Proteomic analysis for Type I interferon antagonism of Japanese encephalitis virus NS5 protein

Japanese encephalitis virus (JEV) nonstructural protein 5 (NS5) exhibits a Type I interferon (IFN) antagonistic function. This study characterizes Type I IFN antagonism mechanism of NS5 protein, using proteomic approach. In human neuroblastoma cells, NS5 expression would suppress IFN-induced responses, for example, expression of IFN-stimulated genes PKR and OAS as well as STAT1 nuclear translocation and phosphorylation. Proteomic analysis showed JEV NS5 downregulating calreticulin, while upregulating cyclophilin A, HSP 60 and stress-induced-phosphoprotein 1. Gene silence of calreticulin raised intracellular Ca2+ levels while inhibiting nuclear translocalization of STAT1 and NFAT-1 in response to IFN, thus, indicating calreticulin downregulation linked with Type I IFN antagonism of JEV NS5 via activation of Ca2+/calicineurin. Calcineurin inhibitor cyclosporin A attenuated NS5-mediated inhibition of IFN-induced responses, for example, IFN-sensitive response element driven luciferase, STAT1-dependent PKR mRNA expression, as well as phosphorylation and nuclear translocation of STAT1. Transfection with calcineurin (vs. control) siRNA enhanced nuclear translocalization of STAT1 and upregulated PKR expression in NS5-expressing cells in response to IFN. Results prove Ca2+, calreticulin, and calcineurin involvement in STAT1-mediated signaling as well as a key role of JEV NS5 in Type I IFN antagonism. This study offers insights into the molecular mechanism of Type I interferon antagonism by JEV NS5.