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

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、最后定位在细胞质中,与阴性对照细胞结果一致;但在该组细胞中,病毒NP蛋白趋向出核以及完成出核的细胞比例均显著低于阴性对照细胞(P<0.05)。将p CAGGS、pCAGGS-PGAM5及pCAGGS-PGAM5+pCAGGS-Importinα1/3/5/7-Flag/PGAM5和pCAGGS-PGAM5、pCAGGS-Importinβ1-Flag以及pCAGGS-PGAM5+pCAGGS-Importinβ1-Flag分别转染HEK293T细胞,24 h后利用Co-IP试验检测PGAM5与核输入蛋白的相互作用;将pCAGGS-PGAM5、pCAGGS-NS2-Flag及pCAGGS-PGAM5+pCAGGS-NS2-Flag和pCAGGS-PGAM5、pCAGGS-M1-Myc及pCAGGSPGAM5+pCAGGS-M1-Myc分别转染HEK293T细胞,24 h后经Co-IP试验检测PGAM5与核输出蛋白的相互作用。结果显示,PGAM5与核输入蛋白Importinα1/α3/α5/α7及Importinβ1及核输出蛋白M1/NS2均无相互作用。提示,宿主因子PGAM5可能并非通过与M1/NS2的互作调控流感病毒vRNP复合体的出核。本研究首次报道宿主因子PGAM5可以促进流感病毒的复制,且其主要参与调控流感病毒vRNP复合体的出核过程。本研究为深入了解PGAM5蛋白功能奠定了研究基础,也为流感的防控提供了新思路。

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 distribution of viral NP protein was detected by laser confocal microscopy at 0,3hpi,4hpi,and 5hpi to determine the impact of downregulated PGAM5 on the nuclear import and export of the viral ribonucleoprotein(vRNP)complex.Image J was used to quantify the proportion of cells containing viral NP protein during v RNP complex nuclear import and export.The results showed that,compared to the control group,after down regulating PGAM5 expression,there was no significant difference in the expression level of NP protein in virus particles attached to the cell surface or endocytosed into the cells.Laser confocal observation showed that in cells with downregulated PGAM5 expression,viral NP protein was first localized on the cell surface,then aggregated in the nucleus,and subsequently completed nuclear export to distribute in the cytoplasm,consistent with the results in negative control cells.However,the proportion of cells with a tendency of viral NP protein to exit the nucleus and complete nuclear export was significantly lower in cells with downregulated PGAM5 expression than in negative control cells.HEK293T cells were transfected with pCAGGS,pCAGGS-PGAM5,pCAGGS-PGAM5+pCAGGS-Importinα1/3/5/7-Flag/PGAM5,pCAGGS-PGAM5+pCAGGS-Importinβ1-Flag,and pCAGGS-PGAM5+pCAGGS-Importinβ1-Flag,respectively.After 24 hours,the interaction between PGAM5 and nuclear import proteins was detected by co-immunoprecipitation(Co-IP)assay.HEK293T cells were also transfected with p CAGGS-PGAM5,pCAGGS-NS2-Flag,pCAGGS-PGAM5+pCAGGS-NS2-Flag,pCAGGS-PGAM5,pCAGGS-M1-Myc,and p CAGGS-PGAM5+pCAGGS-M1-Myc,respectively.After 24 hours,the interaction between PGAM5 and nuclear export proteins was detected by Co-IP assay.The results showed that PGAM5 did not interact with nuclear import proteins Importinα1/α3/α5/α7 and Importinβ1,or with nuclear export proteins M1/NS2.This suggested that the host factor PGAM5 may not regulate the nuclear export of the influenza virus vRNP complex through interaction with M1/NS2.This study is the first to report that the host factor PGAM5 can promote the replication of influenza virus and is mainly involved in regulating the nuclear export process of the influenza virus vRNP complex.This study lays a foundation for further understanding the function of PGAM5 protein and provides new insights into the prevention and control of influenza.