醛糖还原酶AKR1B1调控甲型流感病毒复制机制的初步研究

Study on the mechanism of aldose reductase AKR1B1 regulates influenza A virus replication

醛糖还原酶AKR1B1属于醛酮还原酶家族,是一种NADPH依赖性酶,可催化还原亲水性和疏水性醛。本实验室前期实验发现AKR1B1可以抑制流感病毒复制。为了探究醛糖还原酶AKR1B1调控流感病毒复制的具体机制,本研究通过siRNA干扰下调A549细胞中AKR1B1基因的表达后,经荧光定量RT-PCR(RT-qPCR)检测细胞内AKR1B1 m RNA的转录水平;通过western blot检测siRNA干扰后细胞内AKR1B1蛋白的表达水平;采用噬斑滴定法检测过表达及干扰AKR1B1后对流感病毒滴度的影响;通过western blot检测干扰AKR1B1表达后对细胞内病毒蛋白表达量的影响及甲型流感病毒A/WSN/33(H1N1)(简写为WSN)感染过程中对内源AKR1B1蛋白表达量的影响;采用激光共聚焦试验检测干扰AKR1B1表达对流感病毒NP蛋白核转运的影响;利用双荧光素酶报告系统检测过表达AKR1B1对流感病毒聚合酶活性的影响。RT-qPCR检测结果显示si_AKR1B1_671和si_AKR1B1_721均能极显著下调A549细胞中AKR1B1基因mRNA的转录水平,与对照组相比分别下降95.62%和85.10%(P<0.001),且不影响细胞活力;western blot检测结果显示si_AKR1B1_671能极显著降低细胞中AKR1B1蛋白的表达量(P<0.001),干扰AKR1B1能够增加细胞内病毒蛋白HA、PB2、NP、M1和NS1的表达量;噬斑滴定结果显示,与对照组相比,干扰AKR1B1表达能显著增加流感病毒WSN的病毒滴度,于感染后24 h和48 h病毒滴度分别上升了4.39倍(P<0.01)和5.53倍(P<0.001);与对照组相比,过表达AKR1B1后,流感病毒滴度于感染后24 h和48 h分别下降了62.59%(P<0.01)和82.78%(P<0.001);激光共聚焦试验结果显示,干扰AKR1B1表达不影响流感病毒的入核和出核阶段;Western blot检测结果显示,在流感病毒WSN感染过程中,细胞内源AKR1B1蛋白的表达量较为稳定,不受病毒感染的影响;双荧光素酶报告试验结果显示,与对照组相比,过表达AKR1B1后流感病毒的聚合酶活性下降了26.24%(P<0.001)。上述结果首次表明,醛糖还原酶AKR1B1通过抑制流感病毒聚合酶活性抑制流感病毒复制,干扰AKR1B1蛋白表达对流感病毒NP蛋白的入核和出核均无影响,并且流感病毒感染不影响细胞内源性AKR1B1的表达。本研究初步探究了宿主因子AKR1B1参与流感病毒复制的分子机制,为进一步了解流感病毒的复制调控提供了实验数据。

Aldose reductase AKR1B1,classified within the aldo-keto reductase family,is an NADPH dependent enzyme that catalyzes the reduction of hydrophilic as well as hydrophobic aldehydes.In order to investigate the specific mechanism by which aldose reductase AKR1B1 regulates influenza virus replication,knockdown the expression of AKR1B1 gene in A549 cells was performed by siRNA interference.RT-qPCR showed that si_AKR1B1_671 could effectively reduce the mRNA level of AKR1B1 gene in A549 cells,which decreased by 95.62%(P<0.001)compared with the control group.Western blot showed that si_AKR1B1_671 could significantly reduce the expression of AKR1B1 in A549 cells and did not affect cell viability.The results of plaque assay showed that knockdown AKR1B1 significantly increased the viral titer of influenza virus WSN.Virus titers of 24hpi and 48hpi were increased by 4.39 times and 5.53 times,respectively.The outcome of western blot showed that knockdown AKR1B1 expression significantly increased intracellular viral protein content compared with the control group.Furthermore,overexpression of AKR1B1 could significantly reduce influenza virus WSN titer by plaque assay.Influenza virus titers decreased by 62.59%(P<0.01)and 82.78%(P<0.001)at 24 hours and 48 hours after infection compared with control group,respectively.The results of the polymerase activity of influenza virus showed that overexpression of AKR1B1 decreased influenza virus polymerase activity by 26.24%(P<0.001).In conclusion,the aldose reductase AKR1B1 may inhibit influenza virus replication by suppressing influenza virus polymerase activity.This study preliminarily investigated the molecular mechanism of host factor AKR1B1 involved in influenza virus replication and provided experimental data for further understanding of the regulation of influenza virus replication.