摘要
目的进一步探究甲基转移酶样蛋白3(methyltransferase-like protein 3,METTL3)对抗病毒先天免疫信号转导过程的影响。方法在HEK293T细胞中过表达以及敲低METTL3,利用不同的病毒感染细胞后,通过免疫印迹法检测Ⅰ型干扰素信号通路中上游激酶TANK结合激酶1(TANK binding kinase 1,TBK1)以及下游转录因子干扰素调节因子3(interferon regulatory factor 3,IRF3)和核因子κB(NF-κB)p65亚基活化情况,水疱性口炎病毒(vesicular stomatitis virus,VSV)复制标志分子VSV-G表达水平。通过尾静脉注射VSV建立小鼠感染模型,观察Mettl3敲除小鼠(Mettl3^(F/F;Mx1-Cre))和对照小鼠(Mettl3^(F/F))生存率以及肝系数和肺系数的变化情况;通过ELISA检测小鼠血清Ⅰ型干扰素IFN-α和IFN-β的产生情况。结果HEK293T细胞中过表达METTL3后TBK1、p65以及IRF3的磷酸化减弱,VSV-G的表达下降。小鼠VSV感染模型中,Mettl3^(F/F;Mx1-Cre)小鼠死亡率明显降低,肝系数下降,且血清中IFN-α和IFN-β的含量明显上升。结论过表达METTL3抑制TBK1-IRF3信号通路的激活,促进VSV的复制;在Ⅰ型干扰素反应性细胞中敲除Mettl3后,小鼠对VSV的抵抗力增强。METTL3作为一个负调控因子,抑制抗病毒先天免疫信号转导。
N6-methyladenosine(m6A),as the most common mRNA methylation modification,plays an important role in the development of immune system and the innate and adaptive immune responses.METTL3(methyleransferase-like protein 3),the enzymatic component of the‘writer’complex,adds the m6A modification to mRNAs.In this study,in order to further explore the effects of METTL3 on antiviral innate immune signaling pathway,Western blot was used to detect the activation of TBK1,IRF3,p65 and the expression of vesicular stomatitis virus(VSV)replication marker VSV-G in HEK-293T cells with METTL3 overexpression or knockdown.The mouse model of virus infection,established by tail vein injection of VSV,was employed to monitor the survival of Mettl3 knockout mice(Mettl3^(F/F),Mx1-Cre)and control mice(Mettl3^(F/F)).Liver/lung coefficient was observed at 24 h after VSV infection,and levels of IFN-αand IFN-βin the serum were also measured by ELISA.The results of Western blot showed that METTL3 overexpression inhibited the phosphorylation of TBK1,IRF3 and p65,but enhanced VSVG expression after viral infection,whereas METTL3 knockdown exhibited opposite effects.In line with the in vitro results,compared with Mettl3^(F/F)mice,the Mettl3^(F/F),Mx1-Cre mice were obviously resistant to high dose of VSV-induced lethality,and showed attenuated pathologic lesions in the liver and higher levels of INF-αand IFN-βin the serum after challenged with a moderate dose of VSV.In conclusion,this study proves that METTL3 serves as a native regulator of antiviral innate immune signaling cascades.
作者
邓丽娇
邹滔
张纪岩
董洁
DENG Lijiao;ZOU Tao;ZHANG Jiyan;DONG Jie(Institute of Military Cognition and Brain Sciences,Academy of Military Medical Sciences,Academy of Military Sciences,Beijing 100850,China)
出处
《免疫学杂志》
CAS
CSCD
北大核心
2022年第4期310-315,共6页
Immunological Journal
