METTL3表达水平对口蹄疫病毒复制的影响  

Effect of METTL3 expression level on foot-and-mouth disease virus replication

作　　者：张继燕 李伟伟 范许许 曹雪静 朱兆宇 裴丹诗 王一卓 何路 李熙忠 任青峰 张伟 王川[1] 郑海学[2] ZHANG Jiyan;LI Weiwei;FAN Xuxu;CAO Xuejing;ZHU Zhaoyu;PEI Danshi;WANG Yizhuo;HE Lu;LI Xizhong;REN Qingfeng;ZHANG Wei;WANG Chuan;ZHENG Haixue(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;State Key Laboratory for Animal Disease Control and Prevention/National Foot-and-Mouth Disease Reference Laboratory/College of Veterinary Medicine of Lanzhou University/Lanzhou Veterinary Research Institute,Chinese Academy of A gricultural Sciences,Lanzhou 730000,China;College of Veterinary Medicine,Henan Agricultural University,Zhengzhou 450046,China;College of Life Science and Technology,Gansu Agricultural University,Lanzhou 730070,China)

机构地区：[1]甘肃农业大学动物医学院,甘肃兰州州730070 [2]中国农业科学院,兰州兽医研究所,兰州大学动物医学与生物安全学院,动物疫病防控全国重点实验室,国家口蹄疫参考实验室,甘肃兰州730000 [3]河南农业大学动物医学院,河南郑州450046 [4]甘肃农业大学生命科学技术学院,甘肃兰州730070

出　　处：《中国兽医科学》2024年第8期1010-1018,共9页Chinese Veterinary Science

基　　金：国家自然科学基金青年项目(32202781,32302857);国家重点研发计划项目(2021YFD1801300,2021YFD1800300);甘肃省青年科技基金计划项目(22JR5RA034);中国博士后科学基金项目(2023M743830);国家生猪产业技术体系项目(CARS-35,CARS-39-13);国家生猪技术创新中心先导科技项目(NCTIP-XD/C03);中国农业科学院创新计划项目(CAAS-CSLPDCP-2023002,CAAS-ASTIP-2023-LVRI);“十四五”广东省农业科技创新十大主攻方向“揭榜挂帅”项目(2023SDZG02);兰州兽医研究所基本科研业务费项目(1610312021009)。

摘　　要：利用CRISPR/Cas9系统构建了METTL3基因敲除细胞系(BHK-21),随后用口蹄疫病毒(FMDV)分别感染野生型和METTL3敲除BHK-21细胞,通过q PCR、Western-blot和TCID50方法检测FMDV在上述2株细胞中的复制情况。结果表明,敲除METTL3显著抑制FMDV的复制。为了进一步验证METTL3的表型功能,利用慢病毒包装系统构建METTL3稳定表达细胞系。用FMDV分别感染野生型和METTL3过表达BHK-21细胞,通过q PCR和TCID50试验测定FMDV在这2株细胞中的复制情况。结果显示,过量表达METTL3促进FMDV的复制。此外,激光共聚焦显微镜试验显示,FMDV感染BHK-21细胞后,METTL3从细胞核迁移至细胞质,其细胞定位的改变表明METTL3可能参与调控FMDV基因组的转录过程。本研究中成功构建了METTL3基因敲除和过表达的BHK-21细胞系,首次发现该分子正向调控FMDV的复制。上述实验结果为进一步开展METTL3在宿主细胞中调控FMDV复制的机制研究奠定了可靠的数据基础。METTL3 gene knockout cell line(BHK-21)was firstly constructed utilizing CRISPR/Cas9 system.Subsequently,wild-type and METTL3 knockout cells were infected with foot-and-mouth disease virus(FMDV),and the replication of FMDV in these two cells was detected by q PCR,Western-blot and TCID_(50)experiments.The results indicated that METTL3 deficiency significantly inhibited FMDV replication.To further verify the function of METTL3,a stable METTL3 expression cell line was constructed using the lentivirus packaging system.Wild-type and METTL3 overexpressed cells were infected with FMDV,and the replication of FMDV in these two cells was also measured by q PCR and TCID_(50)assays.The results showed that overexpression of METTL3 promoted FMDV replication.In addition,laser confocal microscopy found that METTL3 migrated from the nucleus to cytoplasm upon FMDV infection,and the change of intracellular localization suggested that METTL3 might be involved in regulating the transcription process of FMDV genome.In this study,we successfully constructed a BHK-21 cell line with METTL3 knockout and overexpression,and found that METTL3 positively regulates FMDV replication.These results provide a reliable data base for further exploration on the mechanism of METTL3 regulation of FMDV replication in BHK-21 cells.

关 键 词：METTL3 m6A修饰 口蹄疫病毒 

分 类 号：S852.659.6[农业科学—基础兽医学]