VIROLOGY

187, 353-359

(1992)

Epitope-Mapped J. DOORBAR,

Monoclonal

Antibodies

S. ELY, N. COLEMAN,

against the HPV16 El AE4 Protein

M. HIBMA,

D. H. DAVIES,* AND L. CRAWFORD’

I.C.R.F. Tumour Virus Group, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge, and *Division of Biomolecular Sciences, King’s College, London, W8 7AH, England Received August 5, 199 1; accepted

November

032 lQP, England:

5, 199 1

The human papillomavirus (HPV) El AE4 protein is the only nonstructural late protein encoded by the virus. We have isolated three hybridomas producing monoclonal antibodies to the El AE4 protein of HPV16, which is the HPV type most frequently associated with cervical cancer. The three antibodies (TVG 401, 402, and 403) detect adjacent epitopes within the major seroreactive region of the molecule and show no reactivity against the E4 proteins of HPVl, HPVP, HPV4, or HPVG. The El AE4 protein migrates as a 1 OK species on SDS-gel electrophoresis and forms cytoplasmic inclusion granules in infected cells in vitro similar in appearance to those produced by HPVl in benign warts. In naturally occurring HPVl g-induced tumors the El AE4 protein was detected in the cytoplasm of cells in the upper layers of the lesion in areas in which HPVl6 DNA replication was occurring, as determined by in situ hybridization. Although the epitopes recognized by these monoclonal antibodies survive brief fixation in 5% formaldehyde, reactivity was destroyed by prolonged fixation. These monoclonal antibodies represent the first against HPVl6 El AE4 and should complement those already available to E7 and Ll for the screening of frozen sections of clinical biopsies o 1992 and will be of value in monitoring the progression of HPV infection from benign lesions to invasive cancer. Academic

Press,

Inc.

visualized by immunostaining in HPVl6-positive premalignant lesions (7) detailed functional and structural analyses have been precluded by lack of suitable detection reagents, and by the low level of expression in naturally occurring tumors. We report here the characterization of three monoclonal antibodies directed against the major seroreactive region of El AE4 (8) and the identification of the El AE4 protein as a 1OK polypeptide which can form cytoplasmic inclusion granules in cultured epithelial cells. Antigen for hybridoma production was expressed as a /I-galactosidase (P-gal) fusion protein using the pEX expression system (9) and was subsequently cleaved to release the El AE4 polypeptide. cDNA species for the El AE4 proteins of HPVl, 6, and 16 were synthesized from cloned viral DNA using pairs of PCR primers hybridizing to the 5’and 3’ends of the E4 open reading frames (ORF). The forward primer (El AE4 F, Fig. 1a) included a 15- to 18-base nonhybridizing segment corresponding to the region of El which would be spliced onto E4 in a naturally occurring cDNA (70). As HPV El AE4 proteins do not contain internal methionine residues, nonfusion protein was generated by digestion of the purified ,&gal El AE4 polypeptide (2) with cyanogen bromide, which cleaves at the carboxy side of methionine residues (1 I). The HPV16 El AE4 protein was identified using an antipeptide sera raised against the N-terminal 12 amino acids of HPV16 El AE4 (7 7) and was used as an im-

The human papillomaviruses (HPVs), which comprise over 60 different types, cause epithelial lesions ranging in severity from benign warts to invasive carcinoma (7). Viral replication and gene expression are closely linked to the normal differentiation pattern of the epidermis, with late proteins being made only after an infected cell has begun its migration toward the surface of the skin. HPV particles are assembled in cells of the granular layerfollowing expression of the Ll and L2 major and minor capsid components (2) while El AE~, a nonstructural late protein, is synthesized in cells of the spinous layer, where it aggregates to form cytoplasmic inclusion granules (3, 4). Although no function has yet been assigned to the El AE4 polypeptides, the importance of their role in the virus life cycle is suggested by the conservation of physical characteristics between the E4 proteins of different HPV types (4). El AE~ does not appear to be a component of the virus particle (2) and in bovine papillomavirus type 1 (BPVl) is not required for transformation or episomal virus replication (5). A role for El AE~ is thus suggested which is not readily assayable in vitro and which may be linked to the epithelial tropism of the virus. Recently, HPV research has centered largely on HPV16 since it is present in the majority of cervical squamous cell carcinomas and in cell lines derived from such tumors (6). Although El AE~ antigen can be ’ To whom reprint requests should be addressed.

353

0042-6822/92

53.00

Copyright 0 1992 by Academic Press. Inc. All rights of reproducton in any form reserved.

354

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a FORWARD HPV 5’ HPV 5’

HPV 5’

1

5’ HPV 5’

HPV 5’

612

627/3200

3241

CG~~CA~~~~~~CAGATAATAAAGd;CCCCAAGGG El 6

a32

3’ E4

04713325

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CGAATTCG~~~“~CCdATGGC~C~~C~~CACTACAC~G~GTATCCA~CCT~TCTACTACATACA~ Eax El E4 16

665

660/3357

1

4039

4013

4460

3’

4434

CGAAiXGGA;;&TCGGGCCCTACTATGTGCCATGGTAi ECXFd E4 16

3’

PRIMER

CGAATTCAGATCTkCAGACTGCCATATTGCAATATllli ECCFU BGLII E4 6

3’

3394

C~~CG~~~C;\TATCCTGCAGC~~C~CG~GTATCCTCTCCTG~~A~AGGCAGCAC~ El E4

REVERSE HPV

PRIMER

4252

CGAArrCGGATCCAGAACGTGXGCA~~~~ EOX BAMHI E4

3’

4226

3’

FIG. 1. Production of expression constructs, (a) Forward and reverse primers used for the generation of El AE4 cDNAs. For HPVl and HPV16, the structure of the primers was based on that of previously reported cDNAs (75, 70), while for HPV6 the primer sequence was established following comparison with HPVl 1 (76). The numbers above each primer indicate the position within the viral genome, while the open reading frame from which each sequence is derived is shown beneath. (b) The complete genome of HPV16 is shown at the top of the figure, with the Hindll-Stul fragment (which was used as a template for PCR) shown beneath. Primers hybridized to either end of the E4 ORF to produce an El AE4 cDNA following 30 rounds of PCR amplification using Tsp polymerase. El AE4 fusion proteins were produced after cloning of the cDNA into the pEX and pGEX expression vectors, with unfused El AE4 being subsequently generated by CNBr cleavage. Monoclonal antibodies raised to this 1 OK El AE4 protein were subsequently screened against the same protein expressed as a glutathione S-transferase fusion.

munogen following extraction from a 20% polyacrylamide gel (Figs. 1 and 2a). Hybridomas were screened against a glutathione S-transferase HPV16 El AE4 fusion protein (expressed from pGEX (12) see Fig. 2), first by ELISA and subsequently by Western blotting. In total, seven hybridomas were obtained from two independent fusions-three of which were established as lines. To characterize the monoclonal antibodies further a series of overlapping octapeptides were synthesized covering the entire El AE4 protein (Fig. 3). Epitope mapping revealed that all three antibodies bind within the seroreactive region (KPSPWAPKKHRRLS, amino acid position 30 to 43 (8)) and recognize related epitopes within this region. TVG 401, which was cloned from an independent fusion from that which generated TVG 402 and 403, reacted with three peptides center-

ing around the epitope WAPKKH (aa position 34 to 39). The epitopes recognized by TVG 402 and 403 both centered around PKKHRR (aa position 36 to 41) although the difference in profiles suggests that they are not identical (Fig. 3). TVG 403, unlikeTVG 402, showed strong reactivity against the peptide KKHRRLSS. No reactivity was observed with a control hybridoma supernatant (A3D2) which was known not to react with HPV16 El AE4 (Fig. 3). Cross-reactivity to the E4 proteins of different HPV types was determined by Western blotting to five pEX fusion proteins containing E4 sequences derived from HPVl, HPV2, HPV4 (4) HPVG, or HPV16. As predicted from amino acid sequence comparisons, all three antibodies were specific for the HPV16 El r\E4 polypeptide and showed no reactivity with the other HPV types (Figs. 2 and 3b).

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355

HPV 16

b

I

I Hindll

-----

StUl

E2

cDNA FRAGMENT

I

m

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mm nl”Tlirn ‘V”“,

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FUSDN PROTEIN

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cleavage

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GLUTATHIONE S TRANSFERASE FUSION PROTEIN

IOK El”E4

FIG. 1-Continued

The HPV16 El AE~ polypeptide was identified as a 10K species following expression in both bacteria (pEX) and eukaryotic cells (vaccinia El r\E4-infected CV-1 cells (13)). This is in accordance with a predicted molecular weight for ElAE4 of 9977 as determined from cDNA analysis (10). Attempts to characterize the ElAE4 protein from clinical samples have not previously been successful due to the low number of E4-expressing cells in HPVl6-positive premalignant lesions (7). Furthermore, only 4% of such lesions are typically E4 positive (7) and we have been able to demonstrate this protein convincingly in only 1 of 16 HPV16-positive cervical intraepithelial neoplasia (CIN) biopsies. The use of these monoclonal antibodies is therefore unlikely to increase the percentage of E4-positive lesions detected, although a

larger series would need to be examined to be certain that this is the case. El AE4 staining was localized to the cytoplasm of cells in the upper layers of the lesion (Fig. 4a) and was similar to that previously reported (7). No staining was observed on sections of normal cervical epithelia or on CIN lesions which were not typed as HPV16 positive. We have not correlated the presence of E4 and Ll in these sections but judging by previous studies we would expect there to be a good correlation and that both would be found in the upper layers of the lesions-E4 in the cytoplasm and Ll in the nuclei. In cultured epithelial cells, the expressed El AE4 protein was also cytoplasmic (expressed from a recombinant vaccinia virus under the control of the 4b promoter) and localized to a cytoplasmic inclusion granule (13) similar to that produced by HPVl in naturally occurring tumors

18.4

TVG

402 BLUE

106 80

49.5

32.5 27.5

A3D2

TVG 403

I

TVG 402

TVG 401

ANTIBODY HPV 1 El”E4 HPV 1 E4 HPV 2 E4 HPV 16 Ei”E4 HPV 4 E4 HPV 6 El “E4 K

FIG. 2. Characterization of monoclonal antibodies. (a) Detection of HPV16 El AE4 as a 1OK polypeptide following cleavage of the p-gal fusion protein with cyanogen bromide. Each set of three lanes contains digestion product from the HPVl El AE~ ,&gal fusion protein (I, El AE4), the HPV16 El AE~ ,&gal fusion protein (76, El AE4), or fusion protein containing no insert (control). The antibodies used for screening are indicated beneath each blot. E4 pep antiserum was raised against E4 synthetic N-terminal peptide. The equivalent antiserum raised against an E7 peptide showed no activity against E4 (first panel). 4.37 is a monoclonal antibody directed against the El AE~ protein of HPVl A Coomassie blue stain of the cleavage products is shown on the right. (b) Reactivity of monoclonal antibodies against the HPV16 ElAE4 protein expressed as a glutathione S-transferase fusion protein (see Fig. 1) Lanes contain extracts of either induced (IND) or uninduced (UN) bacteria, containing the pGEX HPV16 El AE4 expression construct. Antibodies used for each blot are indicated beneath, and the Coomassie blue staining pattern is shown on the right. The fusion protein is identified as a 37K band as predicted from its sequence. (c) Cross-reactivity of the HPV16 El AE4 monoclonal antibodies with the E4 proteins of other HPV types. Lanes contain extracts of bacteria expressing (from left to right) either HPVl El AE4, HPVl E4, HPV2 E4, HPVl6 El AE4, HPV4 E4, or HPV6 El AE4. Antibodies used are indicated beneath each blot and the Coomassie blue staining pattern is shown at the bottom of the figure. 356

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LLGSTWPT GsTwPl-rF TWPTTPPF Pl-rPPRPI TPPRPIPK PRPIPKPS PIPKPSPW PKPSPWAI PSPWAPKI PWAPKKHF APWHRRL KJaiRRLss

LKLLGSTW

MADPAAA DPAAATK’ AAATKYPI ATKYPLLK KYPLLKLL PUKUGS

358

SHORT COMMUNICATIONS

a

CIN 2, TVG 402

CIN 2 , TVG 402

CIN 2 ,TVG 402

NORMAL ECTOCERVIX , TVG 402

FIG. 4. Localization of HPVl6 El AE4 protein in a HPVl6-positive CIN 2 lesion. (a) The El AE~ staining pattern observed with the TVG 402 monoclonal antibody is shown in the bottom left and top panels. No staining was observed on frozen sections of normal cervix (bottom right panel). lmmunolocalization was carried out using a peroxidase-conjugated second antibody and visualization was performed using 3’3’. diaminobenzidine. Counterstaining was with Harris’s hematoxylin. Size bars: 80 Frn (bottom panels), 40 pm (top left panel, and 8 pm (top right panel). (b) Detection of El AE4 inclusion granules. Identification of El r\E4 inclusion granules in Vaccinia E4-infected CV-1 cells (expressing HPV16 El /\E4 from the vaccinia 4b promoter) as previously described (13). The epitope was preserved following fixation in 5%formaldehyde for 5 min (top left) or in methanol at -20” for 10 min (top right) but was lost following longer fixation in formaldehyde for 2 hr (bottom right). No staining was observed with TVG 401, 402, or 403 on cells infected with a recombinant vaccinia expressing the HPV16 Ll protein from the vaccinia 4b promoter (bottom row) (74). The antibodies used are indicated in the panel at the bottom of each photograph, while the recombinant vaccinia used for infection is indicated at the top. Size bars = 10 pm.

(Fig. 4b, 11). No staining was observed with antibodies raised against other HPVI 6 polypeptides (Ll (14) and L2 prepared against a pEX L2 fusion protein) or with a monoclonal antibody raised against the HPVl El AE4 protein (I I). TVG 401 and 402 showed no staining on uninfected cells or on cells infected with wild-type vaccinia. The antibodies described here detect El r\E4 by Western blotting and by immunofluorescence following fixation in both 5% formaldehyde and methanol at -20”. Antibody reactivity was abolished, however, fol-

lowing overnight formalin fixation of vaccinia El AE4infected cells, suggesting that these monoclonal antibodies may only be of use in the routine screening of frozen sections of clinical biopsies. It is particularly intriguing that the El AE~ proteins, when expressed in CV-1 ceils, form inclusion granules with a distribution similar to that observed for E4 in benign tumors (3). Analysis of the El AE4 associations in these structures using the antibodies described here should give us an insight into the functions of the El AE4 polypeptides.

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359

b CV-1, Vacc.ElAE4

Phase Contrast CV-1, Vacc.Li

infected

CV-I,

5% formaldehyde

5 mix.

Infected

Phase Contrast

5% formaldehvde.5

mins.

Vacc.ElAE4

Phase Contrast CV-1 Vacc El”E4

infected

5% formaldehvde

2 hours

infected

Methanol

10 mins.

CV-1 Vacc Ll infected

5% formaldehvde

5 mins

FIG. 4-Continued

REFERENCES 1. BROKER,T. R., and BOTCHAN, M., In “Cancer Cells mour Viruses” (M. Botchan, T. Grodzicker, and Eds.), p. 17-36. Cold Spring Harbor Laboratory, Harbor, NY, 1986. 2. DOORBAR, J., and GALLIMORE, P. H., J. viral. 61,

4: DNA TuP. A. Sharp, Cold Spring 2793-2799

(1987).

3. DOORBAR,J., GRAND, R. J., CAMPBELL, D., and GALLIMORE, P. H., EMBOJ. 5, 355-362 (1986). 4. DOORBAR,J., CONERON, I., and GALLIMORE, P. H., virology 172, 51-62 (1989). 5. NEARY, K., HORWITZ, B. H., and DI MAIO, D., J. !Iirol. 61, 12481252 (1987). 6. ZUR HAUSEN, H., In “Advances in Viral Oncology” (G. Klein, Ed.), Vol. 8, pp. l-26. Raven Press, New York, 1989. 7. CRUM, C. P., BARBER,S., SYMBULA, M., SNYDER, K., SALEH, A. M., and ROCHE, J. K., virology 178, 238-246 (1990). 8. MULLER, M., GAUSEPOHL,H., DE MARP/NOFF, G., FRANK, R., BRASSEUR, R., and GISSMANN, L., J. Gen. Viral. 71, 2709-2717 (1990).

9. STANLEY, K. K., and LUZIO, J. P., EMBO J. 3, 1429-l 434 (1984). 10. DOORBAR,J., PARTON, A., HARTLEY, K. O., BANKS, L., CROOK, T., STANLEY, M., and CRAWFORD, L., Virology 178, 254-262 (1990). 7 1. DOORBAR. J., EVANS, H. S., CONERON, I., CRAWFORD, L. V., and GALLIMORE, P. H., EMBO J. 7, 825-833 (1988). 12. JOHNSON, K. S., HARRISON, G. B., LIGHTOWLERS,M. W., O’Hov, K. L., COUGLE, W. G., DEMPSTER, R. P., LAWRENCE,S. B., VINTON, J. G., HEATH, D. D., and RICKARD, M. D., Name 338, 585-587 (1989). 13. DOORBAR,J., ELY, S., STERLING,J., MCLEAN, C., and CRAWFORD, L. V., Nature 352, 824-827 (1991). 14. MCLEAN, C. S., CHURCHER, M. J., MEINKE, J., SMITH, G. L., HIGGINS, G., STANLEY, M., and MINSON, A. C., J. C/in. Patho/. 43, 488-497 (1990). 15. CHOW, L. T., REILLY,S.-S., BROKER,T. R., and TAICHMAN, L. B., 1. viral.

61, 1913-1918(1987).

16. NASSERI,M., HIROCHIKA,R., BROKER,T. R., and CHOW, L. T., ViralOgy 159,433-439 (1987). 17. JAMESON, B.A., and WOLF, H., CAB/OS4(1), 181-186 (1988).

Epitope-mapped monoclonal antibodies against the HPV16E1--E4 protein.

The human papillomavirus (HPV) E1--E4 protein is the only nonstructural late protein encoded by the virus. We have isolated three hybridomas producing...
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