Japanese Encephalitis Virus NS2B-3 Protein Complex Promotes Cell Apoptosis and Viral Particle Release by Down-Regulating the Expression of AXL

Japanese encephalitis virus(JEV) is a flavivirus transmitted by mosquitoes that causes severe encephalitis in humans and animals. It has been suggested that AXL, a transmembrane protein, can promote the replication of various flaviviruses,such as dengue(DENV), Zika(ZIKV), and West Nile(WNV) viruses. However, the effect of AXL on JEV infection has not yet been determined. In the present study, we demonstrate that AXL is down-regulated after JEV infection in the late stage. JEV NS2B-3 protein specifically interacted with AXL, and promoted AXL degradation through the ubiquitin–proteasome pathway. AXL-degradation increased cell apoptosis by disrupting phosphatidylinositol 3-kinase(PI3 K)/Akt signal transduction. In addition, the degradation of AXL promoted JEV release to supernatant, whereas the virus in the cell lysates decreased. The supplementation of AXL ligand Gas6 inhibited the JEV-mediated degradation of AXL. Altogether,we discover a new function of NS2B-3 during the process of JEV replication, and provide a new insight into the interactions between JEV and cell hosts.

C19orf66 Inhibits Japanese Encephalitis Virus Replication by Targeting-1 PRF and the NS3 Protein

The Japanese encephalitis serogroup of the neurogenic Flavivirus has a specific feature that expresses a non-structural protein NS1'produced through a programmed-1 ribosomal frameshifting(-1 PRF).Herein,C19orf66,a novel member of interferon-stimulated gene(ISG)products,exhibited significant activity of antagonizing Japanese encephalitis virus(JEV)infection.Overexpression of C19orf66 in 293T cells significantly inhibited JEV replication,while knock-down of endogenous C19orf66 in HeLa cells and A549 cells significantly increased virus replication.Notably,C19orf66 had an inhibitory effect on frameshift production of JEV NS1'.The inhibition was more significant when C19orf66 and JEV NS1-NS2A were co-expressed in the 293T cells.Both C19orf66-209 and C19orf66-Zinc^(mut) did not significantly change the NS1'to NS1 ratio and had weaker antiviral effects than C19orf66.Similarly,C19orf66-209 and C19orf66-Zinc^(mut) had no significant effect on the expression of the JEV NS3 protein,whose expression was down-regulated by C19orf66 via the lysosome-dependent pathway.These findings suggest that C19orf66 may possess at least two different mechanisms of antagonizing JEV infection.This study identified C19orf66 as a novel interferon-stimulated gene product that can inhibit JEV replication by targeting-1 PRF and the NS3 protein.The study provides baseline information for the future development of broad-spectrum antiviral agents against JEV.