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• W2979411325 abstract "•The NS3 protein of Zika virus encodes a highly conserved 14-3-3-binding motif•NS3 interacts with 14-3-3ϵ and η to inhibit RIG-I and MDA5 signaling, respectively•NS3 blocks 14-3-3-mediated translocation of RLRs from the cytosol to mitochondria•A mutant virus deficient in 14-3-3 binding is attenuated due to elevated immunity 14-3-3 protein family members facilitate the translocation of RIG-I-like receptors (RLRs) to organelles that mediate downstream RLR signaling, leading to interferon production. 14-3-3ϵ promotes the cytosolic-to-mitochondrial translocation of RIG-I, while 14-3-3η facilitates MDA5 translocation to mitochondria. We show that the NS3 protein of Zika virus (ZIKV) antagonizes antiviral gene induction by RIG-I and MDA5 by binding to and sequestering the scaffold proteins 14-3-3ϵ and 14-3-3η. 14-3-3-binding is mediated by a negatively charged RLDP motif in NS3 that is conserved in ZIKV strains of African and Asian lineages and is similar to the one found in dengue and West Nile viruses. ZIKV NS3 is sufficient to inhibit the RLR-14-3-3ϵ/η interaction and to suppress antiviral signaling. Mutational perturbation of 14-3-3ϵ/η binding in a recombinant ZIKV leads to enhanced innate immune responses and impaired growth kinetics. Our study provides molecular understanding of immune evasion functions of ZIKV, which may guide vaccine and anti-flaviviral therapy development. 14-3-3 protein family members facilitate the translocation of RIG-I-like receptors (RLRs) to organelles that mediate downstream RLR signaling, leading to interferon production. 14-3-3ϵ promotes the cytosolic-to-mitochondrial translocation of RIG-I, while 14-3-3η facilitates MDA5 translocation to mitochondria. We show that the NS3 protein of Zika virus (ZIKV) antagonizes antiviral gene induction by RIG-I and MDA5 by binding to and sequestering the scaffold proteins 14-3-3ϵ and 14-3-3η. 14-3-3-binding is mediated by a negatively charged RLDP motif in NS3 that is conserved in ZIKV strains of African and Asian lineages and is similar to the one found in dengue and West Nile viruses. ZIKV NS3 is sufficient to inhibit the RLR-14-3-3ϵ/η interaction and to suppress antiviral signaling. Mutational perturbation of 14-3-3ϵ/η binding in a recombinant ZIKV leads to enhanced innate immune responses and impaired growth kinetics. Our study provides molecular understanding of immune evasion functions of ZIKV, which may guide vaccine and anti-flaviviral therapy development. Mosquito-transmitted viral pathogens cause significant morbidity and mortality in humans on a global scale (Ferguson, 2018Ferguson N.M. Challenges and opportunities in controlling mosquito-borne infections.Nature. 2018; 559: 490-497Crossref PubMed Scopus (66) Google Scholar, Mackenzie et al., 2004Mackenzie J.S. Gubler D.J. Petersen L.R. Emerging flaviviruses: the spread and resurgence of Japanese encephalitis, West Nile and dengue viruses.Nat. Med. 2004; 10: S98-S109Crossref PubMed Scopus (1009) Google Scholar). Among these pathogens are members of the Flavivirus genus, such as dengue virus (DENV), West Nile virus (WNV), and the recently emergent Zika virus (ZIKV), which can cause a variety of severe diseases in humans, including hemorrhagic shock syndrome (DENV), encephalitis (WNV), and congenital abnormalities in the fetus (ZIKV) (Olagnier et al., 2016Olagnier D. Muscolini M. Coyne C.B. Diamond M.S. Hiscott J. Mechanisms of Zika virus infection and neuropathogenesis.DNA Cell Biol. 2016; 35: 367-372Crossref PubMed Scopus (35) Google Scholar, Pierson and Diamond, 2018Pierson T.C. Diamond M.S. The emergence of Zika virus and its new clinical syndromes.Nature. 2018; 560: 573-581Crossref PubMed Scopus (209) Google Scholar). Flaviviruses are positive-sense single-stranded RNA viruses that replicate in a variety of cell types, where the virus replication complexes are formed at the endoplasmic reticulum (Hasan et al., 2018Hasan S.S. Sevvana M. Kuhn R.J. Rossmann M.G. Structural biology of Zika virus and other flaviviruses.Nat. Struct. Mol. Biol. 2018; 25: 13-20Crossref PubMed Scopus (107) Google Scholar, Mukhopadhyay et al., 2005Mukhopadhyay S. Kuhn R.J. Rossmann M.G. A structural perspective of the flavivirus life cycle.Nat. Rev. Microbiol. 2005; 3: 13-22Crossref PubMed Scopus (892) Google Scholar, Neufeldt et al., 2018Neufeldt C.J. Cortese M. Acosta E.G. Bartenschlager R. Rewiring cellular networks by members of the Flaviviridae family.Nat. Rev. Microbiol. 2018; 16: 125-142Crossref PubMed Scopus (192) Google Scholar). For example, ZIKV can replicate in human astrocytes, neuronal cells, and specific cells of the genital tract (both in females and males) in vivo and in many different cell types in vitro (Miner and Diamond, 2017Miner J.J. Diamond M.S. Zika Virus Pathogenesis and Tissue Tropism.Cell Host Microbe. 2017; 21: 134-142Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar). The positive-sense RNA genome is translated directly into one large polyprotein, which, upon proteolytic cleavage by host and viral enzymes, gives rise to 10 viral proteins (3 structural and 7 nonstructural [NS] proteins). The NS proteins (NS1-5) have enzymatic activities that are crucial for distinct steps in the virus life cycle (Mukhopadhyay et al., 2005Mukhopadhyay S. Kuhn R.J. Rossmann M.G. A structural perspective of the flavivirus life cycle.Nat. Rev. Microbiol. 2005; 3: 13-22Crossref PubMed Scopus (892) Google Scholar). In addition, some of the NS proteins of ZIKV (and also of other flaviviruses) antagonize the innate immune response (e.g., NS5 antagonizes STAT2), thereby indirectly promoting virus replication (Grant et al., 2016Grant A. Ponia S.S. Tripathi S. Balasubramaniam V. Miorin L. Sourisseau M. Schwarz M.C. Sánchez-Seco M.P. Evans M.J. Best S.M. García-Sastre A. Zika virus targets human STAT2 to inhibit type I interferon signaling.Cell Host Microbe. 2016; 19: 882-890Abstract Full Text Full Text PDF PubMed Scopus (538) Google Scholar, Wu et al., 2017Wu Y. Liu Q. Zhou J. Xie W. Chen C. Wang Z. Yang H. Cui J. Zika virus evades interferon-mediated antiviral response through the co-operation of multiple nonstructural proteins in vitro.Cell Discov. 2017; 3: 17006Crossref PubMed Scopus (135) Google Scholar). No licensed antivirals or vaccines are currently available for ZIKV or most other flaviviruses. As such, understanding the molecular details of ZIKV replication and pathogenesis and viral interactions with the human host is crucial for developing effective countermeasures against ZIKV-induced diseases. Among the many different factors that contribute to ZIKV-induced pathogenesis are viral interactions with the mammalian type I interferon (IFN) response, which is essential for the restriction of ZIKV infection both in vitro and in vivo (Lazear et al., 2016Lazear H.M. Govero J. Smith A.M. Platt D.J. Fernandez E. Miner J.J. Diamond M.S. A mouse model of Zika virus pathogenesis.Cell Host Microbe. 2016; 19: 720-730Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar, Yockey et al., 2018Yockey L.J. Jurado K.A. Arora N. Millet A. Rakib T. Milano K.M. Hastings A.K. Fikrig E. Kong Y. Horvath T.L. et al.Type I interferons instigate fetal demise after Zika virus infection.Sci. Immunol. 2018; 3: e1680Crossref Scopus (146) Google Scholar). Several innate immune sensors and key transcription factors of IFN induction are crucial for ZIKV restriction (Aliota et al., 2016Aliota M.T. Caine E.A. Walker E.C. Larkin K.E. Camacho E. Osorio J.E. Characterization of lethal Zika virus infection in AG129 mice.PLoS Negl. Trop. Dis. 2016; 10: e0004682Crossref PubMed Scopus (247) Google Scholar, Lazear et al., 2016Lazear H.M. Govero J. Smith A.M. Platt D.J. Fernandez E. Miner J.J. Diamond M.S. A mouse model of Zika virus pathogenesis.Cell Host Microbe. 2016; 19: 720-730Abstract Full Text Full Text PDF PubMed Scopus (675) Google Scholar, Tripathi et al., 2017Tripathi S. Balasubramaniam V.R. Brown J.A. Mena I. Grant A. Bardina S.V. Maringer K. Schwarz M.C. Maestre A.M. Sourisseau M. et al.A novel Zika virus mouse model reveals strain specific differences in virus pathogenesis and host inflammatory immune responses.PLoS Pathog. 2017; 13: e1006258Crossref PubMed Scopus (164) Google Scholar). Among those detecting ZIKV infection are the cytoplasmic RNA sensors RIG-I and MDA5 of the RIG-I-like receptor (RLR) family, which initiate an IFN response via TBK1, IRF3, and IRF7, ultimately leading to the transcriptional upregulation of a large suite of IFN-stimulated genes (ISGs) (Bowen et al., 2017Bowen J.R. Quicke K.M. Maddur M.S. O’Neal J.T. McDonald C.E. Fedorova N.B. Puri V. Shabman R.S. Pulendran B. Suthar M.S. Zika virus antagonizes type I interferon responses during infection of human dendritic cells.PLoS Pathog. 2017; 13: e1006164Crossref PubMed Scopus (186) Google Scholar, Chazal et al., 2018Chazal M. Beauclair G. Gracias S. Najburg V. Simon-Lorière E. Tangy F. Komarova A.V. Jouvenet N. RIG-I recognizes the 5′ region of Dengue and Zika virus genomes.Cell Rep. 2018; 24: 320-328Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar, Hertzog et al., 2018Hertzog J. Dias Junior A.G. Rigby R.E. Donald C.L. Mayer A. Sezgin E. Song C. Jin B. Hublitz P. Eggeling C. et al.Infection with a Brazilian isolate of Zika virus generates RIG-I stimulatory RNA and the viral NS5 protein blocks type I IFN induction and signaling.Eur. J. Immunol. 2018; 48: 1120-1136Crossref PubMed Scopus (75) Google Scholar). Recently, 14-3-3 protein family members have been implicated in innate immunity, where they function to “translocate” RLR sensors to signaling-permitting organelles. Specifically, 14-3-3ϵ promotes the cytosolic-to-mitochondrial translocation of RIG-I, whereas 14-3-3η facilitates MDA5 translocation to mitochondria, thereby promoting antiviral IFN induction (Lin et al., 2019Lin J.P. Fan Y.K. Liu H.M. The 14-3-3η chaperone protein promotes antiviral innate immunity via facilitating MDA5 oligomerization and intracellular redistribution.PLoS Pathog. 2019; 15: e1007582Crossref Scopus (40) Google Scholar, Liu et al., 2012Liu H.M. Loo Y.M. Horner S.M. Zornetzer G.A. Katze M.G. Gale Jr., M. The mitochondrial targeting chaperone 14-3-3ϵ regulates a RIG-I translocon that mediates membrane association and innate antiviral immunity.Cell Host Microbe. 2012; 11: 528-537Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Given the crucial role of the type I IFN response in restricting ZIKV infection, it is not surprising that ZIKV has evolved to suppress or delay IFN production and IFN-induced signaling, analogous to other flaviviruses. However, the molecular mechanisms and physiological relevance of IFN antagonism by ZIKV have just begun to be elucidated. Here, we show that the NS3 protein of ZIKV antagonizes RIG-I- and MDA5-mediated signaling via molecular mimicry of a cellular 14-3-3-binding motif. A recombinant ZIKV encoding a mutant NS3 protein in which 14-3-3ϵ/η binding was ablated showed attenuated replication capacity and stimulated elevated innate immune responses. Members of the 14-3-3 protein family regulate numerous intracellular processes, such as cell cycling, transcription, apoptosis, and immunity (Tzivion and Avruch, 2002Tzivion G. Avruch J. 14-3-3 proteins: active cofactors in cellular regulation by serine/threonine phosphorylation.J. Biol. Chem. 2002; 277: 3061-3064Crossref PubMed Scopus (425) Google Scholar). 14-3-3ϵ is an essential component of the TRIM25- and RIG-I-mediated innate immune response to infection with several RNA viruses such as Sendai (SeV) and hepatitis C (HCV) viruses (Liu et al., 2012Liu H.M. Loo Y.M. Horner S.M. Zornetzer G.A. Katze M.G. Gale Jr., M. The mitochondrial targeting chaperone 14-3-3ϵ regulates a RIG-I translocon that mediates membrane association and innate antiviral immunity.Cell Host Microbe. 2012; 11: 528-537Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). Furthermore, recent research showed that 14-3-3η promoted antiviral signaling by MDA5 in the context of HCV infection (Lin et al., 2019Lin J.P. Fan Y.K. Liu H.M. The 14-3-3η chaperone protein promotes antiviral innate immunity via facilitating MDA5 oligomerization and intracellular redistribution.PLoS Pathog. 2019; 15: e1007582Crossref Scopus (40) Google Scholar). As such, we tested whether 14-3-3ϵ and 14-3-3η can restrict ZIKV infection in immortalized human fetal astrocytes (SVGA), which are permissive to ZIKV replication and express several key innate immune sensors important for ZIKV detection (Figures S1A and S1B). Ectopic expression of 14-3-3ϵ or 14-3-3η significantly restricted ZIKV infection in SVGA cells, whereas 14-3-3σ did not (Figure 1A ). Ectopically expressed 14-3-3ϵ or 14-3-3η suppressed ZIKV replication to similar levels as ectopically expressed RIG-I or MDA5 did (Figure 1B), suggesting that the anti-ZIKV restriction activity seen for 14-3-3ϵ and 14-3-3η might be due to their roles in RLR signaling. To test this directly, we determined the effect of 14-3-3ϵ or 14-3-3η gene silencing on the abundance of ZIKV-induced cytokine, ISG and chemokine transcripts in infected SVGA cells. In parallel, RIG-I and MDA5 were also silenced (Figures 1C and 1D). Knockdown of endogenous 14-3-3σ, which is not involved in RLR signaling (Lin et al., 2019Lin J.P. Fan Y.K. Liu H.M. The 14-3-3η chaperone protein promotes antiviral innate immunity via facilitating MDA5 oligomerization and intracellular redistribution.PLoS Pathog. 2019; 15: e1007582Crossref Scopus (40) Google Scholar, Liu et al., 2012Liu H.M. Loo Y.M. Horner S.M. Zornetzer G.A. Katze M.G. Gale Jr., M. The mitochondrial targeting chaperone 14-3-3ϵ regulates a RIG-I translocon that mediates membrane association and innate antiviral immunity.Cell Host Microbe. 2012; 11: 528-537Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), served as an additional control. In agreement with previous work (Esser-Nobis et al., 2019Esser-Nobis K. Aarreberg L.D. Roby J.A. Fairgrieve M.R. Green R. Gale Jr., M. Comparative analysis of African and Asian lineage-derived Zika Virus strains reveals differences in activation of and sensitivity to antiviral innate immunity.J. Virol. 2019; 93 (e00640–e19)Crossref Scopus (35) Google Scholar, Hertzog et al., 2018Hertzog J. Dias Junior A.G. Rigby R.E. Donald C.L. Mayer A. Sezgin E. Song C. Jin B. Hublitz P. Eggeling C. et al.Infection with a Brazilian isolate of Zika virus generates RIG-I stimulatory RNA and the viral NS5 protein blocks type I IFN induction and signaling.Eur. J. Immunol. 2018; 48: 1120-1136Crossref PubMed Scopus (75) Google Scholar, Ma et al., 2018Ma J. Ketkar H. Geng T. Lo E. Wang L. Xi J. Sun Q. Zhu Z. Cui Y. Yang L. Wang P. Zika Virus Non-structural Protein 4A Blocks the RLR-MAVS Signaling.Front. Microbiol. 2018; 9: 1350Crossref Scopus (73) Google Scholar), RIG-I or MDA5 depletion strongly diminished IFNB1, CCL5, and ISG (IFIT2 and RSAD2) transcript induction upon ZIKV infection in SVGA cells. Similarly, 14-3-3ϵ or 14-3-3η silencing also markedly reduced virus-induced antiviral gene induction, but 14-3-3σ knockdown did not (Figures 1C and 1D). Together, these results indicate that 14-3-3ϵ and 14-3-3η are crucial for cytokine and ISG induction during ZIKV infection, which ultimately inhibits ZIKV replication. Key molecules in innate immune signaling are targets of viral antagonism (Beachboard and Horner, 2016Beachboard D.C. Horner S.M. Innate immune evasion strategies of DNA and RNA viruses.Curr. Opin. Microbiol. 2016; 32: 113-119Crossref PubMed Scopus (139) Google Scholar, Chan and Gack, 2016bChan Y.K. Gack M.U. Viral evasion of intracellular DNA and RNA sensing.Nat. Rev. Microbiol. 2016; 14: 360-373Crossref PubMed Scopus (289) Google Scholar). As such, one might expect that ZIKV antagonizes RLR signaling by either targeting the sensors or the regulatory proteins critical for the RLR response. Indeed, the NS3 protein of DENV evades RIG-I signaling in a proteolysis-independent manner by binding to 14-3-3ϵ using a negatively charged 64-RxEP-67 motif that mimics a phosphorylatable motif found in cellular 14-3-3 interactors (Chan and Gack, 2016aChan Y.K. Gack M.U. A phosphomimetic-based mechanism of dengue virus to antagonize innate immunity.Nat. Immunol. 2016; 17: 523-530Crossref PubMed Scopus (79) Google Scholar). We evaluated whether ZIKV, which is closely related to DENV, encodes a similar phosphomimetic motif in its NS3 protein. Sequence alignment of the NS3 proteins from different ZIKV strains, including the prototypical African strain isolated in Uganda in 1947 (MR 766) and the more recent epidemic French Polynesian (H/PF/2013) and Brazilian (Brazil Paraiba 2015; hereafter referred to as BRA/2015) strains, revealed that the NS3 proteins of all strains analyzed (total of 673) encode a potential phosphomimetic 14-3-3-binding motif, 64-RLDP-67, containing a central negatively charged Asp residue (D66) (Figures 2A and S2A). Notably, the motif found in ZIKV NS3 is identical to that found in WNV NS3 at the corresponding position (RLDP) and similar to the one in DENV NS3, which harbors a phosphomimetic Glu (E66) residue instead of D66 (motif RIEP or RLEP) (Chan and Gack, 2016aChan Y.K. Gack M.U. A phosphomimetic-based mechanism of dengue virus to antagonize innate immunity.Nat. Immunol. 2016; 17: 523-530Crossref PubMed Scopus (79) Google Scholar). In contrast, none of the analyzed NS3 proteins from yellow fever (YFV), Japanese encephalitis (JEV), tick-borne encephalitis (TBEV) viruses, or the distantly related HCV encoded a phosphomimetic Rx(E/D)P motif at the corresponding location (Figures 2A and S2A). The presence of a potential 14-3-3-interaction motif in ZIKV NS3 led us to hypothesize that ZIKV NS3 might interact with 14-3-3ϵ and thereby antagonize the RIG-I signaling pathway. Indeed, mass spectrometry (MS) analysis of affinity-purified FLAG-NS3 from ZIKV (strain H/PF/2013) identified 14-3-3ϵ as an interaction partner, visible as an ∼30 kDa band present only in the ZIKV NS3 sample but not vector or ZIKV NS1 controls (Figures 2B and S2B). Notably, MS analysis of this band also identified 14-3-3η, although with lower peptide frequency than for 14-3-3ϵ (Figure S2B). Co-immunoprecipitation (co-IP) confirmed that GST-fused NS3 (ZIKV, H/PF/2013) efficiently bound to HA-tagged 14-3-3ϵ, as did GST-NS3 from DENV (Figure 2C). In support of these findings, the NS3 protein from ZIKV (BRA/2015 strain) also readily bound to endogenous 14-3-3ϵ during infection of SVGA astrocytes, where their binding increased with augmented expression of NS3 during the course of infection (Figures 2D and S2C). As the prototypical African lineage strain MR 766 differs in pathogenesis from more recent, epidemic ZIKV isolates (of the Asian lineage) (Pierson and Diamond, 2018Pierson T.C. Diamond M.S. The emergence of Zika virus and its new clinical syndromes.Nature. 2018; 560: 573-581Crossref PubMed Scopus (209) Google Scholar), we assessed whether the NS3 proteins from different ZIKV strains varied in their 14-3-3ϵ-binding capacity. In contrast to YFV NS3, which does not encode a 14-3-3-binding motif at the corresponding site of ZIKV NS3 and thus served as a negative control, the NS3 proteins of all three ZIKV strains tested (MR 766, H/PF/2013 and BRA/2015) bound to endogenous 14-3-3ϵ (Figure 2E), suggesting that 14-3-3ϵ binding is a conserved feature of multiple ZIKV strains. To test whether the ZIKV NS3-14-3-3ϵ interaction requires the phosphomimetic motif, we mutated 64-RLDP-67 to 64-KIKP-67 in ZIKV NS3 (termed hereafter NS3 KIKP), analogous to our 14-3-3ϵ-binding-deficient DENV NS3 KIKP mutant (Chan and Gack, 2016aChan Y.K. Gack M.U. A phosphomimetic-based mechanism of dengue virus to antagonize innate immunity.Nat. Immunol. 2016; 17: 523-530Crossref PubMed Scopus (79) Google Scholar), by substituting R64 and the negatively charged D66 residue for positively charged lysine (K) residues and, in addition, mutating L65 to I65. In contrast to the wild-type (WT) ZIKV NS3 protein, which efficiently bound 14-3-3ϵ, NS3 KIKP showed reduced 14-3-3ϵ-binding capacity (Figure 2F), indicating that the RLDP motif in ZIKV NS3 is important for the interaction with 14-3-3ϵ. Finally, since phosphomimetic motifs have been shown to outcompete cellular (usually phosphorylated) 14-3-3-interacting partners (Chan and Gack, 2016aChan Y.K. Gack M.U. A phosphomimetic-based mechanism of dengue virus to antagonize innate immunity.Nat. Immunol. 2016; 17: 523-530Crossref PubMed Scopus (79) Google Scholar), we tested whether ZIKV NS3 competes with RIG-I for 14-3-3ϵ interaction. A competitive binding assay showed that RIG-I and 14-3-3ϵ interacted upon infection with SeV, a virus that stimulates RIG-I signaling, as previously shown (Liu et al., 2012Liu H.M. Loo Y.M. Horner S.M. Zornetzer G.A. Katze M.G. Gale Jr., M. The mitochondrial targeting chaperone 14-3-3ϵ regulates a RIG-I translocon that mediates membrane association and innate antiviral immunity.Cell Host Microbe. 2012; 11: 528-537Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). However, in the presence of ZIKV NS3, the binding between RIG-I and 14-3-3ϵ was inhibited in a dose-dependent manner (Figure 2G), suggesting that ZIKV NS3 disrupted the physical interaction of the two host proteins. Collectively, these data indicate that ZIKV NS3 encodes a phosphomimetic RLDP motif that allows it to compete with RIG-I for 14-3-3ϵ binding. As 14-3-3ϵ has been shown to mediate the translocation of RIG-I from the cytosol to mitochondria and mitochondria-associated membranes for promoting antiviral signaling (Liu et al., 2012Liu H.M. Loo Y.M. Horner S.M. Zornetzer G.A. Katze M.G. Gale Jr., M. The mitochondrial targeting chaperone 14-3-3ϵ regulates a RIG-I translocon that mediates membrane association and innate antiviral immunity.Cell Host Microbe. 2012; 11: 528-537Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), we assessed endogenous RIG-I protein abundance in the cytosolic and mitochondrial fractions of ZIKV-infected SVGA cells (Figure 3A ). Compared to SeV infection, infection with ZIKV only minimally induced mitochondrial translocation of RIG-I; RIG-I remained primarily in the cytosol during ZIKV infection (Figure 3A), suggesting that RIG-I translocation and thereby its downstream signaling are inhibited by ZIKV. GST-NS3 from ZIKV (strain H/PF/2013) suppressed SeV-induced activation of an IFN-β-promoter-driven luciferase reporter in a dose-dependent manner (Figure 3B), indicating that NS3 of ZIKV is sufficient to block RIG-I signaling and further suggesting that this antagonism is independent of the proteolytic activity of NS3, which requires co-expression of the viral NS2B protein (Kang et al., 2017Kang C. Keller T.H. Luo D. Zika virus protease: an antiviral drug target.Trends Microbiol. 2017; 25: 797-808Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Similarly, ZIKV NS3 (strain H/PF/2013) suppressed IFN-β-promoter activation following ectopic expression of RIG-I-2CARD, the constitutively active form of RIG-I (Gack et al., 2007Gack M.U. Shin Y.C. Joo C.H. Urano T. Liang C. Sun L. Takeuchi O. Akira S. Chen Z. Inoue S. Jung J.U. TRIM25 RING-finger E3 ubiquitin ligase is essential for RIG-I-mediated antiviral activity.Nature. 2007; 446: 916-920Crossref PubMed Scopus (1205) Google Scholar) (Figure 3C). Consistent with their ability to efficiently bind 14-3-3ϵ, GST-NS3 from ZIKV strains MR 766, H/PF/2013, and BRA/2015 also suppressed SeV-mediated IFN-β promoter activation (Figure 3D). Expression of ZIKV NS3 also reduced the protein expression of ISG15, ISG54, and RIG-I (itself an ISG) stimulated by SeV, as did NS3 from DENV (Figure 3E). Moreover, SeV-induced MAVS filament formation was impaired in cells that ectopically expressed ZIKV NS3 (Figure 3F). In comparison, 14-3-3-binding-deficient ZIKV NS3 KIKP minimally blocked endogenous MAVS filament formation (Figure 3F), which supports the idea that RIG-I-signal inhibition by NS3 depends on the RLDP motif. Finally, to corroborate that ZIKV NS3-mediated antagonism primarily occurs at the level of RIG-I, and not downstream of it, we compared the effect of ZIKV NS3 expression on signaling induced by ectopic expression of RIG-I 2CARD or MAVS, which are each sufficient to activate downstream signaling when overexpressed. ZIKV NS3 dampened IFN-β promoter activation induced by RIG-I 2CARD, but it had no effect on IFN-β luciferase activity mediated by FLAG-MAVS (Figure 3G). Taken together, these results indicate that the NS3 protein of ZIKV interferes with the cytosol-to-mitochondria translocation step of RIG-I, thereby dampening downstream signaling and antiviral gene expression. As 14-3-3η was important for ZIKV-mediated cytokine induction (Figure 1C) and since our MS analysis also identified 14-3-3η as a ZIKV NS3-binding partner (Figures 2B and S2B), we hypothesized that ZIKV NS3 might antagonize the function of 14-3-3η in the MDA5-signaling pathway. Indeed, ZIKV NS3 expression effectively downregulated MDA5-mediated IFNB1, IFNL1, CCL5 and ISG (RSAD2, MX1, OAS1) transcript induction in a dose-dependent manner, although not as potently as did the V protein of measles virus (MeV V), a well-characterized MDA5 antagonist (Davis et al., 2014Davis M.E. Wang M.K. Rennick L.J. Full F. Gableske S. Mesman A.W. Gringhuis S.I. Geijtenbeek T.B. Duprex W.P. Gack M.U. Antagonism of the phosphatase PP1 by the measles virus V protein is required for innate immune escape of MDA5.Cell Host Microbe. 2014; 16: 19-30Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) (Figure 4A ). In Co-IP studies, ZIKV NS3 bound to 14-3-3η, but not 14-3-3σ (Figure 4B). NS3 also efficiently interacted with endogenous 14-3-3η in SVGA cells throughout a 72 h time course of ZIKV infection (Figure 4C). The NS3-14-3-3η interaction was dependent on the 64-RLDP-67 motif in ZIKV NS3, as GST-NS3 WT, but not GST-NS3 KIKP, bound to 14-3-3η (Figure 4D). Finally, ZIKV NS3 suppressed the interaction between HA-14-3-3η and FLAG-MDA5 in a dose-dependent manner, suggesting a competitive binding mode (Figure 4E). Of note, the NS3 proteins of DENV and WNV also antagonized MDA5 2CARD-mediated signaling and efficiently bound to 14-3-3η, whereby the flavivirus NS3 proteins showed differential 14-3-3η-binding and MDA5-inhibitory capacities (Figures S3A and S3B). Together, these results indicate that through binding to 14-3-3η, ZIKV NS3 antagonizes MDA5-mediated antiviral gene expression. Furthermore, these data suggest that MDA5/14-3-3η antagonism is conserved also in DENV and WNV NS3, although further studies will be needed to determine the relevance of 14-3-3η targeting for efficient replication and IFN antagonism for DENV and WNV. To further define the relevance of 14-3-3ϵ/η binding in antagonism of the IFN response, we generated a recombinant ZIKV (strain MR 766), termed hereafter ZIKV(KIKP), by introducing the mutations R64→ K64, L65→I65, D66→K66 into NS3. We first compared the replication of ZIKV(KIKP) and the parental virus [ZIKV(WT)] in Vero cells, a cell line deficient in type I IFN genes (Desmyter et al., 1968Desmyter J. Melnick J.L. Rawls W.E. Defectiveness of interferon production and of rubella virus interference in a line of African green monkey kidney cells (Vero).J. Virol. 1968; 2: 955-961Crossref PubMed Scopus (0) Google Scholar) and found only minor differences between the growth of both viruses over a 72 h time course (Figure S4A), ruling out a general growth defect of ZIKV(KIKP). We next analyzed the growth of ZIKV(WT) and ZIKV(KIKP) in A549 cells, a human lung epithelial cancer cell line that has functional IFN signaling. We found an ∼100-fold difference in titers between ZIKV(WT) and ZIKV(KIKP) at 24 h post-infection. The difference in titers between ZIKV(WT) and ZIKV(KIKP) gradually decreased at later time points, likely due to saturation of ZIKV(WT) replication at these times (Figure 5A ). Moreover, the percentage of ZIKV(KIKP)-infected cells was significantly lower than that of ZIKV(WT)-infected cells (Figure S4B). We observed that in A549 cells, ZIKV(KIKP) infection led to greatly enhanced cytokine (IFNB1, IL6, IL8, and TNF), CCL5, and ISG (IFIT1, MX1 and RSAD2) transcript expression in comparison to infection with ZIKV(WT), despite the attenuated growth phenotype (Figure 5B). ZIKV(KIKP) also elicited higher cytokine, chemokine, and/or ISG induction in SVGA (astrocytes) and HMC3 (microglia) cells than did ZIKV(WT) (Figures 5C and S4C). To address whether ZIKV(KIKP), in contrast to ZIKV(WT), is unable to block the translocation of RIG-I and MDA5 to the mitochondria, we infected SVGA cells with ZIKV(WT) or ZIKV(KIKP) and assessed protein abundances for these two sensors in the cytosol and mitochondrial fractions by immunoblot analysis. Cells infected with ZIKV(WT) showed low amounts of RIG-I and MDA5 in the mitochondrial fraction, which were similar to mock-infected cells; in contrast, ZIKV(KIKP)-infected cells exhibited greater abundance of RIG-I and MDA5 at the mitochondria (Figures 5D and 5E). Finally, to confirm that the growth attenuation of ZIKV(KIKP) was due to greater activation of RLR signaling, we infected SVGA cells in which the RLR adaptor MAVS was knocked out using CRISPR-Cas9 gene editing (SVGA MAVS KO) or cells expressing a nontargeting (NT) guide RNA (Figures S4D and S4E). In NT control cells, ZIKV(KIKP) was growth-attenuated in comparison to ZIKV(WT), whereas there was no significant difference between the growth of the two viruses in MAVS KO SVGA cells (Figure 5F). This indicates that the major attenuating feature of ZIKV(KIKP) is its inability to antagonize RLR signaling and, further, that the RLR-MAVS axis is a major sensing pathway of ZIKV infection in these cells. We reasoned that the use of ZIKV(KIKP), which is deficient in antagonism of RIG-I/14-3-3ϵ" @default.
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• W2979411325 date "2019-10-01" @default.
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• W2979411325 title "Zika Virus NS3 Mimics a Cellular 14-3-3-Binding Motif to Antagonize RIG-I- and MDA5-Mediated Innate Immunity" @default.
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• W2979411325 doi "https://doi.org/10.1016/j.chom.2019.09.012" @default.
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