Virology 448 (2014) 238–246

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The epitope structure of Citrus tristeza virus coat protein mapped by 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 China Key Laboratory of Crop Disease Monitoring and Safety Control in Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, People's Republic of China c College of Agronomy and Plant Protection, Qingdao Agricultural University, Qingdao, Shandong 266109, People's Republic of China d Institute of Fruit and Tea, Hubei Academy of Agricultural Sciences, Wuhan, Hubei 430064, People's Republic of China b

art ic l e i nf o

a b s t r a c t

Article history: Received 2 September 2013 Returned to author for revisions 3 October 2013 Accepted 14 October 2013 Available online 31 October 2013

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 using bacterially expressed truncated coat proteins and ten monoclonal antibodies against the native virions of CTV-S4. Site-directed mutagenesis analysis demonstrated that the mutation D98G within the newly identified epitope 97DDDSTGIT104 abolished its reaction to MAbs 1, 4, and 10, and the presence of G98 in HB1-CP also resulted in its failure to recognize the three MAbs. Our results suggest that the conformational differences in the epitope I 48LGTQQNAALNRDLFLT63 between the CPs of isolates S4 and HB1 might contribute to the different reactions of two isolates to MAbs 5 and 6. This study provides new information for the antigenic structures of CTV, and will extend the understanding of the processes required for antibody binding and aid the development of epitope-based diagnostic tools. Crown Copyright & 2013 Published by Elsevier Inc. All rights reserved.

Keywords: Citrus tristeza virus Coat protein Epitope mapping Recombinant protein Monoclonal antibody Structure analysis

Introduction Citrus tristeza virus (CTV) is a member of the genus Closterovirus within the family Closteroviridae (Martelli et al., 2002). Tristeza disease which is caused by the virus has seriously affected the development of the citrus industry in some countries (Moreno et al., 2008). The CTV virion contains a large single-stranded, positive-sense genomic RNA (gRNA) of
19.3 kb, consisting of twelve open reading frames (ORFs). The viral particle has a unique bipolar architecture coated by two coat proteins (CP and CPm) (Febres et al., 1996). CP coats most of the gRNA (genomic RNA), and CPm coats only
630 nt at the 5′ terminus (Satyanarayana et al., 2004). Thus, serological diagnosis of CTV is mainly based on the detection of its CP with specific antibodies. So far, at least twenty complete genomic sequences of CTV isolates have been determined. Nucleotide sequence analysis showed that CTV isolates were highly variable, and could be grouped into six genotypes, namely VT, T3, T30, T36, B165, and

n Corresponding author at: Key Laboratory of Crop Disease Monitoring and Safety Control in Hubei Province, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, People's Republic of China. Tel.: þ 86 27 87283278; fax: þ 86 27 87384670. E-mail address: [email protected] (N. Hong).

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 in the CTV population in China by analyzing the sequences of multiple molecular markers (MMMs) and restriction fragment length polymorphism patterns (RFLP) of the CP gene of CTV isolates (Jiang et al., 2008; Wu et al., 2013). The biological indexing on a set of indicator plants has revealed the pathogenicity differentiation of CTV isolates from different citrus-growing areas (Ballester-Olmos et al., 1993). Many CTV isolates, namely severe strains, are aggressive and are associated with the symptoms of decline and death of citrus trees propagated on sour orange (Citrus aurantium L.) rootstock or stem pitting (SP) of the scion irrespective of the rootstocks. Only a few reported isolates, namely mild strains, induce slight leaf chlorosis or are symptomless on Mexican lime (Moreno et al., 2008). The discrimination between mild and severe strains can provide valuable information for the effective control of the viral disease, and the CP gene sequences have been used extensively to discriminate CTV strains, but other regions can also 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 for the reliable and high-throughput identification of plant viruses, and also for the discrimination of strains or serotypes of some

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

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

viruses. Epitopes play a pivotal role in antigen recognition. The precise localization of an epitope can be essential in the development of serological diagnostic kits for the specific viral strains or variants. The antigenic structures of many plant viruses have been investigated through the identification of epitopes recognized by monoclonal antibodies (MAbs) and polyclonal antisera, which have greatly improved the development of highly specific serological reagents. The serological differentiation has been observed among CTV strains with different biological characteristics. Nikolaeva et al. (1998) described an IDAS-ELISA system developed to distinguish among a wide range of isolates which cause stem pitting in sweet orange indicator plants from those that do not cause sweet orange stem pitting. The development of MAbs significantly improved the differentiation of CTV strains. A previous study identified a monoclonal antibody, MCA-13, which reacted selectively with the majority of CTV severe isolates (Permar et al., 1990). Several monoclonal and polyclonal antibodies have been developed against various CTV isolates (RochaPeña and Lee, 1991; Vela et al., 1986; Wang et al., 2006), which have made it possible to map the epitopes for CTV. Pappu et al. (1993, 1995) found that the amino acids at positions 2 and 124 played crucial roles in the binding of the MAbs 3DF1 and MCA-13, respectively. Nikolaeva et al. (1996) screened 30 CTV-specific MAbs and assigned them into five groups based on epitope specificity. Albiach-Martí et al. (2000) developed a serological analysis procedure which was utilized to produce peptide maps by using protease digestion with MAbs and polyclonal antibodies (PAbs) to detect and discriminate CTV isolates. Recently, Peroni et al. (2009) developed four specific MAbs against the recombinant protein of the most virulent Brazilian CTV genotype “Capão Bonito” (CB) and identified three epitope regions (aa 32–40, 50–61, and 120–131) by ELISA screening of the overlapping recombinant peptides. However, information on the types and distribution of epitopes on the CP of CTV is currently limited as compared with those of other economically important plant viruses, such as Plum pox virus (Candresse et al., 2010; Croft et al., 2008), potyviruses

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.

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(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 and southern China, and the most prevalent CTV isolates are associated with the stem pitting syndrome (Jiang et al., 2008; Zhou et al., 2007). Previously, our group raised a polyclonal antiserum and a set of monoclonal antibodies against virions of two CTV isolates (Wang et al., 2006), respectively. In the course of a serological survey 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 by some MAbs, and bioinformatics analysis indicated that three amino acid sites (S84, G98, and G190) specifically present in the CP of HB1 might affect its recognition by those MAbs (Wang et al., 2007). Comprehensive knowledge of the epitope structures as well as the characterization of new epitope-specific MAbs is necessary for the development of novel epitope-based diagnostic tests. In this study, we initiated a survey of the epitopes of the CTV CP by using ten CTV-specific MAbs raised against a CTV stem-pitting isolate S4, and three epitope regions were identified. Site-directed mutagenesis analysis demonstrated that one important amino acid at the 98 (G98) position of the CP was involved in the antibody binding with HB1-CP. The data obtained contributes to a better understanding of the antigenic structure of the virus and to the improvement of epitope-based diagnostic tools.

Results Reactivity of monoclonal antibodies with the CTV virions and the fulllength and truncated CPs of CTV expressed in Escherichia coli strain BL21 (DE3) The reactivity of ten MAbs with CTV-S4 virions was tested by immune capture (IC) RT-PCR, and compared with that of mixed MAbs and the polyclonal antibody PAb-L5. The results showed that all of the antibodies were able to capture CTV virions, and gave positive results in the RT-PCR tests (Fig. 1). Meanwhile, the full length CP and truncated CPs, named CΔ-1 to -9, were successfully expressed in E. coli strain BL21 (DE3) and visualized by SDS-PAGE analysis (Fig. 2A). However, the expressed amount of full-length CP and two amino-terminal deleted fragments CΔ-4 and CΔ-5 were less than that of the other truncated CPs. The purified extracts of the CP and CΔ-1 to -9 with the expected 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, and 16.2 kDa, respectively, were obtained by using the high-affinity Ni-NTA agarose (Fig. 2A). The CP, CΔ-4, and CΔ-5 were purified under native conditions, while the other seven truncated CPs were

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 after 12% 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 (Lane CK). ‘þ' and ‘ ’ indicate the positive and negative reactions, respectively.

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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 1 Reactivity 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 CP 1–223

Mixed MAb 1 2 3 4 5 6 7 8 9 10

þ þ þ þ þ þ þ þ þ þ þ

a

þ þ þ þ þ þ þ þ þ þ þ

b

Amino acid residue positions of epitopes

CΔ-4

CΔ-5

CΔ-7

CΔ-6

CΔ-8

C-Δ1

CΔ-2

CΔ-9

CΔ-3

1–104

1–146

48–140

84–195

97–195

42–223

64–223

114–223

126–223

þ þ þ  þ þ þ  þ þ þ

þ þ þ þ þ þ þ þ þ þ þ

þ þ þ þ þ þ þ þ þ þ þ

þ þ þ þ þ   þ þ þ þ

þ þ þ þ þ   þ þ þ þ

þ þ þ þ þ þ þ þ þ þ þ

þ þ þ þ þ   þ þ þ þ

þ  þ þ    þ þ þ 

  þ       þ 

þ þ þ  þ þ þ  þ þ þ

þ þ þ þ þ þ þ þ þ þ þ

þ þ  þ þ þ þ þ þ  þ

þ þ  þ þ   þ þ  þ

þ þ  þ þ   þ þ  þ

þ þ þ þ þ þ þ þ þ 7 þ

þ þ þ þ þ   þ þ 7 þ

þ  þ þ    þ þ 7 

  þ       7 

 97–104 ND 114–125 97–104 48–63 48–63 114–125 97–104, 114–125 ND 97–104

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

Western blotting. Indirect ELISA.

purified under denaturing conditions. Western blotting analysis showed that all of the proteins were well recognized by mixed MAbs except for CΔ-3 (Fig. 2B). Meanwhile, all MAbs could react well with the full-length CP in both Western blotting and indirect ELISA assays (Fig. 3, Table 1, Supplementary Table S1). The dimeric patterns of some truncated CPs (especially CΔ-6, 7, 8 and 9) with lower electrophoretic mobility were also detected consistently by some MAbs (1, 3, 4, 7 and 8). Some additional bands below the target 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 with the following experiments. The epitope regions involved in recognition by ten MAbs The full-length CP and nine truncated CPs were subjected to Western blotting analysis by using each of ten MAbs. The results showed 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), and CΔ-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 excluded from the epitope region recognized by all ten MAbs. In addition, CΔ-3 (aa 126–223) showed reactions with MAbs 2 and 9, however, no reaction signal was visible with the other eight MAbs, suggesting that the region containing aa 48–125 was involved in the recognition by those eight MAbs. MAbs 1, 4, and 10 reacted with all of the truncated CPs except for CΔ-3 (aa 126–223), and CΔ-9 (aa 114–223), indicating that the aa region 97DDDSTGIT104 (Epitope II) was required for the epitope to 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 region 114 LSDKLWTDVVFN125 (Epitope III) was required for the epitope to be recognized by MAbs 3 and 7. Both MAbs 5 and 6 exhibited reactions with fragments CΔ-1 (aa 42–223), CΔ-4 (aa 1–104), CΔ-5 (aa 1–146), and CΔ-7 (aa 48–140),

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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 for epitope mapping. Three parameters including surface probability plot-Emini, Antigenic index-Jameson-Wolf, and hydrophilicity plot-Doolittle are used for epitope prediction. Three identified epitopes (I, aa 48-63; II, aa 97-104; III, aa 114-125) are indicated. Black box indicates the hexahistidine tag.

and no reaction with the fragment CΔ-2 (aa 64–223) and other fragments, revealing that they recognized the same epitope located in the aa region 48LGTQQNAALNRDLFLT63 (Epitope I). The interactions between the MAbs (except for MAb 2 and MAb 9) and the CP fragments in the indirect ELISA (Supplementary Table S1) were consistent with those in the Western blotting assays (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 indirect ELISA, which suggested that the epitopes in CΔ-6, CΔ-7, and CΔ-8 which were recognized by the two MAbs might be conformationdependent. MAbs 2 and 9 were able to react with both CΔ-4 (aa 1–104) and CΔ-9 (aa 114–223), which made it difficult to determine the exact epitope regions recognized by these MAbs. Computer–assisted analysis further confirmed the antigenic role of those three identified regions of the CP (Fig. 4A), which are presumably exposed on the particle surface, and exhibited high antigenic and hydrophilic characteristics. Multiple alignments of the deduced amino acid sequences of CP from global

CTV isolates showed that these three regions were relatively conserved 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 in the region containing aa 97–104 of the CP was involved in recognition by MAbs 1, 4, and 10, three oligopeptides, including 97 DDDSTGIT104 and two peptides 97DDDSTGI103 and 97DDDSTG102 with one and two amino acid deletions at the C-terminus of the epitope, were synthesized and used as antigens in indirect ELISA tests (Fig. 5). The results showed that all three oligopeptides were recognized 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 negative control (0.03–0.035) (data not shown). The deletion of one (T104)

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and two (T104 and I103) amino acids resulted in reduced reactions with the three MAbs. The results indicated that the MAbs 1, 4, and 10 recognized the 97DDDSTGIT104 epitope and the deletion of two amino acids (T104 and I103) at the C-terminus had a significant effect on its reaction with those MAbs. Multiple alignments of the amino acid sequences of the CP of the CTV isolate S4 and reference isolates showed that the region containing aa 97-104 is well conserved in each of the CTV groups defined as T36, VT, and HA (Fig. 6A). To evaluate the effect of natural variations of amino acids in the region on its reaction with those MAbs, three mutants (S4-CΔ8/S100A, S4-CΔ8/I103M, and S4-CΔ8/S100T) were constructed by introducing the alanine

Fig. 5. Reactivity of three MAbs to three synthetic oligopeptides in indirect ELISA assays. The three oligopeptides are 97DDDSTGIT104, and the other two contain one or two amino acid deletions at the C-terminus, respectively. Each microplate well is coated 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 data represent the mean absorbance values at a wave-length of 450 nm of three replicates. The wells coated with skim milk are used as negative control.

(A100) specific for the T36 group and threonine (T100) specific for the VT group to replace serine (S100) at the position 100 of the CP of CTV-S4, respectively, and methionine (M103) specific for the HA group to replace isoleucine (I103) at the 103 position of the CP of CTV-S4. All of the mutants were successfully over-expressed in E. coli BL21 (DE3) cells (data not shown). Western blotting showed that S4-CΔ8 and all of the CΔ8 mutants were detected by MAbs 1, 4, and 10 (Fig. 6B). The S4-CΔ8 fragment and the mutant S4-CΔ8/S100A exhibited similar intensities in their reaction signals to MAbs 1 and 4, and a slight reduction was observed for reaction intensity of the mutant S4-CΔ8/S100A against MAb 10. The significantly reduced reactivity for all three MAbs was caused by the S4-CΔ8/I103M and S4-CΔ8/S100T mutations, specific for VT and HA, respectively. Furthermore, a CTV isolate HB1 showing abnormal serological characteristics was also included in this study. Western blotting showed that only five MAbs (2, 3, 7, 8, and 9) reacted with the recombinant HB1-CP (Fig. 7), while MAbs 1, 4, and 10, which recognized the epitope region (aa 97–104) of S4-CP, reacted negatively with HB1-CP. Comparison of the amino acid sequences at the positions 97–104 of CP between isolates HB1 and S4 showed that there were two different amino acids at the positions 98 and 100, respectively (Fig. 6A). Since the S4-CΔ-8/S100T mutant could not abolish the antigen-antibody interaction, therefore, the possible role of aspartic acid at the position 98 (D98) of S4-CP in the antibody-antigen interaction was investigated by site-directed mutagenesis. Two mutants S4-CΔ8/D98G and S4-CΔ8/D98G/ S100T were constructed by introducing the G98 or both G98 and T100 into S4-CΔ-8 to incorporate the corresponding amino acid residues 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. 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). The amino acids highlighted with an asterisk indicate the targeted sites for mutagenesis. ‘Coom’ indicates that equal amounts of those fusion proteins are loaded as indicated by the SDS-PAGE gel stained with Coomassie blue.

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Fig. 7. Western blotting analysis of the CTV-HB1 coat protein expressed in E. coli BL21 (DE3) by using ten MAbs. ‘þ ’ and ‘ ’ indicate the positive and negative reactions, respectively.

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 their interactions with MAbs 1, 4, and 10. Mutation of glycine (G98) in the HB1 CP abolishes the reactivity.

Discussion The generation of MAbs against different CTV isolates has provided an important basis for understanding the antigenic properties of CTV CP. Many methods have been used to identify epitopes 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 expressed fusion proteins (Candresse et al., 2010; Peroni et al., 2009; Saasa et al., 2012), and site-directed mutagenesis (Benjamin and Perdue, 1996). In this study, we constructed nine truncated coat proteins of the CTV isolate S4 and successfully characterized three epitopes by using ten monoclonal antibodies. The lower expression titer of the complete CP and two fragments (CΔ-4 and CΔ-5) containing only the 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 computational methods have been developed for predicting potential epitopes and their aa residues from different antigens (ElManzalawy and Honavar, 2010). Primary analysis indicated that sequences involved in the antigen function of CTV-S4 were distributed across its entire coat protein as indicated by the antigenic index (Fig. 4A). However, there are only three epitopes mapped in this study. Of the ten MAbs, the epitopes recognized by eight MAbs were determined. MAbs 1, 4, and 10 reacted with an epitope consisting of eight amino acids at the 97 to 104 position, which is a newly identified epitope region. The epitope is well conserved among all CTV genotypes except for one or two aa variations present in some isolates. MAbs 3 and 7 recognized an epitope which mapped between aa 114–125, which was within the epitope (aa 118–128) recognized by the MAbs in group III determined by Nikolaeva et al. (1996). In this region, the phenylalanine (F124) at the 124 position of the CP of severe CTV strains plays a crucial role in the recognition by MCA-13 (Pappu et al., 1993). However, these two MAbs 3 and 7 could not discriminate between the severe and mild strains (data not shown), indicating that the key amino acids in the interactions with these two MAbs were different from those recognized by MCA-13. The epitope for MAbs 5 and 6 was mapped to aa residues 48–63, which overlapped with an epitope determined previously (Nikolaeva et al., 1996; Peroni et al., 2009). It is to be expected that all of these MAbs recognize a wide spectrum of CTV strains since the three epitopes mapped in this study are highly conserved in all CTV genotypes (Fig. S1). Coincidentally, there exists high surface probability, antigenic index, and hydrophilicity in these regions (Fig. 4A). Hopp and Woods (1981) and Novotný et al. (1986) reported significant correlations between hydrophilicity, surface probability and

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antigenic determinant. Those results might support the observation that all MAbs used can capture CTV-S4 virions. The finding that all MAbs reacted with the CP of CTV-S4 in Western blotting suggested that the epitopes for those MAbs were largely dependent on the primary structure rather than the CP conformation. The MAbs 2 and 9 strongly recognized CTV virions in IC-RT-PCR, which caused our initial speculation of the presence of conformational epitopes recognized by those two MAbs. However, MAbs 2 and 9 reacted with all of the truncated CPs in Western blotting, which made it impossible to map the exact epitopes recognized by them. The reason for this might be that the two MAbs were mixtures of antibodies secreted by two or more hybrid cell lines. Our results showed that two regions (aa 97–104 and 114–125) might be involved in the recognition of MAb 8. Previously, several studies suggested that the presence of a few key amino acid residues might contribute to multiple epitope determinants (Geysen et al., 1984, 1987), and many residues in linear epitopes can be substituted. A comparison of epitopes II (97DDDSTGIT104) and III (114LSDKLWTDVVFN125) showed that they had the same amino acids D (aspartic acid) and T (threonine). However, it is difficult to conclude the exact role of these amino acids in the antibody-antigen interaction. For the first time, our results revealed that the presence of aspartic acid (D98) in the CP of the majority of CTV isolates played a crucial role in its interaction with MAbs 1, 4, and 10. The mutation of aspartic acid (D98) to gylcine (G98) in S4-CΔ-8 abolished the reactivity, and the reverse mutation of G98 to the D98 in HB1-CΔ-8 recovered the reactivity. Generally, epitope residues should be highly accessible to facilitate their contact with the antibody. The key amino acid D98 in S4-CP exhibited an increased accessible surface area (ASA) (99.562 angstroms^2) when compared with that of G98 (51.917 angstroms^2) in HB1CP (Figs. S2 and S3A and B), which might result in the different reactivity observed when compared with that from previous reports (Novotný et al., 1986; Rubinstein et al., 2008). Previous works reported the amino acid preferences of epitopes, which were generally enriched with charged and polar amino acids (Rubinstein et al., 2008). Although both D98 and G98 are polar amino acids, D98 is charged, and G98 is uncharged, suggesting that the charge of the amino acid at the 98 position of the CP might also affect its binding to antibodies (Pappu et al., 1995). Although other mutations in the epitope region (aa 97–104) did not abolish the antigen-antibody interaction, the variations at positions 100 and 103 might have affected the binding energy (Benjamin and Perdue, 1996), and then reduced the reaction intensity between the epitope region and the MAbs. Whereas, the mutations S4-CΔ8/I103M and S4-CΔ8/S100T showed more significant effects on the reactions with MAbs 1, 4 and 10 than that caused by the mutation S4-CΔ8/S100A (Fig. 5 and Fig. 6B), suggesting different contribution of these amino acids to the reactions between the epitopes and the MAbs. The epitope I (48LGTQQNAALNRDLFLT63) recognized by MAbs 5 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 recombinant HB1-CP and CTV-HB1 virions (Wang et al., 2007), although the amino acids in the epitope I region of S4-CP and HB1-CP are the same (Fig. S1). Epitope prediction analysis showed that the antigenic 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 one alpha helix at the C-terminus of epitope I of S4-CP was replaced by a beta strand at that of the HB1-CP (Fig. S3C and D). A previous report suggested that the amino acid mutations, which are not at positions directly involved in antibody binding, could result in farreaching conformational changes in an antigenic loop, which then destroy the integrity of the epitope (Parry et al., 1990). The

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predicted secondary structure in the region before epitope I showed some differences (Fig. S2), which might result in the different conformations in the epitope regions in S4-CP and HB1CP (Fig. S3E and F), which therefore affect the reactivities of the epitopes with the MAbs 5 and 6. In summary, this study identified a novel conserved linear epitope at the middle region of the CTV coat protein. The importance of individual amino acids for the antibody-antigen interaction was investigated. Binding sites recognized by eight MAbs could be identified, and the epitope regions for MAbs 5 and 6 should be further characterized by constructing the accurate crystal structure of coat proteins for S4 and HB1 (Lassaux et al., 2013). These findings provide new insights into the antigenic structure of the CTV coat protein, and will improve our understanding of antibody-antigen interactions and the development of CTV-specific diagnostic assays. With the development of more MAbs against different plant virus strains, it will be possible to perform a large-scale analysis to define epitope characteristics of plant viruses and reveal new aspects of antigen-antibody recognition.

antigenicity, Emini Surface plot, and secondary structure index analysis. Multiple alignments of predicted amino acid sequences were generated using Clustal X and GeneDoc version 2.7.0 programs. Primers used for the amplification of truncated CPs were designed based on the predicted epitopes (Fig. 4A), and amino acid alignment results (Fig. S1). Initially, five truncated CPs named CΔ1 to CΔ5 were designed, and used for the primary mapping of the epitopes. The other four truncated CPs, named CΔ6 to CΔ9, were designed based on the primary mapping results and used for fine epitope 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 the primers (Supplementary Table S2) to introduce bases corresponding to specific amino acids into CΔ8, and confirmed by sequencing to ensure that the desired mutations were incorporated. The plasmid constructions and expression of the truncated CPs and mutated CΔ8 fragments were conducted by the procedure described above.

Materials and methods

Protein purification and Western blotting

CTV isolates, antibodies, and oligopeptides

E. coli BL21 (DE3) cells containing recombinant plasmids were induced, and expressed recombinant proteins were purified from a biomass of 0.3 L culture by using Ni-NTA agarose (Qiagen, Hilden, Germany). Briefly, bacterial cells were suspend in PBS (0.01 M, pH 7.2) 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) for 30 min at room temperature and then centrifuged at 10,000  g for 20 min. The supernatant containing insoluble proteins was then passed through a pre-equilibrated column. After washing with washing buffer (100 mM NaH2PO4, 10 mM TrisHCl, and 8 M urea, pH 6.3), the proteins bound on the column were eluted out with an 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 elution buffers contained 50 mM NaH2PO4 and 300 mM NaCl with 10, 20, and 250 mM imidazole, respectively, and their pH was adjusted to 8.0. The purified proteins were visualized by 12% SDS-PAGE. Western blotting was carried out by using the protocol previously described by Song et al. (2011) with some modifications. Briefly, the separated proteins on gels were electroblotted onto a polyvinylidene 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 by incubation with AP-labeled goat anti-mouse IgG (Hþ L) (Proteintech Group, Chicago, USA). The immuno-reaction signals were developed with the ready-to-use BCIP/NBT solution (Amresco, OH, USA).

Ten monoclonal antibodies (MAbs, designated as MAb 1 to 10), raised against the CTV-S4 isolate (Wang et al., 2006), were used for epitope mapping. Equal amounts of each MAb were mixed and used as mixed MAb. The polyclonal antibody (PAb) raised against the virion preparation of CTV- L5 named PAb-L5 (Wang et al., 2006) was used in immune capture RT-PCR experiments. Isolate S4 induced stem pitting or vein clearing on C. sinensis plants, and L5 induced stem pitting on Mexican lime [Citrus aurantifolia (Christm.) Swing] plants (Jiang et al., 2008). Both CTV severe isolates 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 Biological Science (Nanjing, China). Plasmid construction and expression of hexahistidine-tagged coat protein in Escherichia coli The full length CPs of CTV-S4 and CTV-HB1 were cloned into the pMD18-T vector (TaKaRa, Dalian, China) and sequenced. For the construction of the expression vector, the cloned CP gene was amplified with the primer set S4-CP-F/-R (Supplementary Table S2), gel purified, digested with the Bam HI and Hind III restriction enzymes (TaKaRa, Dalian, China), and then ligated into the digested expression vector pET-28a ( þ) (Novagen, Madison, WI, USA), which has a His-tag, and transformed into competent E. coli BL21 (DE3) cells. Colonies with the fragment inserted in the correct orientation were identified by PCR and restriction analysis. Three clones of each insert were sequenced by a commercial sequencing service (GenScript Biological Science, Nanjing, China). The recombinant CP with a His-tag was expressed in E. coli strain BL21 (DE3) by incubating at a speed of 250 r/m at 37 1C for 6–8 h using 1 mM isopropyl-β-thiogalactopyranoside (IPTG). Designation of truncated CPs, site-directed mutagenesis and their expression in Escherichia coli The putative epitopes of the CTV coat protein were predicted by using the DNASTAR package software (DNASTAR, Madison, WI, USA) using the Kyte–Doolittle hydrophilic plot, Jameson–Wolf

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

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values at 450 nm were recorded on a Bio-Rad Model 550 Microplate Reader (BioRad, Hercules, CA). Immune capture (IC) RT-PCR Microcentrifuge tubes were coated with 100 ml of each of 10 MAbs (1 mg/ml). CTV-infected leaves were ground in extraction buffer (PBST supplemented with 1% (w/v) Na2SO3 and 2% (w/v) PVP-40) at a 1:10 dilution and the extracts were clarified by centrifugation (10,000  g, 10 min). Then the supernatants were added into each coated tube and incubated at 4 1C overnight. The captured CTV virions were dissolved with DEPC-treated deionized water, vortexed, spun briefly, and incubated at 95 1C for 10 min and chilled immediately on ice for 5 min. Reverse transcription PCR was done by using the T36 CP primer set (Supplementary Table 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 isolates were obtained from GenBank, and other sequences reported by our laboratory are as listed in Supplementary Table S3. Modeling of the 3dimensional structures of the CTV coat protein were carried out using the web based server Phyre 2 (Kelley and Sternberg, 2009). The predicted tertiary and surface structures were constructed using Pymol (http://www.pymol.org/).

Acknowledgments This study was supported by grant no. 30871684 from the National Science Foundation of China.

Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.virol.2013.10.021.

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