Abstract
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.
Highlights
West Nile virus (WNV) is a member of the Japanese encephalitis virus (JEV) serocomplex within the genus Flavivirus of the family Flaviviridae
We demonstrated that residues L51 and A52 of WNV C protein are responsible for the accumulation of the ubiquitinated proteins
A mutation in WNV that impaired the AMPK-C protein interaction attenuated the accumulation of ubiquitinated proteins and neurological symptoms in mice
Summary
West Nile virus (WNV) is a member of the Japanese encephalitis virus (JEV) serocomplex within the genus Flavivirus of the family Flaviviridae. The viral genome constitutes a singlestranded positive-sense RNA of approximately 11 kb that encodes a single polyprotein. This polyprotein is cleaved by cellular and viral proteases into three structural proteins, capsid (C), pre-membrane, and envelope (E) proteins, as well as seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5. Mosquitoes and birds commonly harbor WNV reservoirs, whereas mammals, such as humans or horses, are incidental hosts. Clinical symptoms of WNV infection include febrile illness, fatal meningoencephalitis, and acute flaccid paralysis [1]. Some vaccines are available for use in animals, no vaccine or specific therapy against WNV is currently approved for human use
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