宿主因子ZFAT影响抗流感病毒固有免疫因子MxA表达的研究

ZFAT regulates expression of anti-influenza innate immune factor MxA

黏病毒抵抗蛋白A(MxA)是哺乳动物体内对H5N1高致病性禽流感病毒有着较高敏感度的固有免疫因子,本实验首先对BioGrid数据库的蛋白质互作组学信息进行分析,结果显示包含AT钩结构域锌指蛋白(ZFAT)可能影响MxA的表达活性。为了探究ZFAT对抗流感病毒因子MxA表达调节的分子机制,本实验利用siRNA干扰技术下调A549细胞中ZFAT蛋白的表达,再通过流感病毒滴定试验检测ZFAT沉默对流感病毒复制的影响,荧光定量PCR和蛋白免疫印迹试验检测ZFAT沉默对MxA和干扰素等其他固有免疫因子表达活性的影响。结果显示:下调ZFAT蛋白表达可以显著降低流感病毒的复制能力(p<0.05),且无论流感病毒感染与否,下调ZFAT蛋白表达均可以显著激活A549细胞中MxA的转录与翻译活性(p<0.01);此外,对MxA表达具有严密调控作用的I型(IFNA1、IFNB1)和III型干扰素(IFNL1)以及下游信号分子的转录活性在6 hpi也因ZFAT表达的下调而显著提升(p<0.05)。以上结果表明下调ZFAT蛋白的表达可以提高宿主细胞抗病毒因子的转录水平,并且激活抗流感病毒因子MxA的蛋白表达。本实验为探寻宿主固有免疫系统抵抗流感病毒侵入的分子机制提供了新的视角和依据。

Mammalian myxovirus resistance protein A(MxA) is an innate immune factor with outstanding sensitivity to H5 N1 highly-pathogenic avian influenza(HPAI) virus infection.To explore the molecular mechanism of regulation of MxA expression during influenza virus infection,we performed protein interactome prediction by utilizing the mapping data from BioGrid database,and found that the zinc-finger and AT hook domain containing protein(ZFAT) might affect the MxA expression.To verify this finding,ZFAT-specific siRNA treatment was applied to downregulate the expression of ZFAT protein in A549 cells,followed by H5 N1 HPAI virus infection.We found that viral replication in A549 cells was significantly suppressed(p<0.05) when ZFAT protein expression was inhibited,and significant upregulation(p<0.01) of MxA transcriptional and translational activity were detected by using qPCR and western blot,respectively.Moreover,suppressed ZFAT expression significantly enhanced transcriptional activity of molecules in IFN signal pathway at 6 hours post infection(hpi),including type Ⅰ(IFNA1,IFNB1) and type Ⅲ(IFNL1) IFNs which tightly regulate the expression of MxA.These results indicate that downregulated ZFAT protein expression led to activated MxA protein expression and enhanced mRNA transcription activity of cellular antiviral factors.Our study provides a novel perspective on the molecular mechanism of innate immune response against influenza virus infection.