摘要
为了研究宿主因子转氨醛糖酶1(TALDO1)对流感病毒复制的影响及其作用机制,本研究构建重组质粒pCAGGS-TALDO1-Flag并经PCR和测序鉴定正确后转染HEK293T细胞,24 h后采用western blot检测TALDO1蛋白的表达及反应原性。结果显示在38 ku处出现特异性条带。将TALDO1 siRNA(si_TALDO1)转染A549细胞(干扰组),48 h后采用荧光定量PCR(qPCR)检测TALDO1 m RNA的相对转录水平,分析si_TALDO1的干扰效果,利用细胞活力检测试剂盒检测干扰TALDO1后对细胞活力的影响。结果显示,与转染si_NC组(NC组)相比,干扰组细胞中TALDO1基因的相对转录水平极显著降低(P<0.0001),且与NC组的细胞相比活力无显著差异。将si_TALDO1转染A549细胞,36 h后将A型流感病毒(IAV)WSN株感染上述细胞,分别培养24 h和48 h,收获上清液经噬斑滴定试验测定病毒滴度;将上清液10倍倍比稀释后感染MDBK细胞,通过噬斑计数,检测干扰TALDO1后对流感病毒复制的影响。将si_TALDO1转染A549细胞,48 h后将WSN株感染细胞,分别在感染后0、3 h、6 h和9 h收获细胞后采用western blot检测细胞中流感病毒部分蛋白的表达水平。将pCAGGS-TALDO1-Flag转染HEK293T细胞,36 h后以WSN株感染细胞,采用Co-IP试验检测TALDO1与病毒PB2、PB1、PA和NP蛋白的相互作用。将si_TALDO1转染A549细胞,36 h后将WSN株感染细胞。在感染后早期(3 h~5 h)收获细胞,经qPCR检测细胞中流感病毒NP基因的转录水平;在感染后2 h、3 h、4 h和5 h收获细胞经激光共聚焦试验检测NP蛋白的定位,采用ZEN软件统计WSN株NP蛋白出入核的比例。分析干扰TALDO1后对流感病毒复制早期阶段的影响。结果显示,与NC组相比,流感病毒感染后24 h和48 h,干扰组细胞中的病毒滴度均极显著升高(P<0.01)。流感病毒PB2、PB1、PA和HA蛋白的表达水平均随感染时间的延长呈上升趋势,与NC组相比,病毒感染后6 h干扰组细胞中PB1和PA蛋白的表达量均极显著升高(P<0.0001);病毒感染后9 h PB2、PB1、PA和HA蛋白的表达量均极显著升高(P<0.01、P<0.001、P<0.0001)。未检测到PB2、PB1、PA、NP蛋白与TALDO1蛋白存在相互作用。与NC组相比,各时间点干扰组细胞中NP基因的转录水平均极显著升高(P<0.001、P<0.0001);激光共聚焦试验结果显示,病毒感染后2 h和3 h,干扰组细胞中NP蛋白的入核比例呈上升趋势,且明显高于NC组,感染后4 h与5 h干扰组细胞中的NP蛋白开始出核,且出核的NP蛋白逐渐增多达65%,明显多于NC组细胞。上述结果表明,TALDO1通过影响IAV复制的早期阶段抑制流感病毒的复制,本研究揭示了宿主因子TALDO1对IAV复制的影响及其作用机制,为抗流感病毒提供了潜在作用靶点及有效防控流感奠定了基础。
To investigate the impact of the host factor transaldolase 1(TALDO1)on influenza virus replication and its underly-ing mechanism,the recombinant plasmid pCAGGS-TALDO1-Flag was constructed in this study.After verification by PCR and sequencing,the plasmid was transfected into HEK293T cells.Twenty-four hours post-transfection,western blot was performed to detect TALDO1 protein expression and its reactivity.The results showed a specific band at 38ku.TALDO1 siRNA(si_TALDO1)was transfected into A549 cells(knockdown group).Forty-eight hours post-transfection,fluorescence quantitative PCR(qPCR)was used to detect TALDO1 mRNA relative transcription levels to analyze the interference efficiency of si_TALDO1.A commercial assay kit was used to assess the effect of TALDO1 knockdown on cell viability.The results showed that compared to the si_NC transfected group(NC group),the relative transcription level of the TALDO1 gene in the knockdown group was significantly reduced(P<0.0001),with no significant difference in cell viability compared to the NC group.A549 cells were transfected with si_TALDO1,and 36 hours later,were infected with the WSN strain of influenza A virus(IAV).The culture supernatants were harvested at 24 hours and 48 hours post-infection(hpi),respectively,and viral titers were determined by plaque assay.The supernatants were serially diluted 10-fold and used to infect MDBK cells;plaque counting was performed to assess the effect of TALDO1 knockdown on influenza virus replication.A549 cells were transfected with si_TALDO1,and 48 hours later,were infected with the WSN strain.Cells were harvested at 0,3hpi,6hpi,and 9hpi,and western blot was performed to detect the expression levels of specific influenza virus proteins.HEK293T cells were transfected with pCAGGS-TALDO1-Flag,and 36 hours later,were infected with the WSN strain.Co-immunoprecipitation(Co-IP)assay was conducted to detect the interaction between TALDO1 and the viral PB2,PB1,PA,and NP proteins.A549 cells were transfected with si_TALDO1,and 36 hours later,were infected with the WSN strain.Cells were harvested during the early stage of infection(3hpi-5hpi),and qPCR was used to detect the transcription levels of the influenza virus NP gene.A549 cells were transfected with si_TALDO1,and 36 hours later,were infected with the WSN strain.Cells were harvested at 2 hours,3 hours,4 hours and 5 hours post-infection to analyze the localization of the NP protein by confocal laser scanning microscopy.The ratio of nuclear import and export of the NP protein from the WSN strain was quantified using ZEN software.This experiment was designed to assess the impact of TALDO1 knockdown on the early stages of influenza virus replication.The results showed,compared to the NC group,viral titers in the supernatant of the knockdown group were significantly higher at both 24hpi and 48hpi(P<0.01).The expression levels of influenza virus PB2,PB1,PA,and HA proteins showed an increasing trend over time.Specifically,compared to the NC group,the expression levels of PB1 and PA proteins in the knockdown group were significantly higher at 6hpi(P<0.0001).At 9hpi,the expression levels of PB2,PB1,PA,and HA proteins were all significantly elevated(P<0.01,P<0.001,P<0.0001).No interaction was detected between TALDO1 and the viral PB2,PB1,PA,or NP proteins.Compared to the NC group,the transcription levels of the NP gene in the knockdown group were significantly higher at all tested time points(P<0.001,P<0.0001).The results of confocal microscopy indicated that at 2hpi and 3hpi,the proportion of NP protein localized to the nucleus in the knockdown group showed an increasing trend and was significantly higher than that in the NC group.In the knockdown group,nuclear export of the NP protein was initiated at 4 hours and 5 hours post-infection,with the percentage of exported NP progressively increasing to 65%,which was significantly higher than that in the NC group.This study demonstrates that TALDO1 inhibits IAV replication by affecting the early stages of the viral replication cycle.These findings reveal the impact of the host factor TALDO1 on IAV replication and its mechanism of action,providing a potential target for anti-influenza strategies and laying a foundation for the effective prevention and control of influenza.
作者
李梦雅
王一涵
李奇兵
王波
王一晗
赵普
刘旭伟
单智博
姜丽
陈化兰
李呈军
LI Meng-ya;WANG Yi-han;LI Qi-bing;WANG Bo;WANG Yi-han;ZHAO Pu;LIU Xu-wei;SHAN Zhi-bo;JIANG Li;CHEN Hua-lan;LI Cheng-jun(State Key Laboratory of Animal Disease Control and Prevention/Harbin Veterinary Research Institute Chinese Academy of Agricultural Sciences/Animal Influenza Key Laboratory of the Ministry of Agriculture and Rural Affairs,Harbin 150069,China)
出处
《中国预防兽医学报》
北大核心
2025年第9期885-893,共9页
Chinese Journal of Preventive Veterinary Medicine
基金
黑龙江省杰出青年人才项目(JQ2023C006)。
