temporal usage of multiple promoters during the life cycle of

JOURNAL OF VIROLOGY,0022-538X/98/$04.0010

Apr. 1998, p. 2715–2722 Vol. 72, No. 4

Copyright © 1998, American Society for Microbiology

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

MICHELLE A. OZBUN AND CRAIG MEYERS*

Department of Microbiology and Immunology, The Pennsylvania State University College of Medicine,Hershey, Pennsylvania 17033

Received 29 October 1997/Accepted 23 December 1997

The life cycles of human papillomaviruses (HPVs) are dependent upon the differentiation of the epithelialcells they infect. HPV type 31b (HPV31b) virions can be purified following the growth of a latently HPV-infectedcell line (CIN-612 9E) in the organotypic or raft system. Treatment of the CIN-612 9E raft tissues with proteinkinase C (PKC) activators is required for upregulation of late gene expression and efficient production ofvirions. We employed the raft culture system to study the temporal usage of HPV31b promoters during the virallife cycle. We compared monolayer cultures of CIN-612 9E cells, untreated CIN-612 9E raft tissues, andPKC-induced CIN-612 9E raft tissues harvested at various time points during epithelial differentiation. Wefound that the HPV31b major early promoter precisely maps to nucleotide (nt) 99 (P99). A transcriptional startsite for both early and late gene transcripts mapped upstream of P99 at nt 77 (P77). The P77 and P99 promoterswere used constitutively throughout the HPV31b life cycle; however, initiation from P99 was much stronger thanfrom P77. Mapping of the differentiation-induced P742 promoter revealed multiple start sites. These start siteswere 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 responsiblefor the transcription of unspliced and spliced RNAs containing E5a, E5b, L2, and L1 open reading frames.

Human papillomaviruses (HPVs) are small DNA virusesthat have a tropism for squamous epithelium (45). More than75 types of HPVs have been identified (32), with subsets caus-ing benign and malignant tumors of the anogenital region (26,51). The so-called high-risk types are associated with an in-creased risk of cervical malignancy and include HPV types 16(HPV16), -18, -31, and -33. The low-risk group involved inanogenital lesions includes HPV6 and -11 and is rarely linkedto malignancy. In benign tumors the viral DNA is generallypresent extrachromosomally; however, the viral genome is of-ten 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 thedifferentiation state of the infected cells (8, 30, 42, 45). Thevirions encapsidate a circular DNA molecule containing six toeight early open reading frames (ORFs) and two late ORFs. Anumber of enhancer and promoter elements involved in thecontrol of early gene expression, as well as sequences impor-tant for replication, are contained in the upstream regulatoryregion (URR) of the viral genome. The E6 and E7 transform-ing proteins functionally inactivate the tumor suppressor pro-teins p53 and pRB, respectively (reviewed in reference 50). E1and E2 proteins mediate viral genome replication (6, 48). TheE2 protein also acts as a transcriptional modulator by inter-acting with conserved sequences known as E2 binding sites(E2BSs) located in the URR (1, 12, 14, 22, 41, 46). The E5proteins may augment the effects of E6 and E7 by manipulat-ing the activities of cellular growth factor receptors (11). Ascells from the basal layer divide and migrate up through theepithelium, a complex program of differentiation is initiated.The viral late functions are dependent upon cellular differen-tiation, but the control of these activities is poorly understood.

In suprabasal cells the E4 protein is synthesized as a fusionwith the N-terminal region of E1 (E1^E4) and associates withcytokeratins (13, 33, 37). Concomitant with cellular differenti-ation in virally infected cells are the amplification of viralgenomes in preparation for packaging and the expression ofthe L1 and L2 proteins, which form the viral capsids (4, 16, 17,30).

Historically, the dependence of the HPV life cycle on cellu-lar differentiation has impeded the study of the viral late func-tions. Most cell lines used to study HPVs are derived frommalignancies and contain integrated viral genomes with ob-structed late functions. Continuing advances in organotypic orraft tissue culture systems have permitted the growth of differ-entiated keratinocytes in vitro and provided a permissive en-vironment for the complete HPV life cycle (4, 16, 20, 23, 24, 30,31, 35). Recently, we used DNA transfection techniques cou-pled to the organotypic culture system to purify infectiousstocks of HPV18 (31). These improved molecular and cellulartechniques promise to provide further insights into the enig-matic biology of HPVs.

An understanding of the differentiation-dependent life cy-cles of high-risk HPVs has been enhanced greatly by the studyof the latently infected CIN-612 9E cell line, which containsepisomal copies of HPV31b (4, 23, 24, 35). The growth ofCIN-612 9E monolayer cells and raft tissues has permitted theidentification of the HPV31b early promoter that maps tonucleotide (nt) 97 (P97) and the P742 differentiation-inducedpromoter (23). At least 7 spliced, polycistronic early viralRNAs and 19 polycistronic late viral RNAs have been charac-terized with CIN-612 9E cells and raft tissues (23, 24, 35, 36).A detailed analysis of HPV31b late gene transcripts indicatedthat late gene RNAs initiated from at least three distinct pro-moters; RNA start sites mapped in the region of P97, at mul-tiple sites near P742, and close to the E4 splice acceptor site atnt 3295 (35).

The purpose of this study was to investigate the regulation ofHPV31b gene transcription throughout the viral life cycle by

* Corresponding author. Mailing address: Department of Microbi-ology and Immunology, The Pennsylvania State University College ofMedicine, Hershey, PA 17033. Phone: (717) 531-6240. Fax: (717) 531-6522. E-mail: [email protected].

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precisely mapping the RNA start sites and assessing the tem-poral usage of the viral promoters. Using CIN-612 9E cellsgrown in the raft tissue culture system, we report two novel,constitutively expressed HPV31b promoters, P77 and P3320.Furthermore, precise mapping indicates that the major earlypromoter maps to P99 and that the differentiation-dependentpromoter 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 cervicalintraepithelial neoplasia (CIN) grade I biopsy and contains HPV31b DNA (4). Inthe CIN-612 clonal derivative 9E, the HPV31b genome is maintained episomallyat '50 copies per cell (23). The SCC-13 cell line was established from a squa-mous cell carcinoma of the facial epidermis and does not contain HPV DNA (28,38). Human foreskin keratinocytes (HFKs) were isolated from newborn circum-cisions as previously described (49). Epithelial cell lines were maintained inmonolayer culture with E medium containing 5% fetal bovine serum in thepresence of mitomycin-treated J2 3T3 feeder cells (28, 29). HFKs were grown inthe same manner with the addition of 5 ng of epidermal growth factor per ml tothe cell medium. Epithelial organotypic (raft) tissue cultures for in vitro differ-entiation were maintained as previously described (28–30). Briefly, epithelialcells were seeded onto collagen matrices containing J2 3T3 fibroblast feeders.When the epithelial cells had grown to confluence, collagen matrices were liftedonto stainless steel grids and the epithelial cells were fed by diffusion from underthe matrix. Epithelial tissues were allowed to stratify and differentiate at theair-liquid interface over a 16-day period. Rafts were either untreated or treatedwith 10 mM 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 timepoints starting with day 4 after being lifted to the air-liquid interface (day 4) andextending to day 16 after being lifted.

Nucleic acid extraction and RNA PCR analyses. Total RNAs were extractedfrom rafts and subconfluent monolayer cultures with TRIzol reagent (GibcoBRL, Bethesda, Md.). The RNA samples were treated with DNase I to removecopurifying viral and cellular DNAs (2). RNA concentrations were based onoptical densities; RNA concentrations and qualities were verified by electro-phoresis through agarose gels containing ethidium bromide. DNase I-treatedtotal RNA was reverse transcribed by using random hexamer primers, and PCRwas performed by using a GeneAmp RNA PCR kit as instructed by the manu-facturer (Perkin-Elmer, Branchburg, N.J.). All oligonucleotide primers (Table 1)were synthesized by Operon Technologies (San Diego, Calif.) and were used at0.5 mM for PCR amplification. The thermocycling profile was as follows: 4-mindelay 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.

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

Nuclease protection and primer extension assays. Nuclease S1 (S1) and exo-nuclease VII (exoVII) protection analyses were performed as previously de-scribed (35). Probes were prepared by PCR amplification from cloned segmentsof HPV31 DNAs and cDNAs (35). 59 end-labeled primers complementary to thesense DNA strand (either E6 39 or E4 39) were paired with an unlabeledM13(240) primer complementary to the antisense strand and upstream of thecloned HPV31 URR sequences (Fig. 1). For primer extension reactions 10 pmolof each oligonucleotide primer was 59 end labeled with a solution containing 30mCi of [g-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 T4polynucleotide kinase (New England Biolabs, Beverly, Mass.). The labeled prim-

ers were separated from the unincorporated nucleotides by electrophoresisthrough a 20% polyacrylamide–7 M urea gel. Labeled primers were eluted fromthe gel slices into 10 mM Tris-Cl (pH 8.0)–1 mM EDTA–0.6 M NaCl. Totalcellular RNA was hybridized with 3.5 ml (1 3 104 to 10 3 104 cpm) of elutedprimer in hybridization buffer (150 mM KCl, 10 mM Tris-Cl [pH 8.3], 1 mMEDTA), and the extensions were performed with avian myeloblastosis virusreverse transcriptase (Gibco BRL) as described previously (2). Sequencing lad-ders generated with HPV31b cDNAs (35) and a Sequenase version 2.0 kit wererun as size markers. The samples were analyzed by electrophoresis through a 7%acrylamide–7 M urea gel. Dried gels were subjected to autoradiography withReflection film and intensifying screens. The intensities of protected fragmentswere measured by scanning laser densitometry.

RESULTS

The addition of protein kinase C (PKC) pathway activatorssuch as C8:0 to the culture medium of raft tissues derived fromcervical lesions induces a more complete differentiation pro-gram (30, 34). Specifically, we have shown that PKC activatorsinduce CIN-612 9E rafts to more appropriately express differ-entiation markers, including K10, K14, and filaggrin (30, 34).The enhanced differentiation of the CIN-612 9E tissues isaccompanied by a strong induction of HPV31b late gene ex-pression and the efficient assembly of virions (24, 30, 35). Ourprevious work using S1 and exoVII analyses to characterize thelate transcripts of HPV31b expressed in CIN-612 9E mono-layer cultures and raft tissues indicated that subsets of lategene RNAs initiated at three separate promoters (35). Usingprobes which extended from the late region through the URR,we found that the 59-most RNA start sites for five different lategene RNAs were near P97. Multiple start sites were seen in theregion of P742, and an RNA end was also detected near the E4splice acceptor at nt 3295 (35). To verify and further map these59 RNA start sites, we performed S1 and exoVII analyses usingshorter probes on RNA samples derived from CIN-612 9Emonolayers and PKC-induced, 12-day raft tissues (Fig. 1). Theresults from S1 and exoVII digestion assays were identical foreach of the URR-containing probes, indicating 59 RNA endsrather than splice sites. The exoVII-digested samples migratedslightly slower than the S1-digested samples, a common phe-nomenon when large amounts of RNA are analyzed (5, 35).The use of the probe containing the URR E6 sequences re-vealed protected fragments with a 59 end near HPV31 nt 97 asexpected; an additional 59 end mapped '20 nt upstream at nt77 (Fig. 1A). As in our previous mapping of late gene tran-scripts (35), the probe end labeled in the E4 ORF protected anRNA fragment corresponding to a 59 end near HPV31 nt 3320;multiple protection sites were observed in the region of theP742 promoter (Fig. 1B).

To investigate during the HPV31b life cycle the usage of theP97 promoter and the putative start site '20 nt upstream fromP97, we performed primer extension assays. Total RNAs wereharvested from untreated CIN-612 9E monolayers, from un-

TABLE 1. Oligonucleotide primers used in an analysis of HPV31b gene expression in CIN-612 9E rafts

Primer Sequencea Orientation ORFb HPV31 nta

P77 59 59-GCA CAT AGT CTG TGG TGC AAA CC-39c Sense URR 75–97E6 39 59-GGG TAT TTC CAA TGC CGA GC-39 Antisense E6 173–154E7 39 59-CTG GAT CAG CCA TTG TAG TTA CAG TCT AGT AG-39 Antisense E7E1 874–843E1 39 59-TGT CCT CTT CCT CGT GC-39 Antisense E1 2667–2683E4-2 39 59-CGC CCG CCG CAC ACC TTC ACT GGT GCC CAA G-39 Antisense E4 3409–3380E4 39 59-CTT CAC TGG TGC CCA AGG-39 Antisense E4 3395–3378L2-3 39 59-GTA GAG CGT TTG GAC CGC-39 Antisense L2 4173–4190L1-2 39 59-TAG CAC TGC CTG CGT G-39 Antisense L1 5657–5672

a Corresponding to the sequence and numbering of HPV31 (19).b ORF or region of HPV31.c Underlined bases were altered from the wild-type sequence to increase the melting temperature.

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treated CIN-612 9E raft tissues, and from PKC-induced CIN-612 9E raft tissues. The temporal usage of viral promoters wasassessed by harvesting the raft tissues at 4, 8, 12, and 16 daysafter lifting to the air-liquid interface as previously described(35). The reverse transcriptase-mediated extension of primerE6 39 (Fig. 1C and Table 1) on the RNA samples resulted inthe detection of a strong product corresponding to P97 asexpected (Fig. 2, lanes 3 to 11). However, the inclusion of asequencing ladder synthesized with primer E6 39 on a clonedsegment of HPV31b DNA showed that the primer extensionproducts actually mapped to HPV31b nt 99. Consequently, wewill refer to this promoter as P99. A shorter exposure of theautoradiogram shown in Fig. 2 indicated that the levels oftranscription from this start site were similar regardless ofwhether the RNA was obtained from CIN-612 9E untreatedmonolayer cultures, untreated raft tissues, or PKC-inducedraft tissues. Consistent with the S1 and exoVII data, an E6 39primer extension product also mapped to nt 77 (Fig. 2, lanes 3to 11). The levels of transcripts initiating at nt 77 were gener-ally similar whether the RNA samples were obtained fromCIN-612 9E untreated monolayer cultures, untreated rafts, orPKC-induced raft tissues; however, the levels did appear todecrease between 12 and 16 days in samples from both sets ofraft tissues (Fig. 2, lanes 6, 7, 10, and 11). RNA samplesderived from Saccharomyces cerevisiae, HFK monolayer cul-tures, and SCC-13 raft tissues were used as negative controls.None of these HPV-negative RNA samples gave extensionproducts with primer E6 39 (Fig. 2, lane 12 and data notshown). Densitometry scanning of the autoradiogram indi-cated that the products corresponding to P99 were at least 80 to100 times stronger than the products at nt 77 (data not shown).

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 harvestedat day 12 (R). Yeast RNA samples were included as controls (Y). Thirty micrograms of total RNA or yeast RNA was hybridized with 59-end-labeled probe and analyzedby digestion with S1 or exoVII as indicated. RNA Century Markers (Ambion) and 59-end-labeled fX174 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) ProbeA 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^E4sequences 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 earlyORFs as open boxes and the URR (19). The two reported promoters, P97 and P742 (23), and the potential promoters, PL and PE4 (35), are indicated. The early polyAsite (A1) at nt 4138 to 4143 is shown. Antisense 59-end-labeled probe specificities for nuclease protection assays are indicated at the bottom region of the panel. Filledboxes correspond to HPV31 sequences. Broken lines show sequences spliced out of cDNAs. Thin lines represent plasmid sequences. The positions of the oligonu-cleotide primers used for primer extension analyses are shown, and their orientations are indicated by arrows (Table 1).

FIG. 2. Temporal analyses of HPV31b promoters with primer E6 39 inprimer extension assays. Total RNAs were extracted from CIN-612 9E untreatedmonolayers (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 raftstreated 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 39 (Table 1 and Fig.1C) was 59 end labeled, gel purified, and hybridized to 25 mg of total RNA oryeast RNA (Y). The primers were extended with avian myeloblastosis virusreverse transcriptase, and the RNA was digested with RNase A. Sequencingladders (AG and CT) were generated with the E6 39 primer on the clonedHPV31b DNA template p31U*742L1 (35). The reactions were analyzed byelectrophoresis through a 7% polyacrylamide–7 M urea sequencing gel.

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To help verify that a subset of transcripts was indeed initi-ated upstream of the P99 promoter, a PCR primer was synthe-sized to be specific to the region between HPV31b nt 77 and 99(Table 1). Total RNA from rafts was subjected to reversetranscription, and PCR was performed with primer P77 59paired with various 39 primers (Fig. 3). The PCR products werecloned, and representative clones were sequenced from eachreaction mixture. The structures of the cDNAs are shown inFig. 3. Transcript A (Fig. 3), predicted to use the early poly-adenylation (polyA) site, potentially encodes the E6*, E7,E1*I, E2, and E5a ORFs. This transcript contains the reportedE6* ORF which has the 210ˆ413 splice (23) and a novelHPV31b splice combination. The E1 splice donor (nt 877) isspliced into a consensus acceptor at nt 2646, which results inthe termination of the fused E1 ORF (E1*I) 30 nt prior to theE2 start codon at nt 2693. The E1*I ORF is predicted toencode a 10-amino-acid peptide. This transcript is one of twoviral RNAs we have characterized with the potential to encodethe E2 ORF (36). Transcript B contains the E6Ê4 and E5aORFs, whereas transcript C contains the E6*, E7, E1Ê4, andE5a ORFs. Transcripts B and C reportedly also initiate fromP99 (23). Transcript D in Fig. 3 contains the E6*, E7, E1Ê4,E5, L2, and, presumably, L1 ORFs; we previously reportedthat this transcript initiated near P99 (35). We also used a 39PCR primer specific to the L1 region of the HPV31b genome(L1-2 39) in conjunction with the P77 59 primer (Fig. 3). Wehave used primer L1-2 39 to identify a number of L1 ORF-specific transcripts (35). However, we were not able usingprimers P77 59 and L1-2 39 to amplify and clone HPV31bL1-specific cDNAs which corresponded to those publishedpreviously (24, 35) or which we could verify using other typesof analyses (e.g., nuclease protection). We attribute this inabil-ity to a sensitivity problem commonly encountered when re-searchers try to amplify by PCR late gene cDNAs approaching1 kb in length from a pool of total RNAs containing very low

levels of late gene transcripts (24, 35). S1 and exoVII nucleaseprotection assays of five late gene RNAs showed 59 ends nearP77 or P99; none of the five late gene transcripts had initiationsites upstream of this region (35). Together, the RNA PCRdata, the results of nuclease protection assays, and the primerextensions indicate that these late gene RNAs initiate at P77 orat both P77 and P99 but not upstream of P77. However, we havenot ruled out the possibility that early gene RNAs or addi-tional, uncharacterized late gene RNAs initiate upstream ofP77. It is also possible that some transcripts containing the L1ORF do not initiate upstream of P99. The primer extensiondata (Fig. 2) coupled with the RNA PCR results suggest thatboth early and late transcripts are initiated upstream of P99.

Primer extension reactions were performed with primer E739 (Table 1) to specifically map and determine the temporalusage of the differentiation-inducible P742 promoter during theviral life cycle. The experiments revealed products correspond-ing to nt 737, 742, 750, and 767 (Fig. 4). Start sites at nt 737,742, and 750 were detected in untreated monolayers and raftsbut were strongly induced upon PKC induction in the raftsystem. Densitometry scanning of protected fragments fromthree separate experiments indicated an average of '18-foldupregulation from these start sites in the differentiated tissues(i.e., PKC-induced, 8-day rafts) over levels seen in undifferen-tiated cells (i.e., untreated and treated 4-day rafts and un-treated monolayers [data not shown]). A similar increase in theHPV16 differentiation-inducible P670 promoter was observedin differentiated raft tissues of a cell line containing episomalviral genomes (20). The start site at HPV31b nt 767 appearedto be specifically activated upon epithelial differentiation, as itwas not detected in RNA from monolayer cultures of CIN-6129E cells but was highly upregulated upon PKC induction of rafttissues (Fig. 4, lanes 3 and 9, respectively). Initiation from theP742 start sites was upregulated only an average of approxi-mately threefold in the PKC-induced 8-day rafts compared to

FIG. 3. RNA PCR with primer P77 59 to verify that HPV31b transcripts initiate upstream of P99. Total RNA was extracted from CIN-612 9E rafts treated with thePKC inducer C8:0 every second day for 12 days. RNA (1 mg) was reverse transcribed, and the cDNAs were amplified by PCR with the primer pairs indicated (Table1). The orientations of the primers are indicated by arrows above the primer names. The products from the PCRs were cloned, and representative cDNAs weresequenced. 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 earlypolyA site (A1) (24, 36). (A) RNA PCR with primers P77 59 and E1 39 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 59 and E4 39 yielded cDNAs similar to those previously identified (23). (D) RNA PCR with primers P77 59 and L2-3 39 gavea 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.


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the level of upregulation in the untreated 8-day rafts. However,we have shown that PKC-induced raft tissues at 8 to 12 daysare more differentiated, as was determined from their ability tosynthesize markers of differentiation, and are more efficient inthe production of virions than their untreated counterparts at8 to 12 days (unpublished observations and references 30, 31,and 34). This suggests that the increase in late gene transcriptsand differentiation-specific cellular factors work in concert toincrease virion yield. The negative control RNA samples(yeast, HFK, and SCC-13) yielded no extension products withprimer E7 39 (Fig. 4, lane 12 and data not shown).

Primer E4-2 39 (Table 1) was synthesized to investigate theputative RNA start site in the E4 ORF. We detected a 59 endat nt 3320 by extending the E4-2 39 primer in the E4 ORF onRNA from CIN-612 9E cells and tissues (Fig. 5). The levels ofproducts with this primer were relatively similar among sam-ples from untreated monolayers, untreated rafts, and PKC-induced raft tissues (Fig. 5, lanes 3 to 11). The use of primerE4-2 39 on control RNAs from yeast, HFK monolayer cultures,and SCC-13 raft tissues resulted in no products (Fig. 5, lane 12and data not shown).

The primer extension experiments were representative ofseveral analyses of three separate RNA preparations. To helpcontrol for experimental error, the primer extension analysesshown in Fig. 2, 4, and 5 were all performed with the sameRNA preparations. Further, to establish the differentiation-specific induction of the P742 promoter versus the constitutiveexpression of the P3320 promoter, the primer extension reac-tions shown in Fig. 4 and 5 were performed with the sameamounts and corresponding volumes of RNA preparations andwere processed and analyzed concurrently.

DISCUSSION

High-risk HPVs are etiologic agents of anogenital tumors,including cervical cancers (51). The late viral functions of veg-etative viral genome amplification, late gene expression, andvirion morphogenesis are restricted to the upper, differentiatedkeratinocytes of the epithelium (4, 8, 16, 17, 30, 42). The

organotypic or raft culture system has permitted the study ofcomplete vegetative life cycles of HPVs in vitro (24, 30, 31, 35).We have employed the raft tissue culture system to investigatehow HPV transcript expression is linked to the differentiationstate of the epithelial tissues. This is the first study comparingthe temporal expression patterns from constitutive and differ-entiation-dependent promoters of an HPV during its life cycle.

We have defined two novel HPV31b promoters, P77 andP3320, and have precisely mapped the initiation sites for twopreviously identified HPV31b promoters. P99 is the major earlypromoter, whereas P77 is a minor promoter. Neither P77 norP99 appears to be differentiation responsive; rather, they areexpressed at relatively constant levels throughout the viral lifecycle. This is consistent with our previous work showing equiv-alent levels of late gene transcripts initiating in this region inboth monolayer and differentiated raft tissue cultures (35).Furthermore, others have reported constant levels of RNAsinitiating from the early promoters of HPV31b and HPV16 inmonolayers and differentiated raft tissues (20, 23). Our datafrom S1 and exoVII analyses of the HPV31b late gene tran-scripts with probes which contained the entire URR regionindicated that the 59-most RNA start sites were close to P99(35). Based upon these data in addition to the results of primerextension and RNA PCR analyses (Fig. 2 and 3), we concludethat a subset of late gene transcripts initiate at P77. Whethersome late transcripts also use P99 will be technically difficult totest due to the polycistronic natures of these mRNAs and thefact that the sequences including P99 are contained in all thetranscripts identified initiating from P77. P77 also seems to beinvolved in the transcription of early gene transcripts. How-ever, we have not eliminated the possibility that an additionalRNA start site resides upstream of nt 77. It is noteworthy thatRNA initiation sites analogous in position to P77 have beendescribed for HPV16 (20, 39). Consistent with our previousresults from mapping the 59 ends of HPV31b late gene tran-scripts (35), P742 consists of a cluster of start sites. Expressionfrom this HPV31b P742 promoter cluster is highly dependentupon epithelial differentiation and has been shown to initiateat least six differentially spliced RNA species (23, 24, 35).

FIG. 4. Temporal analyses of HPV31b promoters with primer E7 39 inprimer extension assays. RNA samples, primer extensions, electrophoresis, andabbreviations are as described in the legend to Fig. 2. Primer E7 39 (Table 1 andFig. 1C) was hybridized to 30 mg of total RNA or yeast RNA. Sequencing ladders(AG and CT) were generated with primer E7 39 on the cloned HPV31b DNAtemplate p31U*742L1 (35).

FIG. 5. Temporal analyses of HPV31b promoters with primer E4-2 39 inprimer extension assays. RNA samples, primer extensions, electrophoresis, andabbreviations are as described in the legend to Fig. 2. Primer E4-2 39 (Table 1and Fig. 1C) was hybridized to 30 mg of total RNA or yeast RNA. Sequencingladders (AG and CT) were generated with primer E4-2 39 on the cloned HPV31bDNA template p31U*742L1 (35).


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Multiple start sites used by the differentiation-inducible pro-moters in HPV6, -11, and -16 and in bovine papillomavirustype 1 have also been reported (3, 10, 20). P3320 is a relativelystrong promoter that is utilized at generally constant levelsthroughout the HPV31b life cycle. The first ORFs linearlydownstream of transcripts initiating from P3320 are those ofE5a and E5b (35), leading us to propose that P3320 is respon-sible for the synthesis of RNAs specific for the E5a and E5bgene products. Further, the finding that P3320 is a constitutivepromoter during the viral life cycle is in agreement with addi-tional data from our laboratory indicating that the '10-kDaHPV31b E5a gene product is expressed at constant levels overthis same time frame in the raft system (27). Three additional,spliced HPV31b late gene transcripts also appear to initiate atthe P3320 promoter (35). We have shown that late gene tran-scripts (i.e., viral RNAs with the late polyA signal) initiate fromthe constitutively active P77 and P3320 promoters in undiffer-entiated monolayer cultures of CIN-612 9Es (this study andreference 35). This finding argues against a proposed switch inRNA splicing or polyA during differentiation as the majorcomponent in late gene transcript synthesis (24). Rather, wehave presented data consistent with the idea that PKC-induceddifferentiation results in the synthesis of factors that upregulatethe P742 promoter, which results in an increase in late genetranscription. These temporal analyses show that promotersP77, P99, and P3320 are constitutively expressed during the virallife cycle. Thus, we suggest that the dramatic increase in lategene transcripts and subsequent accumulation of L1 proteinseen in the differentiated raft tissues (16, 17, 30) are substan-tially dependent upon the induction of the P742 promoter clus-ter (24, 35). This suggestion is consistent with the recent workof Cramer et al. demonstrating that the promoter structure ofa differentiation-specific gene affects splice site selection (7).

It is important to note that these experiments give no infor-mation on the spatial expression patterns of the viral promot-ers throughout the epidermal tissues. For example, it is knownfrom immunochemical staining that HPV L1 capsid proteinsare confined to the upper epithelial layers and can be detectedonly in isolated cells (16, 17, 30). In situ hybridization studiesof HPV-containing biopsy material have shown E6 and E7transcripts throughout the epithelial strata; however, a sizableincrease in expression of RNAs containing the E4 and L2-L1ORFs is seen and restricted to the suprabasal layers (8, 42).Based upon these observations, we believe that expressionfrom the differentiation-inducible promoter cluster P742 islikely to be confined to the suprabasal cells. It is also possiblethat the P77, P99, and P3320 promoters may be used differen-tially in the various strata of the epithelium. Our analysesshould detect expression from a promoter with low basal ac-tivity in a small proportion of raft tissue. However, these tech-niques may not efficiently indicate fluctuations in activity froma promoter with relatively high basal expression in a fraction ofcells. A total of 10 differentially spliced transcripts containingHPV31b late ORFs initiate from the P77 or P99 region (24, 35);seven spliced early HPV31b RNAs initiate from this sameregion (23, 36). However, the spatial expression patterns ofthese transcripts are unknown. The polycistronic natures of theHPV transcripts and the lengths of some of the late geneRNAs will make it difficult to investigate the spatial expressionpatterns of these RNAs in situ.

We analyzed the sequences surrounding the HPV31b tran-scriptional start sites for regulatory elements (Fig. 6). The corepromoters of mammalian ORFs often contain a TATA boxsituated 25 to 30 bp upstream of the transcriptional initiationsite and/or an initiator (Inr) element overlapping the start site(reviewed in reference 40). Individually, the elements can di-

rect basal transcription and can determine the start site fortranscription. Together, with the TATA box 25 to 30 bp 59 tothe Inr in the promoter, the elements cooperate to enhance thestrength of the promoter (25, 40). Both P77 and P99 containconsensus Inr sequences overlapping their start sites; an Sp1site is 45 and 68 nt upstream of P77 and P99, respectively (Fig.6A). P99 has a consensus TATA box 32 bp upstream of theinitiation site, whereas P77 has a TATA box 58 bp upstream ofthe initiation site. Cooperation among the properly spacedSp1, Inr, and TATA elements of P99 may account for the muchhigher levels of transcription from P99 than from P77. The HPVE2 proteins bind as dimers to the highly conserved, palin-dromic E2BS (Fig. 6A). The placement and spacing of the Sp1site, the two E2BSs, and the TATA box upstream of the P99promoter are highly conserved among HPVs which infect theanogenital region (18). In vitro analyses suggest that negativeregulation of the P99 promoter occurs via the binding of E2 tothe adjacent E2BSs and the obstruction of the Sp1 and TATAbinding proteins from their respective sites (12, 14, 46). Asthese binding sites are closer to P77 than to P99, E2 proteinsbinding to the E2BSs may repress expression from P77 to agreater extent than from P99. Given a putative role for E2proteins in the negative regulation of expression from P99, wefound it surprising that the P99 promoter was used at relativelyhigh, constant levels for the duration of the viral life cycleincluded in our study. One might expect to see some variationsin the levels of transcripts expressed from P99 depending onthe levels of E2 protein present within the cells. We have foundthe highest levels of E2 transcripts in CIN-612 9E monolayersand in raft tissues harvested at day 12 (36); however, no sig-nificant change in transcriptional initiation from P99 was de-tected over any of these times in the viral life cycle. Thephenomenon of HPV E2-mediated repression may be an arti-fact of the vast amounts of E2 protein used in the nonphysi-ological in vitro systems (12, 14, 46). There is mounting evi-dence that the activity of E2 as either a repressor or activatorof transcription is dependent upon E2 protein dosage. At lowlevels of E2 protein, viral promoters are activated by E2 bind-ing to the distal, high-affinity E2BS, whereas at higher levels ofE2 protein, the promoters are repressed by E2 binding to thepromoter-proximal, low-affinity E2BS (41, 43, 44). However, itmay be that the most critical period for E2-mediated repres-sion is directly after HPV infection. Because the CIN-612 9Ecell line is latently infected with HPV31b, we have been unableto address this possibility. Our ability to purify infectious HPVstocks following DNA transfection of keratinocytes will permitus to investigate viral gene expression immediately followingHPV infection (31). It is also possible that the effects of E2 onP99 and P77 may be dependent upon the localization of themolecules throughout the epithelial strata. As we used wholetissue lysates in these analyses, the data represent an averagingof the effects of spatial expression. Again, changes in the ex-pression from P99 in small compartments of cells will be diffi-cult to detect. It will be important to investigate the levels ofE2 protein expression over the viral life cycle in addressingthese matters.

A CCAAT motif is situated 34 bp upstream of nt 742 (Fig.6B). This sequence is recognized by the CCAAT enhancerbinding protein (C/EBP) (21). C/EBP is expressed at elevatedlevels in a variety of terminally differentiated cells, includingskin cells, and has a function in the differentiation-specificactivation of genes (47). Furthermore, this CCAAT motif isconserved in HPVs; it is present upstream of the differentia-tion-inducible promoters of HPV6, -11, and -16 and of bovinepapillomavirus type 1 (3, 20, 32). Consensus TATA boxes are24 and 29 bp upstream of the start sites at nt 737 and 742,


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respectively. TATA boxes of weak consensus are properlyspaced upstream of the initiation sites at nt 750 and 767. Thelack of Sp1 and Inr elements may contribute to the low expres-sion from the P742 cluster in undifferentiated cells. Differenti-ated tissues may contain, in addition to C/EBP, factors thatovercome the less than optimal cis-regulatory sequences toupregulate expression from this promoter cluster. The E2BSslocated in the URR upstream of P99 (Fig. 6A) may also play afunctional 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 expres-sion from this promoter observed over the course of the virallife cycle. The situation of an Sp1 binding site upstream alsomay contribute to the constitutive expression of the P3320 pro-moter in the undifferentiated monolayer cells and during thedifferentiation process in the raft tissues (Fig. 6C).

In summary, we have shown biochemical evidence for fourHPV31b promoters (Fig. 6D) and their usage throughout theviral life cycle. Our ability to produce infectious HPV stocksfollowing the transfection of HPV genomic DNA into kerati-nocytes will allow us to construct mutant viruses (31). We arein the process of using this technique to gather genetic evi-

dence on how the promoters are regulated and how they con-tribute to the differentiation-dependent life cycle of HPVs.

ACKNOWLEDGMENTS

We thank Carl Baker for advice and helpful discussions.This work was supported by Public Health Service grant CA-66316

from the National Cancer Institute (M.A.O.) and National CancerInstitute grant CA-64624 (C.M.).

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temporal usage of multiple promoters during the life cycle of

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