Human Papillomavirus Type 31 E5 Protein Supports Cell CycleProgression and Activates Late Viral Functions
upon Epithelial DifferentiationFrauke Fehrmann, David J. Klumpp,† and Laimonis A. Laimins*
Department of Microbiology-Immunology, The Feinberg School of Medicine,Northwestern University, Chicago, Illinois 60611
Received 16 September 2002/Accepted 26 November 2002
The function of the E5 protein of human papillomaviruses (HPV) is not well characterized, and controversiesexist about its role in the viral life cycle. To determine the function of E5 within the life cycle of HPV type 31(HPV31) we first constructed HPV31 mutant genomes that contained an altered AUG initiation codon or stopcodons in E5. Cell lines were established which harbored transfected wild-type or E5 mutant HPV31 genomes.These cell lines all maintained episomal copies of HPV31 and revealed similar phenotypes with respect togrowth rate, early gene expression, and viral copy number in undifferentiated monolayer cultures. Followingepithelial differentiation, genome amplification and differentiation-dependent late gene expression were ob-served in mutant cell lines, but at a rate significantly reduced from that observed in cells containing the wild-type genomes. Organotypic raft cultures indicated that E5 does not effect the expression of differentiationmarkers but does reduce expression of late viral proteins. Western analysis and immunofluorescence stainingfor cyclins during epithelial differentiation revealed a decreased expression of cyclin A and B in E5 mutant cellscompared to HPV wild-type cells. Using a replating assay, a significant reduction in colony-forming ability wasdetected in the absence of E5 expression when cells containing wild-type or E5 mutant HPV genomes wereallowed to proliferate following 24 h in suspension-induced differentiation. This suggests that HPV E5 modifiesthe differentiation-induced cell cycle exit and supports the ability of HPV31-positive keratinocytes to retainproliferative competence. In these studies, E5 was found to have little effect on the levels of the epidermalgrowth factor receptor (EGFR) or on its phosphorylation status. This indicates that EGFR is not a target ofE5 action. Our results propose a role for high risk HPV E5 in modulation of late viral functions throughactivation of proliferative capacity in differentiated cells. We suspect that the primary target of E5 is amembrane protein or receptor that then acts to alter the levels or activities of cell cycle regulators.
Human papillomaviruses (HPVs) are small DNA virusesthat induce hyperproliferative lesions of cutaneous and muco-sal epithelia (35). Half of the more than 100 identified types ofHPVs specifically infect the genital epithelium (63). Thesegenital papillomaviruses can be divided into low-risk types,such as HPV type 6 (HPV6) and HPV11, which induce onlybenign lesions, and high-risk types, such as HPV16, -18, and-31, which are associated with cervical carcinoma (32, 36, 63).The productive life cycle of human papillomaviruses is directlylinked to epithelial cell differentiation (25). Following the in-fection of keratinocytes in the basal layer, HPV genomes areestablished as episomes at approximately 50 copies per cell andreplicate in synchrony with cellular DNA replication (28, 33).The establishment and maintenance of HPV genomes is asso-ciated with expression of early HPV transcripts that encode theoncoproteins E6 and E7 as well as the replication proteins E1and E2. Following cell division, infected daughter cells leavethe basal layer, migrate towards the suprabasal regions andbegin to differentiate. In contrast to uninfected keratinocytes,which exit the cell cycle as soon as they detach from the
basement membrane, HPV-infected cells remain active in thecell cycle and enter into S-phase after reaching the suprabasallayer (12, 48). This entry into S-phase results in amplificationof the viral genomes and expression of late transcripts from adifferentiation-dependent promoter (11, 16, 28, 48).
The viral E6 and E7 proteins act as the major oncogenicfactors of high-risk HPVs, binding to cell proteins involved incell cycle regulation. E6 binds the tumor suppressor p53 in acomplex with the cellular ubiquitin ligase E6-AP, which leadsto its degradation (27, 50, 51, 61). In addition, E6 inducestelomerase activity through activation of expression of the cat-alytic subunit, hTert (18, 44, 60). E7 binds and inactivates theretinoblastoma protein (pRB) (5, 13, 37, 41). Another viralprotein, E5, is weakly oncogenic in tissue culture assays andpotentiates the transforming activity of E7 (3, 59). HPV E5proteins are small, extremely hydrophobic, and located mainlyat the endosomal membranes, Golgi apparatus, and, to a lesserextent, the plasma membranes (4, 6). In contrast to bovinepapillomavirus type 1 E5, which has been shown to encode theprimary transforming function, little is known about the bio-logical activity of HPV E5. Abundant mRNA sequences con-taining the E5 open reading frame (ORF) have been identifiedin cervical intraepithelial neoplasial lesions and carcinomas(31, 55). In CIN612 cells, which contain episomal copies ofHPV31, it has been shown that E5 is encoded in most earlyand late transcripts (28). However, E5 is transcribed as a
* Corresponding author. Mailing address: Department of Microbi-ology-Immunology, The Feinberg School of Medicine, NorthwesternUniversity, 303 E. Chicago Ave., Chicago, IL 60611. Phone: (312) 503-0648. Fax: (312) 503-0649. E-mail: [email protected].
† Present address: Department of Urology, The Feinberg School ofMedicine, Northwestern University, Chicago, IL 60611.
part of a polycistronic RNA, and because it is usually the third orfourth ORF, it is not known how efficiently it is translated inundifferentiated cells. In contrast, upon differentiation E5 isthe second ORF present on the majority of late transcripts(Fig. 1B). Direct evidence for the presence of E5 is difficult toobtain, as the protein cannot be detected in cells unless it isoverexpressed from heterologous promoters (7, 29).
In E5-transfected mouse fibroblasts, E5 has been shown toincrease cellular proliferation in the presence of epidermalgrowth factor (EGF) (34, 57). The molecular basis for this effectis not clear, although it has been suggested that E5 associates with
the EGF receptor (EGFR) (29), resulting in increased ligand-dependent activation of the EGFR (9, 57) and enhanced EGFR-mediated mitogen-activated protein (MAP) kinase activity (8,20). Additionally, E5 binds the 16-kDa subunit of the vacuolarproton-ATPase (6) and inhibits the acidification of endosomes(56). This has been suggested to result in a delay in EGFRdegradation and an increased recycling of EGFR to the cell sur-face (57). However, since all these studies have used heterologousexpression systems in absence of other viral proteins, the role ofthe E5 protein during the productive life cycle of HPV remainsunclear.
FIG. 1. Schematic of a linearized HPV31 genome. (A) Diagram representing the HPV31 ORFs, the two major promoters that drive viralexpression (P97 and P742), and the two polyadenylation sites (AE 4140 and AL 7227). (B) Diagram depicting the most-abundant spliced viraltranscripts from the early and late promoter. (C) Diagram showing the HPV31 E5 mutants used in this study (wt, wild type).
The study of the HPV life cycle in tissue culture has beenfacilitated by the development of methods for the genetic anal-ysis of HPV functions in the context of its productive life cycle(15, 16). Differentiation of keratinocytes can be induced inorganotypic raft culture (38) or suspension in methylcellulose(14, 19, 48). Upon differentiation these cells induce viral latefunctions, including activation of late gene expression and ge-nome amplification (16, 48). Using these methods we haveexamined the effects of E5 in the context of the whole viral lifecycle and identified a role in activation of late viral functions.
MATERIALS AND METHODS
Cell culture. Human foreskin keratinocytes (HFKs) were derived from neo-natal human foreskin epithelia as previously described (21) and were maintainedin serum-free keratinocyte growth medium (Clonetics, San Diego, Calif.).HPV31 genome transfectants and control HFKs were grown in serum-containingmedium (E medium) supplemented with mouse EGF (5 ng/ml; CollaborativeBiomedical Products, Bedford, Mass.) in the presence of mitomycin C-treated J23T3 fibroblast feeders, kindly provided by the Howard Green laboratory. Prior toharvesting keratinocytes, total cellular DNA, total cellular RNA, or whole-celllysates, the fibroblast feeders were first removed by EDTA (phosphate-bufferedsaline [PBS] [Gibco BRL, Grand Island, N.Y.] with 0.5 mM EDTA). To analyzeEGFR activation, keratinocytes were cultivated in serum-free E medium for 20 hfollowed by a 3-min induction with 5 ng of EGF/ml prior to cell lysis.
Plasmids. The generation of the E5 mutant genes was performed in a pBR322-derived plasmid, pBRmin. pBRmin was made by cutting out the 1,724-bp frag-ment between ClaI and Eco47III, filling in the 5� overhang to form blunt ends,and religating. The plasmid pBRmin-HPV31 contains the HPV31 genome in-serted into the HindIII site of the pBR322 min. The mutants m5-1 and m5-2 wereconstructed via site-directed mutagenesis by overlap extension using PCR. Themutant m5-1 contains a point mutation in the E5 ATG initiation codon, creatinga lysine-coding triplet (AAG), and m5-2 possesses point mutations at codons 5(AAT) and 7 (TCT) in the E5 ORF to generate stop codons (TAG and TGA,respectively; Fig. 1C). The mutated E5 sequences were subsequently exchangedwith the wild-type sequence into pBRmin-HPV31 by using the single-cuttingrestriction endonucleases EcoRI (nucleotide 3361) and PpuMI (nucleotide4464). pSV2neo carries the neomycin drug resistance gene.
Transfection of HFKs. Ten micrograms of the pBRmin-HPV31 constructs wasdigested with HindIII to release the viral genome. The restriction enzyme washeat inactivated, and genomes were unimolecularly ligated in the same bufferwith T4 DNA ligase (10 U/900 �l). The DNA was then precipitated with iso-propyl alcohol and resuspended in 10 mM Tris–1 mM EDTA (pH 7.5). Onemicrogram of the religated DNA was cotransfected with 1 �g of the selectablemarker, pSV2neo, into HFKs with FuGene (Roche Diagnostics, Mannheim,Germany) as described by the manufacturer. At 1 day posttransfection, cells wereplated onto mitomycin C-treated fibroblast feeders in E medium. Selectionbegan day 2 posttransfection with G418 (Gibco BRL) as follows: G418 (200mg/ml) was added every two days for a total of 4 days, and then G418 at aconcentration of 100 mg/ml was added every two days for 4 more days. Afterselection, pooled populations were expanded for analyses.
Differentiation of keratinocytes in semisolid medium. HFKs and HPV31transfectants were suspended in 1.5% methylcellulose to induce differentiation.The methylcellulose solution was prepared by adding half of the final volume ofE medium containing 5% fetal bovine serum to autoclaved dry methylcellulose(4,000 cps; Sigma, St. Louis, Mo.) and heating in a 60°C water bath for 20 min.The remaining E medium containing 10% fetal bovine serum was added, and themixture was stirred at 4°C overnight. Approximately 1 � 106 to 2 � 106 kera-tinocytes were harvested by trypsinization, resuspended in 1 ml of E medium,and added dropwise to a 10-cm-diameter petri dish containing 25 ml of 1.5%methylcellulose. Cells were stirred with a pipette and incubated at 37°C in aCO2-humidified incubator at the indicated times. Cells in methylcellulose wereharvested by scraping into four 50-ml conical tubes, washing with PBS (50 ml)three times, combining into a 15-ml conical tube for a final PBS wash, andpelleting by centrifugation. Samples were then subjected to Southern analyses todetect HPV31 genomic DNA and Northern analyses to examine transcripts andWestern analysis.
Differentiation of keratinocytes in raft cultures. HFKs as well as HPV31wild-type and E5 mutant transfectants were differentiated in raft cultures aspreviously described (39). Briefly, cells were plated onto a solidified collagenmatrix containing J2 3T3 fibroblasts, allowed to grow to confluency, and then
transferred to a metal grid which provides an air-liquid interface for differenti-ation. Cultures were harvested at 14 days, fixed in 4% paraformaldehyde, paraffinembedded, sectioned, and stained with hematoxylin and eosin for visualization ofdifferentiated raft tissue.
Immunohistochemistry. The expression of E1∧ E4 proteins was examined byimmunofluorescence of cross sections of raft tissue. Thin sections of paraform-aldehyde-fixed, paraffin-embedded tissue on silanized slides were incubated at50°C for 30 min, and this was followed by three 5-min rinses in xylene to removeresidual paraffin. The sections were rehydrated in absolute ethanol and thenincubated in 10 mM citrate, pH 6.0, at 95°C for 20 min, followed by an additional20-min incubation at room temperature. Incubation with the primary antibodyrabbit anti-E1∧ E4, which has been described previously (47), was performedovernight at 4°C. The sections were subsequently incubated with the secondaryanti-rabbit antibody conjugated to fluorescein isothiocyanate (Amersham Phar-macia, Piscataway, N.J.) for 1 h at room temperature. Following 5 min ofDNA-counterstaining with 4�,6�-diamidino-2-phenylindole-2HCl (DAPI) (1 �g/ml; Serva, Heidelberg, Germany) the sections were mounted and sealed.
Southern blot analyses. Total genomic DNA from wild-type and mutantHPV31 transfectants was prepared by resuspension of the cell pellet in lysisbuffer (400 mM NaCl, 10 mM Tris-HCl [pH 7.4], 10 mM EDTA); then, RNaseA (50 �g/ml), proteinase K (50 �g/ml) and sodium dodecyl sulfate (SDS) (0.2%)were added, and this was followed by incubation at 37°C overnight. DNA wassheared by passing through an 18-gauge needle approximately 10 times, ex-tracted with phenol-chloroform, and then precipitated with ethanol. Totalgenomic DNA (5 �g) was digested with DpnI to remove any residual input DNA,and another 5 �g of each sample was additionally linearized with XbaI to serveas a copy number control. Digested DNA was separated on a 0.8% agarose gel,treated, and alkaline transferred onto DuPont GeneScreen Plus nylon mem-brane (NEN Research Products, Boston, Mass.) as described by the manufac-turer. The membrane was prehybridized in a solution containing 50% form-amide, 4� SSC (1� SSC is 0.15 M NaCl plus 0.015 M sodium citrate), 5�Denhardt’s solution, 1% SDS, 10% dextran sulfate, and denatured salmon spermDNA (0.1 mg/ml) for 1 h at 42°C. The HPV31 probe was prepared by gelpurification of the entire HPV31 genome from pBRmin-HPV31 digested withHindIII and labeling with the Ready-To-Go DNA labeling kit (Amersham Phar-macia). Labeled probe was purified with ProbeQuant G-50 Micro columns (Am-ersham Pharmacia), denatured, and added to fresh hybridization solution, whichwas incubated with membrane at 42°C overnight. Membrane was washed twicewith 2� SSC–0.1% SDS for 15 min at room temperature, twice with 0.5�SSC–0.1% SDS for 15 min at room temperature, twice with 0.1� SSC–0.1% SDSfor 15 min at room temperature, and once with 0.1� SSC–1% SDS for 30 min at50°C. Hybridizing species were visualized by autoradiography. Quantitative anal-ysis was done with a PhosphorImager (Molecular Dynamics, Sunnyvale, Calif.).
Analyses of HPV31 late transcripts. Total RNA was isolated from methylcel-lulose-treated normal HFKs and HPV31 transfectants with TRIzol reagent(Gibco BRL) as described by the manufacturer and examined by Northernanalyses as follows. Total RNA (10 �g) was separated on a 1.0% agarose–2.2 Mformaldehyde gel in 1� MOPS buffer (10� MOPS buffer is 0.2 M MOPS, 50 mMNa acetate, 10 mM EDTA) and transferred onto a Zeta-Probe membrane (Bio-Rad, Hercules, Calif.). After cross-linking, the membrane was prehybridized in asolution containing 1 mM EDTA, 0.5 M Na2HPO4, and 7% SDS for 10 min at65°C. The HPV31 probe was prepared by gel purification of the entire HPV31genome from pBRmin-HPV31 digested with HindIII and labeling with theReady-To-Go DNA labeling kit (Amersham Pharmacia). Labeled probe waspurified with ProbeQuant G-50 Micro columns (Amersham Pharmacia), dena-tured, added to fresh hybridization solution, and incubated with membraneovernight at 65°C. The membrane was washed twice with 2� SSC–10% SDS for5 min at room temperature and once with 0.2� SSC–1% SDS for 15 min at 55°C.As a loading control, the levels of 28S and 18S rRNA were examined in the gelafter staining with ethidium bromide. Hybridizing species were visualized byautoradiography. Quantitative analysis was done with a PhosphorImager (Mo-lecular Dynamics).
Western analysis. Whole-cell extracts were prepared with NP-40 lysis buffer(150 mM NaCl, 50 mM Tris-HCl [pH 8], 5 mM EDTA [pH 8], 0.5 mM dithio-threitol, 100 mM sodium fluoride, 200 �M sodium-orthovanadate, 0.5% NP-40,1 mM phenylmethylsulfonyl fluoride) containing a cocktail of protease inhibitors(Complete, Mini; Roche Diagnostic) and quantitated with the Bradford assay(Bio-Rad). Equal amounts of protein were electrophoresed on a SDS-polyacryl-amide gel and subsequently transferred to a polyvinylidene difluoride membrane(Immobilon-P; Millipore, Bedford, Mass.). The membrane was blocked in washsolution (0.1% Tween 20 in PBS) containing 5% nonfat dry milk. The followingantibodies were used: anti-EGFR (Santa Cruz Biotechnology, Inc., Santa Cruz,Calif.), anti-p-Tyr (–horseradish peroxidase (Santa Cruz), anti-pERK (Santa
Cruz), anti-cyclin E (Santa Cruz), anti-cyclin B (Pharmingen, San Diego, Calif.),anti-p21 (Pharmingen), anti-p27 (Transduction Laboratories, Lexington, Ky.),anti-p57 (Oncogene Research, Uniondale, N.Y.), rabbit anti-cyclin A (a gift of R.Assoian). Western analysis employing anti-p-Tyr–horseradish peroxidase wasperformed by using block solution containing 0.25% gelatin instead of 5% nonfatdry milk. Proteins were visualized via enhanced chemiluminescence (AmershamPharmacia).
Replating assay. Normal HFKs as well as HPV31 wild-type and E5 mutant-positive keratinocytes grown in the presence of fibroblast feeders were harvestedat 80% confluence and suspended in 1.5% methylcellulose. Following 24 h ofdifferentiation the cells were collected, washed, and replated onto fibroblastfeeders. After 5 days in culture colonies were visible and counted from at leastfive different randomly chosen fields.
RESULTS
In order to study the role of the E5 protein in the productivelife cycle of HPVs, two mutant HPV31 genomes were con-structed in the context of the plasmid pBRmin-HPV31 (Fig.1A). One of the mutant genomes contains a mutated E5 ATGinitiation codon (m5-1), which is the only ATG present in theE5 ORF, and the other one possesses translation terminationcodons inserted at amino acids 5 and 7 in the E5 ORF (m5-2).Both of these mutations inhibit E5 translation (Fig. 1C). Wild-type as well as E5 mutant HPV genomes were then used totransfect HFKs. After selection for neomycin resistance,pooled colonies were analyzed approximately 1 month aftertransfection.
E5 knockout HPV31 genomes are stably maintained inHFKs and replicate in a manner similar to that of HPV wild-type genomes. We first investigated whether E5 was necessaryfor stable maintenance and replication of HPV31 genomes.Southern blot analysis of total genomic DNA derived fromthree matched sets of independently transfected primary HFKisolates from different donors is shown in Fig. 2A. HarvestedDNA from both wild-type and E5 mutant HPV31 cell lineswere DpnI digested to remove any residual input DNA. Analiquot of each sample was additionally digested with XbaI,which cuts the HPV genome once to linearize the viral DNAand to facilitate copy number analysis (Fig. 2A, lanes 2, 4, 6, 8,10, and 12). The cells transfected with wild-type HPV31 ge-nomes were found to contain significant amounts of episomalcopies of HPV31 DNA (Fig. 2A, lanes 1, 5, and 9). Similaramounts of episomal forms of viral DNA were detected in cellstransfected with E5 mutant genomes (Fig. 2A, lanes 3, 7, and11). The amount of linearized viral DNA copies was also com-parable in matched wild-type and E5 mutant-containing cells(Fig. 2A, lanes 2 and 4, 6 and 8, and 10 and 12), although therewere slight variations in viral copy numbers dependent onindividual primary HFK isolates. No significant differencesconcerning episomal maintenance or viral copy number couldbe observed between cells transfected with one or the other E5knockout mutant HPV genome. These experiments were re-peated a total of six times using different primary HFK isolateswith comparable results. Southern analysis of transfectants atlater passages as well as cells that had been frozen and thawedshowed no differences in the state of viral DNA.
Cell lines immortalized by E5 mutant or wild-type HPVgenomes show similar expression patterns of early viral tran-scripts. We next compared the amount of viral expression inwild-type and E5 mutant HPV-containing undifferentiated ker-atinocytes. The result of a representative Northern analysis is
shown in Fig. 2B. Two major sets of transcripts of approxi-mately 1.5 and 4.3 kb were observed in cells containing eitherwild-type or E5 mutant genomes. The larger transcript repre-sents the unspliced mRNAs initiated at the early promoterP97, whereas the 1.5-kb messages probably encode two of themost abundant spliced mRNAs: E6, E7, E1∧ E4, E5 and E6*,E7, E1∧ E4, E5 (Fig. 1B). HPV wild-type as well as both of theE5 mutant cell lines revealed a similar pattern and level ofearly expression.
E5-deficient HPV containing keratinocytes did not revealany phenotype relative to the wild-type cells in monolayerculture. Since it was possible that E5 affected the growth prop-erties of cells, we next compared the morphology of E5 knock-out keratinocytes to wild-type HPV transfected keratinocytesas well as the relative growth rates and life spans of these cells.No differences concerning these criteria could be detected inundifferentiated monolayer cultures. In heterologous expres-sion systems E5 has been shown to enhance mitogenic signal-ing through the EGFR in the presence of EGF (8, 34, 46, 57)by various mechanisms. Based on these observations we want-ed to evaluate the influence of EGF on the growth rates of thetransfectants. HPV31 wild-type and E5 mutant transfectedcells were split in E medium in absence or presence of 5 ng ofEGF per ml, and cell growth was monitored by counting cells.The results revealed that the growth rates of both HPV31wild-type and E5 mutant-containing cells were reduced by ap-proximately 50% when cultivated in EGF-free medium com-pared to medium supplemented with EGF (data not shown).Surprisingly, the growth rates of HPV31 wild-type and E5mutant-transfected cells in the presence or absence of EGFwere similar (data not shown), suggesting that E5 does notinfluence the growth of HPV-immortalized cells in monolayerculture.
The hyperphosphorylation of EGFR is not due to E5 inHPV31-containing cells. While we did not observe a synergisticeffect of EGF on cell growth mediated by E5, it was stillpossible E5 had an influence on EGFR expression and activa-tion. To investigate this possibility, untransfected HFKs as wellas wild-type and E5 mutant HPV31-containing cells werestarved in EGF- and serum-free medium for 20 h. Total celllysates were then harvested following a 3-min induction with 5ng of EGF per ml, and the levels of EGFR were examined byWestern analysis. Keratinocytes stably transfected with HPV31wild-type or E5 mutant genomes were found to contain levelsof EGFR increased over those in HFKs (Fig. 3A). The levelsof EGFR expression varied moderately between normal HFKsisolated from different donors (data not shown). However, theexpression level of EGFR was found to be consistently higherin HPV-containing cells. No differences concerning EGFR lev-els or their degree of phosphorylation were detected betweenwild-type and E5 mutant-containing cells (Fig. 3B). The down-stream targets of EGFR signaling, the MAP kinases 1 and 2,were also found phosphorylated to the same extent in bothHPV wild-type and E5 mutant-containing cell lines (Fig. 3C).These results suggest that inhibition of HPV31 E5 in the con-text of all other viral proteins does not effect EGFR hyper-phosphorylation in undifferentiated cells growing in monolayerculture.
Amplification of the E5 mutant HPV31 genomes is reduced
upon epithelial differentiation. Since we were not able to de-tect any phenotype due to the lack of E5 in monolayer culture,we next examined whether E5 plays a role in the later phasesof the HPV life cycle. It is not possible to determine in whichphase of the viral life cycle E5 is expressed by measuringprotein levels, because efficient E5 antibodies are not available.While most HPV31 early mRNAs (Fig. 1B) contain the E5ORF, it is most often the fourth ORF in these transcripts andunlikely to be efficiently translated. In contrast, E5 is the sec-ond ORF present in the major late E1∧ E4, E5 transcripts,which may allow it to be more efficiently translated. Thus, itseemed plausible that E5 would exert some effects on late viralfunctions.
We first studied the ability of the E5 mutant HPV31 ge-nomes to undergo productive replication upon epithelial dif-ferentiation following suspension in semisolid medium. It hasbeen previously shown that growth in semisolid medium in-duces the differentiation of HPV-containing keratinocytes, re-sulting in activation of viral late functions (49). Total genomicDNA from wild-type and E5 mutant HPV31 transfected cellscultivated in monolayer culture or in methylcellulose wereharvested and Southern analysis was performed. Followingincubation in methylcellulose for a total of 48 h wild-typeHPV31 DNA was found to increase to over 3-fold in contrastto 1.6-fold increase seen in mutant E5 HPV m5-1 (Fig. 4A).Figure 4B shows a graph of the average results of five inde-
FIG. 2. Replication of HPV31 and E5 mutant HPV genomes in monolayer cultures. (A) Southern blot analysis of three independent trans-fections (#1, #2, and #3) of HFKs derived from different donors stably transfected with HPV31 (wt) and E5 mutant (m5-1) DNA. Total genomicDNA was harvested from monolayer cultures and digested with DpnI to remove residual input DNA. XbaI was additionally used to linearize theHPV31 genomes in lanes 2, 4, 6, 8, 10, and 12. The Southern blot was hybridized with a probe, which includes the complete HPV31 genome.(B) Northern blot analysis of mRNA from monolayer cultures of HPV31 and two E5 mutant-positive cell lines (m5-1 and m5-2). The Northernblot was hybridized with a probe, which includes the complete HPV31 genome. 26S and 18S markers correspond to molecular sizes of approxi-mately 4.7 and 1.8 kb, respectively. Equal loading was monitored by comparing the levels of 28S and 18S rRNA in ethidium bromide-stained gels.The slight change in mobility of the smaller HPV messages observed in lane wt was not seen in repeated experiments.
pendent Southern blot analyses. After 24 h of suspension inmethylcellulose, the wild-type HPV31 DNA was amplified onaverage 2.6-fold in contrast to the two mutant E5 HPV DNAsm5-1 and m5-2, which were increased 1.6- and 1.3-fold, respec-tively. The reduced amplification of E5 mutant HPV upondifferentiation compared to wild-type HPV suggests that atleast some of the effects of E5 are directed against the latephases in the viral life cycle.
Late gene transcription is decreased upon differentiation inE5 mutant HPV transfected keratinocytes. It was next impor-tant to analyze the patterns of viral expression upon differen-tiation in cells maintaining wild-type and E5 mutant genomes.RNA was harvested from cell lines cultured for 24 and 48 h inmethylcellulose, and Northern analysis was performed using aprobe encompassing the whole HPV genome. The probe al-lows for the identification of all early and late transcripts (Fig.5). Following differentiation, three transcripts appeared inHPV31-positive cells which were not seen in undifferentiatedcells (Fig. 5, lanes 1 to 3). Previous analyses indicate that theseencode the late transcripts E1, E4, E5 (3.8 kb), E1∧ E2, E5 (2.2kb) and E1∧ E4, E5 (1.3 kb) (Fig. 1B). While the expression oftranscripts from the early promoter P97 in monolayer cultureis similar in both E5 mutant and wild-type HPV-transfectedcell lines (Fig. 5, dark arrows), expression of differentiation-dependent transcripts from the late promoter P742 is signifi-cantly impaired in E5 mutant cell lines (Fig. 5, open arrow-
heads). Unspliced P97 messages were also upregulated upondifferentiation in wild-type HPV containing cells in contrast toE5 mutant-containing cell lines. Similar results were seen inthree separate experiments. This result further supports theobservation that keratinocytes that stably maintain HPV31 ge-nomes lacking a functional E5 are significantly impaired intheir ability to induce late viral functions.
Low levels of EGFR expression are maintained upon differ-entiation in HPV immortalized cells, but not in HFKs. Uponepithelial differentiation induced by suspension in methylcel-lulose, expression of EGFR decreased dramatically (Fig. 6).However, in HPV wild-type and E5 mutant-positive cells sig-nificant levels of EGFR were observed for periods up to 48 h.In contrast, no EGFR expression was detected in HFKs upondifferentiation. This result indicates that the less pronounceddecrease of EGFR expression upon differentiation in HPV-positive cells is not due to E5 but to the presence of other viralproteins.
HPV31 and E5 mutant HPV transfected keratinocytes ex-hibit the same morphological differentiation pattern in orga-notypic raft cultures. The next set of experiments examinedwhether the cells containing E5 mutant genomes induced anyhistological changes upon differentiation that could correlatewith a reduced ability to induce late viral functions. Normalkeratinocytes rapidly lose nuclei upon differentiation, whilecells that express high-risk HPVs maintain nuclei throughoutthe suprabasal layers. To examine putative effects of E5 onepithelial morphology we performed organotypic raft cultureanalysis with HPV31 and HPV E5 mutant transfectants. Asshown in Fig. 7A untransfected cells demonstrated a normaldifferentiation pattern in the raft cultures, with nuclear stainingpredominantly localized to cells in the basal layer. In contrast,HPV31-positive cells showed an altered differentiation pat-tern, with a thickening of the basal layer and nuclear stainingthroughout all layers (Fig. 7B). Organotypic raft cultures fromHPV E5 mutant-containing cells revealed a morphology simi-lar to that of HPV wild-type transfectants (Fig. 7C). We fur-ther examined if cells with HPV E5 mutant genes exhibitedaltered expression of differentiation markers such as keratin 10(a differentiation-specific keratin), involucrin, and filaggrin,which are expressed beginning in the spinous layer (17). Nosignificant difference in staining of any of these markers couldbe detected in HPV wild-type and E5 mutant transfectants inorganotypic raft culture (data not shown).
E1∧ E4 protein expression is remarkably reduced in E5 mu-tant HPV-transfected organotypic raft cultures compared towild-type transfectants. Since Northern blot analysis indicat-ed that late viral gene expression is reduced in E5 mutant-containing cell lines (Fig. 5), we next wanted to analyze thedistribution pattern of the most-abundant viral late E1∧ E4protein in organotypic raft cultures. Immunohistochemicalanalysis using antibodies against E1∧ E4 protein showed it wasexpressed predominantly in the suprabasal cells of HPV31containing raft cultures, confirming previous reports (Fig. 7E)(47). In contrast, rafts of cells containing HPV E5 mutantsexhibited a consistent reduction in the number of E1∧ E4 pos-itive cells (Fig. 7F). This result was confirmed by immunoflu-orescence staining for E1∧ E4 of cells suspended in semisolidmedium for 24 h. On average 24% of HPV31 wild-type-con-taining cells were positive for E1∧ E4 expression, in contrast to
FIG. 3. Expression of EGFR in HFKs, HPV31, and E5 mutant-positive cells. Equivalent amounts of whole-cell extracts of HFK,HPV31, and E5 mutant-containing cells, not treated (lanes 0) ortreated (lanes 3) with EGF for the indicated times were separated bySDS-polyacrylamide gel electrophoresis and examined by Western blotanalysis with anti-EGFR (A), anti-pTyr (phosphorylated EGFR) (B),and anti-pERK (phosphorylated MAP kinases 1 and 2) (C) antibodies.Proteins were visualized by chemiluminescence.
14.5% of positive cells in E5 mutant cell lines (data not shown).It was not possible, however, to determine in this assay, if thelevel of staining per cell was also reduced.
Cyclin A and B expression is slightly reduced in HPV31 E5mutant-containing cells compared to that in wild type. Nextwe wanted to investigate the mechanism by which the E5protein stimulates viral amplification and late viral geneexpression. One possibility is that E5 assists E6 and E7 inprogression into S-phase to enhance viral late functions.Previous studies indicated that activation of late gene ex-
pression as well as amplification occurs in S-phase (49). Toaddress the E5 effects on cell cycle progression we firstcompared the expression of cyclins in untransfected HFKs,HPV31 wild type, and E5 mutant cell lines upon differenti-ation. Cells were harvested as a function of time followingsuspension in methylcellulose, and Western blot analyses forthe cyclins A, B, and E were performed. The HPV31 wild-type and E5 mutant-containing cell lines both expressedlevels of cyclin A and B about two to three times higher thanHFKs when growing as monolayer culture (Fig. 8A). Fol-
FIG. 4. Differentiation-dependent amplification of HPV31 and E5 mutant HPV DNA following suspension in methylcellulose. (A) Autora-diogram of Southern analysis of HFKs stably transfected with HPV31 (wt) and E5 mutant (m5-1) DNA. Total genomic DNA was harvested fromcells cultured in methylcellulose (MC) for the indicated times and prepared as described before. The Southern blot was hybridized with a probecorresponding to the complete HPV31 genome. (B) Southern blot analysis was performed on total genomic DNA of HPV31 and E5 mutant cells(m5-1 and m5-2), cultured in monolayer culture or in methylcellulose (MC) for 24 h. Equal amounts of total genomic DNA were digested withDpnI to remove residual input DNA and XbaI to linearize the HPV31 genomes. Differentiation-dependent viral amplification was quantified usinga PhosphorImager. Results are the means � standard deviations (error bars) of data from five experiments.
lowing suspension in methylcellulose for 24 h, cyclin A levelswere dramatically reduced in HFKs, whereas HPV31-posi-tive cells retained cyclin A expression. E5 mutant cells alsoretained cyclin A expression, but to a lesser extent thanHPV31 wild-type cells. The pattern of cyclin B expressionduring suspension culture was found to be similar to that ofcyclin A. HPV31 containing cells retained significant levelsof cyclin B at 48 h, whereas in HFKs cyclin B is undetectableafter 24 h. E5 mutant cells exhibited an intermediate phe-notype. These effects were consistently seen in four inde-pendent experiments. When cyclin E expression during dif-ferentiation was examined, it was found to increase in thefirst 24 h and then rapidly decrease in all HPV cells tested,but at rates less rapid than for cyclin A and B. For cyclin Eno differences between wild-type and E5 mutant cell lineswere observed. From these studies, we conclude that E5moderately influences the levels of cyclins A and B but notE in differentiated HPV positive cells.
We next examined the expression of the cyclin–cyclin-dependent kinase complex inhibitors (CKI) p21, p27, andp57 in these lines after suspension in methylcellulose. CKIhave been shown to be upregulated during the differentia-tion of keratinocytes (40, 43). By Western blot analysis wefound that p27 was dramatically induced in methylcellulosein both HFKs and HPV-positive cells. (Fig. 8B). In contrast,the p53-regulated CKI p21 was not induced upon differen-tiation of HFKs but was retained for up to 24 h in HPV-positive cells. There was no change in the level of p57expression following suspension culture in either HFKs orHPV-containing cells. These experiments confirm the induc-tion of CKI during suspension-induced cell cycle exit in
keratinocytes (2, 24, 42, 48) but demonstrate that E5 doesnot influence this process. These experiments were repeatedfive times with similar results. In these studies we observeda modest but consistent reduction of cyclin A and B expres-sion in E5 mutant-containing keratinocytes (Fig. 8A) com-pared to HPV31 wild-type cells which is not due to increasedCKI expression.
The HPV E5 protein supports ongoing mitosis and celldivision following differentiation-induced cell cycle arrest.From the above studies, E5 was suggested to have a modu-latory effect on cell cycle regulators, and we wanted to testif E5 had functional consequences in cell cycle progressionin differentiating cells. For these studies we performed a
FIG. 6. Expression of EGFR in HFKs, HPV31, and E5 mutant-positive cells upon differentiation. Equivalent amounts of whole-cellextracts of HFKs, HPV31, and E5 mutant-positive cells cultured inmethylcellulose (MC) for the indicated times were separated by SDS-polyacrylamide gel electrophoresis and examined by Western blotanalysis with an anti-EGFR antibody. An anti-glyceraldehyde-3-phos-phate dehydrogenase (GAPDH) antibody was used as a loading con-trol. Proteins were visualized by chemiluminescence.
FIG. 5. Northern blot analysis of differentiation-dependent transcripts in HPV31 and E5 mutant positive cell lines following suspension inmethylcellulose (MC). Total RNA was isolated at various times from HPV31 and two E5 mutant-positive cell lines (m5-1 and m5-2). Bands of earlytranscripts are labeled with a black arrow and bands of late transcripts are labeled with arrows with open heads. The Northern blot was hybridizedwith a probe corresponding to the complete HPV31 genome. 26S and 18S markers correspond to molecular sizes of approximately 4.7 and 1.8 kb,respectively. Uniform loading was monitored by comparing levels of 28S and 18S rRNA in ethidium bromide-stained gels. The reduced levels ofP97 unspliced messages in lanes 5 and 8 were not seen in other experiments.
replating assay that measured the ability of cells to remainreplication competent following differentiation. In this assayHPV-positive cell lines were first induced to undergo epi-thelial differentiation by suspension culture in methylcellu-lose. After 24 h the cells were washed and an aliquot of cellswere replated into tissue culture dishes containing fibroblastfeeders. After 5 days colonies appeared and random fields ofcells were counted (Fig. 9). Cells containing HPV31 wild-type genomes were able to form colonies in an efficientmanner, with about 40 (mean � standard deviation, 39.25 �4.15) colonies visible per field at day 5. After an additional
week in culture, the plates became confluent. In contrast,cells containing the E5 mutant genomes formed significantlyreduced numbers of colonies, with about 8 (mean � stan-dard deviation, 8.55 � 4.48) colonies detectable after 5 days.These cells grew poorly and did not become confluent after2 weeks. HFKs did not form colonies after methylcellulose-induced differentiation (mean � standard deviation, 0.2 �0.42). This suggests that HPV E5 modulates the differenti-ation-induced cell cycle exit and supports the ability ofHPV31-positive keratinocytes to remain active in the cellcycle.
FIG. 7. Stained sections of organotypic raft cultures. Normal and transfected HFKs were induced to differentiate in raft cultures as describedin Materials and Methods. Sections were stained with hematoxylin and eosin for visualization of differentiation (A to C) or stained with an antibodyto HPV31 E1∧ E4 protein and examined by immunofluorescence (D to F). As a secondary antibody a fluorescein-conjugated anti-rabbit antibodywas used and the nuclei were counterstained with DAPI. (A and D) Normal HFKs; (B and E) HPV31-transfected HFKs; (C and D) E5mutant-transfected HFKs.
Our studies have examined the function of the E5 protein ofthe high-risk HPV types during the productive viral life cycle.In bovine papillomavirus type 1, E5 encodes a primary trans-forming activity, but in high risk types the E6 and E7 proteinsprovide this function (10, 52). In heterologous expression sys-tems E5 can act as a weak-transforming protein, and it hasbeen suggested that E5 acts to augment E6/E7 action (3).However, it is not clear whether this is a physiologically rele-vant activity during viral pathogenesis. In our studies we ana-lyzed the role of E5 in the productive life cycle by monitoringthe effects of loss of E5 function in the context of the completeviral genome. We examined two different mutations in E5 andobserved similar effects. The lack of E5 expression was foundto have little effect on the growth properties, the ability tomaintain episomes or the expression of viral genes in HFKstransfected with HPV 31 genomes in undifferentiated cells.Wild-type or E5 mutant genomes were equally effective atimmortalization, and transfected cells grew at comparablerates either in the absence or presence of added EGF. This was
consistent with Western blot analysis that showed that the lossof E5 had no effect on the number or phosphorylation status ofthe EGFRs. Our observations were surprising and are in con-trast to previous reports that expression of E5 from heterolo-gous promoters can lead to increased levels of EGFR (57). Inour studies, increased levels of EGFR were detected in cellsthat maintain complete and E5 mutant viral genomes, whichwe believe is due to the action of E6 and E7 rather than E5.These observations are consistent with previous reports on theeffects of E6 and E7 on EGFR levels (1, 26, 30, 53). Weconclude that E5 in undifferentiated cells has no effect on anyof the aspects of viral pathogenesis assays we tested. However,it is possible E5 has effects in other aspects of the life cycle inundifferentiated cells that we have not examined. Previousstudies have analyzed the effects of HPV16 E5 in heterologousoverexpression systems, whereas our studies have focusedon presumably natural expression levels of the HPV31 pro-tein. While these proteins are 77% homologous, it remainspossible that the differential effects on EGFR levels ob-served with HPV16 E5 could be due to either expression
FIG. 8. Expression of cell cycle proteins in HFK, HPV31, and E5 mutant cells. Western blot analyses were performed on whole-cell extractsof HFK, HPV 31, and E5 mutant cells cultured in methylcellulose (MC) for the indicated times. Equivalent amounts of whole-cell extracts wereseparated by SDS-polyacrylamide gel electrophoresis and examined by Western blot analysis with anti-cyclin A, B, and E antibodies (A) oranti-CKI p21, p27, and p57 antibodies (B) as indicated. Proteins were visualized by chemiluminescence.
levels or different functional properties attributed to thissequence variation.
In contrast to the lack of effects due to E5 observed inundifferentiated monolayer cultures, we detected a significantincrease in differentiation-dependent viral amplification andlate gene transcription when E5 was present. The decrease oflate viral transcripts observed in the absence of E5 was not dueto reduced stability of the late RNAs resulting from the mu-tations in the E5 ORF since we observed no difference in thelevels of early transcripts which also contain the E5 ORF. Viralamplification and late gene transcription are strictly dependenton two apparently opposing processes: epithelial differentia-tion and DNA synthesis in suprabasal cells. First, it is possiblethat E5 has an effect on epithelial differentiation, a process thatis necessary to induce efficient transcription from the differen-tiation-dependent late viral promoter and to activate viralDNA amplification. Alternatively, E5 could act to augment theactivity of E6 and E7 in modulating progression through cellcycle in differentiated cells. Similar analyses have been per-formed using HPV16 genomes containing mutant E5 genes(17a) and yielded a quantitative reduction in ability of the E5mutant HPV16 genome to reprogram differentiated cells tosupport DNA synthesis. In our study, we observed effects of E5on late viral functions that were more severe than those seen inthe study on HPV16. This difference may be due to the differ-ent HPV types examined or to the assays used.
We examined whether E5 has any effect on differentiation ofcells containing HPV31 wild-type and HPV E5 mutant ge-nomes in organotypic raft cultures. No differences in morphol-ogy of cells or expression of differentiation markers such askeratin 10, filaggrin, and involucrin were observed. However,we did observe a reduction in E1∧ E4 expression in keratino-cytes that contain E5 mutant genomes, consistent with ourfindings from Northern blot analysis. Our studies support theidea that E5 does not act to modulate late functions throughalteration of epithelial differentiation.
Differentiation of HPV-positive cells results in retention of
high levels of S-phase cyclins, such as cyclins A, E, and B. Thelevels of these cyclins decrease with differentiation, but thepresence of HPV gene products reduces the rate of loss. Anal-ysis of HPV-positive cells lacking E5 consistently revealed amoderately increased rate of loss of cyclin A and B levelscompared to that observed in HPV31 wild-type cells. While wedo not believe E5 directly targets cyclin A and B expression, itis likely that E5’s interaction with a membrane-bound cellularreceptor indirectly leads to altered levels of the cell cycle reg-ulators. The reduced levels of cyclins A and B may be sufficientto contribute to the reduced capability of HPV E5 mutanttransfectants to amplify viral genomes and induce late geneexpression. We examined the number of cyclin A-positive cellsby immunohistochemistry following suspension on methylcel-lulose and found minimal differences between keratinocytescontaining wild-type and E5 mutant genomes (F. Fehrmannand L. A. Laimins, unpublished data). Since HPV late func-tions are activated in S-phase, our observations suggest that E5does not significantly alter the number of cells activating latefunctions but increases the levels of activation in each cell.Other potential targets of E5 action include the CKI, whichhave been shown to be upregulated during differentiation invivo (22, 23, 40, 45, 54, 62). In our study, we observed nodifference in the expression pattern of p21, p27, and p57 be-tween cells transfected with HPV31 wild-type or HPV E5 mu-tant genomes following differentiation. It was previously re-ported using an inducible heterologous system that HPV16and -11 E5 suppressed p21 expression in mouse fibroblasts andimmortalized human keratinocytes (58). Our work indicatesthat when E5 is expressed from its natural promoter in thecontext of the complete viral genome, it does not significantlyrepress p21 transcription. We conclude that effects of E5 onactivation of late viral functions are most likely not mediatedthrough altered expression of CKI.
More direct support for the hypothesis that E5 has a role inthe modulation of cell cycle progression during differentiationof HPV positive cells comes from a replating assay. This assaymeasures the ability of cells to remain competent for prolifer-ation after being induced to differentiate. We observed thatcells that expressed the complete viral genome were able toreinitiate proliferation at four times the rate of cells that lackE5. This further suggests that E5 is involved in overcoming thedifferentiation-induced cell cycle arrest and on maintainingproliferative potential. It is possible that the moderate reduc-tion in cyclin A and B expression in the absence of E5 canexplain this effect or alternatively other cell cycle regulatorsmaybe more significantly impaired. Furthermore, the moder-ate reduction in cyclin A and B levels may be a consequence oftargeting another regulator of the cell cycle. The search forsuch a regulatory target for E5 is ongoing.
The E5 protein has been shown to be a membrane proteinthat is localized either to the Golgi, endoplasmic reticulum, orcytoplasmic membranes (4, 6). The question arises as to how amembrane protein can alter cell cycle activities that are pri-marily localized to the nucleus. We suspect that the primarytarget of E5 is a membrane protein or receptor that then actsto alter the levels or activities of cell cycle regulators. Ourstudies suggest that this target is not the EGFR; however, it ispossible that another member of the EGFR family or someother growth factor receptor is the primary target. Elucidation
FIG. 9. Replating of HPV31- and E5 mutant HPV-positive cellsfollowing suspension in methylcellulose (MC). Monolayer keratino-cytes were induced to undergo epithelial differentiation by suspensionculture in methylcellulose. After 24 h the cells were harvested andreplated onto a feeder layer of J2s. Colonies were visible after a fewdays and were counted on day 5 from five random fields. Results arethe means � standard deviations (error bars) of data from four exper-iments. Approximately 10% of the replated wild-type HPV31 cellsgrew into colonies in these assays.
of the target of E5 action will require screening for alteredlevels or activities of membrane associated proteins in differ-entiated cells.
We conclude from our studies that the E5 protein from highrisk HPVs acts in the late phases of the viral life cycle tomodulate differentiation-induced functions like viral amplifi-cation and late gene expression. Additionally, E5 may play amajor role in retaining proliferative activity following differen-tiation, a process that is essential for high levels of viral pro-duction.
ACKNOWLEDGMENTS
We thank Kathy Rundell, Richard Longnecker, and LawrenceBanks for critical reviews of the manuscript. We gratefully acknowl-edge Walter Hubert and the members of the Laimins laboratory forhelpful discussions.
This work was supported by a grant from the Penny Severns Breastand Cervical Cancer Research Fund from the Illinois Department ofPublic Health and a Gramm Fellowship Award from NorthwesternUniversity to F.F. as well as by a grant from the National CancerInstitute (CA74202) to L.A.L.
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