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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

Alignments of coat protein sequence from five Betanodavirus genotypes, covering the regions possessing the epitope recognized by RG-M18 mAB.The boxed sequences indicate the RG-M18-recognizing epitope. The epitopes are conserved, with the exception that residue 200 is Methionine in SJNNV, but Leucine in the sequences of the other four genotypes. A total of 37 isolates were aligned using ClustalX2 and BioEdit 7.2.3 software. GenBank accession numbers are listed in front of the strain name.
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pone.0126121.g004: Alignments of coat protein sequence from five Betanodavirus genotypes, covering the regions possessing the epitope recognized by RG-M18 mAB.The boxed sequences indicate the RG-M18-recognizing epitope. The epitopes are conserved, with the exception that residue 200 is Methionine in SJNNV, but Leucine in the sequences of the other four genotypes. A total of 37 isolates were aligned using ClustalX2 and BioEdit 7.2.3 software. GenBank accession numbers are listed in front of the strain name.

Mentions: The specificity of the interactions between epitope peptide and RG-M18 was further assessed by Alanine-scanning mutagenesis. Constructs were synthesized in which one of the eight residues of 195VNVSVLCR202 was replaced with Alanine. The Alanine mutation peptide array was placed on a PVDF membrane, and peptide 195–202 aa, peptide 195–214 aa, and VLP were used as positive controls (Fig 3A). Dot blotting indicated that the V199A and C201A mutations reduced the binding affinity of RG-M18, while the V197A mutation abolished binding (Fig 3A). The results of ELISA using RG-M18 were similar to those of dot blot analysis. The lowest OD absorption was observed for the V197A, V199A, and C201A mutations (Fig 3B). Comparative sequence alignment demonstrated that all Betanodavirus coat proteins exhibit high conservation at the region of the neutralizing binding site, with the exception that residue 200 is Methionine in SJNNV, but Leucine in the sequences of the other four genotypes (Fig 4). We thus inspected the binding of RG-M18 to peptide sequence 195VNVSVMCR202 (in which the Leucine at position 200 of the SJNNV genotype sequence is replaced with Methionine) by dot blotting and ELISA. To our surprise, RG-M18 recognized 195VNVSVMCR202 as effectively as it recognizes the RGNNV genotype epitope sequence (Figs 3A, 3B and 4).


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)

Alignments of coat protein sequence from five Betanodavirus genotypes, covering the regions possessing the epitope recognized by RG-M18 mAB.The boxed sequences indicate the RG-M18-recognizing epitope. The epitopes are conserved, with the exception that residue 200 is Methionine in SJNNV, but Leucine in the sequences of the other four genotypes. A total of 37 isolates were aligned using ClustalX2 and BioEdit 7.2.3 software. GenBank accession numbers are listed in front of the strain name.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0126121.g004: Alignments of coat protein sequence from five Betanodavirus genotypes, covering the regions possessing the epitope recognized by RG-M18 mAB.The boxed sequences indicate the RG-M18-recognizing epitope. The epitopes are conserved, with the exception that residue 200 is Methionine in SJNNV, but Leucine in the sequences of the other four genotypes. A total of 37 isolates were aligned using ClustalX2 and BioEdit 7.2.3 software. GenBank accession numbers are listed in front of the strain name.
Mentions: The specificity of the interactions between epitope peptide and RG-M18 was further assessed by Alanine-scanning mutagenesis. Constructs were synthesized in which one of the eight residues of 195VNVSVLCR202 was replaced with Alanine. The Alanine mutation peptide array was placed on a PVDF membrane, and peptide 195–202 aa, peptide 195–214 aa, and VLP were used as positive controls (Fig 3A). Dot blotting indicated that the V199A and C201A mutations reduced the binding affinity of RG-M18, while the V197A mutation abolished binding (Fig 3A). The results of ELISA using RG-M18 were similar to those of dot blot analysis. The lowest OD absorption was observed for the V197A, V199A, and C201A mutations (Fig 3B). Comparative sequence alignment demonstrated that all Betanodavirus coat proteins exhibit high conservation at the region of the neutralizing binding site, with the exception that residue 200 is Methionine in SJNNV, but Leucine in the sequences of the other four genotypes (Fig 4). We thus inspected the binding of RG-M18 to peptide sequence 195VNVSVMCR202 (in which the Leucine at position 200 of the SJNNV genotype sequence is replaced with Methionine) by dot blotting and ELISA. To our surprise, RG-M18 recognized 195VNVSVMCR202 as effectively as it recognizes the RGNNV genotype epitope sequence (Figs 3A, 3B and 4).

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