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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-? promoter by specifically targeting the IFN-stimulated response element but not the NF-?B 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.

SUBMITTER: Ning YJ

PROVIDER: S-EPMC5633133 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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

Ning Yun-Jia YJ Feng Kuan K Min Yuan-Qin YQ Deng Fei F Hu Zhihong Z Wang Hualin H

The Journal of biological chemistry 20170828 40

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 rep ...[more]

PMID: 28848048

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Project description:Heartland bandavirus (HRTV), which is an emerging tick-borne virus first identified in Missouri in 2009, causes fever, fatigue, decreased appetite, headache, nausea, diarrhea, and muscle or joint pain in humans. HRTV is genetically close to Dabie bandavirus, which is the causative agent of severe fever with thrombocytopenia syndrome (SFTS) in humans and is known as SFTS virus (SFTSV). The generation of infectious HRTV entirely from cloned cDNAs has not yet been reported. The absence of a reverse genetics system for HRTV has delayed efforts to understand its pathogenesis and to generate vaccines and antiviral drugs. Here, we developed a reverse genetics system for HRTV, which employs an RNA polymerase I-mediated expression system. A recombinant nonstructural protein (NSs)-knockout HRTV (rHRTV-NSsKO) was generated. We found that NSs interrupted signaling associated with innate immunity in HRTV-infected cells. The rHRTV-NSsKO was highly attenuated, indicated by the apparent absence of symptoms in a mouse model of HRTV infection. Moreover, mice immunized with rHRTV-NSsKO survived a lethal dose of HRTV. These findings suggest that NSs is a virulence factor of HRTV and that rHRTV-NSsKO could be a vaccine candidate for HRTV. IMPORTANCE Heartland bandavirus (HRTV) is a tick-borne virus identified in the United States in 2009. HRTV causes fever, fatigue, decreased appetite, headache, nausea, diarrhea, and muscle or joint pain in humans. FDA-approved vaccines and antiviral drugs are unavailable. The lack of a reverse genetics system hampers efforts to develop such antiviral therapeutics. Here, we developed a reverse genetics system for HRTV that led to the generation of a recombinant nonstructural protein (NSs)-knockout HRTV (rHRTV-NSsKO). We found that NSs interrupted signaling associated with innate immunity in HRTV-infected cells. Furthermore, rHRTV-NSsKO was highly attenuated and immunogenic in a mouse model. These findings suggest that NSs is a virulence factor of HRTV and that rHRTV-NSsKO could be a vaccine candidate for HRTV.

Project description:UnlabelledDengue virus (DENV) replication is inhibited by the prior addition of type I interferon or by RIG-I agonists that elicit RIG-I/MAVS/TBK1/IRF3-dependent protective responses. DENV infection of primary human endothelial cells (ECs) results in a rapid increase in viral titer, which suggests that DENV inhibits replication-restrictive RIG-I/interferon beta (IFN-β) induction pathways within ECs. Our findings demonstrate that DENV serotype 4 (DENV4) nonstructural (NS) proteins NS2A and NS4B inhibited RIG-I-, MDA5-, MAVS-, and TBK1/IKKε-directed IFN-β transcription (>80%) but failed to inhibit IFN-β induction directed by STING or constitutively active IRF3-5D. Expression of NS2A and NS4B dose dependently inhibited the phosphorylation of TBK1 and IRF3, which suggests that they function at the level of TBK1 complex activation. NS2A and NS4B from DENV1/2/4, as well as the West Nile virus NS4B protein, commonly inhibited TBK1 phosphorylation and IFN-β induction. A comparative analysis of NS4A proteins across DENVs demonstrated that DENV1, but not DENV2 or DENV4, NS4A proteins uniquely inhibited TBK1. These findings indicate that DENVs contain conserved (NS2A/NS4B) and DENV1-specific (NS4A) mechanisms for inhibiting RIG-I/TBK1-directed IFN responses. Collectively, our results define DENV NS proteins that restrict IRF3 and IFN responses and thereby facilitate DENV replication and virulence. Unique DENV1-specific NS4A regulation of IFN induction has the potential to be a virulence determinant that contributes to the increased severity of DENV1 infections and the immunodominance of DENV1 responses during tetravalent DENV1-4 vaccination.ImportanceOur findings demonstrate that NS2A and NS4B proteins from dengue virus serotypes 1, 2, and 4 are inhibitors of RIG-I/MDA5-directed interferon beta (IFN-β) induction and that they accomplish this by blocking TBK1 activation. We determined that IFN inhibition is functionally conserved across NS4B proteins from West Nile virus and DENV1, -2, and -4 viruses. In contrast, DENV1 uniquely encodes an extra IFN regulating protein, NS4A, that inhibits TBK1-directed IFN induction. DENV1 is associated with an increase in severe patient disease, and added IFN regulation by the DENV1 NS4A protein may contribute to increased DENV1 replication, immunodominance, and virulence. The regulation of IFN induction by nonstructural (NS) proteins suggests their potential roles in enhancing viral replication and spread and as potential protein targets for viral attenuation. DENV1-specific IFN regulation needs to be considered in vaccine strategies where enhanced DENV1 replication may interfere with DENV2-4 seroconversion within coadministered tetravalent DENV1-4 vaccines.

Dataset Information

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

Publications

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

Similar Datasets

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