26018.fulle7 protein

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THE OURNALF BIOLOGICALHEMISTRY0 1993 by T he Amer ican Society for BiochemistryandMolecular Biology Inc. Vol. 268, No. 34,Isaueof December 5, pp.2601626025,1993Printed in U.S.A.

Structural and Functional Characterizationf theHPV16E7ProteinExpressed in Bacteria*(Received for publication, April 15, 1993,and in revised form, June30, 1993)

Gregory Pahel, Ann Aulabaugh,Steven A. Short, J ulie A. Barnes, GeorgeR. Painter, Paul Ray, andWilliam C. PhelpsSWelcome Research Laboratories,Burroughs Welcome Co., ResearchTrianglePark, North Carolina27709

The E7 gene of the human papillomaviruses (HPV)encodes a 8-amino acid, multifunctional nuclear phos-phoprotein with functional and structural imilaritiesto adenovirus E1A and the papovavirusantigens. E7is a viral oncoprotein, which will cooperate with anactivated ras oncogene to transform primary rodentcells, and can cooperate with the HPV E6 protein forthe efficient immortalization of primary human era-tinocytes. Due to the compelling epidemiological andexperimental association between HPV infection andcervical cancer, we have undertaken a detailed studyof the structure of the HPV16 E7 protein. The E7protein was expressed inEscherichia coli as a native,unfused polypeptide, and soluble protein as purifiedby conventional chromatographic techniques. The pu-rified protein was assessed for various biochemical andbiophysical properties. Purified E7 binds the retino-blastoma protein avidly and specifically, and it candissociate the E2F transcription factor when assayedin vitro. Circulardichroismspectroscopy ndicatedthat E7 reversibly bindsZn2+and Cd2+ resulting in asubstantial ncrease n he a-helical contentof hemetal-bound E7 consistent with the stabilizationof ahydrophobic core in theOOH terminus of the protein.

Of the nearly 70 different HPV types, approximately 20are associated with infection of the oral and genital mucosa(DeVilliers, 1989). Epidemiological and biological data sup-port the division of the mucosal associated HPVs into twogroups: those associated with benign lesions that are atlowrisk for malignant progression (HPV 6and H PV l l ), andthose represented by HPV 16 and HPV18, that are consideredhigh risk due to their strongassociation with cervical cancer(zur Hausen and Schneider, 1987). Approximately 85% ofhuman cervical carcinomas harbor HPVDNA with more thanhalf found to contain HPV16 (Riou et al., 1990; zur Hausen,1991).HPV types 16 and 18are archetypical of the high risk virusgroup, and analysis of cervical carcinoma tissues and ell lineshas revealed that theviral DNA is frequently integrated intothe host cell genome. Although integration of the circularviral genome isnormally accompanied by rearrangements and*The costs of publication of this article were defrayed in part bythe payment of page charges. This article must therefore be herebymarked oduertisement in accordance with 18U.S.C. Section 1734solely to indicate this fact.3To whom correspondence should be addressed Div. of Virology,Burroughs Wellcome Co., 3030 Cornwallis Rd., Research TrianglePark, NC 27709.Tel.: 919-315-4022;Fax:919-315-5243.

retinoblastomaprotein;DTT, dithiothreitol;CR1andCR2,conservedThe abbreviations used are: HPV, human papillomavirus; pRB,region1 and2.

deletions of viral sequences, the E6 and E7 open readingframes are invariably spared (Baker et al., 1987; Matsukuraet al., 1986; Schwarz et al., 1985). Furthermore, biochemicalanalyses have demonstrated that the E6 and E7 genes areconsistently expressed in cervical carcinomas, suggesting thatthese proteins may contribute to the malignant phenotype(Smotkin and Wettstein, 1986).A number of cellular transforming and growth stimulatoryproperties have been attributed to E7 for review, see Mungerand Phelps 1993)). Expression of the HPV16 and HPV 18 E7proteins can induce morphological transformation of estab-lished rodent cells in culture (Bedell etal., 1987; K anda etal.,1988; Phelps et al., 1988; Tanaka et al., 1989; V ousdenet aL,1988; Watanabe and Y oshiike, 1988), and E7 can cooperatewith an activated ras or /os oncogene to transform primaryrodent cells (Crook et al., 1988, Phelps et al., 1988; Storey etal., 1988). The expression of the high risk HPV E6 and E7proteins together can efficiently induce immortalization ofprimary human keratinocytes (Hawley-Nelson et al., 1989;Hudson et al., 1990; M iinger et al., 1989a; Watanabe et al.,1989) which likely represent the most relevant target cell fornatural infection by HPVs. Therefore, both epidemiologicaland biological data suggest that the E6 and 7 proteins playan important role in HPV -associated malignancy.The amino terminus of the HPV E7 proteins shares sub-stantial amino acid sequence similarity with two noncontig-uous portions of the adenovirus E1A proteins (Phelps et al.,1988). In addition, E7 s functionally related toE1A and SV40large antigen (TAg) in that it an cooperate with an activatedras oncogene, trans-activate he adenovirus E2promoter(Phelpsetal., 1988), associate with the retinoblastoma protein(pRB) (Dyson et al., 1989), and abrogate the transforminggrowth factor-p-induced transcriptional repression of the C-mycpromoter in keratinocytes (Pietenpol et al., 1990).The HPV 16 E7 protein is an acidic, nuclear phosphoproteinwith no known enzymatic activity. HPV 16 E7 is 98 aminoacids long with a M , 11,000 based on amino acid compositionand an apparentelectrophoretic M , of 19,000. E7 binds Zn2+through two conserved Cys-X -X -Cys motifs present n theCOOH-terminal half of the protein (Barbosa et al., 1989;Rawlset al., 1990). ntracellular phosphorylation of E7 occurson a conserved serine and is apparently mediated by caseinkinase I1(Firzlaff etal., 1991).Due to the correlation between the expression of the HPVE6 and 7 transforming proteins andhe growth of neoplasticcells, we have ini tiated an effort to examine the tertiarystructure of the HPV16 E7 protein. Here, we present a prelim-inary structural and functional analysis of the HPV 16 E7protein expressed in Escherichia coli as a native polypeptidewithout protein usion. Purification of the soluble protein wasby conventional chromatographic techniques under nonde-naturing conditions.

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26020 HPVl6E7 Characterization1- thi o- f l - D- gal actopyranosi deo the cultureedium. The cellswere harvested and lysed using a French press. Examinationof cell extracts indicated that the E7 protein routinely ac-counted for approximately 30% of the total proteinith about50% of the E7 protein being soluble (Fig.1,lune2, Table I).The soluble E7 protein was isolated essentially according toImai et al. (1991). Proteinpurityas monitored by SDS-polyacrylamide gel electrophoresis was greater than 90%.Typical yield of purified E7 protein was 2 mg/g, wet weight,of cells. Polyclonal rabbit antiserawas produced utilizing thepurified E7 protein, and the ntibody was shown to effectivelyrecognize the bacterially expressed protein as well as E7madein COS-1 monkey cellsor the human cervical carcinoma cellline, Caski (Phelps etal., 1992).

Isoelectric Pointof HPV-16E7The PI of zinc-bound HPV-16 E7 was obtained from iso-electric focusing. TheRFof HPV-16 E7 on pH 4-6.5 gradientisoelectric focusing gel was 0.54 which corresponded to a PIof 5.4 (Fig.2). Based on the amino acid sequence, the predictedPI was 4.05. The difference between the predicted and ob-served PI is consistent with folding of the protein and inac-cessibility of some charges to thesolvent.

Functional Characterization of E7pRB Binding-The ability of the E7 protein to nteractwith the RB tumor suppressor protein appears to correlatewell with cellular transforming functions including ruscoop-erativity (Edmonds and Vousden, 1989; Phelps et al., 1992;Storey etal., 1990; Watanabe etal., 1990). n addition, the E7proteins from the high risk HPVs, HPV16 and HPV18, bindpRB with a greater affinity han the E7 proteinsrom the lowrisk HPVs, types 6 and 11(Barbosa et al., 1990; Gage etal.,

1 2 3 4 5.5

I1

!1.5

14

FIG. 1. Purification of theHPVl 6 E7 protein. Lane 1, lowspeed supernatant from an uninduced culture (16 pg); lune 2, highspeed (100,000X g) supernatant from an induced culture (12.5 pg);lane3, peak fraction from DE52 (4.2 pg); lane4, peak fraction fromSuperDex 75 gel filtration (1pg); lane5,molecular weight markers.Protein was fractionated in a 12% SDS-polyacrylamide gel andstained with Coomassie Blue R-250.

TABLEPurification of HPVl6E7step Percenturifica- Y ieldE7 tionb

w -fold %French press extract 416 125 30.1 100Low speed supernatant 3743.39.6 1.0 58.6100,000X g supernatant 312 63.70.4 1.0 50.9DE52 21.4 15.31.6 3.7 12.2Sephadex G-75 4.08.70.0.6.9The amountof E7 protein was determined by quantitative West-

*Fold purification is of the soluble E7 protein which representsern analysis as described under Materials and Methods.about 50% of the total E7 xpressed.

6.5-6.0-

E 5.5-5.0-4.5-

RFIG. 2. Determinationof isoelectric point. Migration distance( RF) ersuspH of purified, zinc-bound HPV16 E7 andprotein stand-ards obtained from isoelectric focusing. Protein standards and theircorresponding PI included human carbonic anhydrase (6.55), bovinecarbonic anhydrase (5.85), 8-lactoglobin (5.20), soybean trypsin n-hibitor (4.551, and glucose oxidase (4.15).

1990; Munger etal., 1989b). To assess the ability of purified,bacterially expressed E7 protein to nteract with the pRB,increasing amounts of E7 were mixedwith aconstant amount(400 pg) of total cell lysate from Vero cells, which was previ-ously determined to contain high levels of pRB. Complexeswere immunoprecipitated with an excess (see Materials andMethods) of polyclonal antibody to E7, and complexed pRBwas detected by Western blot analysis (Fig. 2, inset). Therelative amount of complexed pRB was determined by phos-phoimage analysis and is plotted as relative pRB concentra-tion (Fig. 3). The control lane shows that no pRB is immu-noprecipitated in theabsence of added E7 protein indicatingthat the E7 antibody has no inherent ability to immunopre-cipitate pRB. As the amount of purified E7 protein is in-creased from 2 to 400 ng, the amount of complexed pRBincreases correspondingly indicating that the urified E7 pro-tein can readily form an immunoprecipitable complex withthe pRBprotein in a dose-dependent manner.Peptide Competition-Mutational analyses of the HPV16E7 protein have indicated that amino acids (see Fig. 7) inconserved region 2 (CR2) are required for interaction withthe pRB protein (Munger et al., 1989b; Banks et dl., 1990;Barbosa et al., 1990; Gage et al., 1990; Firzlaff et al., 1991;Munger et al., 1991; Heck et dl., 1992; Phelps et al., 1992;Sang and Barbosa, 1992). To verify that the E7-pRB com-plexation shown above issequence-specific, a peptide repre-senting the pRBbinding domain of HPV16 E7 was synthe-sized and tested for its ability to inhibit the association. Asshown in Fig. 4, the peptide T20-NBencompassing the pRBbinding domain effectively inhibits binding of the bacteriallyexpressed E7 protein to pRB. In contrast, a nonspecific pep-tide derived from the El open reading frame of HPVll hadno effect on E7-pRB association. Neither peptide had anyeffect on immunoprecipitation of the E7 protein within theconcentration range indicated (data not shown), consistentwith specific nhibition of complexation by the E7 ecapeptide(Fig. 4). Furthermore, it should be noted that, at equimolarconcentrations of E7 peptide and E7protein, no inhibitionofE7-pRB association was observed. Substantial inhibition re-quired greater than a 15-fold molar excess of E7 peptide, withcomplete inhibition equiringa >OO-fold excess. This isconsistent with previous studies which have shown that thenative protein has a much higher binding affinity for pRBthan small inhibitory peptides (J ones et al., 1990, 1992; Pa-trick et ul., 1992). This elevated binding affinity is expectedto be a propertyof the native E7 protein, as other mino acids


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HPVl6E7Characterization 26021

w l o r

u L - -0 100 200 300 400E7 (ng)

FIG. 3. Association of purified E7 with pRB. PurifiedE7protein (1-400 ng) was mixed with an total cell extract fromVerocells (400pg). E7-RB complexeswere immunoprecipitated andfrac-tionated on a polyacrylamide electrophoresis9% gel. Protein waselectroblotted to nitrocellulosendpRB detectedby Western blottingas described under Materials and Methods. The i nset at the topshows the autoradiographof the blot used for quantitationby phos-phoimage anaysis. The amount of pRB which was immunoprecipi-tatedis expressedrelative to background obtainedwith no E7 addedand was normaized to he amountof heavychain Igdetected ineachlane.

loo1\hI I I0 10 100 1000

E7 PEPTIDE molarexcess)FIG.4. Inhibitionof pRB association. Peptide competitionwasas in Fig. 3 using 200 ng of E7 proten, 400 pg of Vero cell lysate(pRB),and1-128-foldmolarexcessof peptides derivedfrom HPV16E7 TZo-NB and HPVll El (Breamet al., 1993).outside of CR2, such as in CR1, may stabilize the secondarystructure and contribute additional interactive sites. There-fore, the additional contact points ormore favorable confor-mation which are responsible for the enhanced affinity arerepresented in this purified E7 preparation.E2F Dissociation-Recent evidence indicates that HPV16E7 mediates transcriptional transactivation hrough modula-tion of theE2F ranscription factor (Phelpset al., 1991;Chellopan et al., 1992). The E2F transcription factor wasoriginally identified through its role in activation of the ade-novirus E2 promoter (Kovesdi et al., 1986; Yee et al., 1989).The E2F transcription actor is found in most cells in heter-omeric protein complexes with several cell cycle associatedproteins including pRB and cyclin A (Bagchi et al., 1991;Bandara and La Thangue, 1991; Chellapan etal., 1991; Chit-tenden et al., 1991). The viral transforming proteins ElA,TAg, and E7 have been recently demonstrated to dissociateE2F from these protein complexes effectively. Release of E2Fis thought to result in activationof several cellular promotersknown to be important for DNA synthesis such as dihydro-folate reductase, c-myc, thymidine kinase, and DNA polym-erasea (Heibert etal., 1991).

Bacterially expressed and purified E7 was tested for itsability to dissociate E2F by mixing increasing amounts of E7protein with partially purified nuclear extracts of U937human

cells. In theabsence of E7 protein, gel shift analysis indicatesthat the E2F cellular transcription factor exists n at leastthree discreet macromolecular complexes. Incubation of theseextracts with deoxycholate reduces the complexity of thepattern to a single shifted species which has been previouslyshown to represent free or uncomplexed E2F (Fig.5). Thehigher molecular weight complexes have been determined torepresent E2F/p107/cdk2/cyclin A E2Fc) and E2F/pRB(E2Fc*). Titration of purified E7 protein into the U937 ex-tracts led to the gradual dissociation of the E2Fc* complexaccompanied by the concordant increase in the amount offree E2F. Indeed, the most noticeable alteration n the geshift pattern s the appearance of substantially more free E2Fas purified E7 protein is titrated nto he reaction. Thisobservation suggests that E7 s able to dissociate a fractionofE2F which is unable to bind the DNA fragment under theseconditions. In contrast, the E2Fc complex appeared to bemore resistant to E7-mediated dissociation as loss of E2Fcwas only noted at thehighest E7 concentrations.Therefore, in agreement with previous studies, the E7pro-tein will selectively target the E2F-RB complex for dissocia-tion of E2F n vitro (Chellappan et al., 1992). Amino acidsequences necessary for this activity include the pRBbindingregion in CR2; however, additional sequences in the carboxylterminus arealso required (Wu etal. 1993). The specific highaffinity interaction with pRB and the n vitro dissociation ofthe E2F transcriptionactor are functional properties scribedto the ative HPV E7protein, and suggest that the E7roteinpurified and characterized in this work is an appropriatesubstrate for structural studies.

Structural AnalysisSecondary Structure from CD Spectra-The avid and spe-cific RB binding and E2F dissociation suggested that thepurified E7 protein was structurally intact and, therefore, agood substrate for structural analysis. As a preliminary meas-ure of global structuralcontent,the purified protein wassubjected to analysis by circular dichroism.HPV16 E7 protein obtainedafter size exclusion chromatog-raphy was judged to be greater than 95% pure by SDS-gelelectrophoresis. Before biophysical studies were initiated withthe purified protein, samples from the size exclusion chro-matography step were submitted for metal analysis. Less than1% (mole %) zinc or any other heavy metal including iron,nickel, copper, and lead was bound to the protein. The CDspectrum of metal-free E7 (Fig. 6) displays a large negativeminimum centered at 200 nm indicative of unfolded structure,and a negative shoulder at 222 nm which is consistent withthe presence of some secondary structure.A marked change inhe ultraviolet CD spectrum of HPV16E7 was observed upon addition of zinc or cadmium. Thismetal-induced change consisted of a decrease in the negativeminimum centered at 202 nm and an ncrease in the positivemaximum at 192 nm and the negative minimum at 222 nm(Fig. 6A). Secondary structure contentcalculations indicatedthe percentage of turn and random coil was the same withinexperimental error in the presence or absence of metal butthere was an increase n a-helixand decrease in@-sheetcontent upon the addition of zinc or cadmium (Table 11).Reversible metal binding s demonstrated in Fig. 6B in whichzinc is first added to E7protein solution, ollowed by additionof EDTA to regenerate the CD spectrum of metal-free E7.Therefore,preliminary structural analysis of the purified

HPV16 E7 protein by CD verified that thisprotein reversiblybinds Zn2+or Cd2+Roth etal., 1992) and that metal coordi-nation induced an alteration n the conformation of the poly-


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26022 HPVl6E7 Characterization

FIG.5. Dissociation of the E2Ftranscription factor. A heparin aga-rose purified E2F-containing lysatekindly provided by Joe Nevins wasmixed with purified HPV16 E7 protein(3-300 pg) and labeled E2F oligonucleo-tide and incubated for 30 min. Com-plexes were fractionated on a 4% non-denaturing polyacrylamide ge in 0.25%TBE. Deoxycholate(DOC)was added toa final concentration of 0.4% and com-petitor DNA was unlabeled oligonucleo-tide at a 100-fold molar excess.

A

- 1 5 o o o 1 , , , , , , , , ,100 1W 200 210 220 230 240 250 20 270 280

Wavelength (nm)

DOCCOMP

- - + + - "+ - + - - - - -30000007 ( ~ 4 ""

NON-SPECIFIC -+

B15Wavelength (nm)peptide. This conformational shift occurs with the additionof either Cd2+or Zn2+esulting in nearly identical spectra.Zinc has been identified as anessential component of some300 different proteins and is important for both catalytic andstructural functions (Vallee and Auld, 1990). X-ray crystal-lographic analyses have indicated that a catalytic Zn2+atomis coordinated by 3 amino acids (His, Glu, Asp, or Cys) andan activated water molecule. In contrast, for Zn2+-containingenzymes, structural Zn2+s coordinated by4cysteine residues.Tetrahedral coordination of Zn2+through cysteine residuesresults in a very stable structural motif similar to that pro-vided by disulfide bonds. Furthermore, Zn2+s inert to oxi-doreduction and, therefore, very stable to an intracellularenvironment that fluctuates in redox potential (Vallee andAuld, 1990).A heterogeneous group of nucleic acid bindingproteinsinitially exemplified by TFIIIA, has been shown to coordinate

FIG. 6. Circular dichroism spec-troscopyof E7. CD spectra of 0.78 p~HPV16 E7 at different EDTA and metalconcentrations. A , CD spectra of E7 inthe absence of metal (-); in the pres-ence of 1mM zinc chloride(- - - - -), andin the presenceof 1mM cadmium chlo-ride(. ... ).B, reversible bindingof zincis demonstrated by addition of zinc chlo-ride to E7 to a final metal concentrationof 1mM (- - - - -); this was followed bythe addition of an excess of EDTA(.. . ).Spectra were recorded at 22 "Cand pH 7.5 in a starting buffer of 1mMTris-HC1, 0.1 mM DTT,and 0.1 mMEDTA.

structurally important Zn2+atoms (reviewed in Berg (1993)).Computer searches configured to locate spatially juxtaposedCys and Hisesiduesortwo Cys pairs has putativelydentifiedroughly 150 different proteins which may have the capacityto bind Zn2+atoms (Valee and Auld, 1990). Despite the lackof experimental verification of Zn2+binding, there has been apromiscuous use of the concept of zinc fingers derived fromthe structuralmodel determined for TFI IIA.With the unusually large spacing between the Cys-X-X-Cys motifs in the COOH terminus of E7, it should not beassumed that coordination of Zn2+induces a classical zincfinger. The amino acid spacing between the Cys-X-X-Cysmotifs (29 amino acids) is rigorously conserved in the se-quences of all E7 proteins analyzed to date. Indeed, exami-nation of the predicted amino acid sequence for theE6pro-teins of the papillomaviruses revealed that this 9-amino acidspacing is also entirely conserved. This is particularly inter-


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HPVl6E7 Characterization 26023AdE 1A ~/////~CR?~//////////////~Similarity

16611183 133354239455758

1I MQDTPTLHXW W O ~ L K DI MQRLVTLKDWWQPMTWDNRQETPTWDNRQHXPTLnI M Q E I T T W DNRQPXPTWENRQETPTLKDIMEWFFWSWWQERPSLSDNRQIWPTLRE

Y N L DL P -PEIVL -DWPPDI V I - D W P P DW L - D W - P LIVLHLEPQNEWL-DLY -P IYVL -Dm-PI ,IVLDLCPYNEI VL FD I PT C EI VL H L EWN BI T L I L S E I P EY I L - D L K P E

-TTDLYCYE-PWLHCYE-PVQLHCYEI - PWL L C H E-ATDLHCYE-PTDLYCYE-ATDLYCYEIQPVDLVCXST -P I DL Y C Y SLDPVDLLCYE1-VDLHCDE-PTDL?CYE

QLNDSSEDSDQLWSSEDEVQLEDSSEDEVQLSDSDSENDQLPDSSDEE-QLSDSSDEDEQLCDSSLSDEQ WXSEDE I DQL-DSSDEDDQLSESLESNDQ?DNSSEDTNQLCDBSDEDE

COnSOnSUS Y-0- _ T L D _VL-.E "DL-C-E QL_DS.EE Q ~ -1-CC- C-L-V-D-R-L- -LL-TL-VP-C-FI G.7. Homology among the mucosal-associatedHPV E7proteins. HPV E7 sequences were obtainedromthe GenBank" database,and alignments were made using the Gap and Pileup programs in the Proten Comparison Modulef GCG. A gap creation penaltyof 3.0 anda gap extension penalty of 0.1 were used. The region of amino acid sequence similarity is indicated at the top of the figure by the shadedboxes and comprises the amino one-third of E7 (Phelpset al , 1988). HPV 16 E 7 is displayed across the top line and the other mucosal-associated viralE7 protens are shownbelow. A consensus sequence s suggested below.

esting since E6 and E7 are oth transforming proteinswhoseexpression is selectively retained in HPV -associatedcarcino-mas Baker et al., 1987; Schneider-Gadicke and Schwarz,1986; Smotkin and Wettstein, 1986).Conseruation of Primary and Secondary Structure-Pri-mary sequence alignments for the E7proteins of 12mucosal-associated HPVs were generated using the Protein Compari-son Module of the Wisconsin Genetics Computer Group.Proper alignment was derived from the Gap and Pileup pro-cedures, and the esults of this alignment aredisplayed in Fig.7. The predicted primary amino acid sequences of the E7proteins are well conserved (Cole and Danos, 1987; Baker,1987) with the greatest divergence occurring in the egion justprior to amino acid residue 50 (HPV 16). In particular, HPVs18,39, and 45 contain a small insertion, and HPV 42,asmalldeletion in this region that falls between the acidic domain(CK II phosphorylation site) and the f irstys-X -X -Cys motif.Moreover, inspection of the cutaneous and epidermodysplasiaverruciformis (EV)-associated HPV E7s ndicates that thisregion is also poorly conserved in these proteins (data notshown). The pRB binding region and the acidic/CK II phos-phorylation site in CR2 are highly conserved among all of theE7 sequences; and asobserved previously, the COOH-termi-nal Cys-X-X -Cys motifs and the 29-amino acid spacing isstrictly conserved (Cole and Danos, 1987).A prediction of secondary structure for these proteins wasdetermined using the Predict-Secondary-Structure programin Sybyl V ersion 5.4. A predicted motif was adopted only if i twas described by at least two of the three algorithms. Aconsensus structure was assembled for the mucosal HPVs andis shown in Fig. 8. Secondary structure analysis of the con-served primary amino acid sequence of the mucosal-associatedHPV E7 proteins predicts that several structural elementsare consistently found, and that HPV 16 is representative ofthis group. From such predictive algorithms, the computedvalues for a-helix and @-sheetare 24% and lo%,respectively.These numbers are in good agreement with those generatedby CD analysis (Table 11)for the Zn2+ orm of E7, suggestingthat metal binding may stabilize the secondary structure ofthe protein.Mutational studiesof the HPV 16 E7 proteinhave indicatedthat substitution of the COOH-terminal Cys residues leads tothe expression of a protein with reduced intracellular stabil ity(Storey etal.,1990;Phelps etal.,1992).It was concluded that

TABLE1Estimates of Secondary Structure of HPV16 E7 byCDSpectralAnalysis (%)Results are accurate2 5%.

Metalr-Helix @-Sheet Turn RandomAPO 16.0 27.4 24.1 32.5+ZnZ+ 29.3 10.5 27.6 32.6+Cd2+ 30.0 10.5 27.7 31.8

the primary role of the COOH terminus and the Cys-X -X -Cys motifs is structural, since the amino acid domains nec-essary for the biological functions of the protein mapped tothe NHderminalhalf of the protein (Edmonds and ousden,1989; Phelps et aL, 1992; Storey et al., 1990; Watanbe etal.,1990). A hydrophobicity plot (Fig. 8) of the HPV 16 E7 se-quence predicts a substantial hydrophobic character for theCOOH terminus of the protein. Therefore, chelation of zincthrough the Cys-X-X -Cys motifs may stabilize a hydrophobiccore in he COOH terminus of the E7protein. Inothersystems, zinc binding has been shown to dramatically stabilizethe a-helical content of small peptides in aqueous solution(Ruan etaL,1990) such that demu0 metal-binding siteshavebeen engineered into some proteins to impart structural sta-bility (Ghardiri and Choi, 1990; Handel and DeGrado, 1990).Binding of Zn2+by HIV integrase leads to an ncrease in thea-helical content of the apoprotein from 14 to 32% (Burke etaL, 1992).

Primary sequence and hydrophobicity comparison of theE7 proteins suggest that the proteins are composed of twodistinctdomains in the NH2- and COOH-terminal halvesseparated by a poorly conserved region preceding amino acid50of HPV 16 E7. Studies with TFII IA and AL4 transcriptionfactors indicate hat zinc may not be directly involved in DNAbinding but instead may stabil ize the binding region to main-tain an optimal local conformation (Christianson, 1991). Byanalogy, the optimal local tertiary structure for interaction ofE7 with various cellular proteins including pRB and p107,may be substantially affected by coordination of Zn2+ n theCOOH terminus. T his may well account for the relativedifference in affinity between the native E7 protein and theE7 peptide encompassing the pRBbinding domain.Recent studies of the viral transforming proteins of theadenoviruses, polyomaviruses, and thepapillomaviruses haveled to the roposal of a conserved biochemical mechanism for


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26024 HPVl6 E7 Characterization

(types6, 11, 16, 18, 31.3 3 , 3 5 . 3 9 , 4 2 , 4 5 .57 .58)Mucosal HP V E7-

HPV-16 E710 16 3300868 7 7 8 4

1ydrophi l ic -5-4

Hydrophobic 0 10 20 30 40 50 60 70 80 90 1005 ' , . . , . . ' . , . , . ' . , , . . ' , . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . ,FI G.8. Secondary structurepredictions for HPV16E7 and theHPV E7consensussequencearebasedon acomparison ofthree different algorithms in Predict-Secondary-Structureof Sybyl Version 6.4. A structural feature, a-helix (cylinder)or & sheet(w), s denoted where at least two of thr ee algorithms are in agreement. The K yte-Doolittle hydropathy profile was obtained using the proteinanalysis module of the Wisconsin Genetics Computer Gr oup.

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their growth stimulatory functionswhich is mediated throughinteractions with tumor suppressor proteins such as p53 andpRB. Infection of quiescent cells by these DNA viruses leadsto the early expression of a group of transforming proteinswhich function to stimulate host cellular growth and DNAsynthesis. Replication of the viral DNA either requires or isenhanced under conditions of rapid cell growth. Cell cyclestimulation by HPV E7 is thought to be mediated at least inpart through interaction with host regulatory proteins suchas pRB and 107, an pRB-related protein Dysonet al., 1992).The interaction of HPV 16 E7 with pRB has been shown toresult in the dissociation of the E2Franscription factor whichregulates several cellular genes important to cellular growthincluding: c-myc, dihydrofolate reductase, thymidylate syn-thetase, thymidine kinase, DNA polymerasea,and epidermalgrowth factor receptor (Heibert et al., 1991). Therefore, theinterface between the E7 protein and cell cycle regulatorsrepresents a cri tical point of intracellular control and is anattractive therapeutic target for antiviral or antitumor inter-vention.A detailed, structural understanding of the interface be-tween the E7 oncoprotein and thecellular regulatory proteinsthat mediate growth of the virus is an important step in thedesign of antiviral inhibitors. Rationaldesign of inhibitors ofHPVE7 would be expected to result in the synthesis ofcompounds which will nterfere with growth of HPV -infectedbenign lesions, as well as malignant tumors.

Acknowledgments-We thank K im Carpenter for help with puri-fication, Barbara M err ill and Bil l Chestnut for protein sequencingand amino acid analysis, Doug Sherman for peptide synthesis, andCarol Ohmstede for help with the gel shift analysis.REFERENCES

Bafter, C. C. (1987) in ThePopouauiridae 2 (Salzman, N. P., and Howley, P.Ba chi, S.,Weinmann, R., and Raychaudhuri, P. (1991) CeU 66,1063-1072Baker, C. e.,Phelps, W. C., L indgren, V., Braun, M. J ., Gonda, M . A,, andBandara L. R., and LaThangue, N. B. (1991) Nature 3 6 1 , 4 9 4 4 9 7Banks, i . ,Edmonds, C., and Vousden, K. H . ( 1990) Oncogene6,1383-1389Barbosa, M. S., Lowy, D. R., and Schiller, J . T. (1989) J . Virol.63,1404-1407Barbosa, M. S., Edmonds, C., Fisher, C., Schiller, J . T., Lowy,D.R., and

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26018.fulle7 protein

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