The epitope structure of Citrus tristeza virus coat protein mappedby recombinant proteins and monoclonal antibodies

Guan-Wei Wu a, Min Tang b, Guo-Ping Wang a,b, Cai-Xia Wang b,c, Yong Liu b,d,Fan Yang b, Ni Hong a,b,n

a National Key Laboratory of Agromicrobiology, Huazhong Agricultural University, Wuhan, Hubei 430070, People's Republic of Chinab Key Laboratory of Crop Disease Monitoring and Safety Control in Hubei Province, College of Plant Science and Technology, Huazhong AgriculturalUniversity, Wuhan, Hubei 430070, People's Republic of Chinac College of Agronomy and Plant Protection, Qingdao Agricultural University, Qingdao, Shandong 266109, People's Republic of Chinad Institute of Fruit and Tea, Hubei Academy of Agricultural Sciences, Wuhan, Hubei 430064, People's Republic of China

a r t i c l e i n f o

Article history:Received 2 September 2013Returned to author for revisions3 October 2013Accepted 14 October 2013Available online 31 October 2013

Keywords:Citrus tristeza virusCoat proteinEpitope mappingRecombinant proteinMonoclonal antibodyStructure analysis

a b s t r a c t

It has been known that there exists serological differentiation among Citrus tristeza virus (CTV) isolates.The present study reports three linear epitopes (aa 48–63, 97–104, and 114–125) identified by usingbacterially expressed truncated coat proteins and ten monoclonal antibodies against the native virions ofCTV-S4. Site-directed mutagenesis analysis demonstrated that the mutation D98G within the newlyidentified epitope 97DDDSTGIT104 abolished its reaction to MAbs 1, 4, and 10, and the presence of G98 inHB1-CP also resulted in its failure to recognize the three MAbs. Our results suggest that theconformational differences in the epitope I 48LGTQQNAALNRDLFLT63 between the CPs of isolates S4and HB1 might contribute to the different reactions of two isolates to MAbs 5 and 6. This study providesnew information for the antigenic structures of CTV, and will extend the understanding of the processesrequired for antibody binding and aid the development of epitope-based diagnostic tools.

Crown Copyright & 2013 Published by Elsevier Inc. All rights reserved.

Introduction

Citrus tristeza virus (CTV) is a member of the genus Closteroviruswithin the family Closteroviridae (Martelli et al., 2002). Tristezadisease which is caused by the virus has seriously affected thedevelopment of the citrus industry in some countries (Morenoet al., 2008). The CTV virion contains a large single-stranded,positive-sense genomic RNA (gRNA) of �19.3 kb, consisting oftwelve open reading frames (ORFs). The viral particle has a uniquebipolar architecture coated by two coat proteins (CP and CPm)(Febres et al., 1996). CP coats most of the gRNA (genomic RNA), andCPm coats only �630 nt at the 5′ terminus (Satyanarayana et al.,2004). Thus, serological diagnosis of CTV is mainly based on thedetection of its CP with specific antibodies.

So far, at least twenty complete genomic sequences ofCTV isolates have been determined. Nucleotide sequence analysisshowed that CTV isolates were highly variable, and could begrouped into six genotypes, namely VT, T3, T30, T36, B165, and

RB based on the phylogenetic analysis of the 5′ proximal half(about 11 kb) of the genome (Harper et al., 2010; Hilf et al., 1999,2005; Roy and Brlansky, 2010). Four genotypes were identified inthe CTV population in China by analyzing the sequences of multi-ple molecular markers (MMMs) and restriction fragment lengthpolymorphism patterns (RFLP) of the CP gene of CTV isolates (Jianget al., 2008; Wu et al., 2013). The biological indexing on a set ofindicator plants has revealed the pathogenicity differentiation ofCTV isolates from different citrus-growing areas (Ballester-Olmoset al., 1993). Many CTV isolates, namely severe strains, areaggressive and are associated with the symptoms of declineand death of citrus trees propagated on sour orange (Citrusaurantium L.) rootstock or stem pitting (SP) of the scion irrespec-tive of the rootstocks. Only a few reported isolates, namely mildstrains, induce slight leaf chlorosis or are symptomless on Mexicanlime (Moreno et al., 2008). The discrimination between mild andsevere strains can provide valuable information for the effectivecontrol of the viral disease, and the CP gene sequences have beenused extensively to discriminate CTV strains, but other regions canalso be used (Cevik et al., 1996; Herrera-Isidron et al., 2009;Sambade et al., 2003).

Serological technique is one of the most widely used tools forthe reliable and high-throughput identification of plant viruses,and also for the discrimination of strains or serotypes of some

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Virology

0042-6822/$ - see front matter Crown Copyright & 2013 Published by Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.virol.2013.10.021

n Corresponding author at: Key Laboratory of Crop Disease Monitoring and SafetyControl in Hubei Province, College of Plant Science and Technology, HuazhongAgricultural University, Wuhan, Hubei 430070, People's Republic of China.Tel.: þ86 27 87283278; fax: þ86 27 87384670.

E-mail address: whni@mail.hzau.edu.cn (N. Hong).

Virology 448 (2014) 238–246

viruses. Epitopes play a pivotal role in antigen recognition. Theprecise localization of an epitope can be essential in the develop-ment of serological diagnostic kits for the specific viral strains orvariants. The antigenic structures of many plant viruses have beeninvestigated through the identification of epitopes recognized bymonoclonal antibodies (MAbs) and polyclonal antisera, whichhave greatly improved the development of highly specific serolo-gical reagents. The serological differentiation has been observedamong CTV strains with different biological characteristics.Nikolaeva et al. (1998) described an IDAS-ELISA system developedto distinguish among a wide range of isolates which cause stempitting in sweet orange indicator plants from those that do notcause sweet orange stem pitting. The development of MAbssignificantly improved the differentiation of CTV strains. A pre-vious study identified a monoclonal antibody, MCA-13, whichreacted selectively with the majority of CTV severe isolates(Permar et al., 1990). Several monoclonal and polyclonal antibo-dies have been developed against various CTV isolates (Rocha-Peña and Lee, 1991; Vela et al., 1986; Wang et al., 2006), whichhave made it possible to map the epitopes for CTV. Pappu et al.(1993, 1995) found that the amino acids at positions 2 and 124played crucial roles in the binding of the MAbs 3DF1 and MCA-13,respectively. Nikolaeva et al. (1996) screened 30 CTV-specificMAbs and assigned them into five groups based on epitopespecificity. Albiach-Martí et al. (2000) developed a serologicalanalysis procedure which was utilized to produce peptide mapsby using protease digestion with MAbs and polyclonal antibodies(PAbs) to detect and discriminate CTV isolates. Recently, Peroniet al. (2009) developed four specific MAbs against the recombi-nant protein of the most virulent Brazilian CTV genotype “CapãoBonito” (CB) and identified three epitope regions (aa 32–40, 50–61,and 120–131) by ELISA screening of the overlapping recombinantpeptides. However, information on the types and distribution ofepitopes on the CP of CTV is currently limited as compared withthose of other economically important plant viruses, such as Plumpox virus (Candresse et al., 2010; Croft et al., 2008), potyviruses

(Desbiez et al., 1997; Shukla et al., 1989), and Tobacco mosaic virus(Dore et al., 1987; Holzem et al., 2001).

CTV infection occurs widely in citrus plants in central andsouthern China, and the most prevalent CTV isolates are associatedwith the stem pitting syndrome (Jiang et al., 2008; Zhou et al.,2007). Previously, our group raised a polyclonal antiserum and aset of monoclonal antibodies against virions of two CTV isolates(Wang et al., 2006), respectively. In the course of a serologicalsurvey of Chinese CTV strains, a CTV isolate from pummelo (HB1),which differed serologically from all previously studied isolates,was identified. The HB1 isolate was unable to be recognized bysome MAbs, and bioinformatics analysis indicated that threeamino acid sites (S84, G98, and G190) specifically present in theCP of HB1 might affect its recognition by those MAbs (Wang et al.,2007). Comprehensive knowledge of the epitope structures as wellas the characterization of new epitope-specific MAbs is necessaryfor the development of novel epitope-based diagnostic tests.In this study, we initiated a survey of the epitopes of the CTV CPby using ten CTV-specific MAbs raised against a CTV stem-pittingisolate S4, and three epitope regions were identified. Site-directedmutagenesis analysis demonstrated that one important amino acidat the 98 (G98) position of the CP was involved in the antibodybinding with HB1-CP. The data obtained contributes to a betterunderstanding of the antigenic structure of the virus and to theimprovement of epitope-based diagnostic tools.

Results

Reactivity of monoclonal antibodies with the CTV virions and the full-length and truncated CPs of CTV expressed in Escherichia coli strainBL21 (DE3)

The reactivity of ten MAbs with CTV-S4 virions was tested byimmune capture (IC) RT-PCR, and compared with that of mixedMAbs and the polyclonal antibody PAb-L5. The results showed thatall of the antibodies were able to capture CTV virions, and gavepositive results in the RT-PCR tests (Fig. 1).

Meanwhile, the full length CP and truncated CPs, named CΔ-1to -9, were successfully expressed in E. coli strain BL21 (DE3) andvisualized by SDS-PAGE analysis (Fig. 2A). However, the expressedamount of full-length CP and two amino-terminal deleted frag-ments CΔ-4 and CΔ-5 were less than that of the other truncatedCPs. The purified extracts of the CP and CΔ-1 to -9 with theexpected molecular weights of 29.0 kDa, 24.4 kDa, 21.9 kDa,14.8 kDa, 15.5 kDa, 20.2 kDa, 16.2 kDa, 14.4 kDa, 14.8 kDa, and16.2 kDa, respectively, were obtained by using the high-affinityNi-NTA agarose (Fig. 2A). The CP, CΔ-4, and CΔ-5 were purifiedunder native conditions, while the other seven truncated CPs were

Fig. 1. The detection of CTV virions captured by MAbs and PAb by IC-RT-PCR.M: DNA marker from Tiangen (Beijing, China); lane CK(�): Normal mouse serum;lanes 1–10: MAbs 1–10, respectively; lane 11: PAb-L5; lane 12: Mixed MAb.CP indicates the CP product with a size of 672 bp.

Fig. 2. Detection of the CTV coat protein and its truncated fragments expressed in E. coli BL21 (DE3). The purified fusion proteins (A) was stained with Coomassie blue after12% SDS-PAGE analysis. The reactivity of those proteins with mixed MAbs was detected by Western blotting (B). Molecular weight markers (Fermentas) are shown in lane M.Lanes CP and 1–9: CP and nine truncated CPs CΔ-1 to -9, respectively. Cell lysates of E. coli transformed with the empty pET28a vector were used for the cell control (LaneCK). ‘þ ' and ‘� ’ indicate the positive and negative reactions, respectively.

G.-W. Wu et al. / Virology 448 (2014) 238–246 239

purified under denaturing conditions. Western blotting analysisshowed that all of the proteins were well recognized by mixedMAbs except for CΔ-3 (Fig. 2B). Meanwhile, all MAbs could reactwell with the full-length CP in both Western blotting and indirectELISA assays (Fig. 3, Table 1, Supplementary Table S1). The dimericpatterns of some truncated CPs (especially CΔ-6, 7, 8 and 9) withlower electrophoretic mobility were also detected consistently bysome MAbs (1, 3, 4, 7 and 8). Some additional bands below thetarget bands of some CPs (CΔ-1 and CΔ-2) were also observed,which might be caused by degradation or incomplete translation.However, the non-specific reactions would not interference withthe following experiments.

The epitope regions involved in recognition by ten MAbs

The full-length CP and nine truncated CPs were subjected toWestern blotting analysis by using each of ten MAbs. The resultsshowed that all ten MAbs were able to recognize the full-length CP,

and three truncated CPs [CΔ-1 (aa 42–223), CΔ-5 (aa 1–146), andCΔ-7 (aa 48–140)] (Fig. 3), suggesting that the N-terminus of the CP(aa 1–47) and C-terminus of the CP (aa 141–223) should be excludedfrom the epitope region recognized by all tenMAbs. In addition, CΔ-3(aa 126–223) showed reactions with MAbs 2 and 9, however, noreaction signal was visible with the other eight MAbs, suggesting thatthe region containing aa 48–125 was involved in the recognition bythose eight MAbs.

MAbs 1, 4, and 10 reacted with all of the truncated CPs exceptfor CΔ-3 (aa 126–223), and CΔ-9 (aa 114–223), indicating that theaa region 97DDDSTGIT104 (Epitope II) was required for the epitopeto be recognized by those MAbs (Table 1, Fig. S1).

MAbs 3 and 7 reacted with all of the fragments except for CΔ-3(aa 126–223) and CΔ-4 (aa 1–104), indicating that the aa region114LSDKLWTDVVFN125 (Epitope III) was required for the epitope tobe recognized by MAbs 3 and 7.

Both MAbs 5 and 6 exhibited reactions with fragments CΔ-1 (aa42–223), CΔ-4 (aa 1–104), CΔ-5 (aa 1–146), and CΔ-7 (aa 48–140),

Fig. 3. Western blotting detection of full-length fusion and truncated CPs of CTV using ten monoclonal antibodies. Lanes CP and 1-9: CP and its truncated fragments CΔ-1 to-9, respectively.

Table 1Reactivity of the full-length CP and truncated CPs with MAbs in the Western blotting and indirect ELISA tests.

Monoclonal antibodies Antibody reactivity with CP and truncated CPs with MAbs Amino acid residue positionsof epitopes

CP CΔ-4 CΔ-5 CΔ-7 CΔ-6 CΔ-8 C-Δ1 CΔ-2 CΔ-9 CΔ-3

1–223 1–104 1–146 48–140 84–195 97–195 42–223 64–223 114–223 126–223

Mixed MAb þa þb þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ � � �1 þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ � � � � 97–1042 þ þ þ þ þ þ þ � þ � þ � þ þ þ þ þ þ þ þ ND3 þ þ � � þ þ þ þ þ þ þ þ þ þ þ þ þ þ � � 114–1254 þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ � � � � 97–1045 þ þ þ þ þ þ þ þ � � � � þ þ � � � � � � 48–636 þ þ þ þ þ þ þ þ � � � � þ þ � � � � � � 48–637 þ þ � � þ þ þ þ þ þ þ þ þ þ þ þ þ þ � � 114–1258 þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ � � 97–104, 114–1259 þ þ þ þ þ þ þ � þ � þ � þ 7 þ 7 þ 7 þ 7 ND10 þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ � � � � 97–104

Absorbance ratios of the CP or ΔCPs/control in the indirect ELISAo2, 2–4 and 44 are indicated with ‘� ’, ‘7 ,’ and ‘þ ’, respectively. Symbols ‘þ ’ and ‘� ’ indicate the positiveand negative reactions of MAbs with CP and truncated CPs in Western blotting, respectively. ND, not determined.

a Western blotting.b Indirect ELISA.

G.-W. Wu et al. / Virology 448 (2014) 238–246240

and no reaction with the fragment CΔ-2 (aa 64–223) and otherfragments, revealing that they recognized the same epitopelocated in the aa region 48LGTQQNAALNRDLFLT63 (Epitope I).

The interactions between the MAbs (except for MAb 2 andMAb 9) and the CP fragments in the indirect ELISA (SupplementaryTable S1) were consistent with those in the Western blottingassays (Table 1). In contrast to the Western blotting results,MAbs 2 and 9 did not react with CΔ-6, CΔ-7, and CΔ-8 in the indirectELISA, which suggested that the epitopes in CΔ-6, CΔ-7, and CΔ-8which were recognized by the two MAbs might be conformation-dependent. MAbs 2 and 9 were able to react with both CΔ-4 (aa1–104) and CΔ-9 (aa 114–223), which made it difficult to determinethe exact epitope regions recognized by these MAbs.

Computer–assisted analysis further confirmed the antigenicrole of those three identified regions of the CP (Fig. 4A), whichare presumably exposed on the particle surface, and exhibitedhigh antigenic and hydrophilic characteristics. Multiple align-ments of the deduced amino acid sequences of CP from global

CTV isolates showed that these three regions were relativelyconserved among isolates belonging to six genotypes (VT, T3,T30, B165, RB, and T36) (Fig. S1).

Effect of amino acid mutations in the epitope region (aa 97–104)of the CP on the antibody-antigen interaction

To confirm that the epitope consisting of eight amino acids inthe region containing aa 97–104 of the CP was involved inrecognition by MAbs 1, 4, and 10, three oligopeptides, including97DDDSTGIT104 and two peptides 97DDDSTGI103 and 97DDDSTG102

with one and two amino acid deletions at the C-terminus of theepitope, were synthesized and used as antigens in indirect ELISAtests (Fig. 5). The results showed that all three oligopeptides wererecognized by all three MAbs. The oligopeptide 97DDDSTGIT104

reacted strongly with MAbs 1, 4, and 10, and their OD450 values(0.38–0.53) were over ten times higher than those of the negativecontrol (0.03–0.035) (data not shown). The deletion of one (T104)

Fig. 4. Epitope prediction of the CTV-S4 coat protein (A) by DNASTAR software and schematic representation of the full-length CP and truncated CP constructs (B) used forepitope mapping. Three parameters including surface probability plot-Emini, Antigenic index-Jameson-Wolf, and hydrophilicity plot-Doolittle are used for epitopeprediction. Three identified epitopes (I, aa 48-63; II, aa 97-104; III, aa 114-125) are indicated. Black box indicates the hexahistidine tag.

G.-W. Wu et al. / Virology 448 (2014) 238–246 241

and two (T104 and I103) amino acids resulted in reduced reactionswith the three MAbs. The results indicated that the MAbs 1, 4, and10 recognized the 97DDDSTGIT104 epitope and the deletion of twoamino acids (T104 and I103) at the C-terminus had a significanteffect on its reaction with those MAbs.

Multiple alignments of the amino acid sequences of the CP ofthe CTV isolate S4 and reference isolates showed that the regioncontaining aa 97-104 is well conserved in each of the CTV groupsdefined as T36, VT, and HA (Fig. 6A). To evaluate the effect ofnatural variations of amino acids in the region on its reaction withthose MAbs, three mutants (S4-CΔ8/S100A, S4-CΔ8/I103M, andS4-CΔ8/S100T) were constructed by introducing the alanine

(A100) specific for the T36 group and threonine (T100) specificfor the VT group to replace serine (S100) at the position 100 of theCP of CTV-S4, respectively, and methionine (M103) specific for theHA group to replace isoleucine (I103) at the 103 position of the CPof CTV-S4. All of the mutants were successfully over-expressed inE. coli BL21 (DE3) cells (data not shown). Western blotting showedthat S4-CΔ8 and all of the CΔ8 mutants were detected byMAbs 1, 4, and 10 (Fig. 6B). The S4-CΔ8 fragment and the mutantS4-CΔ8/S100A exhibited similar intensities in their reaction sig-nals to MAbs 1 and 4, and a slight reduction was observed forreaction intensity of the mutant S4-CΔ8/S100A against MAb 10.The significantly reduced reactivity for all three MAbs was causedby the S4-CΔ8/I103M and S4-CΔ8/S100T mutations, specific for VTand HA, respectively.

Furthermore, a CTV isolate HB1 showing abnormal serologicalcharacteristics was also included in this study. Western blottingshowed that only five MAbs (2, 3, 7, 8, and 9) reacted with therecombinant HB1-CP (Fig. 7), while MAbs 1, 4, and 10, whichrecognized the epitope region (aa 97–104) of S4-CP, reactednegatively with HB1-CP. Comparison of the amino acid sequencesat the positions 97–104 of CP between isolates HB1 and S4 showedthat there were two different amino acids at the positions 98 and100, respectively (Fig. 6A). Since the S4-CΔ-8/S100T mutant couldnot abolish the antigen-antibody interaction, therefore, the possi-ble role of aspartic acid at the position 98 (D98) of S4-CP in theantibody-antigen interaction was investigated by site-directedmutagenesis. Two mutants S4-CΔ8/D98G and S4-CΔ8/D98G/S100T were constructed by introducing the G98 or both G98 andT100 into S4-CΔ-8 to incorporate the corresponding amino acidresidues in CTV-HB1. Using Western blotting analysis and MAbs 1,4, and 10, no reaction signal was observed for the two mutants.However, when aspartic acid (D98) was introduced into the

Fig. 5. Reactivity of three MAbs to three synthetic oligopeptides in indirect ELISAassays. The three oligopeptides are 97DDDSTGIT104, and the other two contain oneor two amino acid deletions at the C-terminus, respectively. Each microplate well iscoated with 20 μg of each oligopeptide, and incubated with the MAbs 1, 4, and 10,respectively, followed by incubation with HRP-conjugated anti-mouse IgG. The datarepresent the mean absorbance values at a wave-length of 450 nm of threereplicates. The wells coated with skim milk are used as negative control.

Fig. 6. Sequence alignment of the epitope region (aa 97–104) (A) of CTV isolates and Western blotting analysis of site-directed mutants of S4-CΔ-8 (B) and HB1-CΔ-8 (C). Theamino acids highlighted with an asterisk indicate the targeted sites for mutagenesis. ‘Coom’ indicates that equal amounts of those fusion proteins are loaded as indicated bythe SDS-PAGE gel stained with Coomassie blue.

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HB1-CΔ-8 mutant to replace the G98 in the HB1-CΔ-8 (HB1-CΔ-8/G98D), its reaction signals with MAbs 1, 4, and 10 were recovered(Fig. 6C). These results indicated that the presence of aspartic acid(D98) in the CP of the majority of CTV isolates was crucial for theirinteractions with MAbs 1, 4, and 10. Mutation of glycine (G98) inthe HB1 CP abolishes the reactivity.

Discussion

The generation of MAbs against different CTV isolates hasprovided an important basis for understanding the antigenicproperties of CTV CP. Many methods have been used to identifyepitopes or key amino acids contributing to specific serotypes,including phage display (Holzem et al., 2001; Peng et al., 2008),synthetic peptides (Commandeur et al., 1994; Dell'Orco et al.,2002; Meyer et al., 2012; Shukla et al., 1989) or bacterial expressedfusion proteins (Candresse et al., 2010; Peroni et al., 2009; Saasaet al., 2012), and site-directed mutagenesis (Benjamin and Perdue,1996). In this study, we constructed nine truncated coat proteins ofthe CTV isolate S4 and successfully characterized three epitopes byusing ten monoclonal antibodies. The lower expression titer of thecomplete CP and two fragments (CΔ-4 and CΔ-5) containing onlythe N terminus suggested that the amino acids at their N terminus(1–41) might affect their expression.

By using the primary amino-acid sequence many computa-tional methods have been developed for predicting potentialepitopes and their aa residues from different antigens (El-Manzalawy and Honavar, 2010). Primary analysis indicated thatsequences involved in the antigen function of CTV-S4 weredistributed across its entire coat protein as indicated by theantigenic index (Fig. 4A). However, there are only three epitopesmapped in this study. Of the ten MAbs, the epitopes recognized byeight MAbs were determined. MAbs 1, 4, and 10 reacted with anepitope consisting of eight amino acids at the 97 to 104 position,which is a newly identified epitope region. The epitope is wellconserved among all CTV genotypes except for one or two aavariations present in some isolates. MAbs 3 and 7 recognized anepitope which mapped between aa 114–125, which was within theepitope (aa 118–128) recognized by the MAbs in group III deter-mined by Nikolaeva et al. (1996). In this region, the phenylalanine(F124) at the 124 position of the CP of severe CTV strains plays acrucial role in the recognition by MCA-13 (Pappu et al., 1993).However, these two MAbs 3 and 7 could not discriminate betweenthe severe and mild strains (data not shown), indicating that thekey amino acids in the interactions with these two MAbs weredifferent from those recognized by MCA-13. The epitope for MAbs5 and 6 was mapped to aa residues 48–63, which overlapped withan epitope determined previously (Nikolaeva et al., 1996; Peroniet al., 2009). It is to be expected that all of these MAbs recognize awide spectrum of CTV strains since the three epitopes mapped inthis study are highly conserved in all CTV genotypes (Fig. S1).Coincidentally, there exists high surface probability, antigenicindex, and hydrophilicity in these regions (Fig. 4A). Hopp andWoods (1981) and Novotný et al. (1986) reported significantcorrelations between hydrophilicity, surface probability and

antigenic determinant. Those results might support the observa-tion that all MAbs used can capture CTV-S4 virions.

The finding that all MAbs reacted with the CP of CTV-S4 inWestern blotting suggested that the epitopes for those MAbs werelargely dependent on the primary structure rather than the CPconformation. The MAbs 2 and 9 strongly recognized CTV virionsin IC-RT-PCR, which caused our initial speculation of the presenceof conformational epitopes recognized by those two MAbs. How-ever, MAbs 2 and 9 reacted with all of the truncated CPs inWestern blotting, which made it impossible to map the exactepitopes recognized by them. The reason for this might be that thetwo MAbs were mixtures of antibodies secreted by two or morehybrid cell lines. Our results showed that two regions (aa 97–104and 114–125) might be involved in the recognition of MAb 8.Previously, several studies suggested that the presence of a fewkey amino acid residues might contribute to multiple epitopedeterminants (Geysen et al., 1984, 1987), and many residues inlinear epitopes can be substituted. A comparison of epitopes II(97DDDSTGIT104) and III (114LSDKLWTDVVFN125) showed that theyhad the same amino acids D (aspartic acid) and T (threonine).However, it is difficult to conclude the exact role of these aminoacids in the antibody-antigen interaction.

For the first time, our results revealed that the presence ofaspartic acid (D98) in the CP of the majority of CTV isolates playeda crucial role in its interaction with MAbs 1, 4, and 10. Themutation of aspartic acid (D98) to gylcine (G98) in S4-CΔ-8abolished the reactivity, and the reverse mutation of G98 to theD98 in HB1-CΔ-8 recovered the reactivity. Generally, epitoperesidues should be highly accessible to facilitate their contact withthe antibody. The key amino acid D98 in S4-CP exhibited anincreased accessible surface area (ASA) (99.562 angstroms^2)when compared with that of G98 (51.917 angstroms^2) in HB1-CP (Figs. S2 and S3A and B), which might result in the differentreactivity observed when compared with that from previousreports (Novotný et al., 1986; Rubinstein et al., 2008). Previousworks reported the amino acid preferences of epitopes, whichwere generally enriched with charged and polar amino acids(Rubinstein et al., 2008). Although both D98 and G98 are polaramino acids, D98 is charged, and G98 is uncharged, suggestingthat the charge of the amino acid at the 98 position of the CPmight also affect its binding to antibodies (Pappu et al., 1995).Although other mutations in the epitope region (aa 97–104) didnot abolish the antigen-antibody interaction, the variations atpositions 100 and 103 might have affected the binding energy(Benjamin and Perdue, 1996), and then reduced the reactionintensity between the epitope region and the MAbs. Whereas,the mutations S4-CΔ8/I103M and S4-CΔ8/S100T showed moresignificant effects on the reactions with MAbs 1, 4 and 10 than thatcaused by the mutation S4-CΔ8/S100A (Fig. 5 and Fig. 6B),suggesting different contribution of these amino acids to thereactions between the epitopes and the MAbs.

The epitope I (48LGTQQNAALNRDLFLT63) recognized by MAbs5 and 6 just overlapped the previous reported epitope(50TQQNAALNRDLF61) recognized by MAb 37.G.11 (Peroni et al.,2009). However, the two MAbs did not react with the recombinantHB1-CP and CTV-HB1 virions (Wang et al., 2007), although theamino acids in the epitope I region of S4-CP and HB1-CP are thesame (Fig. S1). Epitope prediction analysis showed that theantigenic index of the CTV-S4 CP in the region containing aa 48–50 was much higher than that of CTV-HB1 (Fig. S2), and that onealpha helix at the C-terminus of epitope I of S4-CP was replaced bya beta strand at that of the HB1-CP (Fig. S3C and D). A previousreport suggested that the amino acid mutations, which are not atpositions directly involved in antibody binding, could result in far-reaching conformational changes in an antigenic loop, which thendestroy the integrity of the epitope (Parry et al., 1990). The

Fig. 7. Western blotting analysis of the CTV-HB1 coat protein expressed in E. coliBL21 (DE3) by using ten MAbs. ‘þ ’ and ‘� ’ indicate the positive and negativereactions, respectively.

G.-W. Wu et al. / Virology 448 (2014) 238–246 243

predicted secondary structure in the region before epitope Ishowed some differences (Fig. S2), which might result in thedifferent conformations in the epitope regions in S4-CP and HB1-CP (Fig. S3E and F), which therefore affect the reactivities of theepitopes with the MAbs 5 and 6.

In summary, this study identified a novel conserved linear epitopeat the middle region of the CTV coat protein. The importance ofindividual amino acids for the antibody-antigen interaction wasinvestigated. Binding sites recognized by eight MAbs could beidentified, and the epitope regions for MAbs 5 and 6 should befurther characterized by constructing the accurate crystal structure ofcoat proteins for S4 and HB1 (Lassaux et al., 2013). These findingsprovide new insights into the antigenic structure of the CTV coatprotein, and will improve our understanding of antibody-antigeninteractions and the development of CTV-specific diagnostic assays.With the development of more MAbs against different plant virusstrains, it will be possible to perform a large-scale analysis to defineepitope characteristics of plant viruses and reveal new aspects ofantigen-antibody recognition.

Materials and methods

CTV isolates, antibodies, and oligopeptides

Ten monoclonal antibodies (MAbs, designated as MAb 1 to 10),raised against the CTV-S4 isolate (Wang et al., 2006), were used forepitope mapping. Equal amounts of each MAb were mixed andused as mixed MAb. The polyclonal antibody (PAb) raised againstthe virion preparation of CTV- L5 named PAb-L5 (Wang et al.,2006) was used in immune capture RT-PCR experiments. Isolate S4induced stem pitting or vein clearing on C. sinensis plants, and L5induced stem pitting on Mexican lime [Citrus aurantifolia(Christm.) Swing] plants (Jiang et al., 2008). Both CTV severeisolates S4 and L5 were maintained in sweet orange [Citrus sinensis(L.) Osb.] seedlings. The CTV isolate HB1 described previously(Wang et al., 2007) was also included in this study.

Oligopeptides 97DDDSTGIT104, 97DDDSTGI103, and 97DDDSTG102

were synthesized with over 98% purity by GenScript BiologicalScience (Nanjing, China).

Plasmid construction and expression of hexahistidine-tagged coatprotein in Escherichia coli

The full length CPs of CTV-S4 and CTV-HB1 were cloned intothe pMD18-T vector (TaKaRa, Dalian, China) and sequenced. Forthe construction of the expression vector, the cloned CP gene wasamplified with the primer set S4-CP-F/-R (Supplementary TableS2), gel purified, digested with the Bam HI and Hind III restrictionenzymes (TaKaRa, Dalian, China), and then ligated into thedigested expression vector pET-28a (þ) (Novagen, Madison, WI,USA), which has a His-tag, and transformed into competent E. coliBL21 (DE3) cells. Colonies with the fragment inserted in thecorrect orientation were identified by PCR and restriction analysis.Three clones of each insert were sequenced by a commercialsequencing service (GenScript Biological Science, Nanjing, China).The recombinant CP with a His-tag was expressed in E. coli strainBL21 (DE3) by incubating at a speed of 250 r/m at 37 1C for 6–8 husing 1 mM isopropyl-β-thiogalactopyranoside (IPTG).

Designation of truncated CPs, site-directed mutagenesis and theirexpression in Escherichia coli

The putative epitopes of the CTV coat protein were predictedby using the DNASTAR package software (DNASTAR, Madison, WI,USA) using the Kyte–Doolittle hydrophilic plot, Jameson–Wolf

antigenicity, Emini Surface plot, and secondary structure indexanalysis. Multiple alignments of predicted amino acid sequenceswere generated using Clustal X and GeneDoc version 2.7.0 pro-grams. Primers used for the amplification of truncated CPs weredesigned based on the predicted epitopes (Fig. 4A), and amino acidalignment results (Fig. S1). Initially, five truncated CPs named CΔ1to CΔ5 were designed, and used for the primary mapping of theepitopes. The other four truncated CPs, named CΔ6 to CΔ9, weredesigned based on the primary mapping results and used for fineepitope mapping (Fig. 4B).

The site-directed mutagenesis in all of the mutants (S4-CΔ8/S100A, S4-CΔ8/I103M, S4-CΔ8/S100T, S4-CΔ8/D98G, S4-CΔ8/D98G/S100T, and HB1-CΔ8/G98D) were done by PCR using theprimers (Supplementary Table S2) to introduce bases correspond-ing to specific amino acids into CΔ8, and confirmed by sequencingto ensure that the desired mutations were incorporated. Theplasmid constructions and expression of the truncated CPs andmutated CΔ8 fragments were conducted by the proceduredescribed above.

Protein purification and Western blotting

E. coli BL21 (DE3) cells containing recombinant plasmids wereinduced, and expressed recombinant proteins were purified from abiomass of 0.3 L culture by using Ni-NTA agarose (Qiagen, Hilden,Germany). Briefly, bacterial cells were suspend in PBS (0.01 M, pH7.2) and disrupted by sonicating 6�10 s with 10 s pauses at 200–300 W on ice. The pellets were incubated in the lysis buffer(100 mM NaH2PO4, 10 mM Tris HCl, and 8 M urea, pH 8.0) for30 min at room temperature and then centrifuged at 10,000� g for20 min. The supernatant containing insoluble proteins was thenpassed through a pre-equilibrated column. After washing withwashing buffer (100 mM NaH2PO4, 10 mM TrisHCl, and 8 M urea,pH 6.3), the proteins bound on the column were eluted out withan elution buffer (100 mM NaH2PO4, 10 mM TrisHCl, and 8 M urea,pH 5.9 or 4.5). For soluble proteins, the lysis, washing, and elutionbuffers contained 50 mM NaH2PO4 and 300 mM NaCl with 10, 20,and 250 mM imidazole, respectively, and their pH was adjustedto 8.0.

The purified proteins were visualized by 12% SDS-PAGE. Wes-tern blotting was carried out by using the protocol previouslydescribed by Song et al. (2011) with some modifications. Briefly,the separated proteins on gels were electroblotted onto a poly-vinylidene difluoride (PVDF) membrane (Amersham Biosciences,Chiltern, UK). Then, the membrane was blocked overnight at 4 1C,incubated in MAbs at a dilution of 1:5000, and followed byincubation with AP-labeled goat anti-mouse IgG (HþL) (Protein-tech Group, Chicago, USA). The immuno-reaction signals weredeveloped with the ready-to-use BCIP/NBT solution (Amresco,OH, USA).

Indirect ELISA

Microtiter plates were coated with an equal amount of each ofthe purified CP or truncated proteins or oligopeptides (20 μg/well)at 37 1C for 2 h or at 4 1C overnight, and blocked with PBSsupplemented with 5% skim milk and 0.05% Tween-20 (v/v) at37 1C for 2 h. The wells coated with skim milk or empty vectorpET-28a (þ) expression products are used as negative controls.MAbs at 1 mg/ml were added into each well and incubated at37 1C for 2 h, and then detected by using goat anti-mouse IgG(HþL)-HRP (Proteintech Group, Chicago, USA). The detectionsignals were developed by using a mixture of H2O2 and 3,3′,5,5′-tetramethyl benzidine substrate (TMB; Sigma-Aldrich). The reac-tion was then stopped by adding 0.1 M H2SO4, and the absorbance

G.-W. Wu et al. / Virology 448 (2014) 238–246244

values at 450 nm were recorded on a Bio-Rad Model 550 Micro-plate Reader (BioRad, Hercules, CA).

Immune capture (IC) RT-PCR

Microcentrifuge tubes were coated with 100 ml of each of 10MAbs (1 mg/ml). CTV-infected leaves were ground in extractionbuffer (PBST supplemented with 1% (w/v) Na2SO3 and 2% (w/v)PVP-40) at a 1:10 dilution and the extracts were clarified bycentrifugation (10,000� g, 10 min). Then the supernatants wereadded into each coated tube and incubated at 4 1C overnight. Thecaptured CTV virions were dissolved with DEPC-treated deionizedwater, vortexed, spun briefly, and incubated at 95 1C for 10 minand chilled immediately on ice for 5 min. Reverse transcriptionPCR was done by using the T36 CP primer set (SupplementaryTable S2) as described previously (Jiang et al., 2008).

Sequence analysis and structure prediction of coat protein

Nucleotide sequences of the CP gene of the referenced CTV isolateswere obtained from GenBank, and other sequences reported by ourlaboratory are as listed in Supplementary Table S3. Modeling of the 3-dimensional structures of the CTV coat protein were carried out usingthe web based server Phyre 2 (Kelley and Sternberg, 2009). Thepredicted tertiary and surface structures were constructed usingPymol (http://www.pymol.org/).

Acknowledgments

This study was supported by grant no. 30871684 from theNational Science Foundation of China.

Appendix A. Supplementary material

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.virol.2013.10.021.

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