Transcription of the E6 and E7 genes of human papillomavirus type 6 in anogenital condylomata is restricted to undifferentiated cell layers of the epithelium.

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0022-538X/92/084639-08$02.00/0

Transcription

of

the

E6

and

E7

Genes

of

Human

Papillomavirus

Type 6 in

Anogenital

Condylomata

Is

Restricted

to

Undifferentiated Cell

Layers

of

the Epithelium

T.

IFTNER,1

M.

OFT,'

S.

BOHM,'

S. P.

WILCZYNSKI,2

AND H.

PFISTER`*

Institutfur KlinischeundMolekulare Virologie, Friedrich Ale-xander Universitat

Erlangen-Nurnberg,

Schlossgarten 4, 8520Erlangen,

Germany,'

andDepartmentof Pathology, City ofHope,

National Medical Center, Duarte, Califomia 910102

Received 21January1992/Accepted13April 1992

The E6 and E7 genes of human genital papillomaviruses (HPVs) appear to transform cells by different mechanisms. They seem to act

synergistically

but are not equally important when tested under diverse experimental conditions. We were therefore tempted to investigate the E6- and E7-specific transcription pattern inHPV6-infected condylomas separately, byinsituhybridization. Recent studies have identified three promoters within the E6-E7 region of HPV6 andHPVll by applyingSi,exonuclease VII, and cDNA

analyses.

On the basis ofthesedata,weclonedsubgenomicfragments of HPV6 intoplasmidpBS toobtainriboprobes thatdifferentiated between transcripts starting upstream oftheE6 andE7 openreadingframes, respectively. These different species of mRNAs were analyzed in serial thin sections of eight HPV6-positive anogenital condylomas. The E6 probe (nucleotides 7862to241) led to weaksignals within the basallayer.Inthree cases, rather strongsignals were confined to a few basal cells. The E7 probe (nucleotides 242 to 534) gave riseto a morepronounced labeling of all cells within the two to three lowest epidermal layers.Insituhybridization with a riboprobe for human c-fos revealed an expression pattern similar tothat observed with the E7 probe.In contrast tothepreferentialexpression of the transforming E6 and E7 genesinthe lowerepithelium, themajor transcriptional activityof the virus was detectedin themiddle and upper thirdby probes colinear with the 3'

moiety

of the

early

region.

The oncogenic potential of human papillomaviruses

(HPVs) is reflected in vitroby their ability to immortalize

keratinocytes and to change their differentiation behavior

(17). Genetic experiments identified the E6 and E7 genes of

HPV16 asboth necessary andsufficient for this activity (9, 11). However, under certain experimental conditions, E6 or E7 wasable to immortalize humanprimary epithelial cells by itself (1, 8). It was recently suggested that genital HPVs induceproliferation, at least in part, through interaction of the viraloncoproteinsE6 and E7 withthetumorsuppressor

proteins p53 andRb, respectively (7, 12,23).

The E6 and E7 genes were shown to be retained and transcribed in most cervical carcinomas (24), which may indicate that they contributetotheproliferative potential of the cancer cells. The activity of these genes in benign HPV-inducedtumorsisnotyetclearly elucidated. Northern

(RNA)blot studies of HPV16 RNA revealed nodifferences

in the amount of E6 and E7 transcripts in premalignant versus malignant lesions (19). In situ hybridization with HPV6-, HPV11-, and HPV33-infected condylomas showed a differentiation-linked gene expression,however, and itwas

surprising to note that E6 and E7 transcripts were almost

undetectable in the proliferating lower layers (2, 21). The separateexpressionof E6 and E7was notassessed because ofuncertaintiesconcerningthestructureof thegene-specific transcripts.

Recently, two promoters were identified within the E6 open reading frame(ORF) of HPV6 and HPV11 (20). Initi-ationatpromoter P2(Fig. 1) leadsto atranscript with E7as thefirst translatable ORF.Transcripts derived fromP1 (Fig.

* Correspondingauthor.

1) could encode both E6 and E7, but reinitiation of transla-tionatthe E7AUG ishighly unlikely (10, 14); therefore,this

mRNAisprobably E6specific. On thebasis of these data,

we designed probes for in situhybridization which specifi-cally detect the E6 and E7 transcript of HPV6. Care was taken to avoid any overlap with the major E1-E4 mRNA class, which initiatesaround nucleotide(nt)700 in HPV6or betweennt 674 and 714 in HPV11 (Fig. 1) (4, 20).

MATERIALSANDMETHODS

Biological specimens and HPV typing. Specimens from randomly selectedpatientsofdifferentsexand age(Table 1)

were snap-frozen immediately after surgery and stored at

-70°C until processed. High-molecular-weight DNA was

extracted from the frozen tumor tissue. The polymerase chain reaction for HPV classification was performed as described previously (3). Eight condylomas from different sites of the anogenital region (Table 1) thatwereproven to contain HPV6 DNAwere chosen for in situ hybridization

analysis. Normal ectocervical mucosa was obtained from a

patientwithahistoryof breastcancerandwasproventobe

HPVnegative.

Construction ofspecific probes. For the synthesis of spe-cificRNAprobes,subgenomic restrictionfragments (Fig. 1) of HPV6b (6) were cloned into the SmaIsite of the vector

pBS+ (Stratagene) orpGEM1 (Promega). Theidentity and

orientation of each fragmentwere confirmed byrestriction enzyme analysis. The construct togenerate antisense ribo-probes for thec-fos gene is available fromAmersham.

Depending on the choice of the promoter, the in vitro-synthesized RNAs were either in the same polarity as

mRNA (sense) or in the opposite polarity

(antisense).

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4640 IFTNER ET AL.

00CD~~~~~~~~~cc>LOOtr~~~~-- O

123 4

HPV 6

probes

0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 kb

*

Pl1P2 P3 SA

_______

C

-~~~~~~

-FG. 1. Genetic and transcriptional map of the earlyregion of HPV6andlocalization of themRNA-specific probes 1 to 4 used in thisstudy.The ORFs(shaded boxes) and the nucleotide positions of the probe boundaries (at the top) correspond to the published sequence (18). Vertical lines within the ORFs represent the first translational start codon. Themajor transcriptsof theearly region (a toc) aredepictedatthe bottom aslines with differentthicknesses correspondingtotheir relative abundances determined inprevious investigations (13, 20). The promoters areindicatedasP1toP3, and themajorspliceacceptoris indicated asSA.

generateantisenseprobes,thepBS+-based RNA expression vectors were each linearized with HindIll cutting in the multiple cloning site distal from the T3 bacteriophage pro-moter used for transcription. The HPV6 DNA fragment cloned in pGEM1to synthesizeprobe4(Fig. 1) spanned nt 2792 to 4029. After cleavage with AvaIl at nt 3491, the appropriate antisense RNA was transcribed from the T7 promoter. In vitro RNA synthesis in the presence of

[35S]UTP, resultingin probeswithaspecific activityof 3 x

108 cpm/pg, was followedby alkaline hydrolysis to reduce

theprobelength to approximately200nt (5). Thequalityof thetranscribed RNAswastestedbyagarosegel electropho-resis. The shortestprobe (probe 1,withaprobe complexity of 143nt)wasapplied at aconcentrationof 6 x 107cpm per

ml of hybridization mixture (0.15 ,ug/ml/kb of probe

com-plexity), and the others were normalized in relation to the probelength.

[image:2.612.58.297.73.205.2]

RNA in situ hybridization. Serial cryostat sections of biopsy material, mounted on aminopropylsilane-coated slides, were fixed in 4% paraformaldehyde in phosphate-buffered saline anddehydratedthrough gradedethanols. The dry sectionswerethenacetylatedin 0.1M triethanolamine-0.25% acetic anhydride for 10 min, washed in 0.2x SSC(lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate), and preincubated for 2 h at 45°C covered by solution I (50%

TABLE 1. Clinical data

Case Localization Patient data

Pretreatment

no. oflesion Age (yr) Sex'

1 Genital 2 F None

2 Vagina 22 F None

3 Perianal 22 M Unknown

4 Perianal 31 M None

5 Vulva 16 F None

6 Perianal 28 F None

7 Genital 2 M None

8 Penis 41 M None

aF,female; M,male.

formamide, 0.6 M NaCl, 2.5x Denhardt's solution, 10 mM Tris-HCl [pH 7.5], 1 mM EDTA, 0.1% sodium dodecyl sulfate, 0.15 mg of tRNA per ml). After removal of this prehybridizationsolution, the tissue sections were incubated for 16 h at 45'C with 35S-labeled antisense riboprobes in solution I containing 10% dextran sulfate. The incubated slides were washed in 50% formamide-lx SSC at 500C, treated with RNase A(5 ,ug/ml), washed for 1 h each in0.2x SSCat50°C and0.lx SSC at60°C, dehydrated,and finally coated with Kodak NTB-2 emulsion. All slides from one specimenweredeveloped after the same exposure time (10 to 14 days) to allow direct comparison of signals obtained with different probes. Hematoxylin-and-eosin-counter-stained sections were observed and photographed with a Reichart & Jung Polyvar microscope by usage of a dark- or bright-field condenser. The specific detection of RNA by in situ hybridization was established by using sense ribo-probes,which gave only background signals, or by pretreat-mentof the tissue withDNase-free RNase before hybridiza-tion. The intactness of the RNA in the tissue sections could be demonstrated by hybridization with aprobe specific for anepithelial cell adhesion molecule (E-cadherin [16]).

RESULTS

Toobtain riboprobes specific for different mRNA classes ofHPV6, weclonedsubgenomic viral DNAfragments into the RNAexpression vectorpBS+ (Stratagene) or pGEM1

(Promega). Probe 1 is specific for E6 mRNAs starting at

promoterP1(Fig. 1),whereasprobe 2 recognizes all mRNAs starting at promoter P2 or upstream of it. The net signal resulting from the E7 mRNA startingatP2canbeobtained

by subtracting signals generated by probe 1 from signals

generated by probe 2. Probe 3 hybridizes only to mRNA

specieswith thecodingcapacity forafull-length E2protein

(Fig. 1). Antisense probe 4 is complementary to all known

mRNAspecies of HPV6 exceptfor the L2 mRNA.

With the helpof theseprobes,weanalyzed thepatternof HPV6 RNA expression in adjacent serial sections of eight

condylomataacuminata fromdistinctsites of theanogenital

region, whichwereprovenby polymerase chain reaction and in situ hybridization to contain HPV6 DNA (data not

shown). Each of the four different riboprobes resulted in

highly characteristic signal patterns (Fig. 2), which were consistent for allcondylomas tested.

Fivecondylomas hybridized with theE6 probe(probe 1) revealed homogeneously distributed weak signals in the

cytoplasmof basal cells(Fig.3A andB),whereas three other

condylomasexhibited onlyafewheavilylabeled cell nuclei

within the basallayer(Fig. 2B and 3C). In some cases, weak signalswerealso discernible insuprabasal cells.

The E7 probe (probe 2) gave rise to a rather uniform cytoplasmic labeling of all cells within the two to three lowestlayers of thestratum germinativum (Fig. 4A, C, and E). No signals could be observed inhigher layers, with the exception that inonecondyloma,labelingextended through-outthe lower third(Fig. 2C).Thesignals in Fig.2C that seem to appearin the upper third oftheepidermis arelocated in undifferentiated basal cells around a crosswise-cut papilla, as canbeseenbycomparisonwith thebright-field histology

micrograph (Fig. 2A).When adjacent sectionsofall

condy-lomas were analyzedfor transcription of thec-fos gene by hybridization with an antisense probe, the distribution of

signalswassimilartothatobserved with theE7probe(Fig.

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VOL.66, 1992

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.Sv*

*.1;t8> , .4 8@- t*&

t2;

TRANSCRIPTION OF HPV6 E6 AND E7 GENES 4641

E

_

F

_

1

FIG. 2. Insituhybridization analysis ofananal condyloma whosehistopathology is shown by light microscopy after hematoxylin-and-eosinstaining (A). Adjacent tissue sectionswerehybridizedtoHPV6-specific probes1(B),2(C), 3(D), and4(E) (Fig. 1) and withahuman c-fosantisenseriboprobe(F).Thesilvergrainsgenerated in the film emulsion afterexposuretothe

35S-labeled

probesare seen aswhitegrains under dark-fieldillumination (BtoF). Black arrowheads indicatethelocations oftwocrosswise-cutpapillas.

2F and 4). Investigation of normal HPV DNA-negative ectocervical mucosa revealed an identical c-fos expression

pattern, whereasthe HPV6 E7probe didnothybridize(data

not shown).

Hybridizationwith the E2probeyieldedsignals dispersed

through thelower and middle parts of the epithelium, with

somestrongsignalsin thenuclei ofkoilocytes(Fig. 2D and

5A).

The major transcriptional activity of the virus was

detected withprobe4 (Fig. 1)inthe middle and upper third

of theepithelium. The strongestsignalswere seenin koilo-cyte clusters with a perinuclear

cytoplasmic

distribution

(Fig.2Eand

5B).

As therewere noE6-orE7-specific

signals

detectable in these differentiated areas, this massive label

probably representsthehighly abundantE1-E4 transcripts,

which are initiated downstream from the 3' end of the E7

probe (4), and latetranscripts havingashort exonfrom the

E4region

(15).

Hybridizationwithprobe4alsoledto asignal

within the lower third of theepithelium, whichwas

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4642 IFTNER ET AL.

I

w-.

FIG. 3. In situhybridizationofagenitaland twoperianal condy-lomata with the E6-specific riboprobe 1 demonstrated by light microscopy. Shown are details of the basallayersin dark-field(A) andbright-field (BandC) illumination.

rable tothat obtained with the E7 probe in both localization and signal strength (Fig. 2E). This finding was not unex-pected, because E7 mRNAs are known to contain a second exon largely colinear with probe 4 (20). The spatial separa-tion of the E7-specific mRNA (detected by probes 2 and 4) and the E1-E4 mRNA(detected by probe 4 only) becomes particularly obvious in Fig. 6, in which the strong signals generated by probe 4 appearedtobeunusually focal.

DISCUSSION

The infectious cycles of all DNA viruses appear to be

sequentially organized. Early proteins ofpapovavirusesand

adenoviruses drive the host cell into the S phase to allow

viral DNAreplication and then the expression of structural proteins. This activity of the early proteins may lead to an

oncogenic transformation ofabortively infected cells.

Inthe caseofpapillomaviruses, the multiplication cycle is

particularly complicated because of its strong dependence

on thedifferentiation state of the host cells. It starts witha

latent infection of the basal keratinocyte, proceeds with

ongoing cell maturation, and ends with the production of

virus particles in the uppermost layers of the epithelium.

Thisprocess is reflectedbyacell differentiation-dependent

geneexpression, whichcouldbe demonstratedby

morphol-ogy-basedmRNA detectioningenital condylomas (2, 21).

Witheach of fourearly-gene-specific probes,weobserved

a characteristic differentiation-dependent transcription

pat-tern that was consistent in eight condylomas from four distinct sites of the anogenital region. The majority of

transcripts of the early region appeared in the middle and

upperthird of theepithelium.Inaccordancewithpreviousin

situ hybridization studies with subgenomic probes (2, 21),

thebulk of thesignalin this area mostlikelyrepresents the E1-E4 mRNA, whichwasidentifiedpreviously byNorthern

blot hybridization as the most abundant transcript (13). A

minor percentage of thesignalin themidstratumseems tobe

due to mRNA with a coding potential for a full-length E2

protein.

Previousinsituhybridizationstudies withaprobe colinear

with the growth-promoting E6 and E7 genes surprisingly revealed an expression pattern similar tothatof the E1-E4 mRNA, with almostno E6and E7transcripts detectable in

themitoticallyactive basal celllayer(2, 21). In contrast,we

found E6- and E7-specific mRNAs strictly confined to the lowest layers of the epithelium. These differences are

par-tially dueto the fact that the earlier in situ studies applied

ORF-specific probes, whereas

transcript-specific

probes

were used in this study. The ORF-specific probe for the detection of HPV6orHPV11 E6 and E7 mRNA(nt7694to

811 [21]) contains

approximately

100nt thathybridize with

the 5' end of the E1-E4 messages, which were shown to initiate between nt 674 and 714 (Fig. 1, P3 [4, 20]). It will thereforeprimarilydetect thehighlyabundant E1-E4 mRNA transcripts, whichare 50 to 100 times moreprevalent than theE6- andE7-specific transcripts in basal cells.Usingsuch

anORF-specific probefor theanalysisofourspecimens,we

observed prominent signals in the upper part of the

epithe-lium(datanotshown). Theyweresimilar in locationtothose

FIG. 4. Correlationof HPV6 E7 and c-fos expression in two perianal condylomas and one genital condyloma. In situ hybridization was carriedoutwithriboprobe2 inantisense (A, C, and E) or sense (G) orientation, a riboprobe for human c-fos(B,D, andF), and a riboprobe fortheepithelial cell-specificadhesion molecule E-cadherin (H). Details of bright-field photographs of the lower strata are shown as insets inpanelsA, C, andE.

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4644 IFTNER ET AL. J. VIROL.

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r,

;iNO

MAKO

X

^^

;

~~~~~*

'-^ ' * '*+ ;{'t '';;' ,A~~~~~~~~~4't' 4' a *'^z'

FIG. 5. In situhybridizationofagenital condylomawithriboprobes3(A)and 4(B)asdemonstratedby bright-fieldillumination.Shown aredetails of the midstratum of theepithelium.

obtained with probe 4 although lower in intensity, as one would expectfrom the differentlengthsof the 5' and 3'exons of the E1-E4 mRNA. It is not quite clear why previous studies failed todetect major signals in the lowestlayersof

theepithelium,whichweredetected here both withprobe2

(Fig. 2C, 4, and 6) and the E6 and E7 ORF-specific probe mentioned above (datanotshown) aswell aswith probe4 (Fig. 2E and 6B), which recognizes the second exon of E6 andE7transcripts(Fig. 1[4,22]). Thisdiscrepancycould be due to different sensitivities obtained with frozen versus

paraffin-embeddedmaterial and

35S-

versus

3H-labeled

ribo-probes. The exclusiveexpressionof thetransformingE6and E7 genes in the basal cell layers is fully in line with their

expectedrole earlyin the viral lifecycle.

The different signal intensities observed after hybridiza-tionwith eitherprobe 1 orprobe2pointtotheexistence of twoclasses of mRNAs from the E6-E7region. Themajority of thetranscriptsdonotcontain sequences betweennt7862 and241becausetheyare notdetectable withprobe1.These RNAscontain E7asthe first translatable ORF andarelikely

toinitiateat nt270,whichwasidentifiedas amajor capsite ofmRNAsfrom HPV6-infectedcondylomas (20). The

tran-scripts hybridizingtoprobe 1,whichwasdesignedtodetect

E6-specificmRNAs,weremuch less abundant. The relative

signalintensities obtained withprobes1and 2correspondto

the amounts of mRNAs initiated atpromoters P1 and P2,

respectively(20).

Arelatively sharp borderline above whichnoE7-specific

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TRANSCRIPTION OF HPV6 E6 AND E7 GENES 4645

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FIG. 6. In situhybridization ofan anal condyloma with riboprobe 4. The samesection is shownin light-field

(A)

and dark-field

(B)

illumination.Theresultofhybridizationwithriboprobe 2 is visualizedbydark-fieldoptics(C). VOL. 66,1992

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4646 IFTNER ET AL.

transcripts could be observedwas aconsistent feature of all condylomas. That line also formed theboundaryof thearea showing c-fos expression. This coincidence doesnotreflect an influence of E7 on thetranscription of c-fos, because the c-fos expression pattern did notdiffer between condyloma-tous and HPV-negative, normal mucosa. However, one might speculate that only

c-fos-expressing

cellsprovidethe conditions allowing transcription from the E7 promoter. The lackof E6 and E7expression in the more differentiated cells of the condylomas points to a very effective control of the activity of promoters P1 and P2 (Fig. 1). Synthesis of the E6 mRNA seemstobeeven morerestrictedthanthat of theE7 mRNA.Previous studies noted that the total amounts of E6 and E7 mRNAs from HPV-infectedcondylomas differ by 1 order of magnitude (20). Our data provide the additional information that this difference is due not only to a lower abundance of E6 mRNA per cell but alsoto an expression strictly confined to undifferentiated cells of the basallayer. This highly restricted synthesis of the E6 mRNAindicates that the E6proteinactsveryearlyduring the multiplication cycle of the virus.

ACKNOWLEDGMENTS

WethankA. Schmitt andM. Elbel for excellent technical assis-tance, G.Schmid forproviding biopsy material,W.Birchmeier for the E-cadherin plasmid, and T. Leist and P. Schirrmacher for helpful technicaladviceconcerning in situhybridization.

This researchwassupported bygrant 89.042.1from the Wilhelm-Sander-StiftungtoT.I.andby PublicHealth Service grant CA53005 toS.P.W.

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