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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

Coverage of the CHIKV capsid protein by peptides identified via mass spectrometry analysis of different bands excised from SDS-PAGE of boiled and reduced proteins immunoprecipitated by a group 1 mAb, 1.7B2. Indicated in green and blue are the predicted positions of the CHIKV CP NLS and NES, respectively. Sequences for peptides identified within each respective CHIKV CP band (1–7) are bolded and highlighted in yellow.
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viruses-07-02754-f007: Coverage of the CHIKV capsid protein by peptides identified via mass spectrometry analysis of different bands excised from SDS-PAGE of boiled and reduced proteins immunoprecipitated by a group 1 mAb, 1.7B2. Indicated in green and blue are the predicted positions of the CHIKV CP NLS and NES, respectively. Sequences for peptides identified within each respective CHIKV CP band (1–7) are bolded and highlighted in yellow.

Mentions: To determine the identity of the truncated native versions of CP, capsid proteins were immune-precipitated from CHIKV-infected lysates with protein G beads coupled to mAb 1.7B2 (group 1). Resulting pull-downs were resolved on SDS-PAGE and selected Coomassie-stained CPs were analysed by mass spectrometry. These analyses showed that all species of CP recognized by mAb 1.7B2, including the rCap, contained the same C-terminal peptide (Figure 7). However, as the CP molecules were truncated, detection of the N-terminus region was progressively lesser, with observations that peptides toward the N-terminal side of the protein, that were previously identified in larger-sized bands, were missing. No smaller molecules of CP were precipitated, which was consistent with the binding pattern of 1.7B2 and other group 1 mAbs to N-terminally-truncated recombinant CPs; binding to N1 to N4 (35–140 residues removed) but not N5 and N6 (175 and 210 residues removed). Together, these data confirm that the epitopes recognized by 1.7B2, and other group 1 mAbs, were located between residues 140 and 210 in the C-terminal half of CP. Furthermore, the lack of recognition of native CP truncated by 36 or more residues from the N-terminus by group 2 mAbs, also supports their binding to an N-terminal domain—within the first 35 amino acids—of the protein.


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)

Coverage of the CHIKV capsid protein by peptides identified via mass spectrometry analysis of different bands excised from SDS-PAGE of boiled and reduced proteins immunoprecipitated by a group 1 mAb, 1.7B2. Indicated in green and blue are the predicted positions of the CHIKV CP NLS and NES, respectively. Sequences for peptides identified within each respective CHIKV CP band (1–7) are bolded and highlighted in yellow.
© Copyright Policy
Related In: Results  -  Collection

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

viruses-07-02754-f007: Coverage of the CHIKV capsid protein by peptides identified via mass spectrometry analysis of different bands excised from SDS-PAGE of boiled and reduced proteins immunoprecipitated by a group 1 mAb, 1.7B2. Indicated in green and blue are the predicted positions of the CHIKV CP NLS and NES, respectively. Sequences for peptides identified within each respective CHIKV CP band (1–7) are bolded and highlighted in yellow.
Mentions: To determine the identity of the truncated native versions of CP, capsid proteins were immune-precipitated from CHIKV-infected lysates with protein G beads coupled to mAb 1.7B2 (group 1). Resulting pull-downs were resolved on SDS-PAGE and selected Coomassie-stained CPs were analysed by mass spectrometry. These analyses showed that all species of CP recognized by mAb 1.7B2, including the rCap, contained the same C-terminal peptide (Figure 7). However, as the CP molecules were truncated, detection of the N-terminus region was progressively lesser, with observations that peptides toward the N-terminal side of the protein, that were previously identified in larger-sized bands, were missing. No smaller molecules of CP were precipitated, which was consistent with the binding pattern of 1.7B2 and other group 1 mAbs to N-terminally-truncated recombinant CPs; binding to N1 to N4 (35–140 residues removed) but not N5 and N6 (175 and 210 residues removed). Together, these data confirm that the epitopes recognized by 1.7B2, and other group 1 mAbs, were located between residues 140 and 210 in the C-terminal half of CP. Furthermore, the lack of recognition of native CP truncated by 36 or more residues from the N-terminus by group 2 mAbs, also supports their binding to an N-terminal domain—within the first 35 amino acids—of the protein.

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