Limits...
Recognition of Linear B-Cell Epitope of Betanodavirus Coat Protein by RG-M18 Neutralizing mAB Inhibits Giant Grouper Nervous Necrosis Virus (GGNNV) Infection.

Chen CW, Wu MS, Huang YJ, Cheng CA, Chang CY - PLoS ONE (2015)

Bottom Line: Comparative analysis of Alanine scanning mutagenesis with dot-blotting and ELISA revealed that Valine197, Valine199, and Cysteine201 are critical for antibody binding.Substitution of Leucine200 in the RGNNV, BFNNV, and TPNNV genotypes with Methionine200 (thereby simulating the SJNNV genotype) did not affect binding affinity, implying that RG-M18 can recognize all genotypes of Betanodaviruses.The data provide new insights into the protective mechanism of this neutralizing mAB, with broader implications for Betanodavirus vaccinology and antiviral peptide drug development.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan; Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Betanodavirus is a causative agent of viral nervous necrosis syndrome in many important aquaculture marine fish larvae, resulting in high global mortality. The coat protein of Betanodavirus is the sole structural protein, and it can assemble the virion particle by itself. In this study, we used a high-titer neutralizing mAB, RG-M18, to identify the linear B-cell epitope on the viral coat protein. By mapping a series of recombinant proteins generated using the E. coli PET expression system, we demonstrated that the linear epitope recognized by RG-M18 is located at the C-terminus of the coat protein, between amino acid residues 195 and 338. To define the minimal epitope region, a set of overlapping peptides were synthesized and evaluated for RG-M18 binding. Such analysis identified the 195VNVSVLCR202 motif as the minimal epitope. Comparative analysis of Alanine scanning mutagenesis with dot-blotting and ELISA revealed that Valine197, Valine199, and Cysteine201 are critical for antibody binding. Substitution of Leucine200 in the RGNNV, BFNNV, and TPNNV genotypes with Methionine200 (thereby simulating the SJNNV genotype) did not affect binding affinity, implying that RG-M18 can recognize all genotypes of Betanodaviruses. In competition experiments, synthetic multiple antigen peptides of this epitope dramatically suppressed giant grouper nervous necrosis virus (GGNNV) propagation in grouper brain cells. The data provide new insights into the protective mechanism of this neutralizing mAB, with broader implications for Betanodavirus vaccinology and antiviral peptide drug development.

No MeSH data available.


Related in: MedlinePlus

Epitope identification through dot-blotting with synthetic peptides.(A) Left: sequences of the 20-mer synthetic peptides from 195 to 338 aa; each peptide had a 10-mer amino acid overlap with the following peptide. Right: schematic of the peptide array on the PVDF membrane. Virus-like particle (VLP, 1–338 aa) was used as a positive control. Dot-blotting of the 20-mer peptide was performed using RG-M18 mAB. (B) Fine mapping of 8-mer synthetic peptides from 195–206 aa (left). All the assays were performed in triplicate (right).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4418827&req=5

pone.0126121.g002: Epitope identification through dot-blotting with synthetic peptides.(A) Left: sequences of the 20-mer synthetic peptides from 195 to 338 aa; each peptide had a 10-mer amino acid overlap with the following peptide. Right: schematic of the peptide array on the PVDF membrane. Virus-like particle (VLP, 1–338 aa) was used as a positive control. Dot-blotting of the 20-mer peptide was performed using RG-M18 mAB. (B) Fine mapping of 8-mer synthetic peptides from 195–206 aa (left). All the assays were performed in triplicate (right).

Mentions: Subsequently, the 195–338 aa region was further divided into 14 serial sections, each of 20 amino acids (with the exception of the last segment, which consisted of 14 amino acids). The C-terminal sequence of each section overlaps with the N-terminus of the next section by 10 amino acids (Fig 2A). After peptide synthesis, a synthetic peptide array consisting of 13 spots of 20-mer peptides and 1 spot of 14-mer peptide on a PVDF membrane was generated, and subjected to dot blot analysis using RG-M18 mAB. Each synthetic peptide was named according to its location in the coat protein, and the Betanodavirus virus-like particle (VLP, 1–338 aa) was used as a positive control. Peptide 195–214 aa exhibited a strong signal that was similar to the positive control; on the other hand, a signal was not observed between RG-M18 and peptide 205–224 aa, indicating that the peptide 195–204 aa region is the actual site recognized by RG-M18. In addition, a weak signal was observed for the peptide 225–244 aa region; however, similar signals were not observed for peptides 215–234 aa or 235–254 aa, and therefore the weak signal for peptide 225–244 aa may arise from binding at 234 and/or 235 aa. Weak signals were also observed for peptides 275–294 aa and 285–304 aa, indicating that the 285–294 aa region may contribute to the epitope of RG-M18 as well (Fig 2A). The peptide 195–204 aa region was further divided into three serial 8-mer sections, which started at residue 195, 197, and 199, respectively. Dot blot analyses of these three synthetic peptides revealed that only peptide 195–202 aa undergoes a similar interaction with RG-M18 as peptide 195–214 aa and VLP (Fig 2B). Moreover, dot blot analyses of 7-mer peptides 195–201 aa and 196–202 aa showed no binding affinity with RG-M18 (data not shown). Based on these results, the 195VNVSVLCR202 sequence of Betanodavirus coat protein is the epitope site of RG-M18 recognition.


Recognition of Linear B-Cell Epitope of Betanodavirus Coat Protein by RG-M18 Neutralizing mAB Inhibits Giant Grouper Nervous Necrosis Virus (GGNNV) Infection.

Chen CW, Wu MS, Huang YJ, Cheng CA, Chang CY - PLoS ONE (2015)

Epitope identification through dot-blotting with synthetic peptides.(A) Left: sequences of the 20-mer synthetic peptides from 195 to 338 aa; each peptide had a 10-mer amino acid overlap with the following peptide. Right: schematic of the peptide array on the PVDF membrane. Virus-like particle (VLP, 1–338 aa) was used as a positive control. Dot-blotting of the 20-mer peptide was performed using RG-M18 mAB. (B) Fine mapping of 8-mer synthetic peptides from 195–206 aa (left). All the assays were performed in triplicate (right).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4418827&req=5

pone.0126121.g002: Epitope identification through dot-blotting with synthetic peptides.(A) Left: sequences of the 20-mer synthetic peptides from 195 to 338 aa; each peptide had a 10-mer amino acid overlap with the following peptide. Right: schematic of the peptide array on the PVDF membrane. Virus-like particle (VLP, 1–338 aa) was used as a positive control. Dot-blotting of the 20-mer peptide was performed using RG-M18 mAB. (B) Fine mapping of 8-mer synthetic peptides from 195–206 aa (left). All the assays were performed in triplicate (right).
Mentions: Subsequently, the 195–338 aa region was further divided into 14 serial sections, each of 20 amino acids (with the exception of the last segment, which consisted of 14 amino acids). The C-terminal sequence of each section overlaps with the N-terminus of the next section by 10 amino acids (Fig 2A). After peptide synthesis, a synthetic peptide array consisting of 13 spots of 20-mer peptides and 1 spot of 14-mer peptide on a PVDF membrane was generated, and subjected to dot blot analysis using RG-M18 mAB. Each synthetic peptide was named according to its location in the coat protein, and the Betanodavirus virus-like particle (VLP, 1–338 aa) was used as a positive control. Peptide 195–214 aa exhibited a strong signal that was similar to the positive control; on the other hand, a signal was not observed between RG-M18 and peptide 205–224 aa, indicating that the peptide 195–204 aa region is the actual site recognized by RG-M18. In addition, a weak signal was observed for the peptide 225–244 aa region; however, similar signals were not observed for peptides 215–234 aa or 235–254 aa, and therefore the weak signal for peptide 225–244 aa may arise from binding at 234 and/or 235 aa. Weak signals were also observed for peptides 275–294 aa and 285–304 aa, indicating that the 285–294 aa region may contribute to the epitope of RG-M18 as well (Fig 2A). The peptide 195–204 aa region was further divided into three serial 8-mer sections, which started at residue 195, 197, and 199, respectively. Dot blot analyses of these three synthetic peptides revealed that only peptide 195–202 aa undergoes a similar interaction with RG-M18 as peptide 195–214 aa and VLP (Fig 2B). Moreover, dot blot analyses of 7-mer peptides 195–201 aa and 196–202 aa showed no binding affinity with RG-M18 (data not shown). Based on these results, the 195VNVSVLCR202 sequence of Betanodavirus coat protein is the epitope site of RG-M18 recognition.

Bottom Line: Comparative analysis of Alanine scanning mutagenesis with dot-blotting and ELISA revealed that Valine197, Valine199, and Cysteine201 are critical for antibody binding.Substitution of Leucine200 in the RGNNV, BFNNV, and TPNNV genotypes with Methionine200 (thereby simulating the SJNNV genotype) did not affect binding affinity, implying that RG-M18 can recognize all genotypes of Betanodaviruses.The data provide new insights into the protective mechanism of this neutralizing mAB, with broader implications for Betanodavirus vaccinology and antiviral peptide drug development.

View Article: PubMed Central - PubMed

Affiliation: Institute of Cellular and Organismic Biology, Academia Sinica, Taipei, Taiwan; Institute of Fisheries Science, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Betanodavirus is a causative agent of viral nervous necrosis syndrome in many important aquaculture marine fish larvae, resulting in high global mortality. The coat protein of Betanodavirus is the sole structural protein, and it can assemble the virion particle by itself. In this study, we used a high-titer neutralizing mAB, RG-M18, to identify the linear B-cell epitope on the viral coat protein. By mapping a series of recombinant proteins generated using the E. coli PET expression system, we demonstrated that the linear epitope recognized by RG-M18 is located at the C-terminus of the coat protein, between amino acid residues 195 and 338. To define the minimal epitope region, a set of overlapping peptides were synthesized and evaluated for RG-M18 binding. Such analysis identified the 195VNVSVLCR202 motif as the minimal epitope. Comparative analysis of Alanine scanning mutagenesis with dot-blotting and ELISA revealed that Valine197, Valine199, and Cysteine201 are critical for antibody binding. Substitution of Leucine200 in the RGNNV, BFNNV, and TPNNV genotypes with Methionine200 (thereby simulating the SJNNV genotype) did not affect binding affinity, implying that RG-M18 can recognize all genotypes of Betanodaviruses. In competition experiments, synthetic multiple antigen peptides of this epitope dramatically suppressed giant grouper nervous necrosis virus (GGNNV) propagation in grouper brain cells. The data provide new insights into the protective mechanism of this neutralizing mAB, with broader implications for Betanodavirus vaccinology and antiviral peptide drug development.

No MeSH data available.


Related in: MedlinePlus