Japanese encephalitis virus NS1 and NS1′ protein disrupts the blood-brain barrier through macrophage migration inhibitory factor-mediated autophagy

ABSTRACT Flaviviruses in the Japanese encephalitis virus (JEV) serogroup, such as JEV, West Nile virus, and St. Louis encephalitis virus, can cause severe neurological diseases. The nonstructural protein 1 (NS1) is a multifunctional protein of flavivirus that can be secreted by infected cells and circulate in the host bloodstream. NS1′ is an additional form of NS1 protein with 52 amino acids extension at its carboxy-terminal and is produced exclusively by flaviviruses in the JEV serogroup. In this study, we demonstrated that the secreted form of both NS1 and NS1′ can disrupt the blood-brain barrier (BBB) of mice, with NS1′ exhibiting a stronger effect. Using the in vitro BBB model, we found that treatment of soluble recombinant JEV NS1 or NS1′ protein increases the permeability of human brain microvascular endothelial cells (hBMECs) and leads to the degradation of tight junction proteins through the autophagy-lysosomal pathway. Consistently, NS1′ protein exhibited a more pronounced effect compared to NS1 in these cellular processes. Further research revealed that the increased expression of macrophage migration inhibitory factor (MIF) is responsible for triggering autophagy after NS1 or NS1′ treatment in hBMECs. In addition, TLR4 and NF-κB signaling was found to be involved in the activation of MIF transcription. Moreover, administering the MIF inhibitor has been shown to decrease viral loads and mitigate inflammation in the brains of mice infected with JEV. This research offers a novel perspective on the pathogenesis of JEV. In addition, the stronger effect of NS1′ on disrupting the BBB compared to NS1 enhances our understanding of the mechanism by which flaviviruses in the JEV serogroup exhibit neurotropism. IMPORTANCE Japanese encephalitis (JE) is a significant viral encephalitis worldwide, caused by the JE virus (JEV). In some patients, the virus cannot be cleared in time, leading to the breach of the blood-brain barrier (BBB) and invasion of the central nervous system. This invasion may result in cognitive impairment, behavioral disturbances, and even death in both humans and animals. However, the mechanism by which JEV crosses the BBB remains unclear. Previous studies have shown that the flavivirus NS1 protein plays an important role in causing endothelial dysfunction. The NS1′ protein is an elongated form of NS1 protein that is particularly produced by flaviviruses in the JEV serogroup. This study revealed that both the secreted NS1 and NS1′ of JEV can disrupt the BBB by breaking down tight junction proteins through the autophagy-lysosomal pathway, and NS1′ is found to have a stronger effect compared to NS1 in this process. In addition, JEV NS1 and NS1′ can stimulate the expression of MIF, which triggers autophagy via the ERK signaling pathway, leading to damage to BBB. Our findings reveal a new function of JEV NS1 and NS1′ in the disruption of BBB, thereby providing the potential therapeutic target for JE.