Temporal Usage of Multiple Promoters during the Life Cycle of Human Papillomavirus Type 31b

Abstract

The life cycles of human papillomaviruses (HPVs) are dependent upon the differentiation of the epithelial cells they infect. HPV type 31b (HPV31b) virions can be purified following the growth of a latently HPV-infected cell line (CIN-612 9E) in the organotypic or raft system. Treatment of the CIN-612 9E raft tissues with protein kinase C (PKC) activators is required for upregulation of late gene expression and efficient production of virions. We employed the raft culture system to study the temporal usage of HPV31b promoters during the viral life cycle. We compared monolayer cultures of CIN-612 9E cells, untreated CIN-612 9E raft tissues, and PKC-induced CIN-612 9E raft tissues harvested at various time points during epithelial differentiation. We found that the HPV31b major early promoter precisely maps to nucleotide (nt) 99 (P₉₉). A transcriptional start site for both early and late gene transcripts mapped upstream of P₉₉ at nt 77 (P₇₇). The P₇₇ and P₉₉ promoters were used constitutively throughout the HPV31b life cycle; however, initiation from P₉₉ was much stronger than from P₇₇. Mapping of the differentiation-induced P₇₄₂ promoter revealed multiple start sites. These start sites were difficult to detect in monolayer cultures, were induced in untreated rafts, and were greatest in PKC-induced raft tissues at 8 to 12 days. A constitutively active promoter, P₃₃₂₀, was also defined and is responsible for the transcription of unspliced and spliced RNAs containing E5a, E5b, L2, and L1 open reading frames.

Human papillomaviruses (HPVs) are small DNA viruses that have a tropism for squamous epithelium (45). More than 75 types of HPVs have been identified (32), with subsets causing benign and malignant tumors of the anogenital region (26, 51). The so-called high-risk types are associated with an increased risk of cervical malignancy and include HPV types 16 (HPV16), -18, -31, and -33. The low-risk group involved in anogenital lesions includes HPV6 and -11 and is rarely linked to malignancy. In benign tumors the viral DNA is generally present extrachromosomally; however, the viral genome is often integrated into the host cell DNA in malignant lesions (9).

The genomic organization is highly conserved among HPVs, and the life cycles of the viruses are tightly linked to the differentiation state of the infected cells (8, 30, 42, 45). The virions encapsidate a circular DNA molecule containing six to eight early open reading frames (ORFs) and two late ORFs. A number of enhancer and promoter elements involved in the control of early gene expression, as well as sequences important for replication, are contained in the upstream regulatory region (URR) of the viral genome. The E6 and E7 transforming proteins functionally inactivate the tumor suppressor proteins p53 and pRB, respectively (reviewed in reference 50). E1 and E2 proteins mediate viral genome replication (6, 48). The E2 protein also acts as a transcriptional modulator by interacting with conserved sequences known as E2 binding sites (E2BSs) located in the URR (1, 12, 14, 22, 41, 46). The E5 proteins may augment the effects of E6 and E7 by manipulating the activities of cellular growth factor receptors (11). As cells from the basal layer divide and migrate up through the epithelium, a complex program of differentiation is initiated. The viral late functions are dependent upon cellular differentiation, but the control of these activities is poorly understood. In suprabasal cells the E4 protein is synthesized as a fusion with the N-terminal region of E1 (E1^E4) and associates with cytokeratins (13, 33, 37). Concomitant with cellular differentiation in virally infected cells are the amplification of viral genomes in preparation for packaging and the expression of the L1 and L2 proteins, which form the viral capsids (4, 16, 17, 30).

Historically, the dependence of the HPV life cycle on cellular differentiation has impeded the study of the viral late functions. Most cell lines used to study HPVs are derived from malignancies and contain integrated viral genomes with obstructed late functions. Continuing advances in organotypic or raft tissue culture systems have permitted the growth of differentiated keratinocytes in vitro and provided a permissive environment for the complete HPV life cycle (4, 16, 20, 23, 24, 30, 31, 35). Recently, we used DNA transfection techniques coupled to the organotypic culture system to purify infectious stocks of HPV18 (31). These improved molecular and cellular techniques promise to provide further insights into the enigmatic biology of HPVs.

An understanding of the differentiation-dependent life cycles of high-risk HPVs has been enhanced greatly by the study of the latently infected CIN-612 9E cell line, which contains episomal copies of HPV31b (4, 23, 24, 35). The growth of CIN-612 9E monolayer cells and raft tissues has permitted the identification of the HPV31b early promoter that maps to nucleotide (nt) 97 (P₉₇) and the P₇₄₂ differentiation-induced promoter (23). At least 7 spliced, polycistronic early viral RNAs and 19 polycistronic late viral RNAs have been characterized with CIN-612 9E cells and raft tissues (23, 24, 35, 36). A detailed analysis of HPV31b late gene transcripts indicated that late gene RNAs initiated from at least three distinct promoters; RNA start sites mapped in the region of P₉₇, at multiple sites near P₇₄₂, and close to the E4 splice acceptor site at nt 3295 (35).

The purpose of this study was to investigate the regulation of HPV31b gene transcription throughout the viral life cycle by precisely mapping the RNA start sites and assessing the temporal usage of the viral promoters. Using CIN-612 9E cells grown in the raft tissue culture system, we report two novel, constitutively expressed HPV31b promoters, P₇₇ and P₃₃₂₀. Furthermore, precise mapping indicates that the major early promoter maps to P₉₉ and that the differentiation-dependent promoter P₇₄₂ consists of a cluster of transcriptional start sites.

MATERIALS AND METHODS

Cell and tissue cultures.

The CIN-612 cell line was established from a cervical intraepithelial neoplasia (CIN) grade I biopsy and contains HPV31b DNA (4). In the CIN-612 clonal derivative 9E, the HPV31b genome is maintained episomally at ≈50 copies per cell (23). The SCC-13 cell line was established from a squamous cell carcinoma of the facial epidermis and does not contain HPV DNA (28, 38). Human foreskin keratinocytes (HFKs) were isolated from newborn circumcisions as previously described (49). Epithelial cell lines were maintained in monolayer culture with E medium containing 5% fetal bovine serum in the presence of mitomycin-treated J2 3T3 feeder cells (28, 29). HFKs were grown in the same manner with the addition of 5 ng of epidermal growth factor per ml to the cell medium. Epithelial organotypic (raft) tissue cultures for in vitro differentiation were maintained as previously described (28–30). Briefly, epithelial cells were seeded onto collagen matrices containing J2 3T3 fibroblast feeders. When the epithelial cells had grown to confluence, collagen matrices were lifted onto stainless steel grids and the epithelial cells were fed by diffusion from under the matrix. Epithelial tissues were allowed to stratify and differentiate at the air-liquid interface over a 16-day period. Rafts were either untreated or treated with 10 μM 1,2-dioctanoyl-sn-glycerol (C8:0; Sigma Chemical Co., St. Louis, Mo.) in E medium every other day. Raft tissues were harvested at various time points starting with day 4 after being lifted to the air-liquid interface (day 4) and extending to day 16 after being lifted.

Nucleic acid extraction and RNA PCR analyses.

Total RNAs were extracted from rafts and subconfluent monolayer cultures with TRIzol reagent (Gibco BRL, Bethesda, Md.). The RNA samples were treated with DNase I to remove copurifying viral and cellular DNAs (2). RNA concentrations were based on optical densities; RNA concentrations and qualities were verified by electrophoresis through agarose gels containing ethidium bromide. DNase I-treated total RNA was reverse transcribed by using random hexamer primers, and PCR was performed by using a GeneAmp RNA PCR kit as instructed by the manufacturer (Perkin-Elmer, Branchburg, N.J.). All oligonucleotide primers (Table 1) were synthesized by Operon Technologies (San Diego, Calif.) and were used at 0.5 μM for PCR amplification. The thermocycling profile was as follows: 4-min delay at 94°C; 35 cycles of 94°C for 30 s, 58 to 60°C for 1 min, and 72°C for 2 min; and a 15-min extension at 72°C.

TABLE 1.

Oligonucleotide primers used in an analysis of HPV31b gene expression in CIN-612 9E rafts

Primer	Sequencea	Orientation	ORFb	HPV31 nta
P77 5′	5′-GCA CAT AGT CTG TGG TGC AAA CC-3′c	Sense	URR	75–97
E6 3′	5′-GGG TAT TTC CAA TGC CGA GC-3′	Antisense	E6	173–154
E7 3′	5′-CTG GAT CAG CCA TTG TAG TTA CAG TCT AGT AG-3′	Antisense	E7E1	874–843
E1 3′	5′-TGT CCT CTT CCT CGT GC-3′	Antisense	E1	2667–2683
E4-2 3′	5′-CGC CCG CCG CAC ACC TTC ACT GGT GCC CAA G-3′	Antisense	E4	3409–3380
E4 3′	5′-CTT CAC TGG TGC CCA AGG-3′	Antisense	E4	3395–3378
L2-3 3′	5′-GTA GAG CGT TTG GAC CGC-3′	Antisense	L2	4173–4190
L1-2 3′	5′-TAG CAC TGC CTG CGT G-3′	Antisense	L1	5657–5672

^aCorresponding to the sequence and numbering of HPV31 (19). 

^bORF or region of HPV31. 

^cUnderlined bases were altered from the wild-type sequence to increase the melting temperature. 

Cloning and sequencing.

PCR products were cloned with a TA cloning kit (Invitrogen, San Diego, Calif.). Double-stranded DNA sequencing was performed by the dideoxy method with Sequenase version 2.0 (United States Biochemical, Cleveland, Ohio). The reaction products were separated on 8% polyacrylamide–8 M urea sequencing gels. Dried gels were exposed to Reflection film with intensifying screens (DuPont NEN, Boston, Mass.).

Nuclease protection and primer extension assays.

Nuclease S1 (S1) and exonuclease VII (exoVII) protection analyses were performed as previously described (35). Probes were prepared by PCR amplification from cloned segments of HPV31 DNAs and cDNAs (35). 5′ end-labeled primers complementary to the sense DNA strand (either E6 3′ or E4 3′) were paired with an unlabeled M13(−40) primer complementary to the antisense strand and upstream of the cloned HPV31 URR sequences (Fig. 1). For primer extension reactions 10 pmol of each oligonucleotide primer was 5′ end labeled with a solution containing 30 μCi of [γ-³²P]ATP (6,000 Ci/mmol; DuPont NEN) in 50 mM Tris-Cl (pH 7.5), 10 mM MgCl₂, 15 mM dithiothreitol, 0.1 mM spermidine, and 15 U of T4 polynucleotide kinase (New England Biolabs, Beverly, Mass.). The labeled primers were separated from the unincorporated nucleotides by electrophoresis through a 20% polyacrylamide–7 M urea gel. Labeled primers were eluted from the gel slices into 10 mM Tris-Cl (pH 8.0)–1 mM EDTA–0.6 M NaCl. Total cellular RNA was hybridized with 3.5 μl (1 × 10⁴ to 10 × 10⁴ cpm) of eluted primer in hybridization buffer (150 mM KCl, 10 mM Tris-Cl [pH 8.3], 1 mM EDTA), and the extensions were performed with avian myeloblastosis virus reverse transcriptase (Gibco BRL) as described previously (2). Sequencing ladders generated with HPV31b cDNAs (35) and a Sequenase version 2.0 kit were run as size markers. The samples were analyzed by electrophoresis through a 7% acrylamide–7 M urea gel. Dried gels were subjected to autoradiography with Reflection film and intensifying screens. The intensities of protected fragments were measured by scanning laser densitometry.

FIG. 1.

S1 and exoVII nuclease protection analyses of HPV31b transcripts. CIN-612 9E cells were cultured as monolayers (M) or as C8:0-treated rafts harvested at day 12 (R). Yeast RNA samples were included as controls (Y). Thirty micrograms of total RNA or yeast RNA was hybridized with 5′-end-labeled probe and analyzed by digestion with S1 or exoVII as indicated. RNA Century Markers (Ambion) and 5′-end-labeled φX174 DNA digested with HaeIII were used as standards; their sizes (in bases [b]) are indicated at the right of each panel. The reactions were analyzed by electrophoresis through a 4% polyacrylamide–7 M urea sequencing gel. (A) Probe A contains the URR E6 sequences from HPV31 nt 7381 to 173 and was made from plasmid p31URRE1 (35). (B) Probe B contains the URR, E6*, E7, and E1^E4 sequences from HPV31 nt 7238 to 210^413 to 877^3295 to 3395 and was made from plasmid p31U*742L1 (35). (C) HPV31b genome organization showing the early ORFs as open boxes and the URR (19). The two reported promoters, P₉₇ and P₇₄₂ (23), and the potential promoters, P_L and P_E4 (35), are indicated. The early polyA site (A+) at nt 4138 to 4143 is shown. Antisense 5′-end-labeled probe specificities for nuclease protection assays are indicated at the bottom region of the panel. Filled boxes correspond to HPV31 sequences. Broken lines show sequences spliced out of cDNAs. Thin lines represent plasmid sequences. The positions of the oligonucleotide primers used for primer extension analyses are shown, and their orientations are indicated by arrows (Table 1).

RESULTS

The addition of protein kinase C (PKC) pathway activators such as C8:0 to the culture medium of raft tissues derived from cervical lesions induces a more complete differentiation program (30, 34). Specifically, we have shown that PKC activators induce CIN-612 9E rafts to more appropriately express differentiation markers, including K10, K14, and filaggrin (30, 34). The enhanced differentiation of the CIN-612 9E tissues is accompanied by a strong induction of HPV31b late gene expression and the efficient assembly of virions (24, 30, 35). Our previous work using S1 and exoVII analyses to characterize the late transcripts of HPV31b expressed in CIN-612 9E monolayer cultures and raft tissues indicated that subsets of late gene RNAs initiated at three separate promoters (35). Using probes which extended from the late region through the URR, we found that the 5′-most RNA start sites for five different late gene RNAs were near P₉₇. Multiple start sites were seen in the region of P₇₄₂, and an RNA end was also detected near the E4 splice acceptor at nt 3295 (35). To verify and further map these 5′ RNA start sites, we performed S1 and exoVII analyses using shorter probes on RNA samples derived from CIN-612 9E monolayers and PKC-induced, 12-day raft tissues (Fig. 1). The results from S1 and exoVII digestion assays were identical for each of the URR-containing probes, indicating 5′ RNA ends rather than splice sites. The exoVII-digested samples migrated slightly slower than the S1-digested samples, a common phenomenon when large amounts of RNA are analyzed (5, 35). The use of the probe containing the URR E6 sequences revealed protected fragments with a 5′ end near HPV31 nt 97 as expected; an additional 5′ end mapped ≈20 nt upstream at nt 77 (Fig. 1A). As in our previous mapping of late gene transcripts (35), the probe end labeled in the E4 ORF protected an RNA fragment corresponding to a 5′ end near HPV31 nt 3320; multiple protection sites were observed in the region of the P₇₄₂ promoter (Fig. 1B).

To investigate during the HPV31b life cycle the usage of the P₉₇ promoter and the putative start site ≈20 nt upstream from P₉₇, we performed primer extension assays. Total RNAs were harvested from untreated CIN-612 9E monolayers, from untreated CIN-612 9E raft tissues, and from PKC-induced CIN-612 9E raft tissues. The temporal usage of viral promoters was assessed by harvesting the raft tissues at 4, 8, 12, and 16 days after lifting to the air-liquid interface as previously described (35). The reverse transcriptase-mediated extension of primer E6 3′ (Fig. 1C and Table 1) on the RNA samples resulted in the detection of a strong product corresponding to P₉₇ as expected (Fig. 2, lanes 3 to 11). However, the inclusion of a sequencing ladder synthesized with primer E6 3′ on a cloned segment of HPV31b DNA showed that the primer extension products actually mapped to HPV31b nt 99. Consequently, we will refer to this promoter as P₉₉. A shorter exposure of the autoradiogram shown in Fig. 2 indicated that the levels of transcription from this start site were similar regardless of whether the RNA was obtained from CIN-612 9E untreated monolayer cultures, untreated raft tissues, or PKC-induced raft tissues. Consistent with the S1 and exoVII data, an E6 3′ primer extension product also mapped to nt 77 (Fig. 2, lanes 3 to 11). The levels of transcripts initiating at nt 77 were generally similar whether the RNA samples were obtained from CIN-612 9E untreated monolayer cultures, untreated rafts, or PKC-induced raft tissues; however, the levels did appear to decrease between 12 and 16 days in samples from both sets of raft tissues (Fig. 2, lanes 6, 7, 10, and 11). RNA samples derived from Saccharomyces cerevisiae, HFK monolayer cultures, and SCC-13 raft tissues were used as negative controls. None of these HPV-negative RNA samples gave extension products with primer E6 3′ (Fig. 2, lane 12 and data not shown). Densitometry scanning of the autoradiogram indicated that the products corresponding to P₉₉ were at least 80 to 100 times stronger than the products at nt 77 (data not shown).

FIG. 2.

Temporal analyses of HPV31b promoters with primer E6 3′ in primer extension assays. Total RNAs were extracted from CIN-612 9E untreated monolayers (M); untreated rafts harvested at 4 days (4d), 8 days (8d), 12 days (12d), and 16 days (16d) after being lifted to the air-liquid interface; and rafts treated with C8:0 every second day (PKC induced) and harvested at day 4, 8, 12, and 16 after being lifted to the air-liquid interface. Primer E6 3′ (Table 1 and Fig. 1C) was 5′ end labeled, gel purified, and hybridized to 25 μg of total RNA or yeast RNA (Y). The primers were extended with avian myeloblastosis virus reverse transcriptase, and the RNA was digested with RNase A. Sequencing ladders (AG and CT) were generated with the E6 3′ primer on the cloned HPV31b DNA template p31U*742L1 (35). The reactions were analyzed by electrophoresis through a 7% polyacrylamide–7 M urea sequencing gel.

To help verify that a subset of transcripts was indeed initiated upstream of the P₉₉ promoter, a PCR primer was synthesized to be specific to the region between HPV31b nt 77 and 99 (Table 1). Total RNA from rafts was subjected to reverse transcription, and PCR was performed with primer P77 5′ paired with various 3′ primers (Fig. 3). The PCR products were cloned, and representative clones were sequenced from each reaction mixture. The structures of the cDNAs are shown in Fig. 3. Transcript A (Fig. 3), predicted to use the early polyadenylation (polyA) site, potentially encodes the E6*, E7, E1*I, E2, and E5a ORFs. This transcript contains the reported E6* ORF which has the 210̂413 splice (23) and a novel HPV31b splice combination. The E1 splice donor (nt 877) is spliced into a consensus acceptor at nt 2646, which results in the termination of the fused E1 ORF (E1*I) 30 nt prior to the E2 start codon at nt 2693. The E1*I ORF is predicted to encode a 10-amino-acid peptide. This transcript is one of two viral RNAs we have characterized with the potential to encode the E2 ORF (36). Transcript B contains the E6^E4 and E5a ORFs, whereas transcript C contains the E6*, E7, E1^E4, and E5a ORFs. Transcripts B and C reportedly also initiate from P₉₉ (23). Transcript D in Fig. 3 contains the E6*, E7, E1^E4, E5, L2, and, presumably, L1 ORFs; we previously reported that this transcript initiated near P₉₉ (35). We also used a 3′ PCR primer specific to the L1 region of the HPV31b genome (L1-2 3′) in conjunction with the P77 5′ primer (Fig. 3). We have used primer L1-2 3′ to identify a number of L1 ORF-specific transcripts (35). However, we were not able using primers P77 5′ and L1-2 3′ to amplify and clone HPV31b L1-specific cDNAs which corresponded to those published previously (24, 35) or which we could verify using other types of analyses (e.g., nuclease protection). We attribute this inability to a sensitivity problem commonly encountered when researchers try to amplify by PCR late gene cDNAs approaching 1 kb in length from a pool of total RNAs containing very low levels of late gene transcripts (24, 35). S1 and exoVII nuclease protection assays of five late gene RNAs showed 5′ ends near P₇₇ or P₉₉; none of the five late gene transcripts had initiation sites upstream of this region (35). Together, the RNA PCR data, the results of nuclease protection assays, and the primer extensions indicate that these late gene RNAs initiate at P₇₇ or at both P₇₇ and P₉₉ but not upstream of P₇₇. However, we have not ruled out the possibility that early gene RNAs or additional, uncharacterized late gene RNAs initiate upstream of P₇₇. It is also possible that some transcripts containing the L1 ORF do not initiate upstream of P₉₉. The primer extension data (Fig. 2) coupled with the RNA PCR results suggest that both early and late transcripts are initiated upstream of P₉₉.

FIG. 3.

RNA PCR with primer P77 5′ to verify that HPV31b transcripts initiate upstream of P₉₉. Total RNA was extracted from CIN-612 9E rafts treated with the PKC inducer C8:0 every second day for 12 days. RNA (1 μg) was reverse transcribed, and the cDNAs were amplified by PCR with the primer pairs indicated (Table 1). The orientations of the primers are indicated by arrows above the primer names. The products from the PCRs were cloned, and representative cDNAs were sequenced. The ORFs contained in the predicted full-length cDNAs are shown to the right of each cDNA structure. (A to C) Transcripts predicted to end at the early polyA site (A+) (24, 36). (A) RNA PCR with primers P77 5′ and E1 3′ gave a novel cDNA product containing sequences from the ORFs of E6*, E7, E1*I, and E2. (B and C) RNA PCR with primers P77 5′ and E4 3′ yielded cDNAs similar to those previously identified (23). (D) RNA PCR with primers P77 5′ and L2-3 3′ gave a cDNA similar to a reported transcript predicted to end at the late polyA site (24, 35). Open and stippled boxes represent ORFs; thick lines are noncoding regions. Sequences spliced out of transcripts are shown by thin lines.

Primer extension reactions were performed with primer E7 3′ (Table 1) to specifically map and determine the temporal usage of the differentiation-inducible P₇₄₂ promoter during the viral life cycle. The experiments revealed products corresponding to nt 737, 742, 750, and 767 (Fig. 4). Start sites at nt 737, 742, and 750 were detected in untreated monolayers and rafts but were strongly induced upon PKC induction in the raft system. Densitometry scanning of protected fragments from three separate experiments indicated an average of ≈18-fold upregulation from these start sites in the differentiated tissues (i.e., PKC-induced, 8-day rafts) over levels seen in undifferentiated cells (i.e., untreated and treated 4-day rafts and untreated monolayers [data not shown]). A similar increase in the HPV16 differentiation-inducible P₆₇₀ promoter was observed in differentiated raft tissues of a cell line containing episomal viral genomes (20). The start site at HPV31b nt 767 appeared to be specifically activated upon epithelial differentiation, as it was not detected in RNA from monolayer cultures of CIN-612 9E cells but was highly upregulated upon PKC induction of raft tissues (Fig. 4, lanes 3 and 9, respectively). Initiation from the P₇₄₂ start sites was upregulated only an average of approximately threefold in the PKC-induced 8-day rafts compared to the level of upregulation in the untreated 8-day rafts. However, we have shown that PKC-induced raft tissues at 8 to 12 days are more differentiated, as was determined from their ability to synthesize markers of differentiation, and are more efficient in the production of virions than their untreated counterparts at 8 to 12 days (unpublished observations and references 30, 31, and 34). This suggests that the increase in late gene transcripts and differentiation-specific cellular factors work in concert to increase virion yield. The negative control RNA samples (yeast, HFK, and SCC-13) yielded no extension products with primer E7 3′ (Fig. 4, lane 12 and data not shown).

FIG. 4.

Temporal analyses of HPV31b promoters with primer E7 3′ in primer extension assays. RNA samples, primer extensions, electrophoresis, and abbreviations are as described in the legend to Fig. 2. Primer E7 3′ (Table 1 and Fig. 1C) was hybridized to 30 μg of total RNA or yeast RNA. Sequencing ladders (AG and CT) were generated with primer E7 3′ on the cloned HPV31b DNA template p31U*742L1 (35).

Primer E4-2 3′ (Table 1) was synthesized to investigate the putative RNA start site in the E4 ORF. We detected a 5′ end at nt 3320 by extending the E4-2 3′ primer in the E4 ORF on RNA from CIN-612 9E cells and tissues (Fig. 5). The levels of products with this primer were relatively similar among samples from untreated monolayers, untreated rafts, and PKC-induced raft tissues (Fig. 5, lanes 3 to 11). The use of primer E4-2 3′ on control RNAs from yeast, HFK monolayer cultures, and SCC-13 raft tissues resulted in no products (Fig. 5, lane 12 and data not shown).

FIG. 5.

Temporal analyses of HPV31b promoters with primer E4-2 3′ in primer extension assays. RNA samples, primer extensions, electrophoresis, and abbreviations are as described in the legend to Fig. 2. Primer E4-2 3′ (Table 1 and Fig. 1C) was hybridized to 30 μg of total RNA or yeast RNA. Sequencing ladders (AG and CT) were generated with primer E4-2 3′ on the cloned HPV31b DNA template p31U*742L1 (35).

The primer extension experiments were representative of several analyses of three separate RNA preparations. To help control for experimental error, the primer extension analyses shown in Fig. 2, 4, and 5 were all performed with the same RNA preparations. Further, to establish the differentiation-specific induction of the P₇₄₂ promoter versus the constitutive expression of the P₃₃₂₀ promoter, the primer extension reactions shown in Fig. 4 and 5 were performed with the same amounts and corresponding volumes of RNA preparations and were processed and analyzed concurrently.

DISCUSSION

High-risk HPVs are etiologic agents of anogenital tumors, including cervical cancers (51). The late viral functions of vegetative viral genome amplification, late gene expression, and virion morphogenesis are restricted to the upper, differentiated keratinocytes of the epithelium (4, 8, 16, 17, 30, 42). The organotypic or raft culture system has permitted the study of complete vegetative life cycles of HPVs in vitro (24, 30, 31, 35). We have employed the raft tissue culture system to investigate how HPV transcript expression is linked to the differentiation state of the epithelial tissues. This is the first study comparing the temporal expression patterns from constitutive and differentiation-dependent promoters of an HPV during its life cycle.

We have defined two novel HPV31b promoters, P₇₇ and P₃₃₂₀, and have precisely mapped the initiation sites for two previously identified HPV31b promoters. P₉₉ is the major early promoter, whereas P₇₇ is a minor promoter. Neither P₇₇ nor P₉₉ appears to be differentiation responsive; rather, they are expressed at relatively constant levels throughout the viral life cycle. This is consistent with our previous work showing equivalent levels of late gene transcripts initiating in this region in both monolayer and differentiated raft tissue cultures (35). Furthermore, others have reported constant levels of RNAs initiating from the early promoters of HPV31b and HPV16 in monolayers and differentiated raft tissues (20, 23). Our data from S1 and exoVII analyses of the HPV31b late gene transcripts with probes which contained the entire URR region indicated that the 5′-most RNA start sites were close to P₉₉ (35). Based upon these data in addition to the results of primer extension and RNA PCR analyses (Fig. 2 and 3), we conclude that a subset of late gene transcripts initiate at P₇₇. Whether some late transcripts also use P₉₉ will be technically difficult to test due to the polycistronic natures of these mRNAs and the fact that the sequences including P₉₉ are contained in all the transcripts identified initiating from P₇₇. P₇₇ also seems to be involved in the transcription of early gene transcripts. However, we have not eliminated the possibility that an additional RNA start site resides upstream of nt 77. It is noteworthy that RNA initiation sites analogous in position to P₇₇ have been described for HPV16 (20, 39). Consistent with our previous results from mapping the 5′ ends of HPV31b late gene transcripts (35), P₇₄₂ consists of a cluster of start sites. Expression from this HPV31b P₇₄₂ promoter cluster is highly dependent upon epithelial differentiation and has been shown to initiate at least six differentially spliced RNA species (23, 24, 35). Multiple start sites used by the differentiation-inducible promoters in HPV6, -11, and -16 and in bovine papillomavirus type 1 have also been reported (3, 10, 20). P₃₃₂₀ is a relatively strong promoter that is utilized at generally constant levels throughout the HPV31b life cycle. The first ORFs linearly downstream of transcripts initiating from P₃₃₂₀ are those of E5a and E5b (35), leading us to propose that P₃₃₂₀ is responsible for the synthesis of RNAs specific for the E5a and E5b gene products. Further, the finding that P₃₃₂₀ is a constitutive promoter during the viral life cycle is in agreement with additional data from our laboratory indicating that the ≈10-kDa HPV31b E5a gene product is expressed at constant levels over this same time frame in the raft system (27). Three additional, spliced HPV31b late gene transcripts also appear to initiate at the P₃₃₂₀ promoter (35). We have shown that late gene transcripts (i.e., viral RNAs with the late polyA signal) initiate from the constitutively active P₇₇ and P₃₃₂₀ promoters in undifferentiated monolayer cultures of CIN-612 9Es (this study and reference 35). This finding argues against a proposed switch in RNA splicing or polyA during differentiation as the major component in late gene transcript synthesis (24). Rather, we have presented data consistent with the idea that PKC-induced differentiation results in the synthesis of factors that upregulate the P₇₄₂ promoter, which results in an increase in late gene transcription. These temporal analyses show that promoters P₇₇, P₉₉, and P₃₃₂₀ are constitutively expressed during the viral life cycle. Thus, we suggest that the dramatic increase in late gene transcripts and subsequent accumulation of L1 protein seen in the differentiated raft tissues (16, 17, 30) are substantially dependent upon the induction of the P₇₄₂ promoter cluster (24, 35). This suggestion is consistent with the recent work of Cramer et al. demonstrating that the promoter structure of a differentiation-specific gene affects splice site selection (7).

It is important to note that these experiments give no information on the spatial expression patterns of the viral promoters throughout the epidermal tissues. For example, it is known from immunochemical staining that HPV L1 capsid proteins are confined to the upper epithelial layers and can be detected only in isolated cells (16, 17, 30). In situ hybridization studies of HPV-containing biopsy material have shown E6 and E7 transcripts throughout the epithelial strata; however, a sizable increase in expression of RNAs containing the E4 and L2-L1 ORFs is seen and restricted to the suprabasal layers (8, 42). Based upon these observations, we believe that expression from the differentiation-inducible promoter cluster P₇₄₂ is likely to be confined to the suprabasal cells. It is also possible that the P₇₇, P₉₉, and P₃₃₂₀ promoters may be used differentially in the various strata of the epithelium. Our analyses should detect expression from a promoter with low basal activity in a small proportion of raft tissue. However, these techniques may not efficiently indicate fluctuations in activity from a promoter with relatively high basal expression in a fraction of cells. A total of 10 differentially spliced transcripts containing HPV31b late ORFs initiate from the P₇₇ or P₉₉ region (24, 35); seven spliced early HPV31b RNAs initiate from this same region (23, 36). However, the spatial expression patterns of these transcripts are unknown. The polycistronic natures of the HPV transcripts and the lengths of some of the late gene RNAs will make it difficult to investigate the spatial expression patterns of these RNAs in situ.

We analyzed the sequences surrounding the HPV31b transcriptional start sites for regulatory elements (Fig. 6). The core promoters of mammalian ORFs often contain a TATA box situated 25 to 30 bp upstream of the transcriptional initiation site and/or an initiator (Inr) element overlapping the start site (reviewed in reference 40). Individually, the elements can direct basal transcription and can determine the start site for transcription. Together, with the TATA box 25 to 30 bp 5′ to the Inr in the promoter, the elements cooperate to enhance the strength of the promoter (25, 40). Both P₇₇ and P₉₉ contain consensus Inr sequences overlapping their start sites; an Sp1 site is 45 and 68 nt upstream of P₇₇ and P₉₉, respectively (Fig. 6A). P₉₉ has a consensus TATA box 32 bp upstream of the initiation site, whereas P₇₇ has a TATA box 58 bp upstream of the initiation site. Cooperation among the properly spaced Sp1, Inr, and TATA elements of P₉₉ may account for the much higher levels of transcription from P₉₉ than from P₇₇. The HPV E2 proteins bind as dimers to the highly conserved, palindromic E2BS (Fig. 6A). The placement and spacing of the Sp1 site, the two E2BSs, and the TATA box upstream of the P₉₉ promoter are highly conserved among HPVs which infect the anogenital region (18). In vitro analyses suggest that negative regulation of the P₉₉ promoter occurs via the binding of E2 to the adjacent E2BSs and the obstruction of the Sp1 and TATA binding proteins from their respective sites (12, 14, 46). As these binding sites are closer to P₇₇ than to P₉₉, E2 proteins binding to the E2BSs may repress expression from P₇₇ to a greater extent than from P₉₉. Given a putative role for E2 proteins in the negative regulation of expression from P₉₉, we found it surprising that the P₉₉ promoter was used at relatively high, constant levels for the duration of the viral life cycle included in our study. One might expect to see some variations in the levels of transcripts expressed from P₉₉ depending on the levels of E2 protein present within the cells. We have found the highest levels of E2 transcripts in CIN-612 9E monolayers and in raft tissues harvested at day 12 (36); however, no significant change in transcriptional initiation from P₉₉ was detected over any of these times in the viral life cycle. The phenomenon of HPV E2-mediated repression may be an artifact of the vast amounts of E2 protein used in the nonphysiological in vitro systems (12, 14, 46). There is mounting evidence that the activity of E2 as either a repressor or activator of transcription is dependent upon E2 protein dosage. At low levels of E2 protein, viral promoters are activated by E2 binding to the distal, high-affinity E2BS, whereas at higher levels of E2 protein, the promoters are repressed by E2 binding to the promoter-proximal, low-affinity E2BS (41, 43, 44). However, it may be that the most critical period for E2-mediated repression is directly after HPV infection. Because the CIN-612 9E cell line is latently infected with HPV31b, we have been unable to address this possibility. Our ability to purify infectious HPV stocks following DNA transfection of keratinocytes will permit us to investigate viral gene expression immediately following HPV infection (31). It is also possible that the effects of E2 on P₉₉ and P₇₇ may be dependent upon the localization of the molecules throughout the epithelial strata. As we used whole tissue lysates in these analyses, the data represent an averaging of the effects of spatial expression. Again, changes in the expression from P₉₉ in small compartments of cells will be difficult to detect. It will be important to investigate the levels of E2 protein expression over the viral life cycle in addressing these matters.

FIG. 6.

Analysis of the nucleotide sequences surrounding HPV31b promoters based upon the sequence for HPV31 (19). Genome nucleotide numbering is given below each sequence. (A to C) Consensus nucleic acid sequence recognition sites are shown for the TATA binding protein (filled boxes indicate good consensus, and open boxes show weak consensus) (40), Sp1 motifs (15), E2BSs (ACCGN₄CGGT) (1), initiator sequences (underlining is used for consensus sequence YYA₊₁NA/TYY with an ∗ at critical +1 and +3 residues) (25, 40), and CCAAT sites (C/EBP) (21). The promoters are indicated by bent arrows with corresponding nucleotide numbers, and the start codons of ORFs are indicated with shaded boxes. (C) The E4 splice acceptor (E4 S.A.) is indicated at nt 3295. (D) Summary of the HPV31b genome, indicating the URR, the ORFs (open boxes), the four promoters, and the polyA (A+) sites.

A CCAAT motif is situated 34 bp upstream of nt 742 (Fig. 6B). This sequence is recognized by the CCAAT enhancer binding protein (C/EBP) (21). C/EBP is expressed at elevated levels in a variety of terminally differentiated cells, including skin cells, and has a function in the differentiation-specific activation of genes (47). Furthermore, this CCAAT motif is conserved in HPVs; it is present upstream of the differentiation-inducible promoters of HPV6, -11, and -16 and of bovine papillomavirus type 1 (3, 20, 32). Consensus TATA boxes are 24 and 29 bp upstream of the start sites at nt 737 and 742, respectively. TATA boxes of weak consensus are properly spaced upstream of the initiation sites at nt 750 and 767. The lack of Sp1 and Inr elements may contribute to the low expression from the P₇₄₂ cluster in undifferentiated cells. Differentiated tissues may contain, in addition to C/EBP, factors that overcome the less than optimal cis-regulatory sequences to upregulate expression from this promoter cluster. The E2BSs located in the URR upstream of P₉₉ (Fig. 6A) may also play a functional role in the regulation of the P₇₄₂ promoter cluster.

P₃₃₂₀, like P₉₉, contains consensus Inr and TATA motifs (Fig. 6C) which likely stimulate the strong constitutive expression from this promoter observed over the course of the viral life cycle. The situation of an Sp1 binding site upstream also may contribute to the constitutive expression of the P₃₃₂₀ promoter in the undifferentiated monolayer cells and during the differentiation process in the raft tissues (Fig. 6C).

In summary, we have shown biochemical evidence for four HPV31b promoters (Fig. 6D) and their usage throughout the viral life cycle. Our ability to produce infectious HPV stocks following the transfection of HPV genomic DNA into keratinocytes will allow us to construct mutant viruses (31). We are in the process of using this technique to gather genetic evidence on how the promoters are regulated and how they contribute to the differentiation-dependent life cycle of HPVs.