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Journal of Virology, April 2005, p. 4918-4926, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.4918-4926.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Induction of the Human Papillomavirus Type 31 Late Promoter Requires Differentiation but Not DNA Amplification

Kathryn M. Spink and Laimonis A. Laimins*

Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois

Received 26 July 2004/ Accepted 1 December 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The human papillomavirus (HPV) life cycle is linked to the differentiation state of the host cell. In virus-infected undifferentiated basal epithelial cells, HPV genomes are maintained as episomes at low copy number. Upon differentiation, a concomitant increase in viral copy number and an induction of late gene expression from a differentiation-specific promoter is seen. To investigate whether late gene expression was dependent on the amplification of the viral genome, inhibitors of DNA replication and in vitro systems for epithelial differentiation were used in conjunction with cells that stably maintain HPV31 episomes. Treatment of cells induced to differentiate in methylcellulose with the DNA synthesis inhibitor cytosine ß-arabinofuranoside (AraC) blocked viral DNA amplification but did not prevent induction of late transcription. This suggests that late gene expression does not strictly require amplification of the viral genome and that differentiation signals alone are sufficient to activate transcription from the late promoter. However, DNA amplification does appear to be necessary for maximal induction of the late promoter. In order to examine the cis-acting elements that contribute to the activation of the late promoter, a transient reporter assay was developed. In these assays, an induction of late gene expression was seen upon differentiation that was specific to the late promoter. Mapping studies localized important regulatory elements to the E6/E7 region and identified short sequences that could serve as binding sites for transcription factors. Elements within the upstream regulatory region were also found to positively and negatively influence transcription from the late promoter. These results identify mechanisms important for the differentiation-dependent activation of late gene expression of high-risk papillomaviruses.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human papillomaviruses (HPVs) are the causative agents of cervical cancer, which is the second leading cause of death by cancer among women worldwide (44). HPVs are small double-stranded DNA tumor viruses with genomes of approximately 8 kbp in length (57). These viruses infect epithelial cells, and approximately one-third of HPV types specifically target the genital epithelia. The viruses that infect the genital tract can be further divided into two groups: low-risk viruses such as HPV type 6 (HPV6) and HPV11 induce benign genital lesions but are rarely associated with invasive cancers and high-risk types such as HPV16, HPV18, and HPV31 that are the etiological agents of cervical cancer (40). High-risk HPV sequences are found in more than 99% of cervical cancer biopsies (44).

The HPV life cycle is linked to the differentiation state of the host cell (30). Viruses infect cells in the basal epithelia and translocate to the nucleus where viral genomes are established as episomes. In infected basal cells, viral genomes are replicated coordinately with cellular DNA, early viral proteins are synthesized at low levels, whereas late genes are not expressed. After cell division, one daughter cell migrates away from the basal layer and begins to differentiate. Upon differentiation in suprabasal cells, viral DNA amplification is activated, along with late transcription, leading to virion production (23). A link between productive viral replication and late gene expression has been well documented in other DNA tumor viruses such as simian virus 40 (SV40) (53, 54), polyomavirus (8, 9, 27, 31), and adenoviruses (51), but it is not clear if this extends to papillomaviruses.

Elements that regulate early gene expression are located in the upstream regulatory region (URR) of papillomaviruses (57). Many of the elements in the URR that are required for the induction of the early promoter (P97 for HPV31 and HPV16) have been characterized, and our understanding of the transcriptional control of the early promoter is extensive. The elements that regulate early transcription include keratinocyte-specific enhancers, as well as repressor sequences (3, 10, 12, 18, 55). Although there exist variations in the positioning of transcription factor binding sites among the different types of HPVs, most URRs contain binding sites for the same subset of factors, including AP1, C/EBPß, NF1, Oct1, Sp1, TEF-2, and YY1 (1, 2, 6, 7, 11, 19-21, 28, 38, 50, 55, 56). It is believed that these factors act in concert with each other to activate early transcription (4, 29, 32, 35), whereas a subset directly represses transcription or quench the activity of other factors (3, 22, 34, 36, 37, 41, 52). High-risk HPVs contain four binding sites in the URR for the E2 protein, which in bovine papillomavirus acts as the primary regulator of viral transcription (23). Binding of HPV E2 protein to sites distal to the start site of early transcription stimulates low levels of expression from p97 (47). In contrast, binding to promoter-proximal E2 sites inhibits early transcription (47). This inhibition of transcription appears to act by displacement of Sp1, along with TBP from sequences that overlap the promoter-proximal E2 binding site (14, 49). The regulation of early transcription by E2 may contribute to copy number control in undifferentiated basal cells by modulating expression of the viral replication proteins (48).

During the productive viral life cycle, HPV gene products are expressed from polycistronic transcripts (see Fig. 1). In HPV31, the early transcripts initiate upstream of the E6 open reading frame (ORF) at sequences around nucleotide (nt) 97 (P97). Upon differentiation, transcripts initiating at P97 appear to be unchanged or are modestly repressed, whereas a novel, late promoter is activated (26). The HPV31 late promoter initiates transcription at heterogeneous sites around nt 742 (P742) in the E7 ORF (25, 39). Coordinate with the activation of late expression, a change in DNase hypersensitivity around P742 has been observed, suggesting that a change in chromatin configuration occurs upon differentiation (13). Although many studies have investigated the role of various cis-acting elements in the activation of the early promoter, few have examined the factors or sequences responsible for activation of the late promoter. One potential regulator of late transcription has been suggested to be CDP, a factor that represses late transcription in undifferentiated cells but not in differentiated cells (36, 41). Additional potential transcription factor binding sites for Oct-1, SOX5, and SRY were identified around P742 (13), but it is not clear if these actually function to activate late expression.



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  		FIG. 1. HPV31 genome organization and transcript representation. (A) The HPV31 genome is depicted. Early and late ORFs are shown, as well as the early and late promoters and polyadenylation sites. The URR is represented by the solid black bar elements. (B) Some of the major early and late polycistronic messages are shown.

 
In the present study, we demonstrate that activation of late viral transcription does not strictly require productive replication. Viral amplification does, however, contribute to the magnitude of activation of late expression. In addition, we use transient expression assays in cells induced to differentiate by suspension in semisolid medium to localize sequences required for activation of HPV31 late transcription to the E6/E7 coding regions.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture. Cell lines harboring HPV31b genomes, LKP31 (17) and CIN612-9E (5), have previously been described and were maintained in serum-containing medium (E medium) supplemented with mouse epidermal growth factor (5 ng/ml; Collaborative Biomedical Products, Bedford, Mass.) in the presence of mitomycin C-treated J2 3T3 fibroblast feeders. Prior to harvesting total cellular DNA and RNA, the fibroblast feeders were removed by treatment with EDTA (phosphate-buffered saline [Gibco-BRL/Invitrogen, Carlsbad, Calif.] with 0.5 mM EDTA). Differentiation was induced by suspension in 1.5% methylcellulose as previously described (43). For inhibitor studies, 40 µg of cytosine ß-arabinofuranoside (AraC; Sigma, St. Louis, Mo.)/ml was added to the methylcellulose and was stirred overnight at 4°C prior to suspension of cells. After suspension in methylcellulose, cells were washed with phosphate-buffered saline and harvested by centrifugation (15).

DNA and RNA analysis. Total genomic DNA was isolated by using previously described techniques (15). For Southern analysis total genomic DNA (5 µg) was digested with BamHI, separated on a 0.8% agarose gel, treated with alkaline, and transferred onto DuPont GeneScreen nylon membrane (NEN Research Products, Boston, Mass.) as described by the manufacturer. Hybridization was carried out by using the entire HPV31 genome as a probe as previously described (15). Total RNA was isolated with TRIzol reagent (Gibco-BRL/Invitrogen) according to manufacturer's recommendations. For Northern analysis, total RNA (10 µg) was separated on a 1.0% agarose gel and transferred onto a Zeta-Probe membrane (Bio-Rad, Hercules, Calif.). Membranes were then hybridized with probes for the entire HPV31 genome, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and involucrin by previously described methods (15). RNase protection analyses used a probe encompassing the E7 ORF which identifies both early and late transcripts as described by Pena et al. (13).

Plasmids. The H31Luc plasmid has previously been described (24). Briefly, this plasmid contains the entire URR of HPV31 and sequences through the N terminus of E1 (nt 892) upstream of the firefly luciferase gene. The luciferase coding sequences are fused in frame with the E1 coding sequences at the tenth amino acid. Plasmid constructs p{Delta}7045-7557, p{Delta}7045-7816, p{Delta}7045-125, p{Delta}7045-609, p{Delta}7045-683, p{Delta}7557-7816, p{Delta}7816-125, p{Delta}124-611, and p{Delta}613-683 were made by digestion with convenient restriction endonuclease sites for KpnI (nt 7045) in the multiple cloning sites of H31Luc and SpeI (nt 7557), NsiI (nt 7816), PstI (nt 125), Bsu36I (nt 609), and Pvu II (nt 683) located in the URR and sequences upstream of P742 (see Fig. 2). After digestion, a fill-in reaction was performed, followed by blunt end ligation leading to the generation of the deletion mutant. Smaller 10-bp deletion constructs p{Delta}611-620, p{Delta}621-630, p{Delta}631-640, p{Delta}641-650, p{Delta}651-660, p{Delta}661-670, p{Delta}671-680, p{Delta}681-690, p{Delta}691-700, p{Delta}701-711, p{Delta}711-720, p{Delta}721-730, and p{Delta}731-740 were made by using the Gene Editor kit by Promega (Madison, Wis.) according to the manufacturer's instructions. Point mutations TAG*ATAA, TATAG*AA, and TAG*AG*AA in the early promoter, P97, were also created by using the Gene Editor kit. The Renilla vector with a thymidine kinase promoter, pRLTK, was obtained form Promega. All constructs were sequenced to confirm that they contained the desired mutation.



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  		FIG. 2. Southern analysis of viral genomes. LKP31 cells were suspended in methylcellulose without or with 40 µg of AraC/ml for 24 and 48 h. Total DNA was isolated, digested with BamHI (which does not cut the genome), electrophoresed on a 0.9% agarose gel, blotted, and hybridized with a probe for the HPV31 genome. Multimeric, circular/nicked, and supercoiled forms of the genome are indicated.

 
Luciferase assays. LKP31 cells were plated at one million cells per 10-cm dish the day prior to transfection. Cells were cotransfected with 2 µg of a luciferase construct, and 50 ng of the Renilla control vector by using FuGene (Roche Diagnostics, Mannheim, Germany) according to manufacturer's instructions. After overnight incubation, feeders were removed, and the LKP cells were split to two fractions: one fraction was plated onto a new dish containing fresh fibroblast feeders, and the other fraction was suspended in 1.5% methylcellulose. After 48 h, cells from both fractions were harvested for luciferase assays by using the Dual Luciferase kit (Promega) according to the manufacturer's instructions.

Electrophoretic mobility shift assays (EMSAs). Nuclear extracts were prepared as previously described (45). Briefly 2 x 106 cells, either LKP31 cells from monolayer cultures with feeders removed or LKP31 cells harvested after methylcellulose treatment, were collected and washed with TBS (Tris-buffered saline). The cell pellets were resuspended in cold buffer A (10 mM HEPES -pH 7.9], 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol [DTT], 0.5 mM phenylmethylsulfonyl fluoride [PMSF]) and allowed to swell on ice for 15 min. Nonidet NP-40 was added, and pelleted cells were resuspended in cold buffer C (20 mM HEPES [pH 7.9], 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 1 mM PMSF) and rocked for 15 min. The cells were pelleted, and the nuclear extract supernatant was divided into aliquots and stored at –70°C. Duplex DNA oligonucleotides for the following HPV sequences were purchased from IDT and reconstituted according to manufacturer's instructions: nt 625 to 645, 635 to 655, 655 to 675, 675 to 695, 685 to 705, 695 to 715, 705 to 725, 715 to 735, and 725 to 745. EMSA reactions were carried out as previously described (29). Briefly, 15 µg of nuclear extract was incubated with 2 µg of poly(dI-dC) in the presence or absence of a 100-fold molar excess of unlabeled competitor DNA oligonucleotides on ice for 20 min in a 25-µl reaction containing 10% glycerol, 25 mM HEPES (pH 7.9), 50 mM KCl, 4 mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, and 0.5 mM PMSF. After incubation, 10,000 cpm of 32P-end-labeled duplex DNA was added and incubated for 30 min at room temperature. The DNA-protein complexes were separated from free probe on a 4% polyacrylamide gel. The gel was dried and subjected to autoradiography.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
HPV late gene transcription is partially dependent upon viral DNA amplification. We first sought to investigate whether HPV31 late transcription is dependent upon differentiation-induced viral amplification using stable cell lines that maintain HPV31 genomes as episomes. Previous studies have shown that suspension of HPV31-positive cells in 1.5% methylcellulose induces differentiation-dependent amplification within 48 h (43). LKP31 cells, which maintain approximately 100 copies of HPV31 (17), were induced to differentiate in methylcellulose in the absence or presence of the DNA synthesis inhibitor AraC. DNA was isolated at 0, 24, and 48 h and examined for viral DNA amplification by Southern blot analysis. As shown in Fig. 2, the HPV31 genomes are amplified, on average, by three- to fourfold by 24 h and by eightfold by 48 h after suspension in methylcellulose, a finding consistent with previous published reports (43). The addition of AraC was found to inhibit viral amplification so that no increase in copy number was seen upon differentiation in methylcellulose (Fig. 2).

It was next important to examine whether activation of late transcripts occurred in the absence of viral genome amplification. RNA was isolated from both AraC-treated and untreated differentiated cultures and examined for the presence of late transcripts by Northern analysis (Fig. 3). Upon differentiation, the induction of transcripts initiated at p742 was observed, including messages encoding E1/E4/E5, E1/E2/E5, as well as E1^E4/E5. The level of late transcripts produced in the presence of AraC was found to be markedly reduced but still significant upon differentiation. HPV31 late transcripts increased approximately 10- to 15-fold upon differentiation of untreated LKP31 cells, but less than a threefold increase, at both time points, was observed in the presence of the inhibitor. Longer exposure revealed a low level of late transcripts in the untreated sample; however, this is consistent with previous results showing a 1 to 2% spontaneous activation of late transcription in monolayer cultures of HPV-positive cells (42). Analysis of transcripts encoding GAPDH synthesis was also carried out to investigate whether AraC induced a general reduction in transcript levels. Previous studies have shown that GAPDH transcription decreases modestly during differentiation (46), and we observed similar changes with or without AraC (Fig. 3). This suggests that AraC does not alter the transcription patterns in treated cells. This observation is in agreement with previous published reports that demonstrate that AraC does not affect transcription (33). Furthermore, addition of AraC did not alter cellular differentiation as evidenced by a lack of change in the expression of the differentiation marker involucrin (Fig. 3). Involucrin expression has previously been shown to closely parallel induction of DNA amplification (43). We also performed RNase protection assays, and these demonstrated that the late transcripts seen in AraC-treated cells were in fact initiated from the late promoter (Fig. 4). These experiments were repeated three times with LKP31 cells, and similar results were seen in another HPV31-positive cell line, CIN612-9E, which was derived from a cervical cancer biopsy (data not shown). Our results indicate that the induction of late transcription can occur independent of viral amplification but that replication acts to increase the template number, leading to elevated levels of late expression.



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  		FIG. 3. Northern analysis of viral transcripts. LKP31 cells were suspended in methylcellulose without or with 40 µg of AraC/ml for 24 and 48 h. Total RNA was isolated, separated by electrophoresis, blotted, and hybridized with probes for the HPV31 genome, GAPDH, and involucrin. Various early and late HPV31 transcripts are indicated.

 


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  		FIG. 4. RNase protection analysis. CIN612-9E cells were suspended in methylcellulose without or with 40 µg of AraC/ml for 24 and 48 h. Total RNA was isolated and subjected to RNase protection analysis with P742 as a probe. Early and late spliced and unspliced transcripts are indicated.

 
Three regions upstream of the late promoter appear to play a role in its activation during differentiation. The previous experiments indicated that differentiation-specific factors play a primary role in the activation of the late promoter. Importantly, these studies also demonstrate that the elements regulating late gene expression could be studied in the absence of differentiation-induced viral amplification and validated the use of transient reporter assays to study this process. We therefore used transient transfection into LKP31 cells, followed by suspension in methylcellulose to identify the sequences important for regulating late gene expression. To ensure that we included all cis-acting elements that are likely to be involved in the activation of the late promoter, we cloned the entire URR, E6/E7 coding sequences, as well as sequences into the E1 ORF (up to nt 892) upstream of the firefly luciferase gene to generate H31Luc (Fig. 5). The luciferase sequences were cloned in frame to the E1 coding sequences (24).



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  		FIG. 5. H31Luc plasmid constructs. The H31Luc plasmid contains HPV31 sequences from the URR through the N terminus of E1 fused to the firefly luciferase gene (luc). Positioning of the early (P97) and the late (P742) promoters, as well as transcription factor binding sites within the URR, are indicated. Deletion mutants created by restriction endonuclease digestion using sites within the URR and other sequences upstream of P742 are shown.

 
Since both the early promoter (P97) and the late promoter (P742) are present in H31Luc, and both are active upon differentiation (26), we sought to minimize the contribution of transcription initiating at p97 to H31Luc expression. We first investigated the effects of mutating the p97 TATA box sequences with a reporter in which only the p97 promoter was present. Three TATA box mutations—pTAG*ATAA, pTATAG*AA, and pTAG*AG*AA (nt 67 to 74)—were tested in pBAS698, a pGL3-Basic plasmid (promega) that contains the URR sequences upstream of the firefly luciferase gene (Fig. 6A). These constructs were transfected into LKP31 cells, along with an internal Renilla control vector as described in Materials and Methods. We observed an approximate 10-fold reduction in P97 promoter activity when the TATA box was mutated to TAGAGAA mutation (Fig. 6B). We reasoned that introduction of this mutation into H31Luc would eliminate the contribution of the early promoter to late gene expression in this reporter. This mutation was introduced in the H31Luc plasmid to generate H31Luc/P97 M.



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  		FIG. 6. Luciferase activity of early promoter mutants. (A) pBAS698 contains the entire URR and HPV31 sequences through nt 698 fused to the firefly luciferase gene. (B) The activity of pBAS698 early promoter mutants in monolayer cultures is represented. LKP31 cells were transfected with 2 µg of the luciferase plasmid, along with 50 ng of pRLTK. Cells were harvested after 48 h, and the ratio of firefly to Renilla luciferase activity was calculated. (C) The H31Luc plasmid and H31Luc/P97M plasmid were transfected into LKP31 cells, along with an internal Renilla control as described in Materials and Methods. After transfection, cells were induced to differentiate by suspension in methylcellulose. The ratio of firefly to Renilla luciferase activity was calculated for both monolayer and methylcellulose-treated cultures, and the fold induction upon suspension in methylcellulose is shown. The Renilla activity was slightly decreased upon differentiation; however, this decrease was consistent and still allowed from comparison between firefly constructs.

 
Transfection of the H31Luc plasmid into monolayer cultures was found to result in expression that further increased approximately threefold after suspension in methylcellulose (Fig. 6C). The plasmid H31Luc/P97M exhibited slightly decreased levels of activity in undifferentiated monolayer cultures but was similarly activated by approximately threefold after suspension in methylcellulose. This suggests that the p97 promoter contributes minimally to H31luc expression in differentiated cells. We also tested these constructs in HPV-negative SCC13 cells and found similar levels of induction upon differentiation (data not shown). This suggests that HPV gene products are not necessary for induction of late transcription upon differentiation and that cellular factors were regulating expression.

We next constructed a series of mutant reporter plasmids that included successive deletions from the 5' end of the URR proceeding through sequences immediately upstream of nt 742. In addition, a set of reporters containing internal deletions within the URR, as well as E6/E7 coding sequences upstream of p742, were constructed (Fig. 5). After transfection and suspension in methylcellulose, the firefly/Renilla ratios were determined and compared to the levels seen in monolayer cultures. The fold induction was calculated, and the results are shown in Fig. 7. Experiments were repeated at least three times, and most reporters were examined up to 10 times. The values presented in Fig. 7 represent averages of these experiments.



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  		FIG. 7. Induction of H31Luc and mutant plasmids upon differentiation. LKP31 cells were transfected with various deletion plasmids in both the wild type (A) and the P97 TATA box mutation, P97M (B). After transfection, cells were induced to differentiate by suspension in methylcellulose. Firefly luciferase activity was measured, along with the Renilla activity of a cotransfected control. The ratio of firefly to Renilla luciferase activity was calculated for monolayer and methylcellulose cultures, and the fold induction upon differentiation in methylcellulose was determined.

 
Our transient transfection experiments identified several regions that appear to play an important role in the activation of the late promoter. Deletions from the L1 proximal end of the URR revealed minimal effect of sequences between nt 7045 to 7816, though a slight increase in activity was seen upon deletion of nt 7045 to 7557 and 7045 to 7816. This region may therefore have a modest role in exerting a negative effect on late expression, but its significance remains unclear. The most significant reductions in late expression were found when sequences between the E6 and E7 coding regions were deleted. In particular, a comparison of the activity for p7045-609 with that of p7045-683 indicates that a major determinant of activity is in the E6/E7 region. However, deletion of sequences 613 to 683 in the presence of upstream URR sequences reduced activity by only 50%, indicating there may be multiple redundant elements in the URR that contribute to late activation. The effects of these additional elements are seen only in the absence of sequences in the E6/E7 region (Fig. 7A, compare p7045-683 to p613-183). Experiments were also carried out in the presence of the P97M mutation (Fig. 7B), where the early promoter was present. Mutation of the early promoter did not alter the pattern of expression seen with the wild-type P97M promoter seen in Fig. 7A, suggesting that the early promoter is not a contributing factor to the observed results.

It was then important to more closely define regulatory sequences through the use of reporter constructs containing internal deletions in the E6/E7 region. For these analyses, we first focused in the region between nt 613 and 683 since this appeared to be a major regulatory region. This region overlaps sequences mapped by del Mar Pena and Laimins (13) found to contain initiation sites of late transcription, as well as regions that underwent chromatin rearrangement upon differentiation, as evidenced by the appearance of DNase-hypersensitive sites. We therefore constructed a series of plasmids that contained sequential 10-bp deletions of the region from positions 613 to 683 in the context of all upstream sequences. These constructs were tested in our transient assays at least three times, and many were tested up to five times, with the results shown in Fig. 8A. Of the eight reporters tested, we found the most significant effects upon deletion of sequences 661 to 670 and 681 to 690. Deletion of two other regions, positions 631 to 640 and 641 to 650, also appeared to have an effect on the induction of the late promoter, although it was not as pronounced as for the sequences from nt 661 to 670 and nt 681 to 690.



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  		FIG. 8. Induction of the H31Luc and mutant plasmids from nt 613 to 683 (A) and nt 691 to 740 (B). LKP31 cells were transfected with various deletion plasmids. After transfection, cells were induced to differentiate by suspension in methylcellulose. Firefly luciferase activity was measured, along with the Renilla activity of a cotransfected control. The ratio of firefly to Renilla luciferase activity was calculated for monolayer and methylcellulose cultures, and the fold induction upon differentiation in methylcellulose was determined.

 
Finally, we investigated the contribution of the region between nt 690 and 740. Sequential 10-bp deletions were constructed and tested in our transient assays five times, and the results are shown in Fig. 8B. All five deletion plasmids exhibited reduced activity, suggesting this region plays an important role in the activation of the late promoter. We conclude from these experiments that multiple elements within the E6/E7 coding region contribute to the induction of the late promoter.

EMSAs demonstrate factor binding to regions in the E6/E7 coding region. We next sought to investigate the factors that bound to the sequences identified in the E6/E7 coding region to be important for the activation of the late promoter through the use of EMSAs. Oligonucleotides were designed such that the 10-bp sequence examined in the previous luciferase assays was centered in a 20-bp duplex oligonucleotide. The strongest binding activities were detected for duplex sequences from nt 635 to 655 and 655 to 675, whereas oligonucleotides spanning sequences from nt 675 to 695 demonstrated moderate binding. A representative series of EMSAs for regions 625 to 645, 635 to 655, and 655 to 675 are shown in Fig. 9. Finally, duplex sequences from nt 625 to 645, 685 to 705, 695 to 715, 705 to 725, 715 to 735, and 725 to 745 all exhibited weak binding. In these initial studies, no significant differences were observed between binding of nuclear extracts from monolayer cells and cells treated with methylcellulose. However, we cannot exclude the possibility that there are moderate quantitative differences in binding activities.



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  		FIG. 9. Representative EMSA. Oligonucleotide (Oligo.) probes for sequences from nt 625 to 645, 635 to 655, and 655 to 675 were end labeled and incubated with nuclear extracts (N.E.) from monolayer (Mon.) and methylcellulose-treated (Diff.) cultures of LKP31 cells in the presence (+) or absence (–) of unlabeled oligonucleotide competitor (Comp.) DNA. A "B" indicates the specific retarded band, while an "F" indicates the unbound free probe.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The productive phase of the HPV life cycle is linked to epithelial differentiation, with late gene expression restricted to highly differentiated suprabasal cells. Although the activation of late gene expression occurs concomitantly with the amplification of HPV DNA, our study indicates that induction of late gene expression is not strictly dependent upon differentiation-dependent viral DNA amplification. A link between productive replication and late gene expression has been shown for SV40, polyomaviruses, and adenoviruses (8, 9, 27, 31, 51, 53, 54), but it was unclear if similar mechanisms functioned in papillomaviruses. Studies with SV40 have suggested repressors negatively regulate late expression and that replication titrates away these factors, allowing for expression of viral capsid genes (53). Our experiments indicate that this is not the mechanism of activation for HPV31 but rather that the effect of productive replication is to increase the magnitude of late transcripts, presumably by increasing template number. The induction of late gene expression by differentiation alone suggests that cellular factors directly or indirectly activate transcription. Previous studies from our laboratory indicated that late gene expression required episomal DNA templates, since integrated copies of HPV DNA did not express late transcripts despite the expression of differentiation markers such as filaggrin or keratin 10 (16). We suspect this may reflect a role for differentiation-dependent chromatin rearrangement that occurs more efficiently on episomal templates.

Since our studies indicated that productive replication is not strictly required for activation of late transcription, it seemed appropriate to use reporter plasmids in transient reporter assays to study the cis sequences required for activation of the late promoter. Using a protocol in which cells were transfected with reporter plasmids and then induced to differentiate in semisolid media, we observed a low level of late expression in undifferentiated cells that was further enhanced by approximately three- to fourfold after suspension in methylcellulose. The low-level transcriptional activity in undifferentiated cells is consistent with the low-level activation we have observed in some monolayer cultures of cells that maintain viral episomes (42). In the present study, we observed that the major determinants of late transcription were found within the E6/E7 coding sequences. When we analyzed expression of a series of reporters containing deletion from the L1 side of the URR, we found that the most significant reduction in activity occurred upon loss of the E6/E7 sequences. In contrast, when the E6/E7 region was deleted in the presence of URR sequences, we observed a less significant reduction. This suggests there may be redundant elements in the URR that can substitute in activating late expression when the primary regulatory sequences in E6/E7 are deleted. We also identified a moderate negative regulatory element at the 5' end of the URR. Whether the negative element in the URR has physiologic significance is unclear at this time. However, a role for a negative regulator has been suggested in HPV6 where the binding of CAAT displacement protein to this region has been implicated as a regulator of late gene expression, and CEBP binding sites are located in this region (41).

We identified nine short regions within the E6/E7 region that seem to play important roles in the activation of late expression. These regions were also found to bind proteins in nuclear extracts, suggesting potential transcription factor binding. While some of these elements exhibited strong binding activities in our EMSAs, other sequences that scored in transient assays failed to exhibit significant binding activities. This may indicate either that we have not used the complete binding regions in our gel shifts or that spatial separation of sequences plays an important role in mediating transcriptional activation. A more detailed point mutational analysis will allow us to distinguish between these two possibilities.

We also failed to observe significant differentiation-specific changes in the binding activities in our EMSA analysis. This may suggest that either small quantitative differences in factor level may be responsible for differentiation-dependent activation or that constitutively expressed transcription factors act in the context of differentiation-dependent chromatin changes to mediate late expression. A detailed analysis of the factors involved will help to elucidate these possibilities.

A search of potential transcription factor binding sites by using the TRANSFAC database suggested several potential regulating factors (Table 1); however, most of these sites are nonconical, with only 70% homology to their consensus sequences. These factors include CEBP, the Oct-1 family of transcription factors, Ap-4, SOX, SRY, and CREB proteins. It is also possible that since start sites of late transcription are very heterogeneous and utilize initiator elements to direct initiation, some of these sequences may act as initiator elements. The identification of the binding factors involved in the regulation of HPV late expression will help to understand the mechanism by which differentiation-specific activation occurs.


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  		TABLE 1. Potential transcription factor binding sites as determined by a search using the TRANSFAC database

 
The activation of late gene expression is a complex process that involves the activation of initiation of transcription, along with alternative splice site usage and polyadenylation sequence utilization. All of these processes seem to be dependent upon the action of factors whose expression is controlled in a differentiation-specific manner. The control of these factors can be exerted at the level of expression or through differentiation-specific posttranslational modifications. Our study has shown that differentiation-specific factors are the primary regulators of expression and that amplification of viral templates serves primarily to enhance the levels of transcript accumulation. The major regulatory factors were found to require sequences in the E6/E7 region, and future studies are required to identify the specific factors involved. Although a genetic system to study the HPV viral life cycle has been developed, it will be difficult to examine the effects of these sequences in the context of complete viral genomes since E6 and E7 sequences are required for critical early viral events (41). A limited number of conservative changes may be possible in the E6/E7 coding sequences but this will restrict mutant choice. An equally important regulatory mechanism is differentiation-specific chromatin rearrangement, and this has not been examined in the present study. Overall, these studies have provided initial insights into the important mechanisms regulating HPV31 late expression.


   ACKNOWLEDGMENTS
 
This study was supported by a grant from the National Cancer Institute to L.A.L. (CA59655) and by a grant from the American Cancer Society to K.M.S. (PF-04-252-01-MBC).


   FOOTNOTES
 
* Corresponding author. Mailing address: Microbiology-Immunology Department, Northwestern University, 303 E. Chicago Ave, Chicago, IL 60611. Phone: (312) 503-0648. Fax: (312) 503-0649. E-mail: l-laimins{at}northwestern.edu. Back


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Journal of Virology, April 2005, p. 4918-4926, Vol. 79, No. 8
0022-538X/05/$08.00+0     doi:10.1128/JVI.79.8.4918-4926.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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