新型冠状病毒辅助蛋白ORF3a、ORF3b的致病机制研究  

Pathogenic mechanism of severe acute respiratory syndrome coronavirus 2 protein ORF3a and ORF3b

作　　者：高文欣 李希琳 傅煜轩 GAO Wenxin;LI Xilin;FU Yuxuan(Institutes of Biology and Medical Sciences of Soochow University,Suzhou,Jiangsu,215000)

机构地区：[1]苏州大学生物医学研究院,江苏苏州215000

出　　处：《实用临床医药杂志》2022年第11期1-5,共5页Journal of Clinical Medicine in Practice

基　　金：国家自然科学基金青年基金项目(31900679)。

摘　　要：目的探讨新型冠状病毒(SARS-CoV-2)的非结构蛋白ORF3a、ORF3b在宿主细胞中的功能。方法采用细胞免疫荧光法检测SARS-CoV-2假病毒颗粒的组装和释放量,采用实时荧光定量聚合酶链式反应和蛋白质印迹法(Western blot)检测ORF3a、ORF3b对β干扰素(IFN-β)和炎症因子表达的影响,采用Western blot检测ORF3a、ORF3b对干扰素信号通路中蛋白表达的影响。结果荧光显微镜观察结果显示,经SARS-CoV-2假病毒感染的过表达血管紧张素转化酶2(ACE2)的A549细胞(A549-ACE2)中,转染ORF3a表达载体、ORF3b表达载体的A549-ACE2细胞组装和释放出的SARS-CoV-2假病毒颗粒量均高于未转染和空载转染的A549-ACE2细胞,差异有统计学意义(P<0.001),说明ORF3a和ORF3b可促进SARS-CoV-2假病毒颗粒的组装和释放;给予ploy(I∶C)刺激并转染ORF3a或ORF3b表达载体的A549细胞,其IFN-β相对表达量低于单纯给予ploy(I∶C)刺激的A549细胞,差异有统计学意义(P<0.001),说明ORF3a、ORF3b转染表达可显著抑制ploy(I∶C)诱导的IFN-β表达水平;Western blot检测结果显示,与单纯给予IFN-β处理相比,给予IFN-β联合ORF3a或ORF3b转染处理能够抑制干扰素信号通路中关键蛋白MyD88、TRAF6的表达水平;转染ORF3a、ORF3b表达载体的A549细胞中炎症因子(肿瘤坏死因子-α、白细胞介素-1β、白细胞介素-6)相对表达量均高于空载转染的A549细胞,差异有统计学意义(P<0.01或P<0.001)。结论非结构蛋白ORF3能够促进SARS-CoV-2组装和释放,诱导宿主炎症应答,并抑制宿主干扰素表达。Objective To investigate functions of severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)non-structural protein ORF3a and ORF3b in host cells.Methods The assembly and release of SARS-CoV-2 pseudovirus were detected by immunofluorescence assay;real-time polymerase chain reaction and western blot were used to detect the effects of ORF3a and ORF3b on expression of IFN-βand inflammatory factors.Western blot was used to detect the effects of ORF3a and ORF3b on protein expression in interferon signaling pathway.Results Fluorescence microscopic results showed that the number of packaged and released pseudovirus particles in human lung cell line A549[A549-angiotensin converting enzyme 2(ACE2)]cells overexpressing ACE2 infected by SARA-COV-2 pseudovirus and transfected ORF3a expression vector and ORF3b expression vector was higher than those of untreated cells and empty vector cells,indicating that ORF3a and ORF3b could promote the assembly and release of SARA-COV-2 particles;A549 cells were stimulated with ploy(I∶C)and then transfected with ORF3a or ORF3b expression plasmid.The expression of IFN-βin above cells was significantly lower than that single ploy(I∶C)stimulated A549cells(P<0.001),indicating that the transfected expression of ORF3a and ORF3b could significantly inhibit IFN-βexpression in cells induced by ploy(I∶C).Western blot assay showed that the expressions of key proteins such as MyD88 and TRAF6 in interferon signaling pathway were inhibited after cells dealing with IFN-β and transfected with ORF3a or ORF3b as compared with treated by IFN-β cells alone;inflammatory cytokines levels such as (tumor necrosis factor-α, interleukin-1β, interleukin-6) in A549 cells transfected with ORF3a or ORF3b expression vector were significantly increased in ORF3a or ORF3b expression host cells compared with the empty vector A549 cells( P <0.01 or P <0.001). Conclusion Non-structural protein ORF3 can promote viral assembly and release of SARS-CoV-2, induce host inflammatory response, and inhibit host inter

关 键 词：新型冠状病毒 非结构蛋白 ORF3a ORF3b 干扰素 炎症因子 

分 类 号：R373[医药卫生—病原生物学] Q932[医药卫生—基础医学]