宿主因子CENPV与流感病毒NP蛋白相互作用的研究  

Study on the interaction between influenza virus NP protein and host protein CENPV

作　　者：温霞 赵玉辉[1] 李奇兵 王倩[1] 孔凡迪 梁立滨[1] 王广文[1] 李俊平[1] 姜丽[1] 陈化兰[1] 李呈军[1] WEN Xia;ZHAO Yu-hui;LI Qi-bing;WANG Qian;KONG Fan-di;LIANG Li-bin;WANG Guang-wen;LI Jun-ping;JIANG Li;CHEN Hua-lan;LI Cheng-jun(Animal Influenza Key Laboratory of the Ministry of Agriculture,State Key Laboratory of veterinary Biotechnology,Harbin Veterinary Research Institute,Chinese Academy of Agricultural Science,Harbin 150069,China)

机构地区：[1]中国农业科学院哈尔滨兽医研究所,兽医生物技术国家重点实验室/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》2021年第1期1-6,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然科学基金(31672582、31521005);中央级公益性科研院所基本科研业务费专项(1610302017001)。

摘　　要：核蛋白(NP)是流感病毒的主要结构蛋白,其与RNA依赖性的聚合酶蛋白(PB2、PB1和PA)及vRNA构成核糖核蛋白复合体,参与流感病毒复制过程中的多个阶段。宿主因子着丝粒蛋白V(CENPV)在细胞生命周期中发挥重要作用,但是目前尚未见关于CENPV蛋白与流感病毒NP蛋白相互作用的报道。为研究宿主因子CENPV与流感病毒NP蛋白相互作用机制,本实验利用酵母双杂交技术筛选与NP蛋白相互作用的CENPV,利用酵母回交验证,结果显示CENPV与NP诱饵质粒共同转化Y2H酵母感受态细胞,在DDO、QDO和QDO/X/A 3种营养缺陷型的培养基上均能生长,并使菌落呈现蓝色,与阳性对照结果一致,而阴性对照组在QDO和QDO/X/A培养基上均未观察到菌落,表明在酵母系统中宿主因子CENPV与流感病毒NP蛋白存在相互作用。将pCAGGS-CENPV-Myc(1μg)表达质粒与pCAGGS-NP(1μg)质粒共转染293T细胞,通过免疫共沉淀(Co-IP)检测,结果显示鼠抗NP MAb仅可结合CENPV与NP共转染组中的CENPV,表明宿主蛋白因子CENPV与流感病毒的NP蛋白存在相互作用;进一步利用激光共聚焦试验检测,结果显示在A549细胞中宿主因子CENPV与流感病毒的NP蛋白存在共定位。根据人CENPV序列设计合成CENPV的特异性siRNA,利用RNAiMAX将CENPV siRNA转染A549细胞,48 h后进行细胞活力测定和病毒感染试验,结果显示利用siRNA干扰技术下调CENPV表达后对细胞活力无影响,但可促进流感病毒在A549细胞中的复制。以上研究结果首次证实宿主因子CENPV与流感病毒NP蛋白存在相互作用,并且调控流感病毒的复制。本研究进一步完善了流感病毒NP蛋白与宿主因子的相互作用网络,为宿主因子调控流感病毒复制研究提供参考。The nucleoprotein(NP)is the main structural protein of influenza virus,and constitutes the ribonucleo protein complex together with RNA-dependent polymerase proteins(PB2,PB1 and PA)and viral RNA.Moreover,NP protein is involved in multiple stages of the influenza virus replication.The centromere protein V(CENPV)of host factors plays a crucial role in the cell life cycle.Up to now,the interaction between influenza virus NP protein and CENPV has not been reported.In this study,CENPV interacting with NP protein was obtained by Yeast two-hybrid screening.In the yeast backcrossing experiments,Y2H yeast competent cells were co-transfected with CENPV and NP bait plasmids,the treated Y2H yeast cell grew on DDO,QDO and QDO/X/A nutrient deficient media,and the colony showed blue colony,which was consistent with the results of the positive control group.However,no colony was observed in QDO and QDO/X/A medium in the negative control group.The results indicated that the CENPV interacted with NP protein of influenza virus in the yeast system.The plasmids of pCAGGS-CENPV-myc(1μg)and pCAGGS-NP(1μg)were co-transfected into HEK293T cells,and the combination of anti-mouse NP Mab between CENPV and NP was was only observed by Co-IP in the group of co-infection with CENPV and NP,which indicated the interaction between CENPV and NP protein in HEK293T cells.Further studies showed that the protein of CENPV and NP were co-localized in the A549 cells infected with virus by confocal assay.The specific siRNA of CENPV was designed and synthesized targeting the sequence of human CENPV,and CENPV siRNA were transfected the A549 cells by RNAiMAX,the cell viability and virus infection test was carried out at 48 hours post infection.The results indicated that down-regulation expression of CENPV by siRNA had no effects on cell viability,but it promoted the replication of influenza virus in A549 cells.Our data demonstrated for the first time that host factor CENPV interacted with influenza virus NP protein and regulated influenza virus replication.

关 键 词：酵母双杂交 流感病毒 核蛋白 着丝粒蛋白V 相互作用 

分 类 号：S852.65[农业科学—基础兽医学]