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The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition.

Goh LY, Hobson-Peters J, Prow NA, Baker K, Piyasena TB, Taylor CT, Rana A, Hastie ML, Gorman JJ, Hall RA - Viruses (2015)

Bottom Line: Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain.Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP.This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

View Article: PubMed Central - PubMed

Affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland 4072, Australia. l.goh1@uq.edu.au.

ABSTRACT
Chikungunya virus (CHIKV) is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs) previously generated towards the capsid protein (CP) of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1-35 and 140-210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

No MeSH data available.


Related in: MedlinePlus

Reactivity of anti-CHIKV CP mAbs against boiled, reduced and/or carboxymethylated CHIKV cell lysates. Boiled (B), boiled and reduced (B/R) or boiled, reduced and carboxymethylated (B/R/C) lysates of CHIKV (lanes 1–3), RRV (lanes 4–6) and West Nile virus (lanes 7–9) were probed with antibodies 1.7B2 (anti-CP), RRG8 (anti-E1) and 17D7 (anti-E), respectively, in Western blot. RRV and West Nile virus lysates were included as controls to illustrate binding of reference antibodies known to recognize conformational (RRG8) and linear epitopes (17D7) respectively.
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viruses-07-02754-f002: Reactivity of anti-CHIKV CP mAbs against boiled, reduced and/or carboxymethylated CHIKV cell lysates. Boiled (B), boiled and reduced (B/R) or boiled, reduced and carboxymethylated (B/R/C) lysates of CHIKV (lanes 1–3), RRV (lanes 4–6) and West Nile virus (lanes 7–9) were probed with antibodies 1.7B2 (anti-CP), RRG8 (anti-E1) and 17D7 (anti-E), respectively, in Western blot. RRV and West Nile virus lysates were included as controls to illustrate binding of reference antibodies known to recognize conformational (RRG8) and linear epitopes (17D7) respectively.

Mentions: To determine whether a panel of eleven anti-CHIKV CP mAbs bound conformational or linear epitopes, antigens in the form of crude lysates derived from CHIKV-infected C6/36 cells were subjected to reduction with DTT and carboxymethylation of their free sulfhydryl groups so as to prevent the reformation of disulfide bonds. These antigens were separated by SDS-PAGE alongside boiled, unreduced, or boiled, reduced, uncarboxymethylated lysates prior to immunoblotting. Probing with each of the CP-specific mAbs, including representative antibody 1.7B2 (Figure 2), revealed that all eleven antibodies recognized CP under both unreduced (~34 kDa) and reduced (~35 kDa) conditions, even after carboxymethylation (~37 kDa), indicating that the anti-CHIKV CP mAbs recognized linear epitopes that are not dependent on disulfide bonds to provide secondary structure. The increase in molecular weight with each treatment is an indication of successful chemical modification due to the thiol-disulfide exchange during reduction and the addition of iodoacetic acid during S-carboxymethylation of cysteine residues within the CP.


The Chikungunya Virus Capsid Protein Contains Linear B Cell Epitopes in the N- and C-Terminal Regions that are Dependent on an Intact C-Terminus for Antibody Recognition.

Goh LY, Hobson-Peters J, Prow NA, Baker K, Piyasena TB, Taylor CT, Rana A, Hastie ML, Gorman JJ, Hall RA - Viruses (2015)

Reactivity of anti-CHIKV CP mAbs against boiled, reduced and/or carboxymethylated CHIKV cell lysates. Boiled (B), boiled and reduced (B/R) or boiled, reduced and carboxymethylated (B/R/C) lysates of CHIKV (lanes 1–3), RRV (lanes 4–6) and West Nile virus (lanes 7–9) were probed with antibodies 1.7B2 (anti-CP), RRG8 (anti-E1) and 17D7 (anti-E), respectively, in Western blot. RRV and West Nile virus lysates were included as controls to illustrate binding of reference antibodies known to recognize conformational (RRG8) and linear epitopes (17D7) respectively.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4488721&req=5

viruses-07-02754-f002: Reactivity of anti-CHIKV CP mAbs against boiled, reduced and/or carboxymethylated CHIKV cell lysates. Boiled (B), boiled and reduced (B/R) or boiled, reduced and carboxymethylated (B/R/C) lysates of CHIKV (lanes 1–3), RRV (lanes 4–6) and West Nile virus (lanes 7–9) were probed with antibodies 1.7B2 (anti-CP), RRG8 (anti-E1) and 17D7 (anti-E), respectively, in Western blot. RRV and West Nile virus lysates were included as controls to illustrate binding of reference antibodies known to recognize conformational (RRG8) and linear epitopes (17D7) respectively.
Mentions: To determine whether a panel of eleven anti-CHIKV CP mAbs bound conformational or linear epitopes, antigens in the form of crude lysates derived from CHIKV-infected C6/36 cells were subjected to reduction with DTT and carboxymethylation of their free sulfhydryl groups so as to prevent the reformation of disulfide bonds. These antigens were separated by SDS-PAGE alongside boiled, unreduced, or boiled, reduced, uncarboxymethylated lysates prior to immunoblotting. Probing with each of the CP-specific mAbs, including representative antibody 1.7B2 (Figure 2), revealed that all eleven antibodies recognized CP under both unreduced (~34 kDa) and reduced (~35 kDa) conditions, even after carboxymethylation (~37 kDa), indicating that the anti-CHIKV CP mAbs recognized linear epitopes that are not dependent on disulfide bonds to provide secondary structure. The increase in molecular weight with each treatment is an indication of successful chemical modification due to the thiol-disulfide exchange during reduction and the addition of iodoacetic acid during S-carboxymethylation of cysteine residues within the CP.

Bottom Line: Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain.Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP.This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

View Article: PubMed Central - PubMed

Affiliation: Australian Infectious Diseases Research Centre, School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland 4072, Australia. l.goh1@uq.edu.au.

ABSTRACT
Chikungunya virus (CHIKV) is an arthropod-borne agent that causes severe arthritic disease in humans and is considered a serious health threat in areas where competent mosquito vectors are prevalent. CHIKV has recently been responsible for several millions of cases of disease, involving over 40 countries. The recent re-emergence of CHIKV and its potential threat to human health has stimulated interest in better understanding of the biology and pathogenesis of the virus, and requirement for improved treatment, prevention and control measures. In this study, we mapped the binding sites of a panel of eleven monoclonal antibodies (mAbs) previously generated towards the capsid protein (CP) of CHIKV. Using N- and C-terminally truncated recombinant forms of the CHIKV CP, two putative binding regions, between residues 1-35 and 140-210, were identified. Competitive binding also revealed that five of the CP-specific mAbs recognized a series of overlapping epitopes in the latter domain. We also identified a smaller, N-terminally truncated product of native CP that may represent an alternative translation product of the CHIKV 26S RNA and have potential functional significance during CHIKV replication. Our data also provides evidence that the C-terminus of CP is required for authentic antigenic structure of CP. This study shows that these anti-CP mAbs will be valuable research tools for further investigating the structure and function of the CHIKV CP.

No MeSH data available.


Related in: MedlinePlus