摘要
目的探讨严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2)突变株刺突蛋白(Spike,S)蛋白对自噬的影响及其作用机制。方法用多种SASR-CoV-2毒株S蛋白表达载体转染293T,通过中和抗体JS016、抗HR121中和抗体以及巴佛洛霉素A1处理,观察细胞融合情况;细胞免疫荧光检测自噬流;荧光定量PCR和蛋白印迹分别检测LC3的mRNA和蛋白水平,探讨S蛋白对自噬的影响及作用机制。结果δ突变株S蛋白能引起LC3蛋白显著积累,但是其仅增加自噬体的数量,并不影响自噬溶酶体的数量;巴佛洛霉素A1处理不影响δ突变株S蛋白诱导的LC3累积;δ突变株S蛋白过表达并未使LC3的mRNA水平升高;抗HR121中和抗体处理可阻断δ突变株S蛋白诱导的细胞融合,相应的LC3蛋白量减少,而JS016中和抗体处理后细胞融合增加,同时伴随细胞内LC3蛋白量增加;HR2结构域缺陷的δ突变株S蛋白不诱导细胞融合,也不诱导LC3的累积及自噬流的改变。结论δ突变株S蛋白诱导胞内的不完全自噬导致LC3蛋白积累与细胞融合相关,提示作用于细胞自噬的药物可能抑制新型冠状病毒的感染。
ObjectiveTo investigate the effect of Spike(S)protein of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)mutant on autophagy and its mechanism.Methods293T cells were transfected with S protein plasmids of SARS-CoV-2 different strains,then cells were treated with neutralizing antibody JS016,anti-HR121 neutralizing antibody and Bafilomycin A1,and cell fusion was observed;the autophagic flux was detected by cellular immunofluorescence;and the mRNA and protein levels of LC3 were detected by quantitative PCR and Western Blot,to explore the effect of S protein on autophagy and its mechanism.ResultsS protein ofδmutant strain could cause significant accumulation of LC3 protein,but it only increased the number of autophagosomes and did not affect the number of autophagolysosomes;bafilomycin A1 treatment did not affect LC3 accumulation induced by S protein ofδmutant strain;overexpression of S protein ofδmutant strain did not increase the mRNA level of LC3;Treatment with anti-HR121 neutralizing antibody could block the cell fusion induced by S protein ofδmutant strain,and the level of LC3 protein decreased.However,after treatment with JS016 neutralizing antibody,cell fusion increased,accompanied by an increase in the level of LC3 protein in cells;the S protein ofδmutant strain with deletion of HR2 domain did not induce cell fusion,nor did it induce the accumulation of LC3 and the change of autophagic flux.ConclusionsThe S protein ofδmutant strain induces incomplete autophagy in cells,leading to the accumulation of LC3 protein,which is associated with cell fusion,suggesting that drugs acting on autophagy may inhibit SARS-CoV-2 infection.
作者
郝振平
庞伟
路莹
蔡雨俊
杨玉玲
郑永唐
田仁荣
HAO Zhenping;PANG Wei;LU Ying;CAI Yujun;YANG Yuling;ZHENG Yongtang;TIAN Renrong(School of Life Sciences,University of Science and Technology of China,Hefei 230026,China;Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences,Kunming Institute of Zoology,Chinese Academy of Sciences,Kunming 650223,China;College of Pharmacy,Dali University,Dali 671000,China)
出处
《国际免疫学杂志》
CAS
2024年第5期463-470,共8页
International Journal of Immunology
基金
云南省重大研发计划(202303AC100026)
云南省基础研究计划(202101AT070282)。
