Identification of conformational epitopes and antigen-specific residues at the D/A domains and the extramembrane C-terminal region of E2 glycoprotein of classical swine fever virus.
Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies in infected pigs. Previous studies revealed that both conformation-dependent and linear epitopes are most present within domains B/C/D/A in the N-terminal half of E2. However, studies of antigenicity beyond the B/C domains remain limited. This study revealed that conformational epitopes were present on the D/A domains as well as the proximal C-terminal of E2, since the mutation of cysteine abrogated their bindings to monoclonal antibodies (mAbs). The residue R845 at domain A and E902 at the C-terminal region were critical for specific binding to mAbs, further supporting the presence of antigenic determinants on these regions. Substitutions of cysteines in domains D/A not only abrogated the binding to mAbs directed to D/A, but also affected the binding of the downstream C-terminal region to its specific mAbs, suggesting a close interaction between the two conformational epitopes. Mutations on the five proximal cysteines at positions 869, 877, 893, 896 and 930 in the C-terminal region only affected the binding to its specific mAbs binding sites. In addition, mutation on the three distal C-terminal cysteines at positions 945, 966, and 983 resulted in loss of E2 homodimerization. This study demonstrates new antigenic epitopes on D/A domains and C-terminal of E2 that have not been reported before, and that the nine cysteines in the C-terminal function differently in either maintaining the antigenic structure or in intermolecular dimerization of E2.
Animal Health Research Institute, Council of Agriculture, 376 Chung-Cheng Road, Tansui, New Taipei City 25158, Taiwan, ROC.
Chia-Yi
CY
Chang
Chin-Cheng
CC
Huang
Ming-Chung
MC
Deng
Yeou-Liang
YL
Huang
Yu-Ju
YJ
Lin
Hsin-Meng
HM
Liu
Yeou-Liang
YL
Lin
Fun-In
FI
Wang
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000276:immunology; D002462:Cell Membrane / Q000821:virology; D006691:Classical Swine Fever / Q000276:immunology / Q000821:virology*; D006692:Classical Swine Fever Virus / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D018604:Epitope Mapping; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D013552:Swine; D014759:Viral Envelope Proteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*
D000914:Antibodies, Viral; D014759:Viral Envelope Proteins; C087086:glycoprotein E2, classical swine fever virus