Laboratory of Public Health, Faculty of Veterinary Medicine, Hokkaido University, Kita-ku, Sapporo, Japan.
Division of Molecular Pathobiology, Research Center for Zoonosis Control, Hokkaido University, Sapporo, Japan.
PLoS Pathog. 2020 Jan 23;16(1):e1008238. doi: 10.1371/journal.ppat.1008238. eCollection 2020 Jan.
West Nile virus (WNV) belongs to the Flaviviridae family and has emerged as a significant cause of viral encephalitis in birds and animals including humans. WNV replication directly induces neuronal injury, followed by neuronal cell death. We previously showed that accumulation of ubiquitinated protein aggregates was involved in neuronal cell death in the WNV-infected mouse brain. In this study, we attempted to elucidate the mechanisms of the accumulation of protein aggregates in the WNV-infected cells. To identify the viral factor inducing the accumulation of ubiquitinated proteins, intracellular accumulation of ubiquitinated proteins was examined in the cells expressing the viral protein. Expression of capsid (C) protein induced the accumulation, while mutations at residues L51 and A52 in C protein abrogated the accumulation. Wild-type (WT) or mutant WNV in which mutations were introduced into the residues was inoculated into human neuroblastoma cells. The expression levels of LC3-II, an autophagy-related protein, and AMP-activated protein kinase (AMPK), an autophagy inducer, were reduced in the cells infected with WT WNV, while the reduction was not observed in the cells infected with WNV with the mutations in C protein. Similarly, ubiquitination and degradation of AMPK were only observed in the cells infected with WT WNV. In the cells expressing C protein, AMPK was co-precipitated with C protein and mutations in L51 and A52 reduced the interaction. Although the viral replication was not affected, the accumulation of ubiquitinated proteins in brain and neurological symptoms were attenuated in the mouse inoculated with WNV with the mutations in C protein as compared with that with WT WNV. Taken together, ubiquitination and degradation of AMPK by C protein resulted in the inhibition of autophagy and the accumulation of protein aggregates, which contributes to the development of neurological disease.
西尼罗河病毒（WNV）属于黄病毒科，已成为鸟类和动物（包括人类）病毒性脑炎的重要病因。WNV 复制直接诱导神经元损伤，随后导致神经元细胞死亡。我们之前的研究表明，泛素化蛋白聚集体的积累与 WNV 感染小鼠大脑中的神经元细胞死亡有关。在这项研究中，我们试图阐明 WNV 感染细胞中蛋白聚集体积累的机制。为了鉴定诱导泛素化蛋白积累的病毒因子，我们在表达病毒蛋白的细胞中检查了泛素化蛋白的细胞内积累。衣壳（C）蛋白的表达诱导了积累，而 C 蛋白的残基 L51 和 A52 突变则消除了积累。将野生型（WT）或突变型 WNV（在残基中引入突变）接种到人神经母细胞瘤细胞中。LC3-II，一种自噬相关蛋白，和 AMP 激活的蛋白激酶（AMPK），一种自噬诱导剂的表达水平在感染 WT WNV 的细胞中降低，而在感染 C 蛋白突变的 WNV 的细胞中未观察到降低。同样，仅在感染 WT WNV 的细胞中观察到 AMPK 的泛素化和降解。在表达 C 蛋白的细胞中，AMPK 与 C 蛋白共沉淀，并且 L51 和 A52 的突变降低了相互作用。虽然病毒复制没有受到影响，但与感染 WT WNV 的小鼠相比，感染 C 蛋白突变的 WNV 的小鼠脑中泛素化蛋白的积累和神经症状减轻。总之，C 蛋白对 AMPK 的泛素化和降解导致自噬的抑制和蛋白聚集体的积累，这有助于神经疾病的发展。
