Heartland virus NSs protein disrupts host defenses by blocking the TBK1 kinase–IRF3 transcription factor interaction and signaling required for interferon induction

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.

Combinatorial Minigenome Systems for Emerging Banyangviruses Reveal Viral Reassortment Potential and Importance of a Protruding Nucleotide in Genome “Panhandle” for Promoter Activity and Reassortment

“…Act.) were then obtained by normalizing firefly luc activities to Renilla luc activities as described previously (Ning et al, 2014(Ning et al, , 2015(Ning et al, , 2017. Note that Renilla luc activities were comparable across experimental groups in the same DLR assays in our study, indicating constant transfection efficiency (Supplementary Figure S1A and data not shown).…”

“…Although viral infectivity and pathogenicity should be affected by complex virus and host factors, many studies have suggested that the NSs proteins of these bunyaviruses are likely the major virulence determinant by manipulating host biological processes (Schmaljohn and Nichol, 2007;Hollidge Bradley et al, 2011). Indeed, NSs proteins of SFTSV, HRTV, and RVFV all have been identified by us and others as robust antagonists against host innate immunity despite functioning with different mechanisms, whereas the NSs of UUKV was suggested to be inefficient in immune antagonism (Hollidge Bradley et al, 2011;Ning et al, 2014Ning et al, , 2015Ning et al, , 2017Ning et al, , 2019Rezelj et al, 2015;Kainulainen et al, 2016;Brennan et al, 2017). Based on the reassortment potential of the viruses shown in this study, genetic materials (including the potential virulence factors) from banyangviruses and at least some of the related phleboviruses may constitute gene pools, providing chances for emergence of novel pathogenic reassortants with highly genetic and phenotypic diversity.…”

Methods and approaches to disease mechanisms using systems kinomics

“…Kinase signaling is a powerful and central cellular mechanism that mediates signal transduction events and is involved in a wide range of nearly all cellular processes including, but not limited to, the control of cell cycle progression, transcriptional regulation, cell transformation, proliferation, differentiation, and apoptosis. Given its central role in cellular function, aberrant regulation of kinase signaling can profoundly affect homeostasis and has been found to be involved in many disease states including insulin resistance [ 3 , 4 ], autoimmunity [ 5 , 6 ], viral infection [ 7 , 8 ], and oncogenesis [ 9 , 10 ]. Hence, assessing the kinome can provide insight into complex pathological processes across a wide array of diseases and has also been a well-studied target for therapeutics.…”

Immune Modulation and Immune-Mediated Pathogenesis of Emerging Tickborne Banyangviruses

“…Furthermore, two mutations in a PxxP motif in the SFTSV NSs protein that are important for the formation of inclusion bodies reduced, but did not completely abolish IFN antagonism activity of the NSs [102]. Indeed, HRTV NSs also binds to host proteins, but does not form inclusion bodies [99,113,114]. Taken together, role of the inclusion bodies during banyangvirus infection remains to be further investigated.…”

“…Another study implicated the role of the SFTSV NSs in the type II IFN response by demonstrating that SFTSV NSs prevents STAT1 homodimerization, and reduces STAT1 protein level overall thereby reducing IFN-γ production [112]. HRTV NSs, similar to SFTSV NSs, has also been shown to interact with TBK-1 and STAT2 [113,114]. It is hypothesized that HRTV NSs binds STAT2 to inhibit its phosphorylation and subsequent dimerization with STAT1 prior to their nuclear translocation [99,114].…”