Japanese Encephalitis Virus NS5 Inhibits Type I Interferon (IFN) Production by Blocking the Nuclear Translocation of IFN Regulatory Factor 3 and NF-κB

Abstract

The type I interferon (IFN) response is part of the first-line defense against viral infection. To initiate replication, viruses have developed powerful evasion strategies to counteract host IFN responses. In the present study, we found that the Japanese encephalitis virus (JEV) NS5 protein could inhibit double-stranded RNA (dsRNA)-induced IFN-␤ expression in a dose-dependent manner. Our data further demonstrated that JEV NS5 suppressed the activation of the IFN transcriptional factors IFN regulatory factor 3 (IRF3) and NF-B. However, there was no defect in the phosphorylation of IRF3 and degradation of IB, an upstream inhibitor of NF-B, upon NS5 expression, indicating a direct inhibition of the nuclear localization of IRF3 and NF-B by NS5. Mechanistically, NS5 was shown to interact with the nuclear transport proteins KPNA2, KPNA3, and KPNA4, which competitively blocked the interaction of KPNA3 and KPNA4 with their cargo molecules, IRF3 and p65, a subunit of NF-B, and thus inhibited the nuclear translocation of IRF3 and NF-B. Furthermore, overexpression of KPNA3 and KPNA4 restored the activity of IRF3 and NF-B and increased the production of IFN-␤ in NS5-expressing or JEV-infected cells. Additionally, an upregulated replication level of JEV was shown upon KPNA3 or KPNA4 overexpression. These results suggest that JEV NS5 inhibits the induction of type I IFN by targeting KPNA3 and KPNA4.IMPORTANCE JEV is the major cause of viral encephalitis in South and Southeast Asia, with high mortality. However, the molecular mechanisms contributing to the severe pathogenesis are poorly understood. The ability of JEV to counteract the host innate immune response is potentially one of the mechanisms responsible for JEV virulence. Here we demonstrate the ability of JEV NS5 to interfere with the dsRNAinduced nuclear translocation of IRF3 and NF-B by competitively inhibiting the interaction of IRF3 and NF-B with nuclear transport proteins. Via this mechanism, JEV NS5 suppresses the induction of type I IFN and the antiviral response in host cells. These findings reveal a novel strategy for JEV to escape the host innate immune response and provide new insights into the pathogenesis of JEV.

Duck karyopherin α4 (duKPNA4) is involved in type I interferon expression and the antiviral response against Japanese encephalitis virus

“…Furthermore, exogenous expression of duKPNA4 significantly inhibited JEV replication in DEFs, thereby suggesting the involvement of duKPNA4 in the host immune response against viral infection in ducks. This observation was consistent with a previous observation in humans, in which human KPNA4 has been found to have a role in the inhibition of JEV replication (Ye et al, 2017).…”

“…Indeed, the 4b protein of Middle East respiratory syndrome coronavirus binds KPNA4 and impairs the interaction between KPNA4 and the NF-κB-p65 subunit, thus interfering with the host innate immune response (Canton et al, 2018). JEV NS5 protein competitively binds KPNA4 and inhibits the nuclear translocation of IFN transcription factors, thus suppressing IFN expression (Ye et al, 2017). These previous observations together with our results emphasize the importance of KPNA4 in the type I IFN-mediated antiviral response.…”

“…Among the identified KPNAs, KPNA4 in the α3 subfamily is involved in the innate immune response against viral infection Ye et al, 2017). Several KPNA4 orthologues from different species including humans (Köhler et al, 1997), mice (Mihalas et al, 2015), pigs (Chen et al, 2016) and chickens (Caldwell et al, 2005) have been identified.…”

“…JEV infection causes stunted growth and death in ducklings (Xiao et al, 2018). In addition, JEV NS5 protein interacts with human KPNA4 and inhibits the nuclear translocation of IRF3 and NF-κB in human cells (Ye et al, 2017). We therefore cloned the duKPNA4 gene and analyzed its role in type I IFN expression as well as the antiviral response against JEV in primary duck embryo fibroblasts (DEFs).…”

Comparative characterization of flavivirus production in two cell lines: Human hepatoma-derived Huh7.5.1-8 and African green monkey kidney-derived Vero

“…At present, the underlying mechanisms that generate splice variants and their impacts on RIG-I signaling remain to be studied. Although JEV has been restricted by RIG-I in a mouse model [31] and mouse cell lines [32,33], JEV production and susceptibility to JEV-induced cell death were not dramatically altered in the Huh7.5.1-8 and Huh7.5.1 cell lines compared with the HuH-7 cell line ( Fig 1A and 1B), implying that the RIG-I pathway may not work or may be counteracted by viral mechanisms [34] under our experimental conditions.…”