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. 2016 Aug 29;10(8):e0004960. doi: 10.1371/journal.pntd.0004960

Fig 3. The E1-E211K amino acid change enhances neutralization activity of ECSA serum.

Fig 3

(A) Schematic diagram showing the generation of fusion recombinant E2 (amino acids 1–362) and recombinant E1 (amino acids 1–412) with a 16 residue linker which has glycine/ serine spacers and octa-histidine sequence. The rE2 and rE1 in each fusion protein are from either MY/08/065 (ECSA) or MY/06/37348 (Asian) virus isolates. (B) Immunoblotting was performed under non-reducing condition. Mouse anti-E2 and mouse anti-His monoclonal antibodies were used as controls. (C) The relative binding capacity of ECSA and Asian sera (1:1000 dilution) with the fusion E2-E1 proteins were determined in ELISA as (OD samples/mean OD samples tested with rE2-E1-ECSA) × 100. Data are presented as means ± SD (n = 4). *P<0.05, Mann-Whitney U test. (D) Schematic representation of the E1 glycoprotein with the numbers indicating the amino acid positions of the glycoprotein and its domain proteins. The amino acid differences between E1 glycoprotein of ECSA (MY/08/065, ICRES1) and Asian (MY/06/37348, CAR) were tabulated and mapped (from amino acids 1–412). Amino acid differences within a genotype are underlined. FL, fusion loop; C. tail, cytoplasmic tail. (E) Immunoblotting was performed against fusion E2-E1 glycoprotein under non-reducing conditions, with each named amino acid change from the Asian to the ECSA sequence introduced independently. Mouse anti-His antibody was used as a control. (F) Seroneutralization was performed against different constructs carrying indicated mutations in the CAR-2SG-ZsGreen backbone, which were rescued from the corresponding icDNA clones of CHIKV. Data are represented as means ± SD from 4 independent experiments at a serum dilution of 1:800 (pooled sera). *P<0.05, ***P<0.001, Mann-Whitney U test, relative to CAR. (G) Seroneutralization was performed against the CAR-E1-E211K rescued virus at a serum dilution of 1:800 with 23 individual serum samples. ***P<0.0001, Wilcoxon matched-pairs signed rank test. (H) The amino acid position of K211 which affects neutralization activity is localized on the structural E1-E2 heterodimer complex (based on PDB 3J2W).