Sequence Close to the N-terminus of L2 Protein Is Displayed on the Surface of Bovine Papillomavirus...

VIROLOGY 227, 474– 483 (1997)ARTICLE NO. VY968348

Sequence Close to the N-terminus of L2 Protein Is Displayed on the Surfaceof Bovine Papillomavirus Type 1 Virions

WEN JUN LIU, LUTZ GISSMANN, XIAO YI SUN, AMORNRAT KANJANAHALUETHAI,* MARTIN MULLER,JOHN DOORBAR,† and JIAN ZHOU1

Departments of Obstetrics and Gynecology and *Microbiology and Immunology, Loyola University Medical Center, Maywood, Illinois 60153;and †MRC Virology Unit, University of Glasgow, Glasgow G11 SJR, United Kingdom

Received October 11, 1996; returned to author for revision October 24, 1996; accepted November 11, 1996

The bovine papillomavirus type 1 (BPV1) L2 protein purified from Escherichia coli was used as an antigen to producemonoclonal antibodies (MAbs). A total of 26 individual clones which recognized the BPV1 L2 protein were obtained. Usinginfectious BPV1 virus particles, 3 of the MAbs were found to interact with BPV1 virus particles. Binding of the MAbs toBPV1 was confirmed by immunoelectron microscopy. A set of 92 13-mer peptides overlapping by 8 amino acids spanningthe entire BPV1 L2 protein was synthesized on a membrane and used to map the epitopes recognized by these antibodies.Seventeen linear epitopes were identified. Our results revealed that a sequence toward the N-terminus of the L2 protein(aa 61 – 123) is displayed on the virus surface, while the remaining L2 sequences are hidden inside the virus capsid. Althoughthe polyclonal antisera raised against BPV1 L2 neutralized the BPV1 virus, we failed to detect any neutralizing activity forthe 3 L2-specific monoclonal antibodies which bound to the BPV1 particles. This suggests that extra binding sites may beneeded for neutralization. This study prompted us to propose a model about how L1 and L2 proteins may interact duringinfectious papillomavirus assembly. q 1997 Academic Press

INTRODUCTION Because of the difficulties in obtaining infectious hu-man PVs, the bovine papillomavirus (BPV) has been

Papillomaviruses (PVs) are nonenveloped double-widely used as a model system for studying the immune

stranded DNA viruses involved in the induction of benignresponses to papillomavirus infection. In addition, the

or malignant tumors (Galloway and McDougall, 1989; Zurinfectivity of BPV can be measured by a quantitative in

Hausen, 1991). PVs have a capsid of 55 nm in diametervitro assay (Dvoretzky et al., 1980; Roden et al., 1994),with icosahedral symmetry (T Å 7) composed of 72 pen-making it suitable for studying the interaction with cellstameric capsomers (Crawford and Crawford, 1963). Thein culture. Several reports suggested that the papillo-capsid consists of the major capsid protein, L1, and amavirus L2 protein alone contains neutralizing epitopesminor structural component, L2, that migrate on sodium(Roden et al., 1994; Gaukroger et al., 1996; Campo et al.,dodecyl sulfate (SDS) – polyacrylamide gels at approxi-1993) and immunization of animals with Escherichia coli-mately 55 and 75 kDa, respectively (Doorbar and Galli-expressed L2 can induce low levels of neutralizing anti-more, 1987; Pfister and Fuchs, 1987). The L1 protein hasbodies protecting animals from infection with the cotton-the capacity to self-assemble into virus-like particlestail rabbit papillomavirus or BPV (Jin et al., 1989; Lin et(VLPs) when expressed in different eukaryotic systemsal., 1992; Christensen et al., 1991). Using the BPV L2(Hagensee et al., 1993; Kirnbauer et al., 1992, 1993; Zhouprotein fused to glutathione S-transferase (GST), it waset al., 1993; Rose et al., 1993; Sasagawa et al., 1995;found that the neutralizing epitope(s) is located betweenHofmann et al., 1995). Although not required for assem-amino acids (aa) 11 to 200 (Chandrachud et al., 1995). Itbly, L2 is incorporated into VLPs when coexpressed withwas also reported that a polyclonal antibody against theL1 in insect or mammalian cells. The location of the L2N-terminal portion of BPV1 L2 reacts with BPV1 virionsprotein within the capsid and its function are still un-and neutralizes BPV1 transforming capacity (Roden etknown (Baker et al., 1991), although there is some evi-al., 1994). Therefore, the N-terminal region of the L2 pro-dence to suggest that the L2 protein is a DNA-bindingtein of BPV1 may be exposed on the surface of the virionprotein and may be involved in viral DNA encapsidationand contain linear neutralizing epitopes (Roden et al.,(Zhou et al., 1994, 1993; Roden et al., 1996).1994).

In order to define the L2 sequences displayed on the1 To whom correspondence and reprint requests should be ad- surface of BPV1 particles and to elucidate the structuredressed at current address: Papillomavirus Research Unit, Department

of papillomavirus capsids, we have generated a panelof Medicine, Princess Alexandra Hospital, Woollongabba, Queensland,Australia. Fax: /61(7) 3240-2048. of monoclonal antibodies against the BPV1 L2 protein.

4740042-6822/97 $25.00Copyright q 1997 by Academic PressAll rights of reproduction in any form reserved.

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475BOVINE PAPILLOMAVIRUS L2 PROTEIN

BPV1 virions were purified from cattle warts and were Western blottingused to characterize the monoclonal antibodies. The epi- BPV1 virions and purified BPV L2 protein were sepa-topes recognized by the monoclonal antibodies were ex- rated on 10% polyacrylamide – SDS gels and transferredamined by using synthetic peptides that span the whole onto nitrocellulose membranes by electroblotting inBPV1 L2 protein sequence. Here we report the segment transfer buffer (25 mM Tris, 190 mM glycine, 20% metha-of the L2 protein which is localized at the virus surface. nol). Membranes were blocked for 1 hr at 377 with 5%A model of the topology of the L2 protein within BPV1 nonfat milk in PBS containing 0.05% Tween 20. After threeparticles is proposed according to the results. washes, the blots were incubated for 1 hr at 377 with

mouse (from mice immunized with L2 protein) or rabbitanti-BPV1 virus antiserum (Dako) at 1:2000 dilution. AfterMATERIALS AND METHODSthree washes, the membranes were incubated with

Purification of BPV1 virions from cattle warts horseradish peroxidase (HRP)-conjugated sheep anti-mouse IgG or with HRP-conjugated anti-rabbit IgG immu-

Details of purification of BPV1 virions have been pre- noglobulins at 1:10,000 dilution. Specific protein bandsviously described (Zhou et al., 1995). Briefly cattle warts were visualized using the enhanced chemiluminescencewere minced and homogenized in extraction buffer (20 detection system (Amersham).mM HEPES, pH 7.4, 10 mM MgCl2 , 50 mM CaCl2, 150mM NaCl, 0.01% Triton X-100 and 1 mM PMSF). The Production of monoclonal antibodies (MAbs)tissue lysate was then cleared by spinning in a Sorval

Three BALB/c mice were immunized three times at 14-GS3 rotor for 20 min at 6000 rpm, and virions were con-day intervals with 50 mg purified BPV1 L2 protein derivedcentrated by pelleting them in a Sorval AH629 rotor forfrom E. coli. Five days after the final boost, mice were2 hr at 27,000 rpm. The pellets were resuspended in thebled and the spleens were removed. A single-cell sus-extraction buffer followed by centrifugation through 40%pension of mouse spleen cells was obtained by pressingsucrose cushion. The BPV particles were then purifiedthe spleens through a 60-mesh sieve. The spleen cellsthrough a CsCl gradient. The purified BPV viruses werewere fused to myeloma cell lines SP2/0 which wereexamined on a 10% SDS gel and DNA was extractedmaintained in RPMI (Gibco) supplemented with 10% fetalfrom the viruses for virus typing. The results show thatcalf serum (Sigma). The fusion reactions were performedthe BPV was purified to high purity and that the type ofin PEG1500 (Boehringer Mannheim) using the standardthe virus was BPV1.protocol supplied by the manufacturer. The fused cellswere then distributed into 96-well plates (a total of 4000

Preparation of BPV1 L2 protein wells were seeded) and were selected with HAT selec-tion medium (Boehringer Mannheim). After 10 days, theThe BPV1 L2 open reading frame was amplified fromcell culture supernatants were screened for the presencea BPV1 plasmid using primers 5*-GCAGATCTATGAGTGof antibodies directed against the denatured L2 proteinCACGAAAAAGAGT AAAACGT GCCAGT GC and 5*-by ELISA using BPV1 L2 protein (as described above).GCAGATCTGGCATGTTTCCGTTTTTTTCGTTTCC. TheImmunoreactions were visualized using peroxidase-con-downstream stop codon was removed in order to havejugated sheep anti-mouse IgG (Sigma).a His-tag attached. The BglII sites in italics were incorpo-

rated to facilitate the cloning. The PCR reaction was car- Immunofluorescenceried out with Taq DNA polymerase in a Perkin– Elmer

The cell culture supernatants of positive MAb clonesthermocycler. The PCR products were purified andwere also examined by immunofluorescence (IF) stainingcloned into the BglII sites of E. coli expression vectorusing Sf9 cells infected with baculoviruses expressingPQE12 (Qiagen) fusing six histidines to the C-terminus.the BPV1 L2 protein. Briefly, the insect cells infected withThe recombinant plasmid was used to transform E. colirecombinant baculoviruses were grown for 72 hr andDH5a, and L2 protein expression was induced withcells were fixed in methanol. The cells were blocked with1 mM IPTG. Affinity purification of the six-histidine L25% milk for 30 min, then incubated with the monoclonalfusion protein was performed according to the protocolsantibodies for 1 hr at 377, followed by incubation withsupplied by manufacturer (Qiagen). Briefly, the bacterialFITC-linked sheep anti-mouse IgG and examination un-pellets were solubilized by adding 10 ml guanidiniumder a fluorescence microscope (Nikon). Clones positivebuffer. The L2 protein was then added to a Ni2/ columnfor L2 protein in both ELISA and IF were subcloned twiceand washed with 6 M urea. The purified L2 protein wasby limiting dilution.eluted with low pH buffer and examined by 10% polyacryl-

amide gel. The L2 protein was further purified by elec-ELISA using BPV1 particlestrophroetic separation on a 10% SDS gel before the L2

bands were cut out, and the protein was eluted with an The specificity of the MAbs to BPV1 particles wastested by ELISA using BPV viruses prepared from warts.electroeluter (Bio-Rad).


476 LIU ET AL.

Intact BPV1 virus particles in PBS were coated onto PAGE. The purified L2 protein appeared as a single bandon SDS gels with the expected molecular weight of 75ELISA plates overnight at room temperature (Cowsert et

al., 1987). BPV1 viruses were also denatured by alkaline kDa (Fig. 1A). In Western blots, mouse polyclonal antise-rum (collected from mice from which the spleen cellstreatment (0.2 M Na2CO3 buffer, pH 10.6, 0.01 M dithio-

threitol) (Favre et al., 1975) and coated onto ELISA plates. were used to generate anti-L2 MAbs) recognized andlabeled the L2 fusion protein and also the L2 proteinELISA plates containing either native or denatured BPV1

particles were washed and blocked with PBS containing from BPV1 particles without noticeable differences (Fig.1B). A commercially available polyclonal antiserum5% milk. A 1:10 dilution of MAb supernatants was added

to each of the plates followed by HRP-coupled anti- raised against whole BPV1 virus (Dako) recognized boththe BPV1 L2 and the BPV1 L1 protein (Fig. 1C). Thesemouse IgG (Sigma). After color development, the ab-

sorbances were read at 415 nm. results suggested that the BPV1 L2 protein from E. coliwas purified to high degree and the protein was identical

Immunoelectron microscopy of BPV1 virions to the L2 protein derived from infectious BPV1 virions.We therefore used this purified antigen to immunize ani-BPV1 viruses were spotted onto nickel grids and ab-mals for monoclonal antibody production and also tosorbed with 1% BSA – PBS. The grids were incubated withscreen the MAbs by ELISA.the mouse MAbs. After washing in PBS, bound primary

antibodies were detected with a gold-particle-conjugatedIsolation of MAbs to the BPV1 L2 minor capsid(10 nm) anti-mouse IgG antibody (Sigma). After extensiveproteinwashing the virus were submitted to negative staining

with 2% uranyl acetate and examined with a Hitachi H-The BPV1 L2 protein derived from E. coli was used600 transmission microscope at 75 kV.

to immunize mice. Hybridomas obtained using standardprocedures were screened with the L2 protein as de-B-epitope mappingscribed under Materials and Methods. Twenty-six stable

Peptides 13 amino acids long, overlapping by 8 amino hybridoma cell lines secreting BPV1 L2-specific MAbsacids, corresponding to BPV1 L2 amino acids 1 to 469 were obtained. All these MAbs reacted strongly with thewere synthesized by Fmoc amino acid chemistry. The BPV1 L2 protein in ELISA assays. The reactivity of thepeptides were coupled onto a derivatized cellulose mem- MAbs raised against the bacterially expressed proteinbrane according to the manufacturer (Genosys). The were also demonstrated against eukaryotically ex-membrane was blocked with blocking buffer and incu- pressed BPV1 L2 by immunofluorescence. All the MAbsbated with individual hybridoma supernatants. The pep- strongly stained the nuclei of Sf9 insect cells infectedtides which reacted with the MAbs were identified by with a BPV1 L2 recombinant baculovirus by immunofluo-color development using an anti-mouse IgG conjugated rescence (Fig. 2), but did not stain uninfected or baculovi-with b-galactosidase. rus wild-type-infected cells. These results proved that

the MAbs raised against the bacterially derived proteinIn vitro neutralization assay

recognized the native BPV1 L2 protein. Most of the MAbswere found to be of IgG1k subtype. Two MAbs were ofAscites of different MAbs were preincubated for 1 hr

with various amounts of infectious BPV1 virus particles IgG1l (D20 and D25) and one (D2) was IgG2bk (seeTable 1).prior to plating onto monolayers of C127 mouse fibro-

blasts (ATCC) in 35-mm-diameter tissue culture plates. All of the MAbs which were positive for BPV1 L2 weretested in ELISA using intact or denatured BPV1 virus asThe BPV1 virions were then added to the cells and incu-

bated at 377 for 2 hr. After the cells were washed, they described under Materials and Methods. Three MAbs(C1, C3, and C6) were strongly reactive with intact BPV1were maintained in DMEM medium containing 2% fetal

calf serum for 3 weeks with a medium change every 3 virions, suggesting that the epitopes recognized by theseMAbs may be exposed on the surface of the virus. Al-days. The foci produced by BPV infection were stained

with 0.5% methylene blue, and the numbers of foci were though the remaining 23 MAbs were reactive with dena-tured BPV1 particles, they did not bind to the intact BPV1scored.virus (Table 1), indicating that the epitopes recognizedby those MAbs were buried inside of the virus.RESULTS

To demonstrate that MAbs C1, C3, and C6 were indeedProduction of BPV1 L2 fusion proteins

bound to the intact BPV1, immunoelectron microscopywas performed as described under Materials and Meth-To produce the minor capsid protein of BPV1, the en-

tire L2 open reading frame of the BPV1 was cloned and ods. Using monoclonal antibodies and gold-particle-la-beled secondary antibodies, all 26 MAbs directedexpressed in E. coli as a fusion protein with six histidines

attached to its carboxyl end. The L2 protein was purified against BPV1 L2 protein were examined by electron mi-croscope (Fig. 3). Again, only MAbs C1, C3, and C6 whichon an affinity Ni2/ column and further separated by SDS –



FIG. 1. Characterization of purified BPV1 L2 protein. Purified BPV1 L2 protein derived from E. coli (lanes 1 and 2) and denatured BPV virions(lanes 3) were fractionated on 10% polyacrylamide gel. (A) Coomassie blue-stained SDS –polyacrylamide gel, (B) Western blot probed with a mousepolyclonal antiserum specific for BPV-1 L2 protein, and (C) Western blot probed with a rabbit polyclonal antiserum specific for BPV1 virus particles.Molecular weight markers (in kilodaltons) are shown on the left. The asterisks indicate the positions of recombinant L2 and the open circles referto the positions of L1.

were positive in BPV1 particle ELISA assay were able to the virus surface. We also conclude from these resultsthat the majority of the BPV1 L2 protein, including thebind to the surface of the virus (Figs. 3B and 3C). As the

gold particles were clustered around the intact virus first 60 N-terminal aa and most of the C-terminal se-quences, is positioned internally, possibly in close con-(Figs. 3B and 3C), we concluded that the epitopes recog-

nized by these 3 MAbs were indeed displayed on the tact with the major capsid protein.surface of the BPV-1 virus. No specific staining was ob-

In vitro neutralization assayserved with the other 23 MAbs or with the secondaryantibody alone (Fig. 3A). This result suggested that the

Several reports have suggested that polyclonal anti-epitopes recognized by MAbs other than C1, C3, andsera raised against the L2 protein can neutralize BPVC6 are not accessible in the context of the assembledvirus (Roden et al., 1994; Chandrachud et al., 1995; Gauk-capsid.roger et al., 1996). To determine whether the MAbs thatrecognize BPV L2 sequences on the virus surface haveEpitope mapping using synthetic peptidesneutralizing activity, MAbs C1, C3, and C6 were tested

To define the linear epitopes on the BPV1 L2 protein in BPV1 focus assays. MAbs C1, C3, C6, and mouserecognized by these MAbs, we synthesized 92 peptides polyclonal anti-BPV1 L2 antiserum were incubated for 1spanning all the L2 amino acids (from 1 to 469). Each hr with about 200 focus-forming units of infectious BPV1peptide was 13 amino acids in length and overlapped virus prior to plating onto monolayers of mouse fibroblastthe following peptide by 8 amino acids. They were syn- C127 cells. The cells were maintained for 3 weeks, andthesized by Fmoc amino acid chemistry and coupled to the reduction in the number of foci in the presence ofderivatized cellulose membranes. The reactivity of the each antibody was scored. For positive controls, BPV1-MAbs to the L2-specific peptides was detected by mea- neutralizing MAb 5B6 (Roden et al., 1994) (kindly providedsuring the activities of b-galactosidase that was linked by Roden and Schiller, NIH) was included and shown toto the secondary antibody. Four MAbs did not react with have strong neutralization activity even at 1:1000 dilutionany peptides suggesting that they may react with discon- (Fig. 5E). In contrast, none of the BPV1 L2 MAbs hadtinuous epitopes on the BPV1 L2 protein. A total of 17 neutralization activity even at a very high concentrationepitopes were recognized by 22 of the 26 MAbs; these (1:50 dilution of ascites) either individually (Figs. 5B, 5C,17 epitopes were quite evenly distributed on BPV1 L2 and 5D) or in combination (not shown). Mouse BPV1 L2-protein sequence. The three MAbs which bound to BPV1 specific polyclonal antiserum (Fig. 5F) neutralized BPV1particles recognized three epitopes in a small region of at 1:1000 dilution. These results suggested that the threethe L2 protein. MAbs C3 and C1 reacted with 2 peptides MAbs could not block the BPV1 infection although theycontaining the sequences STGRVAAGGSPRY (aa 61– 73) are able to bind to the BPV1 virus.and PRYTPLRTAGSTS (aa 71– 83), while MAb C6 recog-nized 2 peptides that contained an overlapping sequence DISCUSSIONof LRPGVYED (aa 116– 123) (Fig. 4). These results sug-gest that the N-terminal region of the L2 protein (from aa L2 is the minor capsid protein of papillomavirus and

its function and location within the capsid have not been61 to 123) represents the portion which is exposed on


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TABLE 1 graphic data. Therefore, we decided to generate BPV1L2-specific MAbs to map the portion of the L2 proteinGeneral Information on the Monoclonal Antibodiesexposed to the surface of the virus particles. We ex-

Intact BPV Denatured BPV pected to generate useful information toward under-Name Subtype (OD) (OD) IF EM standing the papillomavirus structure and function.

It has been suspected, for a long time, that the N-A3 IgG1k 0.033 0.175 Pos NB terminal section of the PV L2 protein is exposed on theA5 IgG1k 0.106 0.424 Pos NB

papillomavirus surface on the basis of the following ob-A5-1 IgG1k 0.090 0.261 Pos NBA6 IgG1k 0.075 0.286 Pos NB servations: (1) amino acids 45– 173 of BPV1 L2 containA7 IgG1k 0.040 0.291 Pos NB the neutralizing epitopes demonstrated by in vitro trans-A8 IgG1k 0.064 0.283 Pos NB formation assays (Roden et al., 1994); (2) the N-terminusA13 IgG1k 0.063 0.227 Pos NB

of L2, comprising amino acids 11– 200, is necessary andA15 IgG1k 0.050 0.364 Pos NBsufficient for protection of animals from BPV4 infectionA16 IgG1k 0.074 0.164 Pos NB

D1 IgG1k 0.083 0.348 Pos NB (Chandrachud et al., 1995; Gaukroger et al., 1996); (3)D2 IgG2bk 0.081 0.419 Pos NB amino acids 101– 120 of the L2 protein of BPV4 are rec-D3 IgG1k 0.097 0.398 Pos NB ognized by sera from animals resistant to BPV4 infectionD7 IgG1k 0.109 0.523 Pos NB

after immunization with the N-terminal L2 protein (Chan-D9 IgG1k 0.079 0.259 Pos NBdrachud et al., 1995); (4) a MAb directed against aa 102–D10 IgG1k 0.033 0.148 Pos NB

D16 IgG1k 0.064 0.356 Pos NB 108 of the HPV1 L2 protein was obtained by immunizingD20 IgG1l 0.055 0.145 Pos NB animals with intact virions (Yaegashi et al., 1992); andD25 IgG1l 0.062 0.314 Pos NB (5) antibodies to aa 106 – 128 of HPV16 L2 and aa 103–D29 IgG1k 0.056 0.155 Pos NB

127 of HPV11 L2 have been detected in sera from pa-D31 IgG1k 0.039 0.219 Pos NBtients with cervical cancer or genital warts (Yaegashi etD32 IgG1k 0.054 0.330 Pos NB

D33 IgG1k 0.056 0.359 Pos NB al., 1992). In this study we have defined regions of theC1 IgG1k 0.283 0.358 Pos B L2 protein with regard to their locations in the virions byC3 IgG1k 0.293 0.384 Pos B using synthetic peptides, intact BPV1 particles in ELISA,C6 IgG1k 0.312 0.398 Pos B

and electron microscopy. In all, 17 epitopes recognizedby 22 of 26 BPV1 L2-specific MAbs were defined usingNote. OD values from ELISA are listed. Results from immunofluores-

cence using baculovirus expressed L2 are showing under IF. The EM a set of overlapping peptides spanning the entire BPV1results using immunogold are showing under EM. B refers to MAb L2 protein. Four MAbs (D1, A7, D29, and D32) reactedbinding to virus and NB means no binding was observed. to BPV L2 protein expressed by recombinant baculovirus

and denatured BPV1 virions, but they did not recognizeany of the 92 synthetic BPV1 L2 peptides suggesting thatfully elucidated (Baker et al., 1991). The molar ratio ofthey may recognize discontinuous epitopes (Volpers etthe L2 to the L1 protein is approximately 1:10 (Doorbaral., 1995). Three MAbs (C1, C3, and C6) were shown toand Gallimore, 1987) or 1:30 (Schiller and Roden, 1996;bind to the surface of the BPV1 virions and recognizeRoden et al., 1996). L2 has two basic amino acid-richepitopes located between aa 61 and 123 of the L2 proteinregions at the N-terminal and C-terminal ends that are(for summary see Fig. 4). Therefore we conclude thatresponsible for nuclear transport of the protein (Sun etthis part of the protein is displayed on the viral surface.al., 1995). Because the L2 protein is the only PV capsidHowever, it is not clear whether it is exposed on theprotein capable of binding to DNA, and is required tosurface as a continuous sequence because we did notassemble infectious BPV1 (Zhou et al., 1993; Roden etidentify a MAb which recognizes epitopes between aaal., 1996), it has been speculated that the protein is in-83 and 116.volved in viral DNA encapsidation (Zhou et al., 1993)

The N-terminal part (aa 16– 28) is recognized by oneand in capsid stabilization at low pH (Frazer, personalMAb (D2), which does not bind to the virus particles. Incommunication). The configuration of the L2 proteinaddition, the first 12 terminal amino acids of HPV16 L2within a PV capsid has not yet been determined due tomay interact with the viral DNA (Zhou et al., 1994) and arethe low resolution of reconstructed BPV images (Baker

et al., 1991; Hagensee et al., 1994) and lack of crystallo- very conserved among PVs. Therefore it is reasonable to

FIG. 2. Immunofluorescence staining of BPV L2 protein using MAbs. Insect cells (Sf9) were infected with baculovirus expressing BPV1 L2. Thecells were harvested and fixed with ethanol before they were coated onto a slide. The MAb C1 diluted at 1:10 was added followed by fluoresceinisothiocyanate-conjugated anti-mouse IgG (Sigma). The L2 proteins localized in the nucleus were clearly visible. An irrelevant MAb (anti BPV1 L1)used as a control failed to detect the L2 protein (data not shown).

FIG. 7. BPV1 model. The BPV1 L2 model is proposed according to our data. The very first 60 aa of L2 protein are inside of the BPV1 capsid withthe charged amino acids bound to viral DNA. The sequence between aa 61 and 123 is exposed on the virus surface. The exact boundary ofexposed and unexposed L2 is not clear because of small gaps between epitopes recognized by virus-bound and unbound MAbs.


480 LIU ET AL.

FIG. 3. Immunoelectron microscopy of BPV virus using anti-L2 MAbs. Carbon grids coated with BPV1 particles were incubated with (A) MAb A3which recognized a C-terminal sequence and (B) MAb C1 and (C) MAb C6 which are specific for the BPV1 L2 sequence exposed on the virussurface. For detailed sequence, see Table 1. After washes, the gold-conjugated anti-mouse IgG (10 nm) were used as secondary antibody and thegrids were examined using a Hitachi H-600 transmission electron microscope at 75 kV.

FIG. 4. Epitope mapping of MAbs using synthetic peptides. A total of 92 BPV1 L2 peptides were synthesized on one membrane. Each peptidewas 13 amino acids in length and overlapped the following peptide by 8 amino acids. The open box represents BPV1 L2 protein. The amino acidpositions at 1, 100, 200, 300, 400, and 469 are indicated. The MAb binding sites are shown on the top. The three MAbs that recognized the L2epitopes displayed on the virus surface are indicated by stars. The bottom shows detailed information about the MAbs listed: names of the MAbs,epitope locations, and epitope sequences.

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FIG. 5. Neutralization assays by focus test. C127 cells were infected with BPV1 virions that were preincubated with (A) PBS, (B) MAb C1, (C)MAb C3, (D) MAb C6, (E) neutralizing MAb 5B6, and (F) mouse polyclonal antibody to BPV1 L2. The cells were maintained in DMEM mediumsupplied with 2% FCS for 3 weeks and the foci were stained and counted.

speculate that the first 60 aa remain inside of the virion nation experiments with cottontail rabbit papillomavirus(CRPV) (Christensen et al., 1991; Lin et al., 1992) andfor protein/DNA interaction.

Thirteen linear epitopes of BPV1 L2 protein toward the BPV4 (Campo et al., 1993) L2 proteins demonstrated thatprotective L2-specific antibodies can also be induced.C-terminus were identified by 18 MAbs, some of which

recognized the same peptide (e.g., MAbs D7, D9, D20, These results suggest that some sequences of the L2protein are exposed on the viral surface allowing neu-and D25; MAbs A5 and A6) (see Fig. 4). These results

suggest that the C-terminal end of BPV1 L2 is an immune- tralizing antibodies to attach. Immunization of calves withan amino-terminal portion (11 – 200) of the BPV4 L2 pro-dominant region. None of these MAbs bound to the intact

virus particles, indicating that these regions of the L2 are tein expressed as a GST fusion protein has been foundto protect animals from development of mucosal warts,buried inside of the virions. However, the exact bound-

aries are not clear as there are gaps between the epi- whereas immunization with other portions of the L2 donot (Chandrachud et al., 1995). However, the three BPV-topes recognized by MAbs C6 and D10 as well as by C3

and D2 (Fig. 4). Similar results were observed in other binding L2-specific MAbs described here failed to blockBPV1 infection of C127 cells. It is possible that the epi-PVs: Volpers et al. (1995) obtained two MAbs reacting

with linear epitopes aa 153 – 160 and aa 163 – 170 of the topes recognized by these three MAbs are not involvedin the infection process or, alternatively, that more thanHPV33 L2 protein and found that these epitopes have an

internal location. three L2-specific antibodies bound to the virus areneeded to achieve neutralizing effects.The major L1 capsid protein conformational epitopes

have been shown to be located on the external surface The minor L2 capsid protein of papillomavirus has pre-viously been demonstrated to induce type-specific anti-of papillomavirus (Kirnbauer et al., 1992; Christensen et

al., 1990; Cowsert et al., 1987; Roden et al., 1994) and bodies in animals immunized with L2 fusion proteins(Volpers et al., 1993; Komly et al., 1986; Tomita et al.,induce neutralizing antibodies in animals (Christensen

et al., 1990). However, some antibodies (Yaegashi et al., 1987; Yaegashi et al., 1991; Christensen et al., 1991)which is in line with the sequence diversity among PV1991; Beiss et al., 1991) have been shown to recognize

linear antigenic determinants on the papillomavirus L2 L2 proteins. Seven different types of papillomavirus L2protein sequences were selected to compare with theminor capsid proteins and are believed to be neutralizing.

BPV1 L2 fusion proteins induced polyclonal antiserum BPV1 L2 sequence using the Clustal method. Bovine pap-illomavirus types 1, 2, and 4; CRPV; human papillomavi-that could neutralize BPV1 in in vitro neutralization

assays (Pilacinski et al., 1984; Roden et al., 1994). Vacci- rus types 11 and 16; the European elk PV; and the rhesus


482 LIU ET AL.

FIG. 6. Sequence alignment analysis of the papillomavirus L2 proteins. Seven papillomavirus L2 proteins were aligned against BPV1 L2 protein.Amino acids identical to the BPV1 L2 protein sequences are indicated by shadow. The result shows the very first 60 amino acids are more conservedacross different types of papillomaviruses than those of the rest of the protein. ELKPV, European elk PV; RHPV, rhesus monkey PV.

monkey PV were selected for their diversity in host and L2 protein other than aa 61 – 123 is displayed on the viralsurface because of the limitation in numbers of MAbtissue specificities. The alignment results showed that

only the very first 60 amino acids of these PV L2 se- obtained. The relationship between L1 and L2 is specula-tive as no published data are available.quences are highly conserved. A sequence alignment of

the N-terminal region of the PV L2 proteins is shown inFig. 6. We propose that these conserved first 60 amino ACKNOWLEDGMENTSacids play an important role in virus formation, probably

We thank Dr. Delgado for his support and Drs. Schiller and Rodenby interacting with L1 sequences within the capsid. Thefor providing monoclonal antibody 5B6. This project was supported insegment spanning aa 61 – 123 which is displayed on thepart by the Schweppe Foundation.

virus surface, like other regions of L2, is less conservedamong different PVs.

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Sequence Close to the N-terminus of L2 Protein Is Displayed on the Surface of Bovine Papillomavirus...

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