West Nile virus capsid protein inhibits autophagy by AMP-activated protein kinase degradation in neurological disease development
West Nile virus capsid protein inhibits autophagy by AMP-activated protein kinase degradation in neurological disease development
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DOI：
10.1371/journal.ppat.1008238
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发表时间：
2020-01-01
期刊：
影响因子：
6.7
通讯作者：
Kariwa, Hiroaki
中科院分区：
文献类型：
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作者：
Kobayashi, Shintaro;Yoshii, Kentaro;Kariwa, Hiroaki
西尼罗河病毒(WNV)属于黄病毒科，已成为包括人类在内的鸟类和动物病毒性脑炎的重要原因。西尼罗河病毒复制直接导致神经元损伤，继而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西尼罗河病毒感染小鼠脑内神经细胞的死亡有关。在这项研究中，我们试图阐明西尼罗河病毒感染细胞中蛋白质聚集体积累的机制。为了确定导致泛素化蛋白积累的病毒因素，在表达病毒蛋白的细胞中检测泛素化蛋白在细胞内的积累。衣壳(C)蛋白的表达诱导了这种积累，而C蛋白L51和A52残基的突变则抑制了这种积累。将野生型(WT)或突变型WNV接种于人神经母细胞瘤细胞。感染WT WNV的细胞自噬相关蛋白Lc3-II和自噬诱导因子AMPK的表达水平降低，而C蛋白突变的WNV感染细胞未见这种降低。同样，只有在感染WT WNV的细胞中才能观察到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，L51和A52突变减少了这种相互作用。虽然不影响病毒的复制，但与WT WNV相比，接种了C蛋白突变的WNV后，脑内泛素化蛋白的积聚和神经症状都有所减轻。综上所述，泛素化和C蛋白对AMPK的降解导致自噬的抑制和蛋白质聚集体的积累，从而导致神经系统疾病的发生。在此之前，我们已确定西尼罗河病毒感染可诱导神经细胞泛素化蛋白的积聚。然而，这种堆积的详细机制及其与脑内致病性的关联仍不清楚。在这里，我们证明了西尼罗河病毒C蛋白的L51和A52残基是泛素化蛋白积累的原因。这两个C蛋白残基促进了C蛋白与自噬诱导酶AMPK的相互作用，导致AMPK的泛素化和降解，并抑制了自噬。WNV中一种破坏AMPK-C蛋白相互作用的突变可以减轻泛素化蛋白的积累和小鼠的神经症状。我们的发现首次描述了病毒蛋白对AMPK的调节，并代表着在理解西尼罗河病毒感染神经发病的分子机制方面向前迈进了一步。
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.Author summary The elimination of ubiquitinated protein aggregates from neuronal cells occurs via protein degradation systems such as autophagy or proteasome. Previously, we determined that WNV infection induces the accumulation of ubiquitinated proteins in neuronal cells. However, the detailed mechanisms underlying this accumulation and its association with pathogenicity in the brain remain unclear. Here, we demonstrated that residues L51 and A52 of WNV C protein are responsible for the accumulation of the ubiquitinated proteins. The two C protein residues promoted the interaction between C protein and the autophagy-inducing enzyme AMPK, resulting in ubiquitination and degradation of AMPK and inhibition of autophagy. A mutation in WNV that impaired the AMPK-C protein interaction attenuated the accumulation of ubiquitinated proteins and neurological symptoms in mice. Our findings are the first to describe the regulation of AMPK by a viral protein and represent a step forward in understanding of molecular mechanisms of the neuropathogenesis of WNV infection.