Proteomic analysis forTypeIinterferon antagonism ofJapanese encephalitis virusNS5 protein

Abstract

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 IFNβ-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 Ca(2+) levels while inhibiting nuclear translocalization of STAT1 and NFAT-1 in response to IFNβ, thus, indicating calreticulin downregulation linked with Type I IFN antagonism of JEV NS5 via activation of Ca(2+) /calicineurin. Calcineurin inhibitor cyclosporin A attenuated NS5-mediated inhibition of IFNβ-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 IFNβ. Results prove Ca(2+) , 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.

Antiviral Activity of a Novel Compound CW-33 against Japanese Encephalitis Virus through Inhibiting Intracellular Calcium Overload

“…JEV exhibited Type I interferon antagonistic function by blocking JAK/STAT signal via suppression of Tyk2 tyrosine phosphorylation [ 32 , 33 ]. JEV NS5 protein had Type I IFN antagonistic activity to suppress the phosphorylation and nuclear translocation of STAT1 via activating Ca 2+ /calcineurin signaling pathway [ 34 ]. CW-33 significantly inhibited the JEV-induced rise of intracellular Ca 2+ , and activated the JAK/STAT signals ( Figure 6 and Figure 7 ).…”

The Many Faces of the Flavivirus NS5 Protein in Antagonism of Type I Interferon Signaling

“…The proposed role for JEV NS5 is to activate protein tyrosine phosphatases (PTPs) that have normal roles as negative feedback regulators of JAK activation (85). Additional studies suggest that JEV NS5 expression downregulates calreticulin to inhibit nuclear translocation of STAT1 (86). However, how these activities of NS5 result in inhibition of Tyk2 phosphorylation and suppression of STAT nuclear localization and how they might be coordinated are not known.…”

Flaviviral NS4b, chameleon and jack‐in‐the‐box roles in viral replication and pathogenesis, and a molecular target for antiviral intervention

“…Thus, the full picture of IFN antagonist function of NS4b is still missing. The role that different viral (such as NS2b/3, 4a, and 5) and host proteins (e.g., regulators of STAT‐1, such as Ube2i and PIAS‐1) play in this process remains to be explored. Similar to the large impact, subtle changes to either component (such as single‐point mutations in NS4b, see earlier discussion) may have an impact on the overall function of the flaviviral RC, thereby resulting in largely altered viral growth kinetics and pathogenesis.…”