流感病毒PA蛋白与宿主SSR4蛋白的相互作用研究  

Interaction between influenza virus PA protein and host protein SSR4

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

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发实验室,黑龙江哈尔滨150069

出　　处：《中国预防兽医学报》2021年第10期1029-1033,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家重点研发计划(2016YFD0500205);黑龙江省青年科学基金项目(QC2018032)。

摘　　要：PA蛋白是流感病毒聚合酶复合体的重要组成部分,在病毒基因组的转录和复制过程中发挥重要功能,而流感病毒在宿主细胞内的有效复制离不开大量宿主因子的协助。本研究团队前期利用酵母双杂交(Y2H)系统从人源cDNA文库中筛选到大量与流感病毒PA蛋白可能存在相互作用的宿主因子,其中之一为Signal sequence receptor subunit 4(SSR4)。为研究流感病毒PA蛋白与宿主蛋白SSR4的相互作用,本研究采用酵母回交试验验证二者之间是否存在相互作用,结果显示PA蛋白与SSR4之间存在相互作用;在真核细胞中利用免疫共沉淀(Co-IP)试验检测,结果进一步证实了PA蛋白与SSR4蛋白之间的相互作用。利用慢病毒包装系统构建SSR4蛋白的过表达细胞系,并感染病毒后进行噬斑滴定,结果显示SSR4过表达可以抑制流感病毒的复制。瞬时转染过表达SSR4后再转染流感病毒聚合酶复合体表达质粒及荧光素酶报告质粒,利用双荧光素酶报告系统检测SSR4蛋白对聚合酶活性的影响,结果显示SSR4过表达后可以显著抑制流感病毒的聚合酶活性。本研究首次证实SSR4可以抑制流感病毒复制,并可使流感病毒RNP聚合酶活性显著降低,上述结果进一步完善了PA蛋白与宿主因子的互作网络,丰富了对宿主因子参与流感病毒复制周期的认识。PA protein is an important component of influenza virus polymerase complex,which plays an important role in the transcription and replication of viral genome.Effective replication of influenza virus in host cells depends on the participation of a large number of host factors.In this study,yeast two-hybrid(Y2H)system was used to screen host factors interacting with PA protein from human cDNA library,and one of them was identified to be signal sequence receptor subunit 4(SSR4).The interaction between PA protein and SSR4 protein was first verified in yeast system,which was further confirmed by Co-immunoprecipitation(Co-IP)in eukaryotic cells.A lentiviral packaging system was used to construct an SSR4-overexpressing cell line on which the virus growth titers were determined by plaque assay.We found that SSR4 overexpression inhibited the replication of influenza virus.Moreover,the effect of transient overexpression of SSR4 on viral polymerase activity was examined by using a DualLuciferase Reporter System.The results showed that the viral polymerase activity was significantly inhibited when SSR4 was overexpressed.This study further improved the interaction network between viral PA protein and host factors,and enriched the understanding of host factors participating in the replication cycle of influenza virus.

关 键 词：流感病毒 PA蛋白 SSR4蛋白 相互作用 

分 类 号：S825.65[农业科学—畜牧学]