溶酶体酸性环境促进猪血凝性脑脊髓炎病毒的复制  

Lysosomal acidic environment promotes replication of porcine hemagglutinating encephalomyelitis virus

作　　者：李彩丽 王改丽 柳雨竹 艾晓敏 王真真 李姿 关继羽 贺文琦 高丰 兰云刚 陆慧君 LI Caili;WANG Gaili;LIU Yuzhu;AI Xiaomin;WANG Zhenzhen;LI Zi;GUAN Jiyu;HE Wenqi;GAO Feng;LAN Yungang;LU Huijun(College of Veterinary Medicine,Jilin University,Changchun 130062,China;Jilin Province Animal Husbandry and Veterinary Academy of Sciences,Changchun 130062,China)

机构地区：[1]吉林大学动物医学学院,吉林长春130062 [2]吉林省畜牧兽医科学研究院,吉林长春130062

出　　处：《中国兽医学报》2022年第4期626-631,642,共7页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(32172804);中国博士后科学基金资助项目(2020M670858)。

摘　　要：为解析猪血凝性脑脊髓炎病毒(porcine hemagglutinating encephalomyelitis virus,PHEV)复制和溶酶体酸性环境的相关性,本研究以PHEV感染神经瘤母细胞(N2a细胞)为模型,利用Western blot和间接免疫荧光方法进行检测,发现PHEV感染神经细胞后溶酶体关联膜蛋白1(LAMP1)表达显著升高,病毒能与溶酶体共定位,并使溶酶体体积增大。其次,用溶酶体荧光探针Lyso Tracker Red DND-99标记活细胞溶酶体,结果发现病毒感染神经细胞后溶酶体的酸性增强。再者,用巴佛洛霉素A1(BafA1)阻止神经细胞溶酶体酸化后接种病毒,发现与未处理的细胞感染组相比,胞内病毒含量显著降低,表明抑制溶酶体酸化能够阻碍细胞内子代病毒的产生。本研究结果表明PHEV感染神经细胞后能定位于溶酶体,影响溶酶体的酸性环境,并且揭示了子代病毒的产生依赖溶酶体的酸性环境;研究结果为深入解析溶酶体与PHEV复制的相关性提供了依据。Previous studies have found that porcine hemagglutinating encephalomyelitis virus(PHEV)can be recruited to lysosomes after infecting nerve cells,However,it is uncertain whether the internal environment of lysosomes affects virus replication.In this study,neuroblastoma cells(N2 a cells)infected by PHEV was used as a model to explore the correlation between PHEV replication and lysosomal acidic environment.Firstly,Western blot and indirect immunofluorescence were used to detect the influence of PHEV on lysosome-related proteins after N2 a cells were infected.The results showed that the total amount of recombinant protein of lysosomal-associated membrane protein 1(LAMP1)increased significantly after PHEV infection.PHEV co-localized with nerve cell lysosomes and enlarged the lysosome.Secondly,PHEV infection increased the acidity of the lysosome 48 h post infetion in N2 a cells labeled by the lysosome fluorescent probe LysoTrackerRed DND-99.The virus was inoculated after preventing lysosomal acidification of nerve cells with bafomycin A1,It was found that the content of intracellular virus decreased significantly compared with the untreated cell infection group.The results showed that inhibition of lysosomal acidification could hinder the production of intracellular progeny virus,but could promote its release.In conclusion,this study confirmed that PHEV locates in lysosomes and affects the acidic environment of lysosomes after infecting nerve cells,and revealed that the generation of progeny virus depended on the acidic environment of lysosomes,which provides the basis for further analyzing the correlation between lysosome and PHEV replication.

关 键 词：猪血凝性脑脊髓炎病毒 溶酶体 N2A细胞 巴佛洛霉素A1 

分 类 号：S852.65[农业科学—基础兽医学]