LRRC23激活RIG-Ⅰ信号通路对流感病毒复制的影响

Effect of activation of RIG-Ⅰsignaling pathway by LRRC23 on replication of influenza virus

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。

Objective To 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 IFNβactivations 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.