Use of synthetic peptides to map sequential epitopes recognized by monoclonal antibodies on the bovine leukemia virus external glycoprotein

VIROLOGY

185,48-55

(1991)

Use of Synthetic Peptides to Map Sequential Epitopes Recognized by Monoclonal Antibodies on the Bovine Leukemia Virus External Glycoprotein ISABELLE CALLEBAUT,* AR&NE BURNY,* VIKTOR KRCHNAKJ HELENE GRAS-MASSE,* BERNARD WATHELET,* AND DANIEL PORTETELLE*v’ Faculty of Agronomy, Passage des D~portt%, 2, B-5030 Gembloux, Belgium; tResearch Institute for Feed Supplements and Veterinary Drugs, 254 49 Jilovd u Prahy, Czechoslovakia; PChimie des Biomol&ules, lnstitut Pasteur de Lille, 590 19 Lille, France Received April 26, 799 1; accepted July 11, 199 1 Six sequential epitopes (A, B, B’, D, D’, E) were previously defined on the bovine leukemia virus (BLV) envelope glycoprotein gp51 by their reactivity with monoclonal antibodies. A panel of synthetic peptides covering almost the entire sequence of gp51 was tested in enzyme-linked immunosorbent assays in order to localize these epitopes. E was shown to be included in peptide 142-181 (MCF4), B and B’ in peptide 195-205, D and D’ in peptide 218-237 (MCFG), and A in peptide 249-268 (MCF7). These results extend and confirm previous observations suggesting that the sequential epitopes recognized by our battery of monoclonal antibodies are located in the carboxylic half of BLV gp51. Q 1991 Academic

Press, Inc.

INTRODUCTION

lowed identification of eight distinct epitopes, A-H; three other epitopes, B’, D’, and F’, were also described in the vicinity of, respectively, B, D, and F (Bruck et al., 1982a). Among the series of epitopes, three conformational sites, F, G, and H, represent major determinants in the biological activities of the virus, namely infection and syncytia induction (Brucketal, 1982b). Theyrepresent important target epitopes for the serological diagnosis of BLV infection (Bruck eta/., 1984a; Portetelle et a/., 1989c). We previously used the synthetic peptides approach in an attempt to precisely localize these crucial epitopes, F, G, and H. Antibodies directed against three synthetic peptides derived from the NH, part of gp51 could neutralize the virus; none of these peptides, however, was recognized by the MAbs (Portetelle et al., 1989b). In the same experiment, none of the chosen peptides allowed identification of the sequential epitopes A, B, B’, D, D’, and E. The location of these epitopes is of interest, particularly in order to set up efficient serological tests. For example, MAbs against epitopes E, A, or B’ proved to be very efficient binding antibodies in enzyme-linked immunosorbent assays. The serological test using MAb against site E as the binding reagent appeared to be highly sensitive and reproducible; gp51 is probably presented in its native configuration, and interestingly enough, no purification step is required (Portetelle et al., 1989c). A tentative mapping of the sequential epitopes was undertaken by compiling the results obtained after urokinase digestion of gp51 (Brucketa/., 1982b), heterolo-

The bovine leukemia virus (BLV), a retrovirus structurally and functionally related to the human T lymphotropic viruses HTLV-1 and HTLV-2 (Rice et a/., 1987), has been shown to be etiologically associated with chronic lymphatic leukemia and malignant lymphoma in cattle populations (Burny et a/., 1980, 1990). Several lines of evidence suggest that the BLV envelope may be a critical viral target for the host immune system. Indeed, the extramembrane glycoprotein gp51 is the first antigen to be recognized by antibodies in the sera of newly infected hosts (Mammerickx et a/., 1980). These polyclonal antibodies neutralize virus pseudotypes, block induction of syncytia (Zavada et al., 1978; Diglio and Ferrer, 1976), and exhibit complement dependent cytolytic activity (Portetelle et al., 1978). Although BLV is a cell-associated pathogen, sheep passively immunized with such anti-BLV antibodies can resist a virus challenge provided they have sufficiently high anti-gp51 antibody titers (Mammerickx et a/., 1980; Kono et al., 1986). Moreover, protection against infection has been recently achieved via vaccination with env glycoprotein vaccinia recombinants (Portetelle et a/., 1991). Further advances in vaccine development and in diagnostic testing require a better understanding of the antigenic structure of this crucial protein. To that end, a battery of mouse monoclonal antibodies (MAbs) al’ To whom 0042-6822191

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requests

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Copyright Q 1991 by Academic Press. Inc. All right9 of reproduction in any form reserved.

be addressed. 48

ANTIGENIC ANALYSIS OF THE BLV-gp51

GLYCOPROTEIN

49

TABLE 1 CHARACTERIZATION OF gp51 PEPTIDESAND THEIR CORRESPONDING ANTIPEPTIDEANTIBODIES Antipeptide antibodies Reactivity against Peptide code

Amino acid numbering

Amino acid sequence

MCFl MCF2 MCF3 GXl MCM GX2 MCF6

38-57 68-87 98-117 119-132 142-161 168-180 218-237

FCAKSPRYTLDSVNGYPKIY GARAMUYDCEPRCPYVGAD SQADQGSFWNHQILFLHLK CHGIFTLTWEIWGY KIPDPPQPDFPQLNSDWVPS LLNQTARAFPDCA NSSSFN-ITQGWHHPSQRLLF

MCF7 195-205

249-268 195-205

255-268 260-268

255-268 260-268

Rabbit number

Titers

BLV

MAb E-gp5 1

94 89 90 87 92 99 93

1,100,000 >9,900.000 44,000 4,900 9,900,000 >3,300,000 4,900,000

++ + +++ ++ ++

++ + ++ ++ + ++

PISLVNLSTASSAPPTRVRR WNKTlSGSGPb

91 2659

15,000 >9,900,000

++ -

STVSSAPPTRVRR* SAPPTRVRRb

2664 2651

44,000 131,000

-

Epitope

OD indices’

E

6

D>D

D: 24 D’: 111 4 B: 10 El’: 1

A B, B +++ -

Note. All peptide sequences are deduced from nucleotide sequence data of the gp51 variant described by Sagaa et al. (1985). except STVSSAPPTRVRR, which originates from the T15-2 variant (Deschamps er al., 1981). Position 1 in the gp51 corresponds to the first residue after the signal peptide (Trp). Antipeptide antibody titers are expressed as the reverse of the serum dilution for which the opti& c&m&y in ELISA is twofold that of the negative control. Antipeptide antibodies reactivity: (+) weak reaction; (++) good reaction; (+++) strong reaction; (--) no reaction. The significance of the reaction was estimated by comparison of the OD with the OD obtained using rabbit sera before injection or serum from rabbits injected with a nonrelated peptide-KLH conjugate. a OD indices ware calculated for 1 pg of MAb by dividing OD values observed for the reaction conjugate/MAb of interest by the mean OD value observed for the negative controls (interaction between the peptide conjugate and the MAbs not involved in the rec@@iin of the epitope). In each case, OD values for the negative controls were below 0.19. Each well of the plates was coated with 5 rg of cafrier protein. b Peptides previously described in Portetelle et al. (1989b).

gous expression of envelope truncated proteins (Legrain et al., 1989), and careful study of the amino acid sequence variation in the env gene (Bruck et a/., 1984b; Portetelle et al., 1989a). Evidence taken from these data assigned site E in the central part of gp51 and strongly suggested that (a) A, D, and D’ were located after residue 217, (b) B resided between residues 150 and 2 17, and (c) B’ lay in the vicinity of amino acid 202. In the present study, we describe new synthetic peptides and new strategies to complete and refine mapping of the sequential epitopes A, B, B’, D, D’, and E. MATERIAL

AND METHODS

Peptide synthesis The sequences of the peptides used in this study are listed in Table 1. Peptides were synthesized using a solid-phase method according to Merrifield (1963), all on polystyrene 1% divinylbenzene resins with chloromethyl resin for Gx2, benzhydrylamine resin for Gxl , and p-methytbenzhydrylamine resin for MCFl, MCF2,

MCF3, MCF4, MCFG, and MCF7. Synthesis of the peptides 195-205, 255-268, and 260-268 was described in a previous report (PorW&e at al., 1989b). Peptides MCFl, MCF2, MCF3, MCF4, MCFG, and MCF7 were prepared by continuous-flow solid-phase multiple peptide synthesis (Krchn&k and V&$W, 1990) using the Fmoc/t-Bu protection stra’fegy and the coupling reaction was monitored by bromophenol blue (KrchnBk er al., 1988). Side chain protecting groups were removed by trifluoroacetic acid (TFA) and peptides were split from the resin in liquid hydrogen fluoride (HF). For the synthesis of Gxl and Gx2, we used a semiautomatic procedure based on a tSoc/Bzl strategy. Trifunctional amino acids were protected as follows: Asp (0-Bzl), Glu (0-Bzl), Thr (O-E&& Ser (O-&I), Arg (Tos), His (Dnp), Cys (Mebzl), Lys (GIZ), Tyr (0-Br-Cbz), and Trp (For). Side chain protections wefe mmoved by liquid HF when peptides were cleaved from the resins. Crude peptides were purified by gel fWration and reversed phase high-performance tiquid chromatography (RP-HPLC). Peptides were analyzed for homogeneity by thin layer chromatography anei RP-HPLC and their composition was assessed by amino acid analy-

50

CALLEBAUT

sis after total acid hydrolysis. ways above 95%. Preparation

of rabbit antipeptide

Peptide purity was al-

antibodies

Production and characterization of MAbs have been described previously (Bruck er al., 1982a, 1982b). Enzyme-linked

immunosorbent

*) I KLH-PEPTIDE GP51 +

+

>--

+

PEPT’DE +

+

=

I-

B,

I

C)

1 GP51 +

D)

1 +

GP51 + y’,,,

E)

1 _(

~51

PROTAPOD POD

antibodies

Gxl and Gx2 peptides were conjugated to keyhole limpet hemocyanin (KLH, Sigma) using maleimidocaproic N-succinimide ester (MCS, Sigma) as a coupling agent, whereas peptides MCFl , MCF2, MCF3, MCF4, MCFG, and MCF7 were linked to KLH using glutaraldehyde (GA, Merck). The coupling procedure of the 195205, 255-268, and 260-268 peptides was previously described (Portetelle et al., 1989b). Briefly, we prepared, for each peptide, solutions containing 5 mg of KLH in 1 ml of phosphate buffer, 0.1 M(0.9% NaCI, pH 8, for the GA method and 0.1 M NaCI, pH 7, for the MCS technique). In the GA procedure, to the 5 mg of KLH we simultaneously added 40 ~1 of 25% GA and 1 mg of peptide. After 5 hr under continuous stirring, free groups of GA were blocked using an excess of glycine and conjugates were stored at -20” in aliquots containing 1 mg KLH/ml. In the MCS procedure, 128 pg of MCS was diluted in dehydrated dimethylformamide (Gold DMF, Sigma) and added to 5 mg of KLH dissolved in 1 ml of phosphate buffer; the mixture was incubated for 1 hr at 30”. After dialysis on a SV 0.025pm membrane (Millipore) against phosphate buffer, peptide powder (2.5 mg) was added to the activated KLH solution. The mixture was stirred overnight, and aliquots were prepared and stored as described above. Rabbits were injected intradermally at 2 week intervals with 1 mg of equivalent carrier protein emulsified in complete Freund’s adjuvant (Sigma) for the first two injections and in incomplete Freund’s adjuvant (Sigma) for the last one. Blood was collected before each injection and 8 to 12 days after the last one. Serum samples were stored at -20”. Monoclonal

ET AL

assays (ELlSAs)

An ELISA was performed to determine rabbit antipeptide antibody titers using peptides adsorbed onto the wells (Portetelle eta/., 1989b). For antibody titration on whole BLV particles disrupted with N-octylglucoside or on gp51 captured by the MAb E, the binding assays were performed as described previously (Bruck er al., 1982a; Portetelle et a/., 1989b). Positive reactions were monitored with goat anti-rabbit immunoglobulins labeled with peroxydase (POD). Production and

+

+

: MAb;

-J----

: goat anti-mouse

+

PROT. A : S. aureus GP51 from disrupted

+ =

POD

POD

: antipeptide antibody: Ig;

POD : peroxydase

conjugates;

protein A. BLV &ions for B and C

GP51 from BLV producing cell line supematant

for D and E

FIG. 1. Schematic representation of the different ELISA systems used to map the sequential epitopes.

purification of BLV virions were essentially as described by Pot-tetelle et al. (1987). ELlSAs were also performed to test the direct recognition by the MAbs of the peptide conjugated to KLH (Fig. 1A). Wells of the plate were each coated with 5 rg of KLH-peptide in 100 ~1 of phosphate-buffered saline (PBS) and incubated overnight at 4”. Threefold serial dilutions (starting with 3.3 pg) of MAbs were prepared and incubated overnight at 4”. The presence of bound antibodies was revealed by POD-labeled protein A. Two types of competition systems were performed (a) between peptides and BLV for reaction with MAbs (Fig. 1 B) and (b) between rabbit antipeptide antibodies and MAbs for BLV-gp5 1 recognition (Fig. 1C). These assays were performed with BLV virions directly adsorbed onto the wells (200 ng of purified BLV first disrupted with 1% N-octylglucoside and, after 10 min at O”, diluted to 0.002% with PBS). Serial dilutions of peptide solution (starting with 20 pg) or of rabbit sera (starting with 30 ~1) were made before adding onto each well 50 ~1 (200 ng) of the competitor MAbs. The presence of bound antibodies was revealed by PODlabeled goat anti-mouse immunoglobulins. We also took advantage of these two competition systems using BLV gp51 linked to MAb coated on the plate (Figs. 1D and 1E). Briefly the wells of the plate were coated overnight with 300 ng of MAb. Supernatants of FLK (BLV-infected cells) cultures (100 ~1) were added to the wells for 2 days at 4”. The competition assay was then performed as described above except that the competitor MAb (1 pg in 50 ~1) was already conjugated to POD. All the plates used in these assays were 96-well Maxisorp lmmunoplates (Nunc). The PBS was 0.01 M

51

ANTIGENIC ANALYSIS OF THE BLV-gp51 GLYCOPROTEIN

sodium phosphate and 0.15 M NaCI, pH 7.4. After coating of the wells, they were saturated by 100 ~1 of 2% bovine serum albumin (BSA) solution in PBS. A total of 100 ~1 of a 4% Tween 80 solution in PBS was then added. Washings between incubations were done with PBS supplemented with 0.02% Tween 80. Peroxydase activity was determined by adding 100 ~1 of a solution of o-phenylenediamine (0.04%, Sigma) and H,O, (0.006%) in a citrate buffer at pH 5. Reaction was stopped by adding 100 ~1 2 N H,SO,. Optical density (OD) at 492 and 540 nm was measured by a Titertek Multiscan spectrophotometer MCC 340. Computer

analysis of sequences

For selection of some of the peptides used in this study, values of surface exposure, i.e., hydrophilicity, accessible surface, mobility, and flexibility, were considered and calculated according to algorithms developed by Hopp and Woods (1981) Janin (1979) Ponnuswamy and Bhaskaran (1984) and Karplus and Schultz (1985), respectively. The values were averaged over stretches of six amino acid residues for the first three criteria and over seven residues for flexibility; mean values were then attributed to the first residue of the hexa- or heptamer. Predictions of regions implied in loops and turns at the surface of the protein were realized by consideration of a hydrophobic cluster analysis (HCA) diagram (Lemesle-Varloot et a/., 1990), performed on a VAX computer with MANSEK software. This diagram provides a 2D representation of the sequence along a helical plot where, between the hydrophobic clusters characterizing the protein core, one finds loops characterized by high contents in hydrophilic residues as well as in Pro, Gly, Ser, and Thr. Predicted loops and turns combined with the simultaneous occurrence of high values for surface exposure were considered to be good candidates for potential antigenicity (Krchnak et al., 1989). Sequence alignments of the BLV gp51 with other proteins were accomplished using the bestfit (Devereux et al., (1984) and align (Sidman et a/., 1988) programs, correlated with the HCA diagrams of the two sequences. RESULTS Production

of rabbit antipeptide

antibodies

A panel of peptides chosen to cover the major part of the polypeptide backbone, with no consideration for the criterion of surface exposure, were synthesized: their exact sequences are indicated in Table 1. Each of them was injected as a KLH conjugate in rabbits to elicit production of antipeptide antibodies.

It appears that the antipeptide antibody titers are quite variable, depending on the rabbit, the coupling method (MCS or GA), and the coupling efficiency. This last factor may be influenced by the hydrophobic@ of the peptide, as is probably the case for MCF3 and Gxl . Moreover, the antibody titers were determined by coating the peptide directly to the plate; this method, which can hide sites recognized by the antipeptides, may lead to an underestimation of the real titer. Reactivity

of antipeptides

with the whole protein

Even at high titers, antipeptides do not necessarily recognize the whole antigen from which they derive because of (1) the inaccessibility of the region on the protein, (2) the structuration of this part of the antigen, or (3) conformational constraints exercised by the surroundings of the peptide in the protein. The results of the gp51 recognition assays are of interest to know whether the antipeptide antibody can react with the whole antigen and consequently is able to compete with the MAbs. Antipeptide antibodies directed toward MCF2, MCFG, MCF7, Gx2, and especially MCF4 exhibit significant recognition capacities for N-octylglucoside-disrupted BLV directly coated onto the plate (Table 1). The peptide sequence of MCF4 is recognized by the MAb E (see results below): when gp51 is presented by the MAb E as a binding antibody, MCF4 antipeptide weakly reacts with a site already occupied by the “competing” MAb. Antibodies against MCFl, MCF3, Gxl, Gx2, and 255-268 show significant ELISA responses toward gp51 bound to the MAb E (Table 1). Localization

of epitopes

defined by MAbs

Different ELISA systems were performed to localize the sequential epitopes recognized by our anti-gp51 MAbs (Fig. 1). None of the MAbs was able to react with the peptides directly coated onto the plate. Conjugates (KLH + peptide) used for the coating of the plate reacted positively with some MAbs, whereas they didn’t react with unrelated anti-gp51 MAbs at concentrations of MAbs and carrier of 7 and 5 rg, respectively. Some MAbs gave positive reaction with given conjugates without binding with the other conjugates. This system, allowing probably a better presentation of the peptides, led us to map epitope A on MCF7 (249-268) B on 195-205, D and D’ on MCF6 (218-237) and E on MCF4 (142-161) (Fig. 1A and Table 1). Peptides were tested in competition assays for their capacity to inhibit in solution the binding of MAbs to BLV virions adsorbed on the plate (Fig. IS). In these conditions, MCF6 (2 18-237) prevented the binding of

52

CALLEBAUT

log peptide

concentration (pg)

FIG. 2. Binding curves of anti-gp51 MAbs to BLV virions or to MAb E-gp51 in the presence of different gp51 oligopeptide concentrations. Binding to BLV virions (Fig. 1 B): (- A -) MCF7/MAb A; (- 0 -) MCFG/MAb D; (- w -) MCFG/MAb D’. Binding to MAb E-gp51 (Fig. 1D): (Cl) MCFG/MAb D; (0) MCFG/MAb D’. Negative controls (-) are based on the peptide recognition by MAbs not involved in the interaction with the epitope of interest.

MAbs D and D’ and MCF7 (249-268) the binding of MAb A (Fig. 2). At any concentration of peptides, no inhibition was established for the binding of these three MAbs with other gp51 peptides; MCF6 and MCF7 didn’t affect the binding of the other anti-gp51 MAbs. We observed that the MAb D’exhibits a greater affinity for its site on the peptide than the MAb D. Lack of competition for the other epitopes could be explained by a less-favored conformation of the peptide in solution than on a carrier (whole BLV or KLH). In the same way, antipeptide antibodies were tested for their ability to inhibit the binding of MAbs to BLV virions adsorbed on the plate (Fig. 1 C). We succeeded in showing the inhibition of binding of MAbs D and D’ by the anti-MCF6 (218-237) antibody and Mab E by the anti-MCF4 (142-161) antibody (Fig. 3). Negative controls here assessed the noninhibitor character of the other antipeptide antibodies for the reaction of MAbs D, D’, and E with the gp51. The anti-MCF4 and anti-MCF6 antipeptide antibodies did not hinder the gp5 1 recognition by other anti-gp51 MAbs. Finally, peptides and antipeptide antibodies were tested in two different sandwich ELlSAs for their capacity to inhibit the binding of MAbs to the gp51 not on the BLV virions but presented by each of the MAbs (Figs. 1 D, 1 E, 2, and 4).

ET AL.

Interestingly, only this type of system, with the MAb C as the binding antibody, allowed inhibition of the MAb B’ binding by the peptide 195-205 (Figs. 1 D and 4). Gp51 bound to the MAb C led also to the localization of epitope B on 195-205 and D/Don MCF6 (Figs. 1 D and 4). Epitopes D and D’ were also localized on MCF6 using the MAbs B’ and E as binding antibodies (Figs. 2 and 4). In all the systems used in our mapping experiments, the positions D and D’ were easily allocated to MCF6 (2 18-237). Epitope B’ was identified on 195-205 in particular conditions. These involved use of soluble peptide competing with gp51 bound to the MAb C, one of the conformational monoclonal antibodies. The presentation of gp5 1 on the MAb E, a system thought to present the gp51 in its native configuration, appeared in this case curiously unsatisfactory to map epitope B’. However, epitope B’ is one of the best recognized sites by the MAbs on the gp5 1 (Portetelle et al., 1989a). It follows that a decrease of the MAb B’ affinity for gp51 bound to the MAb C could allow in this case peptide recognition and consequently competition with the protein. DISCUSSION In the previous study by our group, using a synthetic peptide approach (Portetelle et al., 1989b), none of the

3

log (l/antipeptide

3.5

4

4.5

dilution)

FIG. 3. Binding curves of anti-gp51 MAbs to BLV virions in the presence of different antipeptide antibody concentrations (Fig. 1C). (- n -) anti-MCFG/MAb D; (- l -) anti-MCFG/MAb D’; (- A -) antiMCFUMAb E. Negative controls (-) are based on competition assays between the antipeptide antibody of interest and other MAbs not involved in the recognition of the concerned epitope.

ANTIGENIC

ANALYSIS

OF THE

FIG. 4. Binding curves of anti-gp61 MAb to gp51 bound to MAb C or MAb B’ in the presence of different gp51 oligopeptide concentrations (Fig. ID). MAb C as the binding antibody: (0) MCFG/MAb 0; (,4) MCFG/MAb D’; (- 0 -) 19%205/MAb B’; (- A -) 195-205/MAb B. MAb 8’ as the binding antibody: (- n -) MCFG/MAb 0; (0) MCF6/ MAb D’. Negative controls (-) are based on peptide recognition by MAbs not involved in the interaction with the epitope of interest.

sequential or conformational epitopes defined by MAbs on the BLV gp51 could be mapped on its amino acid sequence. After compilation of different results coming from gp51 urokinase digestion (Bruck et a/., 1982b), heterologous expression of envelope truncated proteins (Legrain et a/., 1989) and study of the amino acid sequence variation in the env gene (Bruck et al., 1984b; Portetelle et a/., 1989a), we used new peptides and new ELISA strategies that allowed us now to map all the sequential sites of the gp51. We show in this study that E is included in MCF4 (142161), B and B’ in 195-205, D and D’ in MCF6 (218237), and A in MCF7 (249-268) (Fig. 5). With MCF4

FIG. 5. Position the sequence.

of all of the gp51

peptides

available

for the study

BLVgp51

GLYCOPROTEIN

53

(142-l 61) epitope E was confirmed to lie in the central part of gp51, a proline-rich region thought to be a “hinge” between the amino and carboxy parts of the glycoprotein. Although epitope E shows a relatively low accessibility when gp51 is on the surface of the viral particle (Portetelle et a/., 1989a), the presentation of gp51 with MAb E led to a good recognition by the whole battery of MAbs. These two observations lead us to hypothesize that this region could be in interaction with the transmembrane glycoprotein gp30 so that both gp30 and MAb E share a similar effect for gp51 presentation. This property of MAb E is used in indirect and competitive ELlSAs for the titration of anti-gp51 antibodies (Portetelle et a/., 1989c). Despite the carboxy-terminal position of site A (in peptide MCF7: 249-268) a position which generally confers flexibility and can easily be mimicked by a peptide, recognition of the peptide by the MAbs was not easy to detect. It seems that the peptide in solution might adopt a peculiar conformation and may be favored by the relative abundance of serine, threonine, and arginine in this region. This particular behavior might hinder recognition by the corresponding MAb. The alcoholic side chains could also be subject to Oglycosylation; however, this possibility seems to be improbable in this particular case since antibodies against peptide 255-268 succeed in reacting very well with gp51 (Portetelle et al., 1989b). Only the longest peptide in the C-terminal region (MCF7: 249-268) gave significant results in the site A mapping. This fact doesn’t necessarily mean that site A could not lie also on the smaller peptides 255-268 and 260-268. Data supporting this hypothesis are provided by another study showing that two peptides overlapping in sequence but each containing the same epitope had different reactivities with different antibodies (Ralston et al., 1989). The data, coming from the present study and from a previous one (Portetelle et a/., 1989a), suggest that the E

Em

DO’

I

I I

I

+

f

and subsequent

localization

of the sequential

A

epitopes

(A, B, B’, D. D’. E) on

CALLEBAUT

54

extramembranous glycoprotein gp5 1 is divided into two extremely different’entities: the NH, part, apparently highly structured, harboring the conformational and biologically crucial epitopes opposed to the COOH part, carrying the sequential epitopes. All of our epitope localizations correspond to regions which are noticed by various criteria used for the prediction of surface exposure such as hydrophilic@ (Hopp and Woods, 1981), accessible surface (Janin, 1979), mobility (Ponnuswamy and Bhaskaran, 1984), and flexibility (Karplus and Schultz, 1985) (data not shown). Moreover, all of the sequential sites A, B’, B, D, D’, and E are located out of the hydrophobic clusters of the gp5 1, thus in relatively hydrophylic areas typical of loops (data not shown). Finally, the data presented in this report should be useful in predicting B-cell epitopes on the counterpart of gp51 in HTLV-1 and HTLV-2 with the help of alignment procedures. A sequential epitope recognized by a MAb (0.5a) having neutralizing capacities has been described on the glycoprotein gp46 of HTLV-1 (Matsushita et al., 1986). Ralston et al. (1989) have mapped this epitope between residues 186 and 195 of the HTLV-1 gp46. This sequence can be aligned by various procedures (bestfit, align, and hydrophobic cluster analysis) on the region of gp51 corresponding to the epitope E. Although the MAb E is not neutralizing, rabbit sera against peptides 144-l 55 and 144157, both sequences included in MCF4 recognized by the MAb E, show some ability to inhibit VSV-BLV pseudotypes (Portetelle et al., 1989b). This fact is in favor of according some importance to this region in both viruses, BLV and HTLV, for the cell entry mechanism. In conclusion, we succeeded, using synthetic peptides, in identifying and delineating the locations of all the sequential epitopes defined by anti-gp51 MAbs. This approach remains now to be completed by the mapping of the exact positions of conformational sites. This goal will be approached by studying a panel of BLV variants. ACKNOWLEDGMENTS I.C. is grateful to Professor A. Tartar for teaching peptide chemistry in his laboratory, to Dr. R. Kettmann for critical review of the manuscript, and to M. Prbvot for preparation of the figures. I.C. is a research assistant at the Belgian National Fund for Scientific Research (FNRS).

REFERENCES BRUCK,C., MATHOT, S., PORTETELLE,D., BERTE.C., FRANSSEN,J. D., HERION,P., and BURNY,A. (1982a). Monoclonal antibodies define eight independent antigenic regions on the bovine leukemia virus (BLV) envelope glycoprotein gp51. Virology 122, 342-352. BRUCK,C., PORTETELLE,D., BURNY,A., and ZAVAOA, J. (1982b). Topo-

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