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Mapping and structural analysis of B-cell epitopes on the morbillivirus nucleoprotein amino terminus

¹ Animal Production Unit, FAO/AIEA Agriculture and Biotechnology Laboratory, IAEA Laboratories, A-2444 Seibersdorf, Austria
² CIRAD-Département EMVT, UPR ‘Contrôle des maladies animales et exotiques’, TA/30G, Campus International de Baillarguet, F-34398 Montpellier Cedex 5, France

Correspondence
G. Libeau
genevieve.libeau{at}cirad.fr

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
By analysing the antigenic structure of the morbillivirus nucleoprotein (N) using a competitive-binding assay of monoclonal antibodies (mAbs), six different antigenic sites were identified previously. By using Pepscan methodology complemented by analysis of truncated N proteins, a better characterization of five of these antigenic sites was provided: I, II, III, IV and VI. mAbs specific to Rinderpest virus, defining antigenic sites II, III and IV, and those common to four morbilliviruses, delineating sites I and VI, were analysed in the present study. It was found that all but one mapped to the same region, between aa 120 and 149 of N. However, the mAb 3-1 epitope was located in the carboxy-terminal region (aa 421–525). This result may indicate the high immunogenicity of the amino-terminal variable region, at least in the mouse. It was surprising that the epitope of mAb 33-4, antigenic site VI, which recognized all morbilliviruses so far tested, was located in one of the two non-conserved regions between morbillivirus N proteins. It is shown that the conserved amino acid motif ¹²⁶EAD¹²⁸----¹³¹F-------¹⁴⁸EN¹⁴⁹ is critical for epitope constitution and recognition.

The GenBank/EMBL/DDBJ accession numbers for the sequences described in this study are EF186057–EF186059.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Rinderpest is a highly contagious viral disease of animals belonging to the order Artiodactyla, although cattle and buffaloes are the most susceptible hosts. The disease is characterized clinically by pyrexia, erosive then necrotic stomatitis, profuse diarrhoea and death (Plowright, 1968). The causal agent, Rinderpest virus (RPV), belongs to the genus Morbillivirus within the family Paramyxoviridae (Bratt & Hightower, 1977). Other members of this genus are Measles virus (MV), Peste-des-petits-ruminants virus (PPRV), Canine distemper virus (CDV), Phocine distemper virus (PDV) and cetacean morbilliviruses. With the success of the Global Rinderpest Eradication Programme (GREP), the FAO (Food and Agriculture Organization of the United Nations)-led international partnership programme, rinderpest is nearly eradicated from the world, with only an area in East Africa that needs further verification. To facilitate the implementation of that programme, two competitive ELISAs (cELISAs) were developed for both seromonitoring of the vaccination campaign and serosurveillance of the disease (Anderson et al., 1990; Libeau et al., 1992). These assays were designed to differentiate between the two ruminant morbillivirus diseases: rinderpest and peste des petits ruminants. They were based on the use of monoclonal antibodies (mAbs) directed against either the viral haemagglutinin (H) or the nucleoprotein (N). H is the viral protein that binds to the cell receptor and against which most of the virus-neutralizing antibodies are directed (Choppin & Scheid, 1980; Merz et al., 1981; Giraudon & Wild, 1985). N is the most abundant and immunogenic viral protein (Barrett et al., 2006).

Sequence comparison of N within the genus Morbillivirus identified three main regions of differing similarity (Diallo et al., 1994): the amino-terminal region of medium similarity, the highly conserved central region and the poorly conserved carboxy-terminal domain covering the last 105 aa. Buckland et al. (1989) showed that mAbs specific to the amino and carboxy termini of the N protein of MV mapped to the least conserved amino acid sequences. Paradoxically, the carboxy-terminal tail of N may be involved in the immunosuppressive effects of morbilliviruses and the host immune response regulation (ten Oever et al., 2002; Zhang et al., 2002; Laine et al., 2003). The antigenic structure of N of both RPV and PPRV was studied by using the strategy of competition between mAbs. This approach has allowed the identification of six antigenic sites, of which four had epitopes that clearly distinguished between RPV and PPRV strains. Some of these mAbs have helped in the development of tests to differentiate between the two viruses (Libeau et al., 1992, 1994, 1995, 1997). Experience has shown, however, that the cELISA that was developed by using one of the specific RPV N mAbs (Libeau et al., 1992) for the serological diagnosis of rinderpest cross-reacts with PPRV antisera. Therefore, we decided to perform a more specific analysis of the antigenic and immunogenic properties of the RPV N protein.

The objective of this study was to use a combined strategy of truncated mutants and overlapping synthetic dodecapeptides, covering aa 115–150 (amino terminus) and 415–495 (carboxy terminus) corresponding to the RBOK strain (GenBank accession no. CAA83177 [GenBank] ), to map the epitope structure of sites I–IV and VI defined by mAbs on the N protein sequence more precisely.

	METHODS

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Virus and cells.
The different RPV strains used in this study were the attenuated vaccine RBOK strain (Plowright, 1962), RPV Saudi, RPV Egypt, RPV Kuwait and RPV RBT1 (Taylor, 1986). They were propagated in Vero cells cultivated in Eagle’s minimal essential medium (MEM) supplemented with 5 % fetal bovine serum and 1 % antimycotics and antibiotics.

mAbs and sera.
Five antigenic site-specific mAbs, IIH2 (I), 48-5 (II), 3-1 (III), IVB2 (IV) and 33-4 (VI) directed against the N protein of RPV RBOK strain, were used along with the anti-PPRV N mAb 38-4 (Libeau et al., 1997). The reactivity of positive RPV antisera [a rabbit rinderpest hyperimmune serum (RHS; a gift from J. Anderson, Institute for Animal Health, UK), a cattle serum against the Kudu strain (lineage II, gift from H. Wamwayi, Kenya Agricultural Research Institute, Kenya) and a naturally infected cattle serum from Chad] were analysed. Additionally, a goat serum against the vaccine strain of PPRV, Nigeria 75-1, was used in this study.

Cloning of the RPV RBOK N gene and transient expression in Vero cells.
Vero cells were infected with the attenuated vaccine RBOK strain of RPV (Plowright, 1962). At 40–50 % cytopathic effect, the cells were lysed and total RNA was extracted by using an RNeasy RNA extraction kit (Qiagen) according to the manufacturer’s instructions. Following RNA extraction, cDNA was synthesized by reverse transcription using a First Strand cDNA synthesis kit and random primers (Amersham Biosciences). This cDNA was used to amplify the full open reading frame of the N gene by using the forward and reverse primers N-PR7 (5'-GATCCTATCGACTGGAGCAAGCTTA-3') and N-PR8 (5'-GGCCTTTGTTGACATGGTAGGCT-'3) with Taq polymerase. The PCR product was cloned into the pGEM-T plasmid (Promega) and the sequence of the insert was confirmed by sequencing. This insert was released by double digestion with restriction enzymes DraI/NotI and recloned into the eukaryotic expression vector pcDNA4/HisMax.B (Invitrogen) previously digested with EcoRV and NotI. In the resulting plasmid, pCN-RBOK, the insert encoding the full-length RPV N protein was placed under the control of the cytomegalovirus promoter. From this plasmid, two truncated mutants named pCN121–146 and pCN421–525 were generated by PCR mutagenesis (Ailenberg & Silverman, 1997) using phosphorylated primers. Reverse and forward primers 120 R (5'-P-ACCCCTAGAGGCAAATGTC-3') and 146 N (5'-P-TGGTTTGAGAATCGAGATA-3') led to the deletion of aa 121–145 in the amino-terminal half, whilst reverse and forward primers 420 R (5'-P-AAGGAATGAAACCTGGGCCTGTTTGG-3') and RTAG (5'-P-AACTGAGTGAGTGCCCCGCA-3') led to the deletion of aa 421–525 in the carboxy-terminal half. Escherichia coli DH5 cells were used to propagate the various plasmids generated in this study. For downstream use, all plasmids were purified from bacteria by using a Qiagen EndoFree plasmid DNA preparation kit. For the transient expression of RPV N protein, Vero cells in 96-well plates (10 000 cells per well) were transfected with either pCN-RBOK plasmid or the truncated mutants by using Fugene 6 transfection reagent (Roche) according to the manufacturer’s instructions. After 72 h incubation at 37 °C, the transfected cells in the 96-well plate were fixed with 80 % acetone for 30 min at –20 °C and subjected to indirect immunofluorescence assay (IFA) to assess RPV N protein expression using the following antibodies: RHS, anti-RPV N mAbs IIH2, 48-5, 3-1, 33-4 and IVB2 and anti-PPRV N protein mAb 38-4 (Libeau et al., 1997). mAbs and RHS were used at a dilution of 1/100. The cells were incubated with 50 µl test antibody for 30 min at 37 °C, then washed with PBS before incubation with 50 µl anti-mouse and anti-rabbit fluorescein isothiocyanate conjugates (Bio-Rad) diluted 1/80. After three washes with PBS, they were examined under a Canon inverted fluorescence microscope.

Western immunoblot.
Cells transfected with the plasmid encoding the full-length RPV N protein, pCN-RBOK, or the truncated mutants pCN120–146 or pCN421–525 were lysed in RIPA buffer [100 mM Tris/HCl (pH 7.4), 150 mM NaCl, 1 % deoxycholate, 1 % Triton X-100, 0.1 % SDS, 1 mM PMSF] and analysed by SDS-PAGE. The proteins were transferred to a PVDF membrane 0.2 µm (Invitrogen) followed by immunostaining. The proteins were probed for 1 h with the primary antibody, RHS or mAb, at an appropriate dilution in PBS (pH 7.4) containing 0.5 % Tween and 5 % skimmed milk. Immune complexes were detected by using a secondary anti-rabbit antibody or anti-mouse immunoglobulin conjugated to horseradish peroxidase and subsequent detection using the ECL system (Amersham Biosciences) according to the manufacturer’s instructions.

Nucleotide sequencing of the N gene from wild-type strains of RPV.
RNA was extracted from Vero cells infected with different RPV strains (Saudi, Egypt, Kuwait or RBT1). The extraction was done as indicated above. The first-strand cDNAs were synthesized using random primers. For each virus, the coding sequence of the N gene was amplified by RT-PCR using forward and reverse N-PR7 and N-PR8 primers and cloned into the pGEM-T vector. The sequence of the entire N gene was obtained by using different internal primers. Forward primers were N-PR9, 5'-CAGCATTAAATTGGTGGAGGTA-3'; N-PR11, 5'-AACAAATGGGTCAACTGGCTC-3'; and N-PR13, 5'-ACAGGCCCAGGTTTCATTCCTGCG-3'; and reverse primers were N-PR6, 5'-GGTAGGCTTGCTCCTCTGCCAT-3'; N-PR14, 5'-ACCTCCACCAATTTAATGCGA-3'; and Nad2, 5'-GATTGAGTTCTCTAAGATCACCAT-3'. Cycle sequencing was performed by using dye-labelled terminators and Taq DNA polymerase followed by analysis on an ABI Prism 377 automatic sequencer (Applied Biosystems). By using the Vector NTI 9 package (Informax), amino acid changes between aa 115 and 150 of the N protein were evaluated by alignment of deduced amino acid sequences obtained for the above RPV strains with those of RPV RBOK, RPV lapinized strain, RPV Kuwait, PPRV 75-1, CDV Onderstepoort and MV Edmonston (GenBank accession numbers CAA83177 [GenBank] , P37708 [GenBank] , Z34262 [GenBank] , CAA52454 [GenBank] , P04865 [GenBank] and AAF85675 [GenBank] , respectively). GenBank accession numbers for sequences obtained in this study are EF186057 [GenBank] –EF186059 [GenBank] for RPV Egypt, RBT1 and Saudi, respectively.

Computer-aided analysis.
The sequence–structure relationship of RPV strain RBOK N protein was built automatically by different programs that allowed the prediction of protein secondary structure according to amino acid sequence: PROFSEC (Rost, 2001), SUBSEC (Rost, 2001), GOR4 (Garnier et al., 1996), PREDATOR (Frishman & Argos, 1996), PSIPRED (Jones, 1999), JNET (Cuff & Barton, 2000) and SSPRO (Baldi et al., 1999). The consensus secondary structure from all of these programs was used for further analysis.

Immunoassay with cellulose-bound peptides (Pepscan).
Cellulose-bound peptides were obtained by Fmoc amino acid chemistry (Synt : em). The Pepscan method used to locate the antibody-reactive peptides has been described previously (Mahé et al., 2000). Synthetic dodecapeptides overlapping by one residue and covering the sequence of N of the RBOK vaccine strain of RPV at positions 115–150 (amino terminus) and 415–495 (carboxy terminus) were examined. Antibody reactivity with Pepscan peptide was measured by indirect ELISA according to the manufacturer’s instructions (SYNT : em) using mAb ascitic fluids (1 : 50–1 : 100) and sera (1 : 10–1 : 50) in blocking buffer (Genosys). Bound antibodies were detected on the membrane with an anti-mouse, anti-goat or anti-cattle alkaline phosphatase conjugate (Sigma) according to the origin of the primary antibody involved. The binding of the conjugate was further revealed by a precipitating phosphatase substrate (BCIP sodium salt, MMT and MgCl₂) that produced blue-coloured spots. A known peptide and its antiserum were used as a positive control and were subjected systematically to the different steps of the immunoassay. Reuse of the membrane was made possible by treatment with dimethylformamide, 6 M urea and 10 % acetic acid in ethanol.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Transient expression of deletion mutants of the N protein
We speculated that the different antigenic sites defined on the N proteins of RPV and PPRV (Libeau et al., 1997) might be located within one of the two non-conserved regions of the N protein: aa 121–145 or 421–490. In order to map these antigenic sites, two plasmids able to express truncated proteins in eukaryotic cells were constructed. Vero cells were transfected with these plasmids and another expressing the full-length RPV N. Control of protein expression was assessed by indirect immunofluorescence using RHS. Each of the three transfected plasmids gave positive signals. No staining was observed with the negative serum or mAb 38-4, which is specific to the PPRV N protein. All of the other five mAbs specific to RPV N exhibited immunofluorescence staining on cells transfected with the full-length N protein plasmid pCN-RBOK and with one of the truncated mutant plasmids. Four of these mAbs (IIH2, 48-5, 33-4 and IVB2) reacted with the carboxy-terminally truncated mutant pCN421–525, whilst mAb 3-1 reacted with the amino-terminally truncated mutant pCN120–146 (Fig. 1). We confirmed the epitope localization of mAbs 33-4, 3-1 and IVB2 by Western blotting analysis of the different RPV N proteins expressed in Vero cells (Fig. 2). The antigenic characteristics of the full-length and truncated N proteins were examined on the basis of their reactivity with RHS (Fig. 2e), showing a strong cross-reaction. The reactivity pattern of the mixed mAbs was comparable to that of RHS (Fig. 2d). Neither mAb IIH2 nor mAb 48-5 bound to the N proteins in this test under denaturing conditions, an indication that they may be directed against a conformational epitope. We concluded that mAbs delineating sites I, II, IV and VI were located in the region comprising aa 121–145, whereas the mAb 3-1 epitope, defining site III, was located in aa 421–525 (see Table 1). To confirm these results, a peptide-mapping study in the amino-terminal part of the N protein was conducted.

View larger version (105K): [in this window] [in a new window]   Fig. 1. Indirect immunofluorescence microscopy of Vero cells analysed for intracellular transient expression of the full-length N protein of the RBOK strain of RPV (pCN-RBOK), the amino-terminally truncated protein pCN120–146 or the carboxy-terminally truncated protein pCN421–525. Seventy-two hours after transfection, cells were permeabilized and stained with the panel of anti-RPV mAbs (IIH2, 48-5, 3-1, IVB2 and 33-4). Non-transfected Vero cells were used as a negative control.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Location of mAbs by Western blotting on the full-length N (lane 1) and on the deleted mutant proteins, N120–146 (lane 2) and N421–525 (lane 3), in transfected Vero cells. RBOK N proteins were separated by gel electrophoresis, blotted onto a membrane and detected by various antibodies: (a) mAb 33-4, (b) mAb 3-1, (c) mAb IVB2, (d) a mix of mAbs 3-1, 33-4 and IVB2, (e) RHS.

View larger version (99K): [in this window] [in a new window]   Fig. 3. Reactivity of consecutive overlapping dodecapeptides spanning aa 115–150 (amino terminus) and aa 415–495 (carboxy terminus) of the N protein of the RBOK strain of RPV with mAbs. Membranes were incubated with an RPV-specific mAb, IIH2 (a), or a broadly reactive mAb, 33-4 (b). Dark-coloured spots indicate the contribution to binding of the mAb. A positive control (isolated peptide) and its antiserum was submitted systematically to the different steps of the immunoassay.

View larger version (37K): [in this window] [in a new window]   Fig. 4. Determination of the fine specificity of mAbs by indirect ELISA . Synthetic dodecapeptides (overlapping by one residue) spanning aa 115–150, corresponding to the variable amino-terminal sequence in the N protein of RPV, were tested on a derivative membrane to identify reactive peptides. Shaded areas correspond to significant binding. Residues in red delineate the recognized epitope.

View larger version (22K): [in this window] [in a new window]   Fig. 5. Indirect immunofluorescence of RPV with mAbs (b) has been published previously (Libeau et al., 1997). Alignment sequence of aa 115–150 of N proteins of different strains of RPV (a). The sequence data for RPV Egypt, RBT1 and Saudi were determined in this study (GenBank accession nos EF186057–EF186059, respectively) and those for RPV RBOK, RPV Kuwait, PPRV 75-1, CDV Onderstepoort and MV Edmonston were obtained from GenBank with accession numbers CAA83177, Z34262, CAA52454, P04865 and AAF85675, respectively. The sequence of the lapinized strain of RPV was published by Kamata et al. (1991) with GenBank accession no. P37708. Blocks in dotted lines delimit a common epitope and shaded areas are shared amino acid sequences.

The delimitation of the mAb interfaces and the residues identified above as important for the recognition of RBOK N are represented schematically in Fig. 5(b). Important residues were also located on peptide 6 (¹²⁰G) and on peptide 23 (¹³⁷N) for mAb IIH2. Therefore, two antigenic motifs were defined. The major one was delimited on peptide DEADRYFTYEEPND from ¹²⁵D to ¹³⁸D, defined as a common antigenic peptide recognized by all mAbs. In the case of mAb IIH2, it was extended to ¹²⁰G. Close to this main sequence, two residues (¹⁴⁷F and ¹⁴⁸E) contributed to the epitope recognition defined below.

Identification by sequence analysis of residues required for efficient binding of the N protein of different RPV strains
mAbs IIH2, 48-5, IVB2 and 33-4 were mapped onto the aa 120–149 sequence of N deduced from the RBOK strain. The pattern of reactivity of different strains of RPV and representatives of PPRV, CDV and MV to mAbs in the IFA was described in a previous publication (Libeau et al., 1997) and is summarized in Fig. 5(b). The four mAbs reacted with the N protein of all of the RPV strains, although mAb 48-5 failed to recognize that of RBT1. Except for mAb 33-4, none of these mAbs reacted with PPRV. mAbs IIH2 and 33-4 reacted with CDV and MV. Consequently, to demonstrate whether the sequence variation among the strains was helpful to determine framework residues contributing to the difference in reactivity, the cDNA corresponding to the targeted area for the different RPV strains was amplified and sequenced. The following RPV strains were used: Saudi, Egypt, Kuwait and RBT1. The deduced amino acid sequences were aligned with published data of the N protein of RPV (RBOK and lapinized strain), PPRV, CDV and MV (GenBank accession numbers CAA83177 [GenBank] , P37708 [GenBank] , CAA52454 [GenBank] , P04865 [GenBank] and AAF85675 [GenBank] , respectively) (Fig. 5b). Reactive amino acids that were shared between all morbilliviruses were not considered as effective when the mAb had a differential reactivity. The remaining residues were then compared to define the minimum amino acid sequence involved in the difference in reactivity among strains.

mAb IIH2 reacts with RPV, CDV and MV. It gave a negative result with PPRV 75-1, but reacted with some other PPRV strains (G. Libeau, unpublished data). Therefore, a conserved sequence could be involved in the delineated epitope with the contribution of a variable residue. ¹²⁴D and/or ¹²⁵D, which are conserved among RPV, PPRV, CDV and MV strains, could be involved. Taken together with the previously identified residues, the contributing motif for IIH2 was found to be ¹²⁰G----¹²⁵D---¹³¹F-¹³³Y---¹³⁷ND¹³⁸ and ¹⁴⁸E.

mAb 48-5 reacted with all RPV strains except RBT1. In the area of peptides recognized by this mAb, there are three main amino acid changes between RBT1 and other RPV strains: ¹³¹L, ¹³⁶S and ¹⁴⁴F in the protein sequence alignment. In the same region, four other amino acids are not conserved between different RPV strains: ¹³⁴E for RBOK, ¹³⁵F for RPV Saudi, ¹³⁸G for RPV lapinized and ¹⁴³R for RPV Egypt. In total, within the sequence between aa 131 and 144, there are seven changes. In addition to this region, the cooperation of the motif ¹⁴⁷FE¹⁴⁸ was found to be necessary for an efficient reaction with the RPV N protein. The motif recognized by mAb 48-5 was therefore determined to be ¹²⁵D---RYFTY--PND^{138 144}S-FE¹⁴⁸. As this does not react with RPV RBT1 strains, the three amino acid changes noted for this strain, ¹³¹L, ¹³⁶S and ¹⁴⁴F, might be critical for the formation of the epitope

By using the same deductive method, the contributing motifs for mAb IVB2 were found to be ¹²⁵D---RYFTY---ND^{138 145}Y-F¹⁴⁷. mAb 33-4 is different from the preceding mAbs in that it recognizes different morbilliviruses, including MV, CDV and PPRV (DMV was not tested). Alignment analysis between the four viruses suggests that the amino acid sequence implicated in the interaction of antigen with this mAb was ¹²⁵DEAD--F-Y---ND^{138 146}WFEN¹⁴⁹.

Antibody-binding sequence comparison between mouse mAbs and sera from immunized or convalescent goats and cattle: correlation with the secondary structure of the epitopes
To determine whether the epitopes that we identified within the amino-terminal region of RBOK N are immunodominant in ruminants, we compared the peptide-reactivity pattern obtained with the different mAbs studied with those with sera from immunized or convalescent goats and cattle. To this end, five different sera were analysed. The history of the sampled animals is as follows: a bovine that recovered from rinderpest during an outbreak in Chad in 1987, an RPV RBOK-vaccinated bovine, a bovine infected experimentally with an RPV strain isolated from an antelope (RPV Kudu strain; H. Wamwayi, personal communication), a goat inoculated with RPV Saudi strain and another vaccinated with the attenuated PPRV strain 75-1. Sera were analysed by indirect ELISA on the peptide-spotted membrane and the results are summarized in Table 2. The convalescent serum had a generally strong reaction, with maximum reactivity in the region encompassing residues 126–133, whereas residues 145–147 did not appear to be involved in the binding. The other sera had weak (RBOK), moderate (lineage II) or strong (Saudi) overall reactivity; they also reacted with peptides located in the region of aa ¹²⁹RYFTY¹³³, demonstrating that these amino acids were strongly immunodominant for the humoral response to RPV infection. The similar reactivity observed for the four anti-RPV animal sera suggests that the immunogenicity of the region encompassing aa 129–133 is conserved among the virus strains and corresponds to the recognition sites of the mAbs. On the other hand, the anti-PPRV goat serum had weak or no reaction with the region encompassing these residues, confirming the difference in the amino acid sequence. The slight reactivity observed with peptides covering the variable sequence 137–143 could be explained by a similar amino acid conformation or a cross-reactivity with an unknown RBOK sequence. Overlapping synthetic peptides covering aa 415–495, corresponding to the variable sequence at the carboxy terminus of the N protein of the RPV RBOK strain, analysed with the ruminant sera also indicated that this region induced a strong immune response (data not shown). The efficiency of a mAb in cELISA to detect the humoral response to RPV is primarily due to the sharing of the same recognition epitope between mAbs and immune sera. Hypothetically, the immune response against a different and non-competitive epitope should not be taken into account.

View larger version (27K): [in this window] [in a new window]   Fig. 6. Secondary-structure prediction of the N protein of the RBOK strain of RPV. A sequence consensus predicting probable secondary structures and folding classes along the polypeptide chain was deduced from the programs PROFSEC (Rost, 2001), SUBSEC, GOR4 (Garnier et al., 1996), PREDATOR (Frishman & Argos, 1996), PSIPRED (Jones, 1999), JNET (Cuff & Barton, 2000) and SSPRO (Baldi et al., 1999). Structure elements (-sheets, -helices and loops) are mapped onto the sequence.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

For all mAbs studied except one, the epitopes that they bind to are within the same area, although they defined different binding sites by absence of mutual competition. mAbs IVB2 and 33-4 were directed against linear epitopes, whereas mAbs IIH2 and 48-5 demonstrated anti-conformational reactions. These two last mAbs may therefore recognize residues distant in sequence from the short, variable amino-terminal cluster, but near their three-dimensional position. The sequential epitope defined by peptide mapping is part of a more complex conformational epitope and explains the lack of competition between these two mAbs and the anti-sequential mAb as defined in the precedent work (Libeau et al., 1997). Nevertheless, we defined for these four mAbs a major antigenic motif, DEADRYFTYEENPND, from ¹²⁵D to ¹³⁸D. To confirm the prominent immunogenic role of this short sequence in the amino-terminal part of RPV N, in silico sequence–structure analysis was performed and this identified an -helical configuration between ¹²⁴D and ¹³³Y, thus defining overlapping residues as essential for mAb recognition. The presence of structures such as short -helices or -turns seems to be critical for increased flexibility and enhanced immunogenic properties of epitopes, due to a better activation of the immune system. Indeed, Alba et al. (2003) demonstrated for Plasmodium falciparum that peptides presenting an -helical fragment between residues 5 and 10 maintained greater flexibility in the rest of the molecule and were immunogenic and protective. Although the region comprising aa 115–150 of N has low similarity between morbilliviruses, we mapped the epitope recognized by mAb 33-4 (site VI) in that region. This antibody is the only mAb to recognize all morbilliviruses and its binding site is composed of residues that are, in fact, well conserved within the genus: ¹²⁶EAD¹²⁸----¹³¹F-------¹⁴⁸EN¹⁴⁹.

Among our mAbs, those determining sites II, III and IV were the most interesting for use in a cELISA for differential serodiagnosis between rinderpest and peste des petits ruminants. Indeed, mAb IVB2, belonging to the group directed against site IV, has already been used in such a test (Libeau et al., 1992). However, whilst unable to react with PPRV strains in an immunofluorescence test, mAb IVB2 showed a cross-reaction of approximately 10 % with anti-PPRV sera in the cELISA format (data not shown). Cross-reactivity among morbilliviruses is known to be important and has hampered the development of highly sensitive and specific serological tests to differentiate between RPV and PPRV infections, two serologically related infectious agents of ruminants that also give rise to similar clinical symptoms. When we developed cELISA tests for the detection of the humoral response to RPV or PPRV, high sensitivity and specificity depended primarily on the correspondence of recognition sites of the specific mAb and antibody from natural immunity response to the virus. In this study, sharing of the recognition epitope between mAb IVB2 and anti-RPV sera from ruminants was demonstrated, whereas it was shown not to be the case with PPRV antisera. Indeed, looking at the 10 aa that are critical for the antigenic site recognized by mAb IVB2, only three residues, ¹³⁰Y, ¹³¹F and ¹⁴⁷F, are conserved between RPV and PPRV and are insufficient to create an antigenic epitope for cross-reactivity. Thus, in the absence of sequence identity between PPRV and RPV in the so-defined immunodominant region of the amino-terminal variable region of N, the serological cross-reactivity observed between RPV and PPRV with the IVB2 mAb-based cELISA could be explained by steric hindrance for the recognized epitope, due to the proximity of an epitope common to both RPV and PPRV in the conserved regions upstream of aa 120 and downstream of aa 145. In an attempt to alleviate the steric hindrance, three histidine residues were inserted between aa 120 and 121, and three others between aa 145 and 146. The N mutant that was obtained was not recognized by mAb IVB2 (S. C. Bodjo, unpublished data). The insertion of the six histidine residues has probably introduced a dramatic change in the conformation of the epitope. The major antigenic motif delimited on peptide DEADRYFTYEEPND bearing the -helix structure is therefore a promising candidate for consideration as an antigen for peptide-based ELISA diagnostic tools. Practically, the diagnostic capacity of small polypeptides or synthetic peptides composed of this antigenic motif remains to be validated in the indirect ELISA format by using extended RPV and PPRV sera.

	REFERENCES

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Ailenberg, M. & Silverman, M. (1997). Site-directed mutagenesis using a PCR-based staggered re-annealing method without restriction enzymes. Biotechniques 22, 624–630.[Medline]

Alba, M. P., Salazar, L. M., Puentes, A., Pinto, M., Torres, E. & Patarroyo, M. E. (2003). 6746 SERA peptide analogues immunogenicity and protective efficacy against malaria is associated with short alpha helix formation: malaria protection associated with peptides alpha helix shortening. Peptides 24, 999–1006.[CrossRef][Medline]

Anderson, J., McKay, J. A. & Butcher, R. N. (1990). The use of monoclonal antibodies in the competitive ELISA for the detection of antibodies to rinderpest and peste des petits ruminants viruses. In The Seromonitoring of Rinderpest throughout Africa – Phase One (Proceedings of the Final Research Coordination Meeting of the FAO/IAEA/SIDA/OUA/IBAR/PARC Coordinated Research programme). IAEA-TECDOC vol. 623, pp. 43–45. Edited by M. H. Jeggo. Vienna: Joint FAO/IAEA Division.

Baldi, P., Brunak, S., Frasconi, P., Soda, G. & Pollastri, G. (1999). Exploiting the past and the future in protein secondary structure prediction. Bioinformatics 15, 937–946.[Abstract/Free Full Text]

Barrett, T., Banyard, A. & Diallo, A. (2006). Molecular biology of the morbilliviruses. In Rinderpest and Peste des Petits Ruminants, pp. 31–67. Edited by T. Barrett, P.-P. Pastoret & W. Taylor. London: Elsevier Academic Press.

Bratt, M. A. & Hightower, L. E. (1977). Genetics and paragenetic phenomena of paramyxoviruses. In Comprehensive Virology, vol. 9, pp. 457–533. Edited by H. Frankel-Conrat & R. R. Wagner. New York: Plenum.

Buckland, R., Giraudon, P. & Wild, F. (1989). Expression of measles virus nucleoprotein in Escherichia coli: use of deletion mutants to locate the antigenic sites. J Gen Virol 70, 435–441.[Abstract/Free Full Text]

Choi, K. S., Kwon, C. H., Choi, C. U., Lee, J. G. & Kang, Y. B. (1998). Biological properties of attenuated rinderpest virus (LATC strain) adapted in Vero cell. RDA J Vet Sci 40, 61–70.

Choi, K. S., Nah, J. J., Ko, Y. J., Choi, C. U., Kim, J. H., Kang, S. Y. & Joo, Y. S. (2003a). Characterization of antigenic sites on the rinderpest virus N protein using monoclonal antibodies. J Vet Sci 4, 57–65.[Medline]

Choi, K. S., Nah, J. J., Ko, Y. J., Kang, S. Y. & Joo, Y. S. (2003b). Localization of antigenic sites at the amino-terminus of rinderpest virus N protein using deleted N mutants and monoclonal antibody. J Vet Sci 4, 167–173.[Medline]

Choi, K. S., Nah, J. J., Ko, Y. J., Kang, S. Y., Yoon, K. J. & Joo, Y. S. (2004). Characterization of immunodominant linear B-cell epitopes on the carboxy terminus of the rinderpest virus nucleocapsid protein. Clin Diagn Lab Immunol 11, 658–664.

Choppin, P. W. & Scheid, A. (1980). The role of viral glycoproteins in adsorption, penetration, and pathogenicity of viruses. Rev Infect Dis 2, 40–61.[Medline]

Cuff, J. A. & Barton, G. J. (2000). Application of multiple sequence alignment profiles to improve protein secondary structure prediction. Proteins 40, 502–511.[CrossRef][Medline]

Diallo, A., Barrett, T., Barbron, M., Meyer, G. & Lefevre, P. C. (1994). Cloning of the nucleocapsid protein gene of peste-des-petits-ruminants virus: relationship to other morbilliviruses. J Gen Virol 75, 233–237.[Abstract/Free Full Text]

Frishman, D. & Argos, P. (1996). Incorporation of non-local interactions in protein secondary structure prediction from the amino acid sequence. Protein Eng 9, 133–142.[Abstract/Free Full Text]

Garnier, J., Gibrat, J.-F. & Robson, B. (1996). GOR method for predicting protein secondary structure from amino acid sequence. Methods Enzymol 266, 540–553.[Medline]

Giraudon, P. & Wild, T. F. (1985). Correlation between epitopes on hemagglutinin of measles virus and biological activities: passive protection by monoclonal antibodies is related to their haemagglutination inhibiting activity. Virology 144, 46–58.[CrossRef][Medline]

Giraudon, P., Jacquier, M. F. & Wild, T. F. (1988). Antigenic analysis of African measles virus field isolates: identification and localisation of one conserved and two variable epitope sites on the NP protein. Virus Res 10, 137–152.[CrossRef][Medline]

Heaney, J., Barrett, T. & Cosby, S. L. (2002). Inhibition of in vitro leukocyte proliferation by morbilliviruses. J Virol 76, 3579–3584.[Abstract/Free Full Text]

Heaney, J., Cosby, S. L. & Barrett, T. (2005). Inhibition of host peripheral blood mononuclear cell proliferation ex vivo by Rinderpest virus. J Gen Virol 86, 3349–3355.[Abstract/Free Full Text]

Jones, D. T. (1999). Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol 292, 195–202.[CrossRef][Medline]

Kamata, H., Tsukiyama, K., Sugiyama, M., Kamata, Y., Yoshikawa, Y. & Yamanouchi, K. (1991). Nucleotide sequence of cDNA to the rinderpest virus mRNA encoding the nucleocapsid protein. Virus Genes 5, 5–15.[CrossRef][Medline]

Karlin, D., Longhi, S. & Canard, B. (2002). Substitution of two residues in the measles virus nucleoprotein results in an impaired self-association. Virology 302, 420–432.[CrossRef][Medline]

Kerdiles, Y. M., Cherif, B., Marie, J. C., Tremillon, N., Blanquier, B., Libeau, G., Diallo, A., Wild, T. F., Villiers, M. B. & Horvat, B. (2006). Immunomodulatory properties of morbillivirus nucleoproteins. Viral Immunol 19, 324–334.[CrossRef][Medline]

Laine, D., Trescol-Biemont, M. C., Longhi, S., Libeau, G., Marie, J. C., Vidalain, P. O., Azocar, O., Diallo, A., Canard, B. & other authors (2003). Measles virus (MV) nucleoprotein binds to a novel cell surface receptor distinct from FcRII via its C-terminal domain: role in MV-induced immunosuppression. J Virol 77, 11332–11346.[Abstract/Free Full Text]

Libeau, G., Diallo, A., Calvez, D. & Lefevre, P. C. (1992). A competitive ELISA using anti-N monoclonal antibodies for specific detection of rinderpest antibodies in cattle and small ruminants. Vet Microbiol 31, 147–160.[CrossRef][Medline]

Libeau, G., Diallo, A., Colas, F. & Guerre, L. (1994). Rapid differential diagnosis of rinderpest and peste des petits ruminants using an immunocapture ELISA. Vet Rec 134, 300–304.[Abstract]

Libeau, G., Prehaud, C., Lancelot, R., Colas, F., Guerre, L., Bishop, D. H. & Diallo, A. (1995). Development of a competitive ELISA for detecting antibodies to the peste des petits ruminants virus using a recombinant nucleoprotein. Res Vet Sci 58, 50–55.[CrossRef][Medline]

Libeau, G., Saliki, J. T. & Diallo, A. (1997). Caractérisation d’anticorps monoclonaux dirigés contre les virus de la peste bovine et de la peste des petits ruminants: identification d’épitopes conservés ou de spécificité stricte sur la nucléoprotéine. Rev Elev Med Vet Pays Trop 50, 181–190. (in French).

Liston, P., Batal, R., DiFlumeri, C. & Briedis, D. J. (1997). Protein interaction domains of the measles virus nucleocapsid protein (NP). Arch Virol 142, 305–321.[CrossRef][Medline]

Longhi, S., Receveur-Brechot, V., Karlin, D., Johansson, K., Darbon, H., Bhella, D., Yeo, R., Finet, S. & Canard, B. (2003). The C-terminal domain of the measles virus nucleoprotein is intrinsically disordered and folds upon binding to the C-terminal moiety of the phosphoprotein. J Biol Chem 278, 18638–18648.[Abstract/Free Full Text]

Mahé, D., Blanchard, P., Truong, C., Arnauld, C., Le Cann, P., Cariolet, R., Madec, F., Albina, E. & Jestin, A. (2000). Differential recognition of ORF2 protein from type 1 and type 2 porcine circoviruses and identification of immunorelevant epitopes. J Gen Virol 81, 1815–1824.[Abstract/Free Full Text]

Merz, D. C., Scheid, A. & Choppin, P. W. (1981). Immunological studies of the functions of paramyxovirus glycoproteins. Virology 109, 94–105.[CrossRef][Medline]

Plowright, W. (1962). Rinderpest virus. Ann N Y Acad Sci 101, 548–573.[CrossRef][Medline]

Plowright, W. (1968). Rinderpest virus. In Virology Monographs vol. 3, pp. 26–110. Edited by S. Gard, C. Hallauer & K. F. Meyer. New York: Springer-Verlag.

Rost, B. (2001). Protein secondary structure prediction continues to rise. J Struct Biol 134, 204–218.[Medline]

Taylor, W. P. (1986). Epidemiology and control of rinderpest. Rev Sci Tech Off Int Epizoot 5, 407–410.

tenOever, B. R., Servant, M. J., Grandvaux, N., Lin, R. & Hiscott, J. (2002). Recognition of the measles virus nucleocapsid as a mechanism of IRF-3 activation. J Virol 76, 3659–3669.[Abstract/Free Full Text]

Zhang, X., Glendening, C., Linke, H., Parks, C. L., Brooks, C., Udem, S. A. & Oglesbee, M. (2002). Identification and characterization of a regulatory domain on the carboxyl terminus of the measles virus nucleocapsid protein. J Virol 76, 8737–8746.[Abstract/Free Full Text]

Received 28 July 2006; accepted 6 November 2006.

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