淋巴细胞抗原6复合物基因座E抑制新型冠状病毒不同变异株刺突蛋白介导的感染侵入  

LY6E inhibit the entry of different variants of SARS-CoV-2 mediated by the spike protein

作　　者：刘永梅 郑双丽 陈丹瑛 宋焱君 李星霖 邱雅若 宋川[1] 张媛媛[1] 王玺 赵学森 Liu Yongmei;Zheng Shuangli;Chen Danying;Song Yanjun;Li Xinglin;Qiu Yaruo;Song Chuan;Zhang Yuanyuan;Wang Xi;Zhao Xuesen(Institute of Infectious Diseases,Beijing Ditan Hospital,Capital Medical University,Beijing 100015,China;Peking University Ditan Teaching Hospital,Beijing 100015,China)

机构地区：[1]首都医科大学附属北京地坛医院传染病研究所,北京100015 [2]北京大学地坛医院教学医院,北京100015

出　　处：《首都医科大学学报》2023年第4期652-662,共11页Journal of Capital Medical University

基　　金：国家自然科学基金面上项目(81971916)。

摘　　要：目的研究淋巴细胞抗原6复合物,基因座E(lymphocyte antigen 6 complex,locus E,LY6E)对新型冠状病毒(severe acute respiratpry syndrome coronavirus 2,SARS-CoV-2)不同变异株刺突蛋白(spike protein,S蛋白)介导的感染侵入的抑制作用。方法构建来源于人的血管紧张素转换酶2(angiotensin-converting enzyme 2,ACE2)表达质粒;建立Flp-In T-Rex 293/LY6E诱导表达细胞系和Flp-In T-Rex 293/氯霉素乙酰转移酶(chloramphenicol acetyltransferase,CAT)诱导表达细胞系,转染人ACE2表达质粒,并通过免疫印迹方法检测ACE2和LY6E在T-Rex 293细胞中的表达情况;构建SARS-CoV-2(Wuhan-Hu-1株系、D614G突变株系、Delta突变株系,以及Omicron BA.1、BA.2、BA.2.12.1、BA.3、BA.4/5、BF.7突变株系)和拉沙热病毒(Lassa fever virus,LASV)假病毒感染系统,利用假病毒荧光素酶报告基因检测LY6E对SARS-CoV-2及其突变株刺突蛋白介导侵入的抑制作用;通过Nano-Glo活细胞检测系统,检测LY6E对SARS-CoV-2突变株刺突蛋白介导的合胞体形成的抑制作用;构建甲型流感病毒(influenza A virus,IAV)假病毒感染系统,经两性霉素B处理后,利用假病毒荧光素酶报告基因检测LY6E对SARS-CoV-2及其突变株刺突蛋白介导侵入的抑制作用。结果构建的Flp-In T-Rex293/LY6E诱导表达细胞系对SARS-CoV-2(Wuhan-Hu-1株系、D614G突变株系、Delta突变株系,以及Omicron BA.1突变株系)假病毒侵入细胞有明显抑制作用,SARS-CoV-2假病毒感染四环素(tetracycline,Tet)处理组与非处理组的相对感染率差异有统计学意义[SARS-CoV-2野生型株系假病毒(WTpp):t=33.920,P<0.001;SARS-CoV-2突变株系假病毒(D614Gpp):t=31.478,P<0.001;Deltapp:t=30.257,P<0.001;Omicronpp:t=21.041,P<0.001];LY6E对SARS-CoV-2不同突变株S蛋白介导的合胞体形成有明显抑制作用,LY6E表达组与未表达组的相对荧光单位差异有统计学意义(D614G:t=18.90,P<0.001;Delta:t=22.28,P<0.001;BA.1:t=8.995,P<0.001;BA.2:t=13.57,P<0.001;BA.2.12.1:t=15.48,P<0.001Objective To investigate the inhibitory effect of lymphocyte antigen 6 complex,locus E(LY6E),on the entry of(severe acute respiratpry syndrome coronavirus 2,SARS-CoV-2)mediated by the spike protein(S)of different variant strains.Methods Plasmid encoding human angiotensin-converting enzyme 2(ACE2)molecules was constructed.We established Flp-In T-Rex 293/LY6E inducible expression cell lines and Flp-In T-Rex 293/chloramphenicol acetyltransferase(CAT)inducible expression cell lines,transfected human ACE2 expression plasmid,and detected the expression of ACE2 and LY6E in T-Rex 293 cells by Western blotting.The pseudoviral infection systems of SARS-CoV-2(Wuhan-Hu-1 strain,D614G variant,Delta variant,and Omicron BA.1,BA.2,BA.2.12.1,BA.3,BA.4/5,BF.7 variant)and Lassa fever virus(LASV)were established to detect the activity of LY6E for inhibiting viral entry by using the luciferase reporter gene.The inhibitory effect of LY6E on SARS-CoV-2 mutant strain spike-mediated syncytium formation was detected by the Nano-Glo Live Cell Assay System.We constructed the pseudoviral infection systems of influenza A virus(IAV)to detect the inhibitory effect of LY6E on SARS-CoV-2 and its mutants spike protein-mediated entry by using pseudovirus luciferase reporter gene after amphotericin B treatment.Results Flp-In T-Rex 293/LY6E inducible expression cell line could significantly inhibit the infection of SARS-CoV-2(Wuhan-Hu-1 strain,D614G strain,Delta strain,and Omicron BA.1 mutantstrain),with statistical differences in relative infection rate between the pseudovirus infection of tetracycline(Tet)treated and non-treated groups(WTpp:t=33.920,P<0.001;D614Gpp:t=31.478,P<0.001;Deltapp:t=30.257,P<0.001;Omicronpp:t=21.041,P<0.001).LY6E significantly inhibited S protein-mediated syncytium formation in different mutant strains of SARS-CoV-2,and the differences in relative fluorescence units between the LY6E-expressing and non-expressing groups were statistically significant(D614G:t=18.90,P<0.001;Delta:t=22.28,P<0.001;BA.1:t=8.995,P<0.001;BA.2:t=13

关 键 词：淋巴细胞抗原6复合体 基因座E 新型冠状病毒 Wuhan-Hu-1株 D614G株 Delta株 Omicron株 抗病毒效应 

分 类 号：R394[医药卫生—医学遗传学]