Abstract
Heartland virus (HRTV) is a pathogenic phlebovirus related to the severe fever with thrombocytopenia syndrome virus (SFTSV), another phlebovirus causing life-threatening disease in humans. Previous findings have suggested that SFTSV can antagonize the host interferon (IFN) system via viral nonstructural protein (NSs)-mediated sequestration of antiviral signaling proteins into NSs-induced inclusion bodies. However, whether and how HRTV counteracts the host innate immunity is unknown. Here, we report that HRTV NSs (HNSs) also antagonizes IFN and cytokine induction and bolsters viral replication, although no noticeable inclusion body formation was observed in HNSs-expressing cells. Furthermore, HNSs inhibited the virus-triggered activation of IFN-β promoter by specifically targeting the IFN-stimulated response element but not the NF-κB response element. Consistently, HNSs blocked the phosphorylation and nuclear translocation of IFN regulatory factor 3 (IRF3, an IFN-stimulated response element-activating transcription factor). Reporter gene assays next showed that HNSs blockades the antiviral signaling mediated by RIG-I-like receptors likely at the level of TANK-binding kinase 1 (TBK1). Indeed, HNSs strongly interacts with TBK1 as indicated by confocal microscopy and pulldown analyses, and we also noted that the scaffold dimerization domain of TBK1 is required for the TBK1-HNSs interaction. Finally, pulldown assays demonstrated that HNSs expression dose-dependently diminishes a TBK1-IRF3 interaction, further explaining the mechanism for HNSs function. Collectively, these data suggest that HNSs, an antagonist of host innate immunity, interacts with TBK1 and thereby hinders the association of TBK1 with its substrate IRF3, thus blocking IRF3 activation and transcriptional induction of the cellular antiviral responses.
Highlights
Heartland virus (HRTV) is a pathogenic phlebovirus related to the severe fever with thrombocytopenia syndrome virus (SFTSV), another phlebovirus causing life-threatening disease in humans
HRTV is genetically related to the severe fever with thrombocytopenia syndrome virus (SFTSV), a highly pathogenic phlebovirus emerging in China and neighboring countries (8 –13)
As shown in the following DLR assays, HRTV NSs (HNSs) inhibited IFN- promoter activation induced by the overexpression of MAVS and TANK-binding kinase 1 (TBK1) (Fig. 5, C and D), suggesting that HNSs acts at the level of TBK1 or further downstream
Summary
SNSs is localized to SNSs self-induced IBs potentially with versatile roles [35]. To elucidate the function of HNSs, we first compared the subcellular localization of HNSs and SNSs. Neither of the two NSs proteins exhibited such an inhibitory effect on the NF-B-responsive element (Fig. 3B) These results suggest that HNSs likely targets the ISRE activation to block IFN/cytokine induction by viral infection. Contrast, the nuclear accumulation of IRF3 was substantially blocked in cells expressing EGFP-tagged HNSs (Fig. 4, B and C) These findings indicate that HNSs prevents IRF3 signaling to repress ISRE activation and, the induction of type I IFNs and other cytokines. As shown in the following DLR assays, HNSs inhibited IFN- promoter activation induced by the overexpression of MAVS and TBK1 (Fig. 5, C and D), suggesting that HNSs acts at the level of TBK1 or further downstream. These data suggest that the HNSs-TBK1 interaction likely impedes the association of TBK1 with its substrate IRF3 and impairs the activation of IRF3 and the induction of interferons and inflammatory cytokines
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