters relative to the E6 and E7 ORFs is shown in the top left. The DNA fragments tested are illustrated below the map on the left; those with the same initiation site are filled in with the same pattern. Start site nt 97, 28–102E; start site nt 482, 272–496E and 448–496E; 272–448E contains the upstream fragment; start site nt 542, 272–556E and 489–556E; start site nt 611, 549–622E; start site nt 670, 615–676E. Luciferase activity measured relative to the pGL3-Enhancer vector in all three cell lines is shown on the right and each column is the mean of at least 12 measurements. The negative control is the modified pGL3-Basic vector. SD is marked for each column. Filled-in bars, SiHa cells; open bars, HeLa cells; hatched bars, C33A cells.

 
{blacksquare} Luciferase assay.
Two days after transfection, the cells were washed with PBS and lysed for 90 min in 300 µl Cell Culture Lysis reagent (Promega) per dish. Cell lysate (20 µl) was mixed with 50 µl luciferin substrate (Promega) and measured in a TD-20/20 luminometer (Turner Designs) with 3 s delay and 12 s integrate period. Each plasmid construct was tested in triplicate in at least two independent experiments with two different DNA preparations (a minimum of 12 transfections). The promoter activities of the pGL3-Basic constructs were all calculated relative to the activity of the 97LCR construct which was included in every experiment. The activities of the pGL3-Enhancer constructs were calculated relative to the empty pGL3-Enhancer vector.

{blacksquare} In vitro transcription.
The plasmid pBRHPV-16 [pBR322 containing the full-length HPV-16 genome linearized with BamHI (nt 6153)] was digested with PstI (nt 7010), NdeI (nt 281), SpeI (nt 1463) and BstXI (nt 2900). The three HPV-16 genome fragments covering the early ORFs were purified from an agarose gel. Each fragment (1·5 µg) was incubated with 0·4 mM ATP, 0·4 mM UTP, 0·4 mM CTP, 0·016 mM GTP, 10 µCi [{alpha}-32P]GTP, 3 mM MgCl2 and 8 units HeLa nuclear extract (Promega) at 30 °C for 1 h. The RNA was extracted with phenol–chloroform, the pellet was resuspended in nuclease-free water and the products were analysed on a 6% urea–polyacrylamide gel. Markers were made by T7 transcription of the pALTER-1 vector (Promega) linearized with EcoRV (nt 642), and the pALTER-Ex2 vector (Promega) linearized with HindIII (nt 164) and EcoNI (nt 1259) in the presence of [{alpha}-32P]GTP.

{blacksquare} CapFinder PCR cDNA synthesis and cloning.
The CapFinder PCR cDNA Synthesis kit (Clontech) is designed to amplify full-length cDNA from mRNA with the included oligo(dT) primer and a special 5'-primer (Chenchik et al., 1998 ). The 5'-primer has at its 3'-end three guanosines which are able to bind to the small (dC) stretch synthesized by the MMLV reverse transcriptase. The kit was used with 2 µg total RNA from the HPV-16-positive cell line CaSki and specific HPV-16 antisense primers instead of the included oligo(dT) primer (see Table 1). The best result was achieved by using Pfu polymerase for the secondary amplification steps. Otherwise, the standard protocol was followed. The specific HPV-16 antisense primers contained a NheI site. The cDNA was digested with NheI and cloned to XbaI and SmaI in the MCS of pBluescript II SK(+) (Stratagene), blue/white selected and sequenced.

{blacksquare} Sequencing.
For sequencing, the T7 Sequenase DNA polymerase kit version 2.0 (Amersham) was used according to the manufacturer’s protocol. Products were separated on 6 % urea–polyacrylamide gels and exposed to a Bio Max MR X-ray film (Kodak).

{blacksquare} Total RNA purification.
Total RNA was prepared using Ultraspec (BioTecx Laboratories) according to the instructions given by the manufacturer.

{blacksquare} Plasmid purification.
Plasmids were purified with the Qiagen Plasmid Midi kit following the procedure of the manufacturer. The concentration of the DNA was determined spectrophotometrically (Uvikon 922; Kontron Instruments) and verified by agarose gel electrophoresis.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Transcriptional analysis of the HPV-16 early genome
To search for promoters in the early region of the HPV-16 genome, three fragments of DNA (nt 7010–281, nt 281–1463 and nt 1463–2900) covering the LCR and part of the early region of the genome were analysed for initiation of transcription in a HeLa cell nuclear extract. The DNA fragments were obtained by restriction enzyme digestion of the HPV-16 genome as described in the Methods. A linearized control DNA construct with a CMV promoter (Promega) produced a transcript of the predicted size (363 bp) (Fig. 1, lane 1). The HeLa nuclear extract was free of endogenous transcriptionally active DNA as proven in a reaction free of externally added DNA (Fig. 1, lane 2). The DNA fragment nt 281–1463, which covers part of the E6, the E7 and the 5'-end of the E1 ORFs, showed a product of approximately 1000 nt indicating a transcriptional start site(s) in the region of nt 500 of the HPV-16 genome (Fig. 1, lane 3). The fragment nt 1463–2900 did not appear to have any transcriptional start sites (data not shown). This region covers the 3'-end of the E1 ORF and the beginning of the E2 ORF.



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Fig. 1. In vitro transcription of HPV-16 DNA using HeLa nuclear extract. Transcription was monitored by incorporation of [{alpha}-32P]GTP in transcripts initiated from promoters present in the HPV-16 genome. The positive CMV control fragment supplied with the HeLa nuclear extract gave the expected fragment of 363 nt (lane 1). The negative control without DNA is shown in lane 2. The DNA fragment covering nt 281–1463 gave a transcript of approximately 1000 nt (lane 3). The DNA fragment nt 201–622 made by PCR gave a transcript of 120–140 nt (lane 4). The marker was made by in vitro transcription with [{alpha}-32P]GTP of digested pALTER-1 and pALTER-Ex2 vectors and the fragment sizes are marked to the left of lane 1 and in the lane marked M. The transcripts were assayed on a 6% polyacrylamide gel.

 
To confirm the presence of a transcriptional start site in the vicinity of nt 500, a second in vitro transcription experiment was conducted using a Pfu PCR-amplified fragment of DNA. The fragment nt 201–622 gave rise to a transcript of approximately 120–140 nt in two separate experiments (Fig. 1, lane 4). This transcript corresponded to a transcriptional start site (on the sense strand) at nt 480–500, which is in agreement with the previous in vitro transcription experiment (Fig. 1, lane 3).

Identification of transcriptional start sites by CapFinder
The transcriptional start points in the E6/E7 region of the genome were further analysed with the aid of the CapFinder technique (Clontech) using total RNA isolated from CaSki cells. The method is similar to the RACE technique. Instead of the 3' oligo(dT) primer supplied by the manufacturer, we used specific antisense HPV-16 primers: 712b, 565b and 293b (Table 1). We performed three separate Pfu PCR amplifications of the cDNA with the CapSwitch II primer as the upstream primer and each of the three downstream primers. The PCR amplifications identified several DNA fragments corresponding to possible transcription initiation sites when analysed by agarose gel electrophoresis. The fragments amplified with primer 712b (corresponding to initiation sites nt 542 and nt 611), 565b (corresponding to nt 482) and primer 293b (corresponding to nt 97) are shown in Fig. 2(a). Sequence analysis of the cloned fragments amplified with the 293b primer showed the main transcriptional start site at nt 97 (Fig. 2b). The cap sites at nt 482 and nt 542 were found in PCR fragments amplified with the 565b primer and the 712b primer (Fig. 2c, d). As for the nt 97 cap site, these transcripts started with an adenine. The transcriptional start site at nt 542 is situated immediately upstream of the E7 ORF. Three cDNA clones revealed this transcriptional start site, which has not been shown previously. A new transcriptional start site in the E7 ORF at nt 611 was identified and the first nucleotide was a cytosine (Fig. 2e).



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Fig. 2. Amplification and sequencing of CapFinder cDNA clones. (a) Total CaSki RNA was extracted and used for RT–PCR using specific HPV-16 antisense primers and a specialized sense primer (CapSwitch II). This was followed by 35 cycles of PCR using the same primers. Lanes: 1, DNA fragment obtained with the downstream primer 712b (sequenced in d); 2, fragment obtained with primer 712b (sequenced in e); 3, fragment obtained with primer 565b (sequenced in c); 4, fragment obtained with primer 293b (sequenced in b). Molecular size markers (bp) are shown. (b) Transcriptional start site at nt 96/97 corresponding to the main promoter P97 of HPV-16. (c) Transcriptional start site at nt 482 (this clone has two consecutive CapSwitch II primers). (d) Transcriptional start site at nt 542. (e) Transcriptional start site at nt 611. The arrows show the first nucleotide of the HPV-16 sequence.

 
Promoter activity in keratinocyte cell lines
All DNA constructs were made by cloning of PCR-amplified DNA fragments of the HPV-16 genome into two different promoter-free luciferase reporter vectors, pGL3-Basic and pGL3-Enhancer containing the simian virus 40 (SV-40) enhancer. The pGL3-Basic was modified by insertion of a ‘nonsense’ segment, as described in the Methods, which reduced the luciferase activity caused by transcription initiation in the vector itself to about one-third of the activity of the empty vector. This was the negative control in Figs 4, 5 and 6. The activity of the pGL3-Control plasmid, which has an SV-40 promoter and enhancer, when measured relative to the 97LCR construct, was shown to be dependent on the cell line used (Fig. 3). The pGL3-Control plasmid was approximately threefold more active than the 97LCR construct in SiHa and HeLa cells, but in C33A cells the ratio was 18-fold (Fig. 3). The pGL3-Control plasmid was, however, 17-fold more active than the empty pGL3-Enhancer vector in all three cell lines (also shown in Fig. 3). The cell lines C33A, SiHa and HeLa were selected for the present study.



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Fig. 6. Activity of P542 from the 489–556E construct. Luciferase activity obtained from the wild-type fragment and the fragments with the mutations Mut1, Mut2 and Mut1+2 was measured in the three cell lines. The nucleotide sequences of the 5'- and 3'-ends of the fragment are shown on the left. The mutated nucleotides are shown in bold and underlined. Luciferase activity of the constructs is shown on the right. Activity is presented relative to the activity of the pGL3-Enhancer vector. The negative control is the modified pGL3-Basic vector. The value of each column represents the mean of at least 12 measurements; the SD is marked on each column. Filled-in bars, SiHa cells; open bars, HeLa cells; hatched bars, C33A cells.

 


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Fig. 3. Activity of the pGL3-Control and pGL3-Enhancer vectors. Luciferase activity was measured relative to the activity of the 97LCR construct. Activity is shown for all three cell lines tested. Each column represents the mean value obtained from at least 12 measurements. SD is marked for each column. Filled-in bars, SiHa cells; open bars, HeLa cells; hatched bars, C33A cells.

 
Transcriptional activity of the promoters
To characterize the responsible promoters and gain information on their activity, a series of reporter constructs was designed carrying different upstream regions in front of each identified cap site. The HPV-16 DNA constructs designed for each cap site and cloned into the pGL3-Basic vector are shown in Fig. 4. Two series of constructs were tested. One began at nt 7103 with the entire LCR and the early region including the cap site; these were named after the cap site followed by LCR. The second began at nt 272 of the HPV-16 genome; these were named after the cap site followed by L.

The constructs were transiently transfected into C33A, SiHa and HeLa cells and the luciferase activity, reflecting the transcriptional activity, was measured relative to the activity of 97LCR. In the HPV-negative C33A cells, the promoter activity of both 482LCR and 542LCR was about 60% (Fig. 4). The activity was similar for the L constructs. The 611LCR construct had approximately 20% activity. An analogous construct made for the known start site at nt 670 (670LCR) also showed 20% activity as illustrated.

When the same constructs were analysed after transfection into SiHa or HeLa cells, only the 482LCR and 542LCR constructs were active with a relative luciferase activity of 20–40% (Fig. 4). To gain information about the impact of upstream ATG codons on translational activity, the 3'-end of the 97LCR construct was extended to nt 141, thus including one ATG codon upstream of the luciferase start codon, and to nt 222 where four ATG codons were present. The presence of four ATG codons reduced the luciferase activity threefold (Fig. 4).

Promoter activity in the presence of the SV-40 enhancer
To characterize the promoter regions, DNA fragments of approximately 60–70 bp containing the identified cap sites were cloned into the pGL3-Enhancer vector with the SV-40 enhancer. All activities were measured relative to the empty vector. The activity of the P97 promoter in the absence of the LCR (28–102E construct) was 150–200% of the activity of the empty vector in all three cell lines tested (Fig. 5). Analysis of the P482 promoter activity using the 272–496E construct and the truncated fragment in 448–496E showed that the upstream sequences were necessary for activity. The promoter activity of the 272–496E construct in C33A and HeLa cells was approximately 200% and in SiHa cells was approximately 600%, whereas the truncated fragment was inactive in all three cell lines. The upstream sequence in 272–448E was as active as the full-length fragment in 272–496E in C33A and SiHa cells, but in HeLa cells, the 272–496E construct was less active than the 272–448E construct.

The P542 promoter in the 489–556E construct showed an activity of approximately 350% in C33A, SiHa and HeLa cells. When the P482 promoter region was present (272–556E), the SiHa cells showed an activity of approximately 700%. The activity of this fragment in C33A and HeLa cells was approximately 350% and thus equivalent to the activity of the 489–556E construct. The 549–622E construct with the transcription initiation site at nt 611 showed no activity in the cell lines tested (Fig. 5). The fragment might be too short but if more of the upstream sequence was added, the sequence would overlap the P542 region. The P670 promoter in the 615–676E construct showed the same activity (200%) as the P97 construct (28–102E) in SiHa and HeLa cells but it was less active in C33A cells (Fig. 5).

Mutational analyses of P542
A computer search of the HPV-16 sequence showed that the fragment nt 489–556 had potential binding sites for several transcription factors. Point mutations were introduced into a region that had abundant possibilities for consensus sequences as follows. Mut1 was mutated at positions nt 532 and 535 (A to C) and Mut2 was mutated at positions nt 552 (A to C) and nt 553 (G to T). Mut1+2 was mutated in all four positions. The three mutated nt 489–556 fragments were cloned into the pGL3-Enhancer vector and analysed in all three cell lines. The mutation Mut1 reduced promoter activity strongly only in SiHa cells; this was also the case for Mut2. The Mut1+2 abolished activity in all three cell lines (Fig. 6). These data indicate that the activity of P542 was dependent on binding of transcription factors to specific sequences.


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
CaSki cells have approximately 600 integrated copies of the HPV-16 genome linked head-to-tail and this line was therefore selected as the source of HPV-16 RNA (Smotkin & Wettstein, 1986 ; Smits et al., 1991 ). Our investigations have revealed two novel putative intragenic promoters in HPV-16, which have not been described previously. These putative promoters are located upstream of and in the E7 ORF, respectively. Transcriptional start sites in the E6/E7 region have been previously reported for HPV-16, but the corresponding promoter regions have not been characterized (Doorbar et al., 1990 ; Higgins et al., 1992 ; Grassmann et al., 1996 ). The major cap site at nt 97 was first discovered in CaSki cells (Smotkin & Wettstein, 1986 ) and later also in the HPV-16-positive cell lines W12 (Doorbar et al., 1990 ) and HPKII (Rohlfs et al., 1991 ).

Our in vitro transcription experiments identified transcriptional start sites corresponding to approximately nt 100 (data not shown) and nt 500. However, CapFinder experiments using total RNA from CaSki cells confirmed both the presence of the P97 promoter and the existence of additional transcriptional start sites at nt 482, nt 542 and nt 611. The nt 482 site is probably identical to the nt 480 transcriptional start site observed in raft cultures of the HPV-16-positive cell line KG with both primer extension and RACE cDNA analysis (Grassmann et al., 1996 ). The promoter corresponding to the nt 480 transcriptional start site was not further characterized. We therefore included this region of the genome in the promoter analysis using the luciferase reporter gene and the cell lines SiHa, HeLa and C33A. Our data support the proposal that within fragments nt 272–496 (P482) and nt 489–556 (P542) there are two individual, transcriptionally active promoters.

The analysis of the promoter constructs in the pGL3-Basic vector where the entire upstream region of the genome was present including the LCR, showed activity of the promoter constructs 482LCR and 542LCR in all three cell lines. It should be noted, however, that the HPV-negative cell line C33A was less prone for transcription of HPV promoter-driven sequences than the HPV-positive cell lines SiHa and HeLa. This was supported by several observations. Firstly, the activity of the modified pGL3-Basic vector relative to that of the 97LCR showed a high background activity of 10% in C33A cells (Fig. 4). Secondly, the activity of the pGL3-Control vector, which was driven by the SV-40 promoter and enhancer relative to the 97LCR, indicated that the 97LCR was more active in the HPV-positive cells than in the C33A cells (Fig. 3). Thirdly, the LCR constructs were 10–19 times more active than the 482L and 542L constructs in HPV-positive cells demonstrating a strong influence of the HPV enhancer region in these cells (Fig. 4). All three observations indicate that cells already adapted to HPV give the most reliable results for HPV-16 promoter activity. This is in agreement with the observation that the activity of the HPV-31b LCR correlates to the level of AP1, which is reported to be low in C33A cells (Kyo et al., 1997 ).

The presence of ATG codons upstream of the reporter gene start codon in the LCR constructs also influenced the transcription of the reporter gene to some extent. The presence of one ATG codon (construct 97LCR+1ATG) did not affect the promoter activity, but four ATG codons (construct 97LCR+4ATG) did reduce the transcription initiation to approximately one-third in all three cell lines (Fig. 4). This was in accordance with the observations of Stacey et al. (1995) , who found that the presence of four upstream ATG codons present in a polycistronic messenger for the HPV-16 E7 gene left a residual activity of only 10% compared to that of a monocistronic E7 messenger. This observation revealed that the activities measured for the 482LCR and 542LCR constructs are most likely due to promoters other than P97, as at least four or more ATG codons are present upstream of the two transcription initiation sites. The promoter activity of the short DNA fragments nt 272–496 (with the nt 482 cap site) and nt 489–556 (with the nt 542 cap site) inserted into the pGL3-Enhancer vector confirmed the presence of two new promoters which were active in all three cell lines (Fig. 5). The 28–102E construct with P97 showed a surprisingly low activity, which could not be increased if the construct was made longer (1–102E construct; data not shown). This might be because of the presence of a recently identified inhibitory element from nt 7903–15 (O’Connor et al., 1998 ).

The construct 272–496E with the nt 482 cap site was twice as active in SiHa cells as in C33A and HeLa cells and it should be noted that the truncated construct 272–448E was at least just as active (Fig. 5). It is therefore likely that there is an additional promoter present in this region.

As one of our goals was to characterize this P542 promoter, the 489–556E construct was mutated in regions where consensus-like sequences for transcription factors were predicted by a computer search. The two mutations Mut1 and Mut2, as well as the combination of these, reduced or completely abolished activity thus indicating that transcription factors are bound in this region of the promoter, although they are still not characterized. The data thus indicate that the transcription start site at nt 542 identifies a separate TATA-less promoter. In HPV-11, a promoter located in the E6 ORF (P264) was reported to be responsible for E7 protein expression (Cheng et al., 1995 ) and the HPV-16 P542 could very well govern expression of the E7 protein, since the first ATG codon following the 542 cap site is the E7 start codon positioned at nt 562. Considering the consensus sequence for initiation of translation in higher eukaryotes (for review, see Kozak, 1989 ), the ATG at nt 562 is regarded to be in a quite strong initiator context. The transcription initiation site identified at nt 611 within the E7 ORF has not been shown before, but a potential cap site at nt 626 has been suggested (Doorbar et al., 1990 ). The 611LCR construct, as well as the nt 549–622 fragment, had no significant activity in the cell lines tested but the fragment might be either too short or the promoter might be differentiation-dependent, as reported by others for P670 (Grassmann et al., 1996 ).

In conclusion, our data show the presence of a potential new E7-specific promoter, P542, from which an E7 transcript might be initiated. The previously identified P480 is also a potential E7 promoter where the transcript holds two minicistrons upstream of the E7 ATG codon at nt 562 (Grassmann et al., 1996 ). If the E7 protein is translated from both, transcripts will need to be further analysed.


   Acknowledgments
 
The work was supported by scholarships from The Danish Cancer Society to T.H.B. and B.G. Equipment and materials for the study were made available with support from The Danish Medical Research Council, Lawyer G. C. Kremer and wife Foundation, The Danish Cancer Society, The Biotechnology Program, The Novo Foundation, Bent Bøgh and wife Inge Bøgh Foundation and The A. P. Møller and Chastine Mc-Kinney Møllers Foundation.


   References
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Abstract
Introduction
Methods
Results
Discussion
References
 
Belaguli, N. S., Pater, M. M. & Pater, A. (1997). Identification and location of human papillomavirus type 16 antisense early promoter and characterisation of antisense RNA. Journal of Medical Virology 51, 344-354.[Medline]

Chenchik, A., Zhu, Y. Y., Diatchenko, L., Li, R., Hill, J. & Siebert, P. D. (1998). Generation and use of high-quality cDNA from small amounts of total RNA by SMARTTM PCR. In The Biotechniques Series on Molecular Laboratory Methods Vol. 1, Gene Cloning and Analysis by RT–PCR, pp. 305-319. Edited by P. D. Siebert & J. W. Larrick. Natick, MA: BioTechniques Books.

Cheng, S., Schmidt-Grimminger, D.-C., Murant, T., Broker, T. R. & Chow, L. T. (1995). Differentiation-dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiated keratinocytes. Genes & Development 9, 2335-2349.[Abstract]

Chow, L. T., Nasseri, M., Wolinsky, S. M. & Broker, T. R. (1987). Human papillomavirus types 6 and 11 mRNAs from genital condylomata acuminata. Journal of Virology 61, 2581-2588.[Medline]

Conrad, M., Bubb, V. J. & Schlegel, R. (1993). The human papillomavirus type 6 and 16 E5 proteins are membrane-associated proteins which associate with the 16-kilodalton pore-forming protein. Journal of Virology 67, 6170-6178.[Abstract]

Crook, T., Morgenstein, J., Crawford, L. & Banks, L. (1989). Continued expression of HPV 16 E7 protein is required for maintenance of the transformed phenotype of cells co-transformed by HPV-16 plus EJ-ras. EMBO Journal 8, 513-519.[Abstract]

de Villiers, E. M. (1994). Human pathogenic papillomavirus types: an update. Current Topics in Microbiology & Immunology 186, 1-12.

DiLorenzo, T. P. & Steinberg, B. M. (1995). Differential regulation of human papillomavirus type 6 and 11 early promoters in cultured cells derived from laryngeal papillomas. Journal of Virology 69, 6865-6872.[Abstract]

Doorbar, J., Parton, A., Hartley, K., Banks, L., Crook, T., Stanley, M. & Crawford, L. (1990). Detection of novel splicing patterns in a HPV16-containing keratinocyte cell line. Virology 178, 254-262.[Medline]

Geisen, C. & Kahn, T. (1996). Promoter activity of sequences located upstream of the human papillomavirus types 16 and 18 late regions. Journal of General Virology 77, 2193-2200.[Abstract]

Grassmann, K., Rapp, B., Maschek, H., Petry, K. U. & Iftner, T. (1996). Identification of a differentiation-inducible promoter in the E7 open reading frame of human papillomavirus type 16 (HPV-16) in raft cultures of a new cell line containing high copy numbers of episomal HPV-16 DNA. Journal of Virology 70, 2339-2349.[Abstract]

Hawley-Nelson, P., Vousden, K. H., Hubbert, N. L., Lowy, D. R. & Schiller, J. T. (1989). HPV 16 E6 and E7 proteins cooperate to immortalize human foreskin keratinocytes. EMBO Journal 8, 3905-3910.[Abstract]

Higgins, G. D., Uzelin, D. M., Phillips, G. E., McEvoy, P., Marin, R. & Burrell, C. J. (1992). Transcription patterns of human papillomavirus type 16 in genital intraepithelial neoplasia: evidence for promoter usage within the E7 open reading frame during epithelial differentiation. Journal of Virology 73, 2047-2057.

Hummel, M., Hudson, J. B. & Laimins, L. A. (1992). Differentiation-induced and constitutive transcription of human papillomavirus type 31b in cell lines containing viral episomes. Journal of Virology 66, 6070-6080.[Abstract]

Hummel, M., Lim, H. B. & Laimins, L. A. (1995). Human papillomavirus type 31b late gene expression is regulated through protein kinase C-mediated changes in RNA processing. Journal of Virology 69, 3381-3388.[Abstract]

Johnsen, C. K., Stanley, M. & Norrild, B. (1995). Analysis of human papillomavirus type 16 E5 oncogene expression in vitro and from bicistronic messenger RNAs. Intervirology 38, 339-345.[Medline]

Karlen, S. & Beard, P. (1993). Identification and characterization of novel promoters in the genome of human papillomavirus type 18. Journal of Virology 67, 4296-4306.[Abstract]

Karlen, S., Offord, E. A. & Beard, P. (1996). Functional promoters in the genome of human papillomavirus type 6b. Journal of General Virology 77, 11-16.[Abstract]

Kozak, M. (1989). The scanning model for translation: an update. Journal of Cell Biology 108, 229-241.[Abstract]

Kyo, S., Klumpp, D. J., Inoue, M., Kanaya, T. & Laimins, L. A. (1997). Expression of AP1 during cellular differentiation determines human papillomavirus E6/E7 expression in stratified epithelial cells. Journal of General Virology 78, 401-411.[Abstract]

Leptak, C., Cajal, S. R., Kulke, R., Horwitz, B. H., Riese, D. J., Dotto, G. P. & DiMaio, D. (1991). Tumorigenic transformation of murine keratinocytes by the E5 genes of bovine papillomavirus type 1 and human papillomavirus type 16. Journal of Virology 65, 7078-7083.[Medline]

Maki, H., Fujikawa-Adachi, K. & Yoshie, O. (1996). Evidence for a promoter-like activity in the short non-coding region of human papillomaviruses. Journal of General Virology 77, 453-458.[Abstract]

Münger, K., Phelps, W. C., Bubb, V., Howley, P. M. & Schlegel, R. (1989). The E6 and E7 genes of the human papillomavirus type 16 together are necessary and sufficient for transformation of primary human keratinocytes. Journal of Virology 63, 4417-4421.[Medline]

Nasseri, M., Hirochika, R., Broker, T. R. & Chow, L. T. (1987). A human papillomavirus type 11 transcript encoding an E1–E4 protein. Virology 159, 433-439.[Medline]

O’Connor, M. J., Stünkel, W., Zimmermann, H., Koh, C.-H. & Bernard, H.-U. (1998). A novel YY1-independent silencer represses the activity of the human papillomavirus type 16 enhancer. Journal of Virology 72, 10083-10092.[Abstract/Free Full Text]

Ozbun, M. A. & Meyers, C. (1997). Characterization of late gene transcripts expressed during vegetative replication of human papillomavirus type 31b. Journal of Virology 71, 5161-5172.[Abstract]

Ozbun, M. A. & Meyers, C. (1998). Temporal usage of multiple promoters during the life cycle of human papillomavirus type 31b. Journal of Virology 72, 2715-2722.[Abstract/Free Full Text]

Ozbun, M. A. & Meyers, C. (1999). Two novel promoters in the upstream regulatory region of human papillomavirus type 31b are negatively regulated by epithelial differentiation. Journal of Virology 73, 3505-3510.[Abstract/Free Full Text]

Roberts, S., Ashmole, I., Rookes, S. M. & Gallimor, P. H. (1997). Mutational analysis of the human papillomavirus type 16 E1{wedge}E4 protein shows that the C terminus is dispensable for keratin cytoskeleton association but is involved in inducing disruption of the keratin filaments. Journal of Virology 71, 3554-3562.[Abstract]

Rohlfs, M., Winkenbach, S., Meyer, S., Rupp, T. & Durst, M. (1991). Viral transcription in human keratinocyte cell lines immortalized by human papillomavirus type-16. Virology 183, 331-342.[Medline]

Smits, H. L., Cornelissen, M. T., Jebbink, M. F., van den Tweel, J. G., Struyk, A. P., Briet, M. & ter Schegget, J. (1991). Human papillomavirus type 16 transcripts expressed from viral–cellular junctions and full-length viral copies in CaSki cells and in a cervical carcinoma. Virology 182, 870-873.[Medline]

Smotkin, D. & Wettstein, F. O. (1986). Transcription of human papillomavirus type 16 early genes in a cervical cancer and a cancer-derived cell line and identification of the E7 protein. Proceedings of the National Academy of Sciences, USA 83, 4680-4684.[Abstract]

Smotkin, D., Prokoph, H. & Wettstein, F. O. (1989). Oncogenic and nononcogenic human genital papillomaviruses generate the E7 mRNA by different mechanisms. Journal of Virology 63, 1441-1447.[Medline]

Sousa, R., Dostatni, N. & Yaniv, M. (1990). Control of papillomavirus gene expression. Biochimica et Biophysica Acta 1032, 19-37.[Medline]

Stacey, S. N., Jordan, D., Snijders, P. J., Mackett, M., Walboomers, J. M. & Arrand, J. R. (1995). Translation of the human papillomavirus type 16 E7 oncoprotein from bicistronic mRNA is independent of splicing events within the E6 open reading frame. Journal of Virology 69, 7023-7031.[Abstract]

Thierry, F., Heard, J. M., Dartmann, K. & Yaniv, M. (1987). Characterization of a transcriptional promoter of human papillomavirus 18 and modulation of its expression by simian virus 40 and adenovirus early antigens. Journal of Virology 61, 134-142.[Medline]

Vousden, K. (1993). Interactions of human papillomavirus transforming proteins with the products of tumor suppressor genes. FASEB Journal 7, 872-879.[Abstract/Free Full Text]

Zhao, W., Chow, L. T. & Broker, T. R. (1997). Transcription activities of human papillomavirus type 11 E6 promoter-proximal elements in raft and submerged cultures of foreskin keratinocytes. Journal of Virology 71, 8832-8840.[Abstract]

zur Hausen, H. (1996). Papillomavirus infections – a major cause of human cancers. Biochimica et Biophysica Acta 1288, F55-78.[Medline]

Received 23 June 1999; accepted 12 August 1999.