NMRAL1对流感病毒复制的调控机制  

Mechanism of NMRAL1 Regulating Influenza Virus Replication

作　　者：严娅 王广文[1] 孔凡迪 王旭远 王一涵 李俊平[1] 赵玉辉[1] 李呈军[1] 陈化兰[1] 姜丽[1] YAN Ya;WANG GuangWen;KONG FanDi;WANG XuYuan;WANG YiHan;LI JunPing;ZHAO YuHui;LI ChengJun;CHEN HuaLan;JIANG Li(State Key Laboratory Veterinary Biotechology/Harbin Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Harbin 150069)

机构地区：[1]中国农业科学院哈尔滨兽医研究所/兽医生物技术国家重点实验室,哈尔滨150069

出　　处：《中国农业科学》2022年第10期2067-2076,共10页Scientia Agricultura Sinica

基　　金：国家自然科学基金创新群体项目(31521005);中国博士后科学基金(2019M660897)。

摘　　要：【目的】流感病毒是一种人兽共患病原,常引起大流行,给人类健康造成巨大威胁,且流感病毒易发生变异,能不断逃逸宿主细胞的免疫反应,对现有抗流感药物产生耐药性,因此寻找抵抗流感的新方法迫在眉睫。研究通过探索NMRAL1(NmrA-like family domain-containing protein 1)对流感病毒复制的影响,并揭示其发挥作用的分子机制,为抗流感药物研发提供潜在靶点。【方法】采用siRNA干扰技术在A549细胞中下调表达NMRAL1,并通过Western Blot检测siRNA干扰后NMRAL1的表达水平;在下调表达NMRAL1的细胞中,分别感染A/Anhui/2/2005(AH05)(H5N1)和A/WSN/33(H1N1)两株不同亚型流感病毒,利用蚀斑试验检测感染病毒后24和48 h细胞上清中的病毒滴度。为确定NMRAL1影响流感病毒复制的具体阶段,在HEK293T细胞中瞬时转染NMRAL1-Myc-pCAGGS质粒过表达NMRAL1,通过双荧光素酶报告系统检测过表达NMRAL1对流感病毒聚合酶活性的影响;使用免疫荧光技术对流感病毒NP蛋白进行染色,通过激光共聚焦试验观察下调表达NMRAL1对感染病毒后3、4、5、6和8 h NP蛋白在被感染细胞中的定位情况的影响,判断下调表达NMRAL1是否影响流感病毒的入核和出核过程;利用Western Blot检测下调表达NMRAL1对流感病毒各病毒蛋白表达的影响和对流感病毒激活I型干扰素通路下游IFN刺激基因(ISGs)表达的影响,利用间接免疫荧光试验进一步研究NMRAL1对流感病毒复制的影响。【结果】Western Blot检测发现NMRAL1 siRNA能显著下调NMRAL1表达,在下调表达NMRAL1的A549细胞中分别感染H5N1和H1N1病毒,并通过蚀斑试验检测感染病毒后细胞上清中的病毒滴度,结果显示在下调表达NMRAL1的细胞中,感染流感病毒后24和48 h收取的细胞上清中病毒滴度显著下降,表明NMRAL1能促进不同亚型流感病毒的复制;为进一步探索NMRAL1调控流感病毒复制的具体机制,利用双荧光素酶报告系统检测流感�【Objective】Influenza virus is a zoonotic pathogen that often causes a pandemic and poses a great threat to human health,and the influenza viruses are prone to variants and can constantly escape the host cell immune response and develop resistance to existing anti-influenza drugs,so the search for new ways to fight influenza is imminent.This study aimed to explore the effect of NMRAL1(NmrA-like family domain-containing protein 1)on influenza virus replication,and to reveal the molecular mechanism by which it functioned,so as to provide a potential target for anti-influenza drugs development.【Method】In this study,si RNA interference technology was used to down regulate the expression of NMRAL1 in A549 cells,and the expression levels of NMRAL1were detected by Western Blot.Virus titers in cell supernatants at 24 h and 48 h after infection with two different subtypes influenza viruses,including a/Anhui/2/2005(AH05)(H5N1)and a/WSN/33(H1N1),were detected using the plaque assay.To determine the specific stage at which NMRAL1 affected influenza virus replication,NMRAL1 was overexpressed by transiently transfecting NMRAL1-Myc-pCAGGS plasmid in HEK293T cells,and the effect of overexpressing NMRAL1 on influenza virus polymerase activity was examined by luciferase reporter system.The influenza virus NP protein was stained by using immunofluorescence,and the down-regulated expression of NMRAL1 on the localization of NP protein at 3,4,5,6 and 8 h post infection was assessed respectively by confocal assay to determine whether down-regulated expression of NMRAL1 affected the process of influenza virus vRNP import and export.Western Blot was used to detect the effect of NMRAL1 knockdown on the expression of viral proteins and on the expression of IFN stimulated genes(ISGs)downstream of type I interferon pathway activated by influenza virus.Indirect immunofluorescence assay was utilized to further verify the effect of NMRAL1 on influenza virus replication.【Result】Western Blot assay showed that NMRAL1 siRNA could signifi

关 键 词：NMRAL1 流感病毒 病毒复制 IFN-Β 抗病毒基因 

分 类 号：S852.65[农业科学—基础兽医学]