Analysis of potato virus Y coat protein epitopes recognized by three commercial monoclonal antibodies

Abstract

Background: Potato virus Y (PVY, genus Potyvirus) causes substantial economic losses in solanaceous plants. Routine screening for PVY is an essential part of seed potato certification, and serological assays are often used. The commercial, commonly used monoclonal antibodies, MAb1128, MAb1129, and MAb1130, recognize the viral coat protein (CP) of PVY and distinguish PVYN strains from PVYO and PVYC strains, or detect all PVY strains, respectively. However, the minimal epitopes recognized by these antibodies have not been identified.

Methodology/principal findings: SPOT peptide array was used to map the epitopes in CP recognized by MAb1128, MAb1129, and MAb1130. Then alanine replacement as well as N- and C-terminal deletion analysis of the identified peptide epitopes was done to determine critical amino acids for antibody recognition and the respective minimal epitopes. The epitopes of all antibodies were located within the 30 N-terminal-most residues. The minimal epitope of MAb1128 was 25NLNKEK30. Replacement of 25N or 27N with alanine weakened the recognition by MAb1128, and replacement of 26L, 29E, or 30K nearly precluded recognition. The minimal epitope for MAb1129 was 16RPEQGSIQSNP26 and the most critical residues for recognition were 22I and 23Q. The epitope of MAb1130 was defined by residues 5IDAGGS10. Mutation of residue 6D abrogated and mutation of 9G strongly reduced recognition of the peptide by MAb1130. Amino acid sequence alignment demonstrated that these epitopes are relatively conserved among PVY strains. Finally, recombinant CPs were produced to demonstrate that mutations in the variable positions of the epitope regions can affect detection with the MAbs.

Conclusions/significance: The epitope data acquired can be compared with data on PVY CP-encoding sequences produced by laboratories worldwide and utilized to monitor how widely the new variants of PVY can be detected with current seed potato certification schemes or during the inspection of imported seed potatoes as conducted with these MAbs.

Figure 1. Mapping of the epitopes detected by monoclonal antibodies in three PVY strains.

A. Each CP of Potato virus Y (PVY) strains O (PVY^O-SASA207), N (PVY^N-605) and C (PVY^C-Adgen), and Potato virus V (PVV), was represented by 84 overlapping 20-residue long peptides synthesized on the membrane. Each row of the membrane contained 20 peptides. Asterisks indicate the position of the most N-terminal peptide of each virus. The membrane was probed with each of the following antibodies against CP: MAb1128, MAb1129, and MAb1130 (panels to the left). Binding of the MAbs to peptides was detected with horseradish peroxidase–labeled anti-mouse antibodies and visualized by capturing the enhanced chemiluminescence signals on X-ray film. The peptides detected with the respective MAb are aligned to the right (+, positive detection; –, negative). The sequence common to all peptides detected with each MAb is demarcated with dashed vertical lines. The positions of the amino acid residues in CP are indicated on top of the alignment. B. Complete amino acid sequences of PVY^N-605, PVY^O-SASA207, PVY^C-Adgen, and PVV-Suomi aligned using Clustal X 2.0. Dots indicate identical residues, and dashes indicate gaps.

Figure 2. Detection of the PVY and PVV CP-derived peptides with polyclonal antibodies against CP of PVY.

The membrane shown in Fig. 1 was probed with polyclonal rabbit anti-PVY CP antibodies and signals were detected using an alkaline phosphatase–conjugated pig anti-rabbit antibody. The membrane sections containing the peptides of different viruses are framed.

Figure 3. Fine-mapping of the MAb epitopes.

Alanine scanning and deletion mapping of selected peptides were used to define the minimal epitope detected by each MAb. In alanine scanning, each residue at a time was replaced with an alanine, whereas in deletion mapping one residue at a time was removed from the N- or C-terminus of the peptide. The peptides subjected to the analyses were N-P5 (MAb1128), O-P3 (MAb1129), and N-P1 (MAb1130) shown in Fig. 1. Probing of the peptides with the MAbs was done as in Fig. 1. The chemiluminescence signals were quantified. Each bar and line represents the relative signal intensity compared with the corresponding wild-type peptide.

Figure 4. Effects of mutations in the conserved DAG motif of PVY CP on recognition with MAb1130.

The N-terminal 35 residues of CP in PVY^O-SASA207, PVY^N-605 and PVY^C-Adgen are aligned, and the residue D6, which was mutated, is indicated by an arrow. Red highlighting denotes the minimal epitope recognized by MAb1130, and the conserved DAG motif is underlined. Crude proteins extracted from E. coli expressing an individual recombinant PVY^N-605 CP or mutant were analyzed by SDS-PAGE and stained with Coomassie Blue to detect the proteins. Positions of molecular size markers (in kilodaltons) are shown to the left. Virions of the wild-type viruses PVY^N-605 and PVY^O-UK and proteins of E. coli transformed with an empty expression vector were included as positive and negative controls, respectively. DAG, AAG, and NAG indicate the bacterial lysates containing the recombinant CPs of the wild-type PVY^N-605 and CPs carrying substitution D6A or D6N, respectively (wild-type CP carrying the DAG motif is marked with an asterisk). The corresponding western blot using MAb1130 is shown below the gel.

Figure 5. Recognition of PVY CP and mutants by the polyclonal antibody or various MAbs differs depending on specific mutations in the CP.

Crude proteins extracted from E. coli expressing the recombinant PVY^O-UK or PVY^N-605 CP and their mutants were analyzed by western blotting using a polyclonal antiserum (PAb) and the three MAbs against PVY CP. Positions of molecular size markers (in kilodaltons) are shown to the right. Virions of the wild-type viruses PVY^O-UK and PVY^N-NTN Nevski, and proteins of E. coli transformed with an empty expression vector [E. coli (-)] were included as positive and negative controls, respectively. The amino acid substitutions introduced to CPs and their positions are specified in the names of the mutant proteins shown at the top of the blot. Detection of virions with PAb (and PVY^O-UK virions with MAb1129 and MAb1130) revealed dimers (ca. 70 kDa) of the CP besides the expected monomers (ca. 35 kDa), indicating the CP-CP interactions essential for virion formation were not fully denatured by the SDS treatment.