Functional identification of Q33/Y145 sites in the active center of Senecavirus 3C protease and their impact on viral replication

JIN Xinshun; RAN Xuhua; WEN Xiaobo

doi:10.13557/j.cnki.issn1002-2813.2025.13.020

Abstract：This study aimed to investigate the impact of specific amino acid mutations in the 3C protein on the replication capacity of Senecavirus A(SVA).Using the wild-type strain CHN/SVA/HB/2018(WT)as the research material,and combining structural information of the 3C protein,key sites were screened via bioinformatic analysis.Changes in the hydrogen bond network and protein structure before and after mutation were predicted.Specific-pathogen-free IBRS-2 cells were used to rescue mutant viruses.Viral titers and growth curves were determined,and transcriptional levels of mutant strains were detected using realtime fluorescence quantitative PCR(RT-qPCR).The results showed that the 33 amino acid(Q33)exhibited the highest folding free energy,while the 145 amino acid(Y145)occupied the most spatially distal position.Mutation of Q33 and Y145 to lysine increased the isoelectric point,decreased the instability index,reduced the hydrogen bond network,and altered the microstructure of the 3C binding pocket.Although the growth trend of mutant viruses resembled WT,their viral titers and transcriptional capacity were extremely lower than WT(P＜0.01).This study indicates that Q33 and Y145 in the 3C protein of WT are likely key sites affecting viral replication.Substitution mutations introduced into the 3C protein via reverse genetics reduced protein structural stability,thereby impairing 3C protease activity and diminishing viral proliferative and replicative capacities,making it a potential candidate strain for SVA attenuated live vaccine.