宿主细胞磷酸甘油酸变位酶5调控A型流感病毒复制机制的研究  

Study on regulation of influenza A virus replication by PGAM5

作　　者：王雨琴 李奇兵 王波[1] 王一涵 刘旭伟 单智博 王一晗 李梦雅[1] 李呈军[1] 陈化兰[1] 姜丽 WANG Yu-qin;LI Qi-bing;WANG Bo;WANG Yi-han;LIU Xu-wei;SHAN Zhi-bo;WANG Yi-han;LI Meng-ya;LI Cheng-jun;CHEN Hua-lan;JIANG Li(Animal Influenza Key Laboratory of the Ministry of Agriculture and Rural Affairs, State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences)

机构地区：[1]中国农业科学院哈尔滨兽医所动物疫病防控全国重点实验室/农业农村部动物流感重点开放实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2024年第9期885-894,922,共11页Chinese Journal of Preventive Veterinary Medicine

基　　金：黑龙江省杰出青年基金项目(JQ2023C006)。

摘　　要：为探究宿主因子磷酸甘油酸变位酶5(PGAM5)调控A型流感病毒复制的机制,本研究设计并合成PGAM5 siRNA和阴性对照Scrambled siRNA(NC siRNA),将二者分别转染A549细胞后,采用荧光定量PCR(qPCR)和western blot(WB)检测该siRNA的干扰效果,利用细胞活力试剂盒检测下调PGAM5表达对细胞活力的影响。结果显示,与转染NC siRNA的阴性对照细胞相比,PGAM5 siRNA能够极显著抑制PGAM5在A549细胞中的转录及表达水平(P<0.001、P<0.01)且对细胞活力基本无影响。为探究PGAM5对流感病毒复制的影响,将PGAM5siRNA和NC siRNA分别转染A549细胞36 h后经流感病毒A/WSN/1933(WSN,H1N1)株感染,分别于感染后24 h、48 h及72 h收获上清经噬斑滴定测定各组细胞中的病毒滴度;分别于感染后3 h~5 h采用WB检测病毒各蛋白的表达水平。结果显示,与阴性对照细胞相比,随着感染时间的延长,转染PGAM5 siRNA细胞中的病毒滴度均极显著下降(P<0.001),尤其在转染后72 h病毒滴度下降最多;且该细胞中流感病毒PB2、PB1、PA、HA、NP、NA、M1和NS1蛋白的表达水平均呈下降趋势,以感染后3 h上述蛋白表达水平的降低最为显著。为探究PGAM5调控流感病毒复制的机制,将PGAM5 siRNA和NC siRNA分别转染A549细胞,36 h后以MOI 10 WSN株感染细胞,1 h后分别采用WB检测病毒NP蛋白的表达水平,分析PGAM5对流感病毒吸附和内吞的影响,并于感染后0、3 h、4 h及5 h通过激光共聚焦试验检测细胞内病毒NP蛋白的定位,以确定下调PGAM5对流感病毒vRNP复合体入核及出核的影响,采用Image J统计含病毒NP蛋白的细胞在v RNP复合体入核及出核中的比例。结果显示,与对照组相比,下调PGAM5基因的表达后,吸附在细胞表面或内吞进入各组细胞中的病毒粒子中NP蛋白的表达水平均无显著差异。激光共聚焦观察可见,在下调PGAM5表达的细胞中,病毒NP蛋白先定位于细胞表面,再向细胞核内聚集,而后完成出核、最后定In order to investigate the regulatory role of host factor phosphoglycerate mutase 5(PGAM5)in influenza A virus(hereafter influenza virus)replication,this study designed and synthesized siRNA-PGAM5 and Scrambled siRNA(NC siRNA).After transfecting A549 cells with the two siRNAs,fluorescence quantitative PCR(qPCR)and western blot were employed to assess the interference efficiency of siRNA,and a cell viability kit was used to evaluate the impact of of down-regulating PGAM5 transcription and expression on cell viability.The results showed that compared with negative control cells,siRNA-PAGM5 effectively reduced the expression level of PGAM5 in A549 cells and had no significantly effect on cell viability.To investigate the effect of PGAM5 on influenza virus replication,A549 cells were transfected with PGAM5 siRNA and NC siRNA,respectively,followed by infection with the influenza virus A/WSN/1933(WSN,H1N1)strain 36 hours after transfection.The supernatants were harvested 24,48,and72 hours after infection,and the virus titers in each group of cells were determined by plaque assay.The expression levels of various viral proteins were detected by western blot at 3hpi-5hpi.The results showed that compared to the negative control cells,the virus titers in cells transfected with PGAM5 siRNA were significantly reduced as the infection time prolonged,especially at 72hpi,and influenza virus proteins PB2,PB1,PA,HA,NP,NA,M1,and NS1 all showed a decreasing trend,with the most significant reduction observed at 3hpi.To further explore the mechanism of PGAM5 in regulating influenza virus replication,A549 cells were transfected with PGAM5 siRNA and NC siRNA,respectively,and then infected with the WSN strain(H1N1)at MOI 10 after 36 hours of transfection.The expression level of viral NP protein was detected by western blot at 1hpi,and the effects of PGAM5 on virus attachment and endocytosis were analyzed.The above two siRNAs were transfected into A549 cells,respectively,and then infected with WSN strain 36 hours later.The intracellular

关 键 词：流感病毒 复制 磷酸甘油酸变位酶5 

分 类 号：S852.65[农业科学—基础兽医学]