朱艳慧,杨芳芳,王鑫,邢雅玲,刘雅琳
ZHU Yanhui,YANG Fangfang,WANG Xin,XING Yaling,LIU Yalin

• 1:河南中医药大学
• 2:天津中医药大学
• 3:军事科学院军事医学研究院辐射医学研究所

摘要(Abstract)：

目的 探讨LRRC23(leucine rich repeat containing 23)激活RIG-Ⅰ信号通路对流感病毒复制的影响。方法 通过在A549细胞中过表达和敲低LRRC23，探讨其对流感病毒复制的影响。将pcLRRC23质粒和siLRRC23(small interfering LRRC23)分别转染A549细胞，24 h后感染A/京防/1/86(H1N1)，空斑试验和ELISA法分别检测细胞上清中流感病毒滴度和HA蛋白表达水平；qRT-PCR和Western blot法分别检测LRRC23、RIG-Ⅰ、MAVS、流感病毒M1基因及HA蛋白的表达水平；免疫荧光和免疫共沉淀试验（co-immunoprecipitation,Co-IP）检测LRRC23与RIG-Ⅰ的相互作用；双荧光素酶报告基因分析试验检测IFN-β-luc和NF-κB-luc活性。结果 过表达LRRC23可显著降低流感病毒A549细胞上清中流感病毒滴度，抑制HA蛋白的表达。过表达LRRC23通过激活RIG-Ⅰ-MAVS信号通路，增强流感病毒刺激的IFN-β和NF-κB激活，抑制流感病毒M1基因和HA蛋白的表达。相反，敲低LRRC23可增加流感病毒感染A549细胞上清中HA蛋白的表达；上调流感病毒M1基因相对表达量，下调RIG-ⅠmRNA和MAVS mRNA相对表达量。结论 LRRC23通过激活RIG-Ⅰ信号通路抑制流感病毒复制，在抗病毒天然免疫中发挥重要作用。
ObjectiveTo investigate the effect of activation of RIG-Ⅰsignaling pathway by leucine rich repeat containing23(LRRC23)on replication of influenza virus.MethodsOverexpression and knock-down of LRRC23 were performed in A549 cells to investigate its effect on influenza virus replication. A549 cells were transfected with pcLRRC23 plasmid or siLRRC23(small interfering LRRC23)for 24 h and then infected with influenza virus A/jingfang/1/86(H1N1). The virus titer and HA protein expression level in the cell supernatant were determined by plaque assay and ELISA respectively.The expression of LRRC23,RIG-Ⅰ,MAVS,M1 and HA at gene and protein levels were determined by qRT-PCR and Western blot respectively. The interactions between LRRC23 and RIG-Ⅰwere analyzed by co-immunopre-cipitation(Co-IP)and immunofluorescence assay(IFA). IFN-β-luc and NF-κB-luc activities were determined by dual-luciferase reporter assay.ResultsThe LRRC23 overexpression significantly decreased the influenza virus titer and inhibited the expression of HA protein in the supernatant of A549 cells,while enhanced the NF-κB and IFNβ activations by activation of RIG-Ⅰ-MAVS signaling pathway,resulting in the inhibition of expressions of M1 gene and HA protein. Conversely,the knock-down of LRRC23 increased the protein expression level of HA in the supernatant of A549 cells,up-regulated the relative expression level of M1 gene and down-regulated those of RIG-ⅠmRNA and MAVS mRNA.ConclusionLRRC23 plays an essential role in innate antiviral response by inhibiting influenza virus replication through activation of RIG-Ⅰsignaling pathway.

关键词(KeyWords)： 流感病毒;LRRC23;RIG-Ⅰ信号通路;HA蛋白
Influenza virus;LRRC23;RIG-Ⅰ signaling pathway;HA protein

Abstract:

Keywords:

基金项目(Foundation): 河南省中医药科学研究专项课题（20-21ZY2147）;; 河南中医药大学博士科研启动基金（RSBSJJ2019-08）

作者(Author): 朱艳慧,杨芳芳,王鑫,邢雅玲,刘雅琳
ZHU Yanhui,YANG Fangfang,WANG Xin,XING Yaling,LIU Yalin

DOI: 10.13200/j.cnki.cjb.003551

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