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Volume 51, Number 4, August 2018

Anti-apoptotic activity of Japanese encephalitis virus NS5 protein in human medulloblastoma cells treated with interferon-β 


Jing-Ru Weng, Chun-Hung Hua, Chao-Hsien Chen, Su-Hua Huang, Ching-Ying Wang, Ying-Ju Lin, Lei Wan, Cheng-Wen Lin


 

Background and purpose: 

Japanese encephalitis virus (JEV) non-structural protein 5 (NS5) exhibits type I interferon (IFN) antagonists, contributing to immune escape, and even inducing viral anti-apoptosis. This study investigated the anti-apoptotic mechanism of JEV NS5 protein on type I IFN-induced apoptosis of human medulloblastoma cells. 



 

Methods:

Vector control and NS5-expressing cells were treated with IFN-β, and then harvested for analyzing apoptotic pathways with flow cytometry, Western blotting, subcellular localization, etc. 



 

Results:

Annexin V-FITC/PI staining indicated that IFN-β triggered apoptosis of human medulloblastoma cells, but JEV NS5 protein significantly inhibited IFN-β-induced apoptosis. Phage display technology and co-immunoprecipitation assay identified the anti-apoptotic protein Hsp70 as a NS5-interacting protein. In addition, Western blotting demonstrated that NS5 protein up-regulated the Hsp70 expression, and reduced IFN-β-induced phosphorylation of ERK2, p38 MAPK and STAT1. Hsp70 down-regulation by quercetin significantly recovered IFN-β-induced apoptosis of NS5-expressing cells, correlating with the increase in the phosphorylation of ERK2, p38 MAPK, and STAT1. Inhibiting the ATPase activity of Hsp70 by VER-155008 resulted in the elevated IFN-β-induced apoptosis in vector control and NS5-expressing cells.

 



 

Conclusion:

The results indicated Hsp70 up-regulation by JEV NS5 not only involved in type I IFN antagonism, but also responded to the anti-apoptotic action of JEV NS5 protein through the blocking IFN-β-induced p38 MAPK/STAT1-mediated apoptosis. 



 

Key words:

Japanese encephalitis virus, NS5, Interferon, Anti-apoptosis, Hsp70