Abstract
Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, responsible for yellow fever was believed to target host cell by the interaction of an envelope protein with a highly sulfated heparin sulfate. Thus, the heparin binding activity of JEV may play an important role in its function on cellular surfaces. Glycosaminoglycans (GAGs) are linear sulfated polysaccharides consisting of several repeating disaccharides units of uronic acid and hexosamines, such as heparin, which maintain a large of negative charges. It is known that the GAG-binding region of JEV envelop protein is located in E279-297. Hence, we propose that the four positive-charged amino acids, K279, K286, R288, and K290 may contribute to the direct interaction with GAGs. In order to demonstrate this hypothesis, we created four JEV E261-402 mutants (K279A, K286A, R288A, K290A) by site- directed mutagenesis, and then we employed NMR and fluorescence experiments to analyze the GAG-binding affinity of wild type JEV E261-402 and four mutants. In addition, we also synthesize a peptide fragment (JEV E279-297) to mimic the GAG-binding ability of wild type JEV E261-402. Furthermore, we employed ELISA analysis to identify the monoclonal antibody E3.3 competes with heparin to bind JEV E261-402. The results showed that the GAG-binding affinity of synthetic peptide is comparable to that of wild type, but which of four mutants are all declined. The ELISA experiments show that mAb E3.3 has significant competition with heparin. Taken together, we conclude that the four positive-charge amino acids are all important for direct interaction with heparin, and the completion of this study will provide a structural basis for design of vaccines.