自噬对尼尔森湾正呼肠孤病毒复制的影响。
The effects of autophagy on the replication of Nelson Bay orthoreovirus.
机构信息
Department of Pathogenic Microbiology, College of Basic Medical Sciences, China Medical University, No. 77, Puhe Road, Shenyang North New Area, Shenyang City, 110013, Liaoning Province, People's Republic of China.
Department of Pathogenic Microbiology, College of Basic Medical Sciences, Jinzhou Medical University, No. 40, the Third Section of SongPo Rd, Jinzhou City, 121200, Liaoning Province, China.
出版信息
Virol J. 2019 Jul 18;16(1):90. doi: 10.1186/s12985-019-1196-7.
BACKGROUND
Nelson Bay orthoreovirus (NBV) was first isolated over 40 years ago from a fruit bat in Australia. Normally, NBV does not cause human diseases, but recently several NBV strains have been associated with human respiratory tract infections, thus attracting clinical attention. Autophagy, an evolutionarily conserved process in eukaryotic cells, degrades intracellular substrates, participates in multiple physiological processes, and maintains cellular homeostasis. In addition, autophagy is intimately involved in viral infection.
METHODS
A new strain of NBV, isolated from a patient with a respiratory tract infection who returned to Japan from Bali, Indonesia, in 2007, was used in this study. NBV was rescued using a reverse genetics system involving cotransfection of BHK cells with 11 plasmids (pT7-L1 MB, pT7-L2 MB, pT7-L3 MB, pT7-M1 MB, pT7-M2 MB, pT7-M3 MB, pT7-S1 MB, pT7-S2 MB, pT7-S3 MB, pT7-S4 MB, and pcDNA3.1-T7), yielding NBV-MB. Recovered viruses were confirmed by immunofluorescence. The effect of NBV-MB on autophagy was evaluated by measuring the LC3-I/II proteins by immunoblot analysis after infection of BHK cells. Furthermore, after treatment with rapamycin (RAPA), 3-methyladenine (3-MA), chloroquine (CQ), or plasmid (GFP-LC3) transfection, the changes in expression of the LC3 gene and the amount of LC3-I/II protein were examined. In addition, variations in viral titer were assayed after treatment of BHK cells with drugs or after transfection with plasmids pCAGM3 and pCAGS3, which encode virus nonstructural proteins μNS and σNS, respectively.
RESULTS
NBV-MB infection induced autophagy in host cells; however, the level of induction was dependent on viral replication. Induction of autophagy increased viral replication. By contrast, inhibiting autophagy suppressed NBV replication, albeit not significantly. The NBV-MB nonstructural protein μNS was involved in the induction of autophagy with viral infection.
CONCLUSIONS
NBV-MB infection triggered autophagy. Also, the NBV nonstructural protein μNS may contribute to augmentation of autophagy upon viral infection.
背景
纳尔逊湾异嗜性呼肠孤病毒（NBV）于 40 多年前在澳大利亚的一种果蝠中首次分离出来。通常情况下，NBV 不会引起人类疾病，但最近有几株 NBV 与人类呼吸道感染有关，因此引起了临床关注。自噬是真核细胞中一种进化上保守的过程，可降解细胞内底物，参与多种生理过程，并维持细胞内稳态。此外，自噬与病毒感染密切相关。
方法
本研究使用了一种从一位 2007 年从印度尼西亚巴厘岛返回日本的呼吸道感染患者中分离出的新型 NBV 株。使用涉及共转染 BHK 细胞的 11 个质粒（pT7-L1 MB、pT7-L2 MB、pT7-L3 MB、pT7-M1 MB、pT7-M2 MB、pT7-M3 MB、pT7-S1 MB、pT7-S2 MB、pT7-S3 MB、pT7-S4 MB 和 pcDNA3.1-T7）的反向遗传学系统拯救了 NBV，产生了 NBV-MB。通过免疫荧光确认了恢复的病毒。通过感染 BHK 细胞后免疫印迹分析测量 LC3-I/II 蛋白来评估 NBV-MB 对自噬的影响。此外，在用雷帕霉素（RAPA）、3-甲基腺嘌呤（3-MA）、氯喹（CQ）或质粒（GFP-LC3）转染后，检查 LC3 基因表达的变化和 LC3-I/II 蛋白的量。此外，在用药物处理 BHK 细胞或转染分别编码病毒非结构蛋白 μNS 和 σNS 的质粒 pCAGM3 和 pCAGS3 后，测定病毒滴度的变化。
结果
NBV-MB 感染诱导了宿主细胞中的自噬；然而，诱导水平取决于病毒复制。自噬的诱导增加了病毒的复制。相反，抑制自噬抑制了 NBV 的复制，但不显著。NBV-MB 的非结构蛋白 μNS 参与了病毒感染诱导的自噬。
结论
NBV-MB 感染触发了自噬。此外，病毒非结构蛋白 μNS 可能有助于在病毒感染时增强自噬。
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