JOURNAL OF INTERFERON AND CYTOKINE RESEARCH 15:495-501 (1995)Mary Ann Liebert, Inc., Publishers

Interferon-a Elicits Downregulation of Human Papillomavirus 18mRNA in HeLa Cells by Selective Repression of Endogenous

Viral TranscriptionSILVIO E. PEREA, OMAR LÓPEZ-OCEJO, ROLANDO GARCÍA-MILIAN, and MANUEL DE J. ARAÑA

ABSTRACT

We have reported that interferon-a inhibits HPV-18 mRNA in HeLa cells. Here we examine mechanisms bywhich IFN could modulate HPV expression. In northern blot experiments, we observed that interferon-a2btreatment reduced HPV-18 mRNA levels in a time- and dose-dependent manner, with a maximal effectachieved at 48 h. Simultaneously, induction of 2-5A synthetase mRNA was verified as indicative of IFN action.The IFN regulatory effect on HPV-18 mRNA at 48 h required de novo protein synthesis. We performed run-on

experiments to determine whether the IFN regulatory effect was at the transcriptional level. HPV-18 endoge-nous transcription was repressed using 200 and 1000 11/ml. Interferon treatment did not affect HPV-18mRNA stability, at least under our experimental conditions. To verify whether HPV-18 enhancer sequenceswere involved in the interferon effect, we transfected a construct containing the chloramphenicol acetyltrans-ferase driven by the HPV-18 upstream regulatory region. The enzyme activity was unmodified on humankeratinocytes and HeLa cells by interferon exposition. Our data demonstrate that interferon-a downregulatesHPV-18 mRNA levels on HeLa cells by repressing nascent viral transcripts, possibly through regulatorycellular flanking regions.

INTRODUCTION

Human PAPILLOMAviruses (HPVs) are useful in controlmechanism studies of gene expression because their tran-

scriptional program appears to be closely linked to specificevents in the control of growth and differentiation of squamousepithelium/1,2' DNAs from several HPV types have been de-tected in intraepithelial neoplasias of cervix/3' vulva,<4> andpenis,(5) as well as in carcinomas,<6) suggesting that this virusmay be an important factor in the development of these tumors.Particularly HPV-16 and 18, are usually integrated into hostchromosomes in more than 90% of cervical tumor biopsies'7' ina specific way, ensuring that the E6 and E7 early genes are

selectively retained and transcribed/8' Furthermore, the HPVE6 and E7 genes cooperate to induce immortality in primarycells cultured from genital epithelia/9' On the molecular level,these two viral proteins usually complex with products fromp53<10) and Rb genes/1 " respectively, and such events seem tobe determinant for cellular transformation produced by theseDNA viruses.

Attempts to develop an ideal model to reproduce HPVs incell cultures in vitro have failed. Therefore, most studies con-

cerning the regulation of HPV gene expression have been per-formed on both HPV-immortalized cells and in cervical carci-noma cell lines containing mainly HPV-16 and 18 DNAs.Thus, the epidermal growth factor has been shown to reduceHPV-16 mRNA through a silencer response element in theupstream regulatory region (URR)/12' and transforming growthfactors ß, and ß2 transcriptionally regulate HPV-16 early geneexpression in HPV-immortalized human genital epithelialcells/13'

Furthermore, 5-azacytidine, a demethylating agent, inhibitedHPV-18 gene transcription in nontumorigenic but not in tumor-

igenic HeLa hybrid cells or cervical carcinoma cell lines/14' Incontrast, retinoic acid repressed HPV-18 transcription in bothnontumorigenic and tumorigenic HeLa hybrid cells through thecentral fragment of the HPV-18 URR/15'

Interferon-a (IFN) has been used effectively to treat HPV-related diseases, such as cervical intraepithelial neoplasia/16'condylomata acuminata/1718' and laryngeal papillomatosis'19';

Departamento de Biología Celular, Centro de Investigaciones Biológicas, La Habana, Cuba.

495

496 PEREA ET AL.

however, the mechanisms by which IFN-ot produces papillomaregression remains to be determined. In cell cultures in vitro,IFN-a differentially reduced the HPV-16 E6-E7 transcript lev-els in the SiHa cervical carcinoma cell line/20' Furthermore,IFN-v and leukoregulin inhibited HPV-16 gene transcription inHPV-immortalized human cervical cells/2" We and othergroups have reported that IFN-a downregulates HPV-18mRNA in HeLa cells/22'23' although the mechanisms involvedin such IFN-mediated repression were not examined. In thispaper, we analyzed the possible viral targets for IFN action andwe found that IFN-a downregulates HPV-18 mRNA by directviral transcriptional repression. Our results also indicate thatneither the viral enhancer sequences nor posttranscriptionalmechanisms are involved on the HPV-18 mRNA downregula-tion produced by IFN in HeLa cells.

MATERIALS AND METHODS

CellsHeLa cells were grown in 5% C02 at 37°C in Dulbecco's

modified Eagle's medium (GIBCO) supplemented with 50(xg/ml of gentamycin and 10% newborn calf serum (NCS; CU-BAVET, Havana). The human epidermal keratinocyte cell line(RHEK-1 cells, HPV negative)'24' was maintained in RPMI1640 medium supplemented with 10% NCS and 1 p-g/ml ofhydrocortisone (Sigma).IFN

Recombinant human IFN-a2b was produced in Escherichiacoli at the Center for Genetic Engineering and Biotechnology inHavana/25' with a specific activity of 2 x 108 IU/mg.RNA isolation and northern blot analysis

Cells were lysed in a solution containing 4 M guanidiniumthiocyanate (Fluka); 25 mM sodium citrate, pH 7; 0.5% sarco-

syl (Sigma); and 0.1 M 2-mercaptoethanol (Merck). Total RNAwas obtained by a single-step method previously described/26'For northern blot analysis, 20 jxg of the different total RNApreparations was fractionated by electrophoresis in 1.0% aga-rose gels containing 2.2 M formaldehyde. Gels were blotted bycapillary transfer onto nylon filters (Hybond-N; Amersham,Buckinghamshire, UK), cross-linked by ultraviolet irradiation,and subsequently hybridized with DNA fragments representingthe HPV-18 genome, 2-5A synthetase, or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes that were previouslylabeled by a random priming method/27' Autoradiographs were

subsequently analyzed by densitometric scanning on a Hewlett-Packard scanner.

Recombinant plasmids and DNA probesPlasmid pi8 URR was obtained by cloning the HPV-18 URR

BamHI fragment into pBLCAT3 vector/28' Plasmid expressingß-galactosidase contains LacZ gene driven by the cytomegalo-virus promoter/enhancer element (CMV-LacZ)/29' TheHPV-18 DNA probe was originally provided by Dr. Harald zur

Hausen (DKFZ, Heidelberg). Constructs containing cDNA in-

serts from 2-5A synthetase and GAPDH genes were generouslyprovided by Dr. Brian R.G. Williams (Cleveland Foundation,Cleveland, OH).

Transient transfection and CAT assayTransfection of cells was performed by the DEAE-dextran

method/30' Briefly, 2 p.g of pl8 URR-CAT plasmid was

cotransfected with 0.7 p,g of the construct expressing ß-galac-tosidase for normalizing the transfection efficiency betweenplates, and 0.5 mg/ml of DEAE-dextran was added. HeLa or

RHEK-1 cells (2 x 107) in 0.1 ml medium without serum were

incubated with the DEAE-dextran and DNA solution for about40 minutes at 37°C in 5% C02. Then, 10% dimethylsulfoxidewas added to each transfection tube and further incubated for 1minute. IFN-a2b was added and the incubation was prolonged15 h, 24 h posttransfection. Cell extracts for measuring ß-ga-lactosidase and CAT activities were prepared by resuspendingthe transfected cells in 0.1 ml 0.25 M Tris-HCl, pH 7.5, as

previously described/30' From each cell extract, 200 and 1000units ß-galactosidase activity were employed in the CAT reac-

tion. Each transfection was performed with two different plas-mid preparations.

Nuclear run-on analysisHeLa cells were cultured for 48 h in the presence or absence

of 200 and 1000 IU/ml of IFN-a2b. After washing with phos-phate-buffered saline, cells were harvested in 4 ml cold RSB(10 mM Tris-HCl, pH 7.4, 10 mM NaCl, and 3 mM MgCl2)with a rubber policeman and incubated for 10 minutes on ice.The nuclear pellet was dissociated by 30 strokes of a tight pestlein a Dounce homogenizer and centrifuged at 4000 rpm for 5minutes at 4°C. Nuclei (2 X 107) in 200 p.1 mix reaction [20mM Tris-HCl, pH 7.4, 140 mM KC1, 10 mM MgCl2, 20%glycerol, 0.5 mM NTPs, 200 u.Ci 32P-labeled UTP (3000 Ci/mmol) and 1 mM dithiothreitol] were incubated at 32CC for 20minutes with gentle agitation. DNase 1 (100 U, Boehringer,Germany) was added, and the mixture was incubated for an

additional 5 minutes at room temperature. 32P-labeled RNAswere dissolved in 500 p.1 cold TE (10 mM Tris-HCl, pH 8, and0.2 mM EDTA) and hybridized at 65°C for 36 h to immobilizedplasmid DNAs in 2 ml TESS (20 mM TES, pH 7.4, 10 mMEDTA, 400 mM NaCl, 0.2% sodium dodecyl sulfate, and two-fold Denhardt's). Following hybridization, the filters were

washed and further exposed at —70°C for about 7 days.

RESULTS

IFN-a elicits downregulation of HPV-18 mRNA

On HeLa cells, two cDNAs coding for a 3.4 kb transcriptcontaining open reading frames (ORFs) E6, E7, and the 5'portion of El and a 1.6 kb transcript that contains ORFs E6 andE7 were respectively well characterized/3" In our northern blotexperiments, IFN-a efficiently inhibited HPV-18 mRNA at 36h, and it was further maintained until 48 h of IFN incubation(Fig. la). IFN-mediated inhibition of the 1.6 kb transcript was

produced in a dose-dependent manner at 36 h and a maximal

IFN-a REPRESSES ENDOGENOUS HPV-18 TRANSCRIPTION 497

2h 12h 24h

°?g|°?°°° *8! IFN(lU/ml)

18S

GAPDH

IFN(lU/ml)

GAPDH

m *

03

Mr

01

a Controla 40 u/mlO 200 u/ml 1000 u/ml

D Control 40 u/ml 200 u/ml 1000 u/ml

FIG. 1. Time course of IFN effect on HPV-18 mRNA levels, (a) HeLa cells were seeded at 2 x 105 cells/125 cm2, total RNAwas isolated after IFN-a treatment during 2, 12, 24, 36, 48, and 72 h using 40, 200, and 1000 IU/ml and further subjected tonorthern blot analysis as described (Materials and Methods). Normalization analysis for HPV-18 1.6 kb (b) and 3.4 kb (c)transcripts. GAPDH expression was used as housekeeping gene.

effect (more than 95% of reduction) was achieved after 48 h atany IFN dose (Fig. lb). However, the 3.4 kb transcript was

affected only after 48 h of IFN incubation (Fig. lc). Theseresults show a differential IFN regulation of both HPV-18 tran-scripts on HeLa cells.

HPV-18 mRNA inhibition by IFN-a depends on

ongoing protein synthesisTo exclude the possibility that the IFN regulatory effect on

HPV-18 mRNA was mediated by preformed proteins, we

treated the cells with cycloheximide for 5 h during IFN incuba-tion. The results, shown in Fig. 2a, clearly indicate that inhibi-tion observed of both HPV-18 transcripts after 48 h of IFNincubation is completely abrogated when the protein synthesisinhibitor is included in the culture medium. In cells treated with

cycloheximide alone, HPV-18 transcript levels increased withrespect to control cells, suggesting either a viral mRNA stabili-zation or an increase in HPV gene transcription (Fig. 2b).

IFN-a represses endogenous HPV-18 transcriptionRun-on experiments indicated that endogenous HPV-18 na-

scent transcripts were selectively repressed when 200 and 1000IU/ml were employed (Fig. 3a). The inhibition rate of HPV-18transcription was from 40 to 50% when indicated IFN doseswere used (Fig. 3b). Simultaneously, induction of the endoge-nous transcription from the 2-5A synthetase gene was clearlyobserved in IFN-treated cells when both IFN doses were ap-plied (Fig. 3a). As expected, neither chick actin nor GAPDHgene transcription was modified after IFN exposition, and per-

IFNCHX

28S

18S

GAPDH

ControlG IFNQIFN »CHXB CHX

a

FIG.2. Requirements of de novo protein synthesis, (a) HeLa cells were seeded at 2 x 105 cells/125 cm2 and were cultured for 48h in the presence or absence of 1000 IU/ml of IFN-a. In parallel, incubations with 10 u.g/ml of cycloheximide (CHX) plus IFN andCHX alone were performed during the first 5 h of culture and further removed from cell cultures. IFN or medium alone was againadded and the incubation continued until 48 h. CHX was added 1 h before starting the IFN incubation. At the end of incubation,total RNA was isolated and analyzed by northern blot as described (Materials and Methods), (b) Normalization analysis for 1.6 and3.4 kb.

498 PEREA ET AL.

IFN (IU/ml) ooCM ~

a

Ch. Actin

HPV-18

pBR322(2'-5')OASGAPDH

200 u/ml 1000 u/ml

IFN

FIG. 3. Effect of IFN on the HPV-18 transcription, (a) HeLa cells were cultured for 48 h in the absence or presence of 200 and1000 IU/ml of IFN-a2b. Isolation of nuclei and run-on assay were performed as described in Materials and Methods. The32P-labeled RNAs from untreated cells and IFN-treated cells were hybridized to filters containing plasmid DNA from HPV-18,2-5A synthetase, chick actin, and GAPDH genes, (b) Normalization analysis with respect to chick actin gene.

mitting us to normalize the transcription rates observed forHPV-18 (Fig. 3b).

Induction of 2-5A synthetase mRNAThe inducibility of 2-5A synthetase gene expression after

IFN treatment was analyzed using an IFN response marker.mRNA from the different 2-5A synthetase isoforms'32' was

evident (Fig. 4a). Upregulation of enzyme mRNA on IFN-treated cells was observed after 12, 24, and 36 h of incubation atthe different IFN doses (Fig. 4b).

IFN-a does not affect HPV-18 mRNA stabilityTo analyze whether IFN treatment leads to destabilization of

HPV-18 mRNA, HeLa cells were cultured in the presence or

absence of 1000 IU/ml of IFN-a for 8 and 32 h. Northern blotanalysis (Fig. 5) showed similar curves of HPV-18 mRNAdecay. The viral mRNA half-life calculated by regression anal-ysis was around 3 h in both IFN-treated and untreated cells.This suggests that IFN treatment does not promote the degrada-tion of existing HPV-18 transcripts in IFN-treated cells, sug-gesting that posttranscriptional mechanisms affecting mRNAstability are not involved in the reduction of HPV-18 mRNA

levels in HeLa cells treated with IFN-a. In these experiments,the p53 cDNA probe was used for normalizing the RNAamounts.

HPV-18 enhancer sequences are not involved inIFN-mediated viral repression

Transient transfection experiments using the pl8URR con-

struct demonstrated that reporter gene expression was not mod-ified on neither HeLa or RHEK-1 cells using different IFNdoses (Fig. 6). These results indicate that HPV-18 regulatorysequences are probably not involved in the viral repressionmechanism produced by IFN on HeLa cells, at least under theseexperimental conditions.

DISCUSSION

We reported earlier that the long-term treatment of HeLacells with IFNs a could partially revert the malignant phenotypetoward a more differentiated stage, along with HPV-18 mRNAdownregulation/22' According to these previous findings, we

a

12h 24h 36hIFN

?8§o?|8o?§§ CU/ml)

•••<••*#••» *fc«-

28S

-

18S

• -» - •••«•«è. GAPDH

D Control040 u/mlH 200 u/mlQ 1000 u/ml

FIG. 4. Induction of 2-5A synthetase mRNA. (a) Total RNA from HeLa cells treated with 40, 200, and 1000 IU/ml after 12, 24,and 36 h of IFN incubation was analyzed by northern blot as described (Materials and Methods), (b) Normalization analysis withrespect to GAPDH gene.

IFN-a REPRESSES ENDOGENOUS HPV-18 TRANSCRIPTION 499

ABC ABCABCACABC

14 hours with |actynomicin D I)

-ControlIFN ( 32 hours )

IFN ( 8 hours )

05 2 5Hours with

actynomicin D

FIG. 5. Effect of IFN-a on HPV-18 mRNA stability, (a) HeLa cells were seeded at 2 x 105 cells/125 cm2 and were cultured for8 and 32 h with 1000 IU/ml of IFN-a. After IFN incubation, 10 u.g/ml of actinomycin-D (Sigma) was added and total RNA wasisolated after 0.5, 2, 5, 9, and 14 h of incubation and further analyzed for HPV-18 mRNA levels by northern blot as described(Materials and Methods), (b) Normalization analysis of p53 gene expression.

next studied the IFN-a effect in short-term assay and some

possible IFN targets involved in the regulatory effect.Our results from northern blot experiments demonstrated that

IFN-a2b efficiently downregulated HPV-18 mRNA in HeLacells after 48 h of incubation. However, there was a differentialregulation of both HPV-18 transcripts that was in turn time anddose dependent. Downregulation of 1.6 kb mRNA levelsstarted after 36 h of IFN incubation, and it was strictly dosedependent. Interestingly, the inhibitory effect was maximal(more than 95%) after 48 h of incubation for the different IFNdoses tested. Otherwise, 3.4 kb mRNA was inhibited only after48 h of incubation, and the effect was also dependent on the IFNdose. Previous findings have indicated that both 1.6 and 3.4 kbmRNAs are transcribed from the HPV-18 genome at the same

integration site with different splicing/3" Our results do noteliminate a possible differential IFN action in HPV-18 splicingprocessing that leads to the two major viral transcripts on HeLacells.

The results from run-on experiments that used double-stranded plasmid probes clearly demonstrate that IFN-a down-regulates HPV-18 mRNA levels by affecting the viral transcrip-tion rate about 50%, thus providing an estimate of the density oftranscribing polymerases over the HPV-18 DNA sequences.There is increasing evidence that HPV-18 transcription on

HeLa cells utilizes cellular promoters'3"; however, it is difficult toestablish from these experiments whether cellular or viral promot-ers were affected by the IFN-a exposition on these cells.

We next determined whether de novo protein synthesis was

required for the downregulation of HPV-18 mRNA levels elic-ited by IFN-a. When cycloheximide was included, HPV-18mRNA reached higher levels than those observed in controlcells, indicating that the synthesis of one or some factors isneeded for the observed IFN effect. HPV-18 mRNA upregula-tion in the presence of cycloheximide could be explained by an

increase in HPV-18 transcript stability, similar to that previ-ously found for c-myc transcripts in HeLa cells treated with thechemical alone/33'

Among the HPV transcriptional control mechanisms, a variantof the E2 gene product was the only viral identified repressor in theexpression of papillomavirus early genes/34' Because disruptionof the E2 gene has been found in HeLa and in several tumor cellscontaining integrated HPV sequences/35' it is unlikely that the E2gene product could mediate the IFN regulatory effect.

Because IFN treatment could also affect viral mRNA levelsat the posttranscriptional level by mechanisms involving the2-5A synthetase system/36' we analyzed HPV-18 mRNA sta-

bility. The results indicated that IFN-a did not affect HPV-18mRNA stability, so that this could indicate a noncausal relation

RHEK-1 HeLa

40 200 1000 C 40 200 1000 IFN (U/ml)FIG. 6. Effect of IFN on HPV-18 viral regulatory sequences. HeLa and RHEK-1 cells were transfected by DEAE-dextranmethod (Materials and Methods) with 2 p,g pl8 URR plasmid along with 0.70 |xg CMV-LacZ construct. IFN (1000 IU/ml) wasadded to the culture medium 24 h after transfection, and after 15 h, cell extracts were prepared for CAT and ß-galactosidase assays.

500 PEREA ET AL.

between 2-5A synthetase induction and viral mRNA downregu-lation produced by IFN in HeLa cells. Finally, to determinewhether viral regulatory sequences were involved in the repres-sion mechanisms exhibited by IFN-a treatment, we first per-formed transient transfection experiments on HeLa cells using a

construct containing the HPV-18 URR fused to CAT gene. Wealso used RHEK-1 cells to avoid possible transactivation of theexogenous HPV-18 URR produced by endogenous viral geneproducts expressed on HeLa cells/37' We were unable to dem-onstrate any IFN effect on the HPV-18 regulatory sequences.The lack of correlation between run-on and transient transfec-tion experiments further suggests that IFN exposition affectsHPV-18-transcribing cellular promoters. Thus, IFN-a appearsto downregulate HPV-18 mRNA by mechanisms other thanthose observed for retinoic acid on HPV-18 in HeLa cells or

epidermal growth factor on HPV-16-transformed keratinocytesin which specific sequences within viral URR for each virusmediate the silencer response. According to our results, itseems that IFN-a specifically blocks the synthesis of nascentHPV-18 transcripts, and cellular flanking regions to viral inte-grated sequences could play a central role in the repression ofHPV-18 expression produced by IFN-a in HeLa cells.

ACKNOWLEDGMENTS

We thank Mathias Durst for providing pi8 URR construct.We also thank Professor Paula M. Pitha for critical review ofthe manuscript and Dr. Alex Von Gabain for stimulating dis-cussions. This work was supported by grants from UNIDO(Contract No. 92/050; CRP/CUB 91-04) and SAREC (S/2.CUB.00).

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Address reprint requests to:Dr. Silvio E. Perea Rodriguez

Departamento de Biología CelularCentro de Investigaciones Biológicas

Apartado 6996La Habana, Cuba

Received 1 June 1994/Accepted 30 December 1994