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Analysis of Amino Acid Sites for HEV Binding to Cells |
Ya-jie LIN,Chang LIU,Shao-qi GUO,Zi-hao YU,Ming-yu LI,Jun-fei LIU,Zi-zheng ZHENG*(),Ning-shao XIA |
National Institute of Diagnostics and Vaccine Development in Infections Diseases, School of Public Health, Xiamen University, Xiamen 361102, China |
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Abstract Objective: In this study, we constructed a model for evaluating the binding of hepatitis E virus (HEV) mutated capsid proteins to cells in a way that allowed us to evaluate the role of various amino acid sites in the cell binding process. This method helps us understand the invasion mechanism of HEV and lays a foundation for studying the receptor of HEV. Methods: We used a genotype IV recombinant virus-like particle-like antigen called D66, which contains the key domain of a.a.459-606.We performed site-specific mutations of amino acids at key D66 domain sites, and then confirmed the normal conformation of all mutated proteins by SDS-PAGE and ELISA methods. After confirming the conformation of the mutated proteins, we analyzed the effect of these mutated proteins on adsorption by flow cytometry to find out the key sites that affect the absorption of virus into the cell. Results: We successfully prepared and ide.pngied 45 correctly folded and conformationally normal mutant proteins by polymerase chain reaction site-specific mutagenesis. ELISA results showed that the single point mutation of recombinant viral particle-like antigen D66 did not affect the conformation of the protein. In the application of the model to simulate the binding process of mutant proteins to cells, we found that the mutant proteins of T484A, S488A, T489A, P491A, R512A, Y561A, N562A and T585A significantly affected their adsorption of C3A. Conclusion: In this study, a model was established to evaluate the cell binding of HEV capsid protein, and it was found that alanine mutations at T484A, S488A, T489A, P491A and R512A significantly weakened the cell binding of HEV capsid proteins. These sites may be the key sites that affect HEV binding to cells.
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Received: 18 January 2023
Published: 03 August 2023
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