The Zika virus (ZIKV) is a flavivirus that was discovered in 1947 near Entebbe, Uganda. In humans, ZIKV infection has been associated with a mild disease characterized by fever, rash, arthritis, and conjunctivitis [1
]. However, the recent epidemic in the Americas in 2013–2015 in an immunologically naïve population revealed that ZIKV can also cause severe neurological symptoms, such as Guillain–Barré syndrome and microcephaly [2
]. ZIKV is primarily spread by Aedes
species mosquitoes, and vertical and sexual transmission among humans was confirmed during the last epidemic. Multiple infection routes of ZIKV are facilitated by its ability to productively infect several types of human cells, such as skin fibroblast and dendritic cells [3
], Sertoli cells [4
], trophoblast progenitor cells and cytotrophoblasts, as well as placental macrophages [5
]. ZIKV also replicates in human brains and cells of the neuronal origin [7
Infected cells respond to virus infections by activating innate immune responses. In RNA virus infection, especially the RIG-I-like pattern recognition receptors, RIG-I and MDA5 recognize ssRNA and dsRNA molecules of invading and replicating viruses. RIG-I-like receptors activate signaling cascades involving cellular kinases that eventually phosphorylate and activate transcription factors IRF3, IRF7, and NF-ĸB. These factors translocate into the nucleus and initiate the expression of type I (IFN-α/β) and type III interferon (IFN-λ1-4) and other inflammatory cytokine genes. IFN-α/β is produced by several cell types, whereas IFN-λs are produced by immune cells and cells of epithelial origin [9
]. Virus-infected cells secrete IFNs that bind to their specific receptors, IFNAR1–IFNAR2 (IFN-α/β) and IL28Ra–IL10Rb (IFN-λ1-4) initiating a signaling cascade leading to the phosphorylation, activation, and dimerization of transcription factors STAT1 and STAT2. STAT1/STAT2 dimers associate with IRF9 to form the so-called ISGF3 complex, which then translocates into the nucleus where it activates the transcription of interferon-stimulated genes (ISGs). This initiates cellular antiviral responses via the production of antiviral proteins such as MxA, Viperin, and IFIT proteins [10
]. Most, if not all, pathogenic viruses encode proteins that interfere with the activation host innate immune responses.
ZIKV has a positive sense ssRNA genome that encodes for one large polyprotein including three structural (C, M, and E) proteins and seven non-structural (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) proteins. The polyprotein is cleaved by cellular and viral proteases into individual proteins. ZIKV infection leads to the production of interferons and antiviral proteins [14
] and ZIKV RNA has been shown to stimulate both RIG-I and MDA5 receptors [15
]. In order to replicate more efficiently in virus-infected cells, ZIKV has mechanisms to evade or delay the activation of innate immune responses. Recent studies have shown that ZIKV can interfere with interferon-induced responses: ZIKV infection inhibits STAT1 and STAT2 phosphorylation [16
], and especially ZIKV NS5 protein inhibits STAT1 phosphorylation [15
] and induces the proteasomal degradation of STAT2 [15
]. ZIKV NS2B-NS3 protein complex promotes the degradation of Jak1 resulting in reduced STAT1 phosphorylation [20
]. ZIKV E, NS4A, and NS5 proteins inhibit the expression of IFIT1 gene [18
], and NS1, NS2B3, NS4B, and NS5 proteins have been shown to inhibit the expression of IFIT2 gene [20
]. In addition, NS1, NS2B, NS4A, and NS5 proteins inhibit IFN-β-induced, and NS5 protein also IFN-λ1-induced ISRE activation [15
]. ZIKV also interferes with the production of IFNs: ZIKV infection prevents the translation of type I and III IFNs in dendritic cells [16
]. Several ZIKV proteins (NS1, NS2A, NS2B, NS4A, NS4B, NS5) have also been shown to inhibit the activation of IFN-β promoter [15
]. NS1, NS4A, and NS5 proteins were demonstrated to inhibit the activation of IRF3 and NS5 was shown to inhibit NF-κB reporters [18
]. In most of the events described above the exact molecular mechanisms are not known. However, it has been suggested that some ZIKV proteins block TBK1 function [20
] leading to reduced IRF3 phosphorylation [21
In the present study, we analyzed the potential inhibitory effect of individual ZIKV proteins on the activation of interferon promoters, specifically, a less-well studied type III IFN-λ1 promoter. We found that the ZIKV NS5 protein efficiently inhibits RIG-I-induced IRF3 phosphorylation, leading to a reduction in type I and type III interferon promoter activation. We show here that ZIKV NS5 interacts with IKKε, an important downstream kinase of the RIG-I pathway. The data indicates that this interaction leads to impaired ability of IKKε to phosphorylate and activate IRF3 leading to reduced activation of IFN promoters including that of the IFN-λ1 gene.
2. Materials and Methods
2.1. Cloning of ZIKV Genes and Construction of Expression Plasmids
Viral genes coding for structural proteins (capsid, pro-membrane, membrane, and envelope) were constructed synthetically (GeneArt, Thermo Fisher Scientific, Waltham, MA, USA) as one continuous sequence, based on Zika virus isolate BeH819966 (GenBank accession no: KU365779.1). The individual structural genes were subsequently amplified by PCR for expression constructs using the Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) with time and temperature parameters adjusted according to the manufacturer’s instructions. The non-structural genes were either constructed synthetically (NS1 and NS5) or amplified from complementary DNA generated by RT-PCR from viral RNA that was isolated from a Zika virus stock propagated for four days in Vero E6 cells with inoculum of 10-3
of isolate FB-GWUH-2016 (Genbank accession no: KU870645.1). The non-structural genes were amplified with the Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) or DreamTaq DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA). The NS5 MTase and RdRp subunits were amplified with DreamTaq DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) and Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA), respectively. Primers used in this study are listed in Supplementary Materials Table S1
. ZIKV genes were subcloned into a mammalian expression plasmid pEBB-HA (kindly provided by Drs. D. Baltimore and K. Saksela) which contains an N-terminal HA-tag.
Luciferase reporter plasmids pIFN-β-Luc, pIFN-λ1-Luc, pIFN-λ1-ISREmut-Luc, pIFN-λ1-NF-κBmut-Luc, and pMxA-Luc with the indicated promoter areas for the cytokine genes upstream of firefly luciferase gene, Renilla luciferase gene under Rous sarcoma virus promoter (RSV-Renilla), and expression plasmids for the wild type (wt) RIG-I, wtIRF3, the constitutively active form of RIG-I (∆RIG-I), FLAG-tagged MAVS, FLAG-tagged TBK-1, FLAG-tagged IKKε, constitutively active form of IRF3 (IRF3-5D), and FLAG-tagged HCV NS3/4A have been described previously [23
]. All plasmids were prepared for transfections with endotoxin-free maxipreps (Sigma-Aldrich, St. Louis, MO, USA or Qiagen, Hilden, Germany).
For the baculoviral expression of GST-tagged ZIKV NS5, the pBVboost vector [29
] was modified to create a GST-pBVboost vector by replacing the Bam
III multiple cloning site with a segment from the pGEX-2T(+) bacterial expression vector (GE Healthcare Europe GmbH, Finland; GenBank: U13850.1). Inserted segment encodes the glutathione S-transferase gene and contains a thrombin recognition site and the multiple cloning site with a Bam
HI recognition site. The production of pAc/YM1-based GST, GST-NS2 (HCV) and GST-NP (IAV) baculoviral expression plasmids have been described previously [30
Rabbit anti-IRF3 (FL-425; Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-IRF3 (SL-12; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-phospho-IRF3 (Ser396) (4D4G; Cell Signaling Technology, Danvers, MA, USA), mouse anti-HA1.1 Epitope Tag (BioLegend, San Diego, CA, USA), mouse anti-GAPDH (6C5; Santa Cruz Biotechnology, Dallas, TX, USA), mouse anti-FLAG (M2; Sigma-Aldrich, St. Louis, MO, USA), rabbit anti-TBK-1/NAK (Cell Signaling Technology, Danvers, MA, USA), rabbit anti-phospho-TBK-1/NAK (Ser172) (D52C2; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-IKKi/IKKε (Abcam, Cambridge, UK), and rabbit anti-phospho-IKKε (Ser172) (D1B7; Cell Signaling Technology, Danvers, MA, USA) were used according to the manufacturers’ instructions. Rabbit polyclonal antibodies against RIG-I was as previously described [31
]. For blocking the activity of type I interferons, a 1:50 dilution of Human Type I IFN Neutralizing Antibody Mixture (PBL Assay Science, Piscataway, NJ, USA) was used. In-house guinea pig anti-ZIKV-NS5 was prepared against ZIKV NS5 based on FB-GWUH-2016 sequence: A synthetic NS5 gene was ordered from GenArt (Thermo Fisher Scientific, Waltham, MA, USA), the NS5 coding sequence was cloned into a baculovirus expression vector GST-pBVboost to produce a chimeric GST-NS5 coding expression vector. Detailed gene expression, protein production, purification, and immunization protocols are described in Melén et al., 2017 [32
Human embryonic kidney 293 (HEK293; ATCC, www.atcc.org
) cells were maintained in Dulbecco’s modified Eagle’s medium (D-MEM) supplemented with HEPES (MP Biomedicals, Santa Ana, CA, USA), and human hepatocellular carcinoma HuH7 cells [33
] were maintained in minimum Eagle’s medium-α (Invitrogen, Carlsbad, CA, USA). Cell media were supplemented with 10% heat-inactivated fetal bovine or calf serum (Biowest, Nuaillé, France or Integro, Zaandam, The Netherlands), penicillin/streptomycin and D-MEM also with L-glutamine. Spodoptera frugiperda
9) cells, used for baculovirus expression, were maintained in TNM-FH medium (Sigma-Aldrich, St. Louis, MO, USA) as described previously [34
2.4. Transfections, Cell Stimulations, and Infections
Plasmids were transfected into HEK293 cells with Lipofectamine2000 (Invitrogen, Carlsbad, CA, USA) or with TransIT-LT1 Reagent (Mirus Bio, Madison, WI, USA) and into Huh7 cells with TransIT-LT1 Reagent (Mirus Bio, Madison, WI, USA). To activate MxA promoter, IFN-α2b (IntronA; Merck Sharp & Dohme, Kenilworth, NJ, USA) IFNβ-1b (Bayer, Leverkusen, Germany), IFN-γ (Gibco), IFN-λ1 (Invitrogen), and TNF-α (Gibco, Thermo Fisher Scientific, Waltham, MA, USA ) were added in indicated concentrations into the cell culture media. For polyI:C stimulation, HEK293 cells were transfected overnight with TransIT-LT1 Reagent with indicated expression plasmids and pIFN-λ1-Luc reporter construct, followed by 7.3 µg/mL polyI:C (LMW, InvivoGen, San Diego, CA, USA) transfected with Lipofectamine2000. PolyI:C-induced cells were incubated overnight, and luciferase activities were measured.
2.5. Reporter Gene Assay
HEK293 cells were grown on 96-well plates and transfected at 70–80% confluency with promoter-luciferase constructs (20 ng/well), with RIG-I pathway expression plasmids (30 ng/well, except 3 ng/well for IKKε), and with ZIKV or HCV NS3/4A expression constructs (3–30 ng/well). RSV-Renilla (50 ng/well) was included as an internal control. Cells were harvested at indicated time points for Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA) or for twinlite Firefly and Renilla Luciferase Reporter Gene Assay System (Perkin Elmer, Waltham, MA, USA) according to the manufacturer’s instructions. The firefly luciferase results were normalized with the values of Renilla luciferase. All experiments were done in triplicates and repeated at least three times.
For immunoblotting, cells were transfected on 12-well plates with 400 ng per well expression plasmids for ∆RIG-I, IRF3, MAVS, TBK-1, and IRF3-5D, and 30 ng or 40 ng per well expression plasmid for IKKε. NS3, NS5, and HCV NS3/4A expression plasmids were transfected with 40 ng and 400 ng per well (or other indicated amounts). After overnight incubation, the cells were lysed on ice with Passive Lysis Buffer provided in the Dual Luciferase Assay Kit (Promega, Madison, WI, USA) or with a native lysis buffer as described previously [35
]. Lysis buffers were supplemented with Complete Protease Inhibitor Cocktail (Roche, Basel, Switzerland) and PhosStop Phosphatase Inhibitor Cocktail (Roche, Basel, Switzerland). Proteins were separated on 4–12% or Any kD SDS-PAGE (BioRad, Hercules, CA, USA) and transferred onto an Amersham Protran 0.2 μm nitrocellulose blotting membranes (GE Healthcare Europe GmbH, Finland). Additionally, ZIKV E was also run under non-reducing conditions. Immunoblotting was done according to the manufacturer’s instructions for a particular antibody. Secondary antibodies were IRDye 800CW goat anti-rabbit IgG and IRDye 680RD goat anti-mouse IgG (LI-COR Biosciences, Lincoln, NE, USA). Membranes were scanned and analyzed with Odyssey Fc Imaging System (LI-COR Biosciences, Lincoln, NE, USA).
2.7. Immunofluorescence Microscopy
Huh7 cells were grown on glass coverslips and fixed with 3% paraformaldehyde in PBS at RT for 30 min. Cells were blocked and permeabilized with 0.5% BSA and 0.1% Triton X-100 in PBS at RT for 30 min. For localization studies in transfected cells, mouse anti-HA1.1 Epitope Tag (BioLegend, San Diego, CA, USA) was diluted 1:500 into 1% BSA in PBS and incubated at RT for 60 min. Secondary FITC-labeled anti-mouse antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) were used according to the manufacturers’ instructions. After washing, coverslips were mounted on Mowiol (Sigma-Aldrich, St. Louis, MO, USA) and images were taken with a Leica TCS NT confocal microscope (Leica Microsystems, Wetzlar, Germany).
2.8. Production of GST Fusion Proteins in Sf9 Cells and GST-Pull-Down Assay
The production of GST and GST-tagged HCV NS2 and IAV NP in Sf
9 cells was done using a baculovirus expression system that has been described previously [30
]. The production of GST-tagged ZIKV NS5 in Sf
9 cells was done according to the instructions by Airenne et al. [29
]. For protein production, the cells were infected with GST and GST-tagged ZIKV NS5, HCV NS2, and IAV NP expressing baculoviruses for 48–72 h. Whole-cell extracts were prepared by lysing the cells in a lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100) and protease inhibitors (Roche, Basel, Switzerland) on ice for 10 min and suctioning five times through a 25 G needle to disrupt DNA, followed by centrifugation (10,000× g
, + 4 °C, 5 min). For pull-down experiments, 1 mg of soluble cellular protein samples were bound to 25 μL of Glutathione Sepharose 4B beads (GE Healthcare Europe GmbH, Finland) for 1 h at + 4 degrees and washed three times with the binding buffer (same as above but with 0.5% Triton X-100). The purity and quantity of each fraction were verified with Coomassie Blue staining on 12% SDS-PAGE (compared to known standard protein, bovine serum albumin). To produce radioactively labeled proteins for binding studies, in vitro-translated (TnT®
Quick Coupled Transcription/Translation Systems; Promega, Madison, WI, USA) MAVS, IKKε, and TBK1 were [35
S]-labeled (Easy TagTM Express Protein Labeling Mix, PerkinElmer, Waltham, MA, USA) and allowed to bind to Sepharose-immobilized GST or GST-fusion proteins on ice for 60 min followed by washing three times with the binding buffer. GST and GST-fusion protein-bound, [35
S]-labeled proteins were separated on 12% SDS-PAGE. The gels were fixed and treated with Amplify reagent (Amersham Biosciences, Little Chalfront, UK) as specified by the manufacturer followed by autoradiography.
For analysis of GST and GST-fusion protein-bound proteins by immunoblotting, proteins were separated on SDS-PAGE gels and transferred onto Immobilon-P membranes (polyvinylidene difluoride; Merck Millipore, Burlington, MA, USA) with an Isophor electrotransfer device (Hoefer Scientific Instruments, Holliston, MA, USA). The proteins were detected with rabbit anti-FLAG (F7425; Sigma-Aldrich, St. Louis, MO, USA), followed by secondary peroxidase-conjugated anti-rabbit IgG (DAKO). Detection was done on HyperMax films using an ECL Plus system (GE Healthcare Europe GmbH, Finland).
2.9. Statistical Analyses
For the luciferase assay data, the Student’s t-test in SPSS (IBM, Armonk, NY, USA ) was used to determine the statistically significant differences between the observed levels of inhibition compared with the control following the transfection of different amounts of ZIKV plasmids.
ZIKV induces innate immune responses in infected cells since the expression of TLR3, RIG-I, and MDA5, as well as IFN-α, IFN-β, IFN-λ1, and MxA genes are enhanced [3
]. Secreted IFNs protect cells from virus infection, and ZIKV infection is inhibited in cells pretreated with IFN-α, IFN-β, IFN-λ1, or IFN-γ [3
]. Of these, IFN-λ1 is especially important in protecting epithelial barriers from virus infection [9
] and, as shown earlier, ZIKV infection is prevented in placental trophoblasts which are secreting IFN-λ1 [45
]. Thus, IFN-λ1 is important in protection from ZIKV infection since ZIKV spreads through epithelial barriers such as the skin, the blood-brain barrier, and the placenta. However, like other flaviviruses, ZIKV has the means to inhibit or delay the activation of innate immune responses. So far, most studies have focused on type I IFNs. In this study, we systematically analyzed the effect of all ZIKV proteins on the RIG-I pathway and type III IFN-λ1 promoter activation. We found that ZIKV NS5 protein efficiently inhibited the RIG-I pathway by interacting with IKKε followed by inhibition of phosphorylation of IRF3 and reduced activation of IFN promoters.
In previous studies, it has been shown that ZIKV NS5 inhibits polyI:C or RNA-induced activation of IFN-β [15
] and NF-ĸB promoters [18
]. Here, we also observed a strong inhibition of ∆RIG-I-induced activation of IFN-β promoter and of IFN-λ1 promoter and of polyI:C-induced activation of IFN-λ1 promoter by ZIKV NS5 protein. The inhibitory effect on the activation of IFN-λ1-promoter required a full-length NS5 protein, as has also been shown for TBK-1-related inhibition of IRF3 phosphorylation by the African lineage ZIKV NS5 [22
]. We found that ZIKV NS5 inhibited ∆RIG-I-induced activation of both IRF3- and NF-ĸB transcription factors that coordinately regulate IFN-λ1 promoter activation (27). Consistent with a previous study on NS5-mediated inhibition of IFN-β promoter [21
], we observed that ZIKV NS5 can also inhibit IFN-λ1 promoter activation induced by all components of the RIG-I pathway upstream or at the level of IRF3.
In addition to ZIKV NS5, we observed a weak inhibition of RIG-I-induced IFN-λ1 promoter activation by NS2A protein, whereas the other proteins were devoid of any inhibitory activity. Other researchers have, however, shown that ZIKV NS1, NS2A, NS2B, NS4A, and NS4B can inhibit poly(I:C)-induced activation of IFN-β promoter [18
]. Our observations are well in line with the results of Hertzog and coworkers, who have shown that no other proteins apart from NS5 inhibit RNA-induced IFN-β promoter activation [15
]. Some of the flavivirus NS proteins function together, like DENV NS2B-NS3 proteins which act as an active protease complex [47
]. The functionality of different ZIKV protein combinations or complexes on innate immunity is presently not known, but, at least in our analyses, the p2K peptide did not provide any inhibitory effect on innate immunity for NS4A or NS4B proteins.
The exact molecular mechanisms of ZIKV proteins on the inhibition of IFN promoter activation are to some extent not known. Wu and coworkers have shown that ZIKV NS1 and NS4B inhibit IFN-β promoter activation by interacting with TBK-1 preventing its dimerization and phosphorylation [20
]. Xia et al. [21
] reported that IFN-β promoter activation was inhibited by NS2A, NS2B, and NS4B via blocking the phosphorylation of TBK-1. In addition, ZIKV NS4A suppressed IRF3 phosphorylation and NS5 was suggested to function on IRF3, possibly by interfering with the nuclear import of IRF3 [21
]. Lin et al. reported that NS5 prevented IRF3 phosphorylation by direct binding to TBK-1 [22
]. Our data on ZIKV NS5 inhibiting the activation of the IFN-λ1 promoter is well in line with the results described above, suggesting a function for ZIKV NS5 at or after activation of IRF3. However, in contrast to the results by Xia et al. [21
], we observed that ZIKV NS5 inhibited the phosphorylation of IKKε and IRF3. In addition, we found a reduction in the amounts of IKKε with increasing expression levels of NS5, and furthermore, IKKε was also found to bind to NS5. In contrast to the data from Lin et al. [22
] who demonstrated that NS5 can interact with TBK-1, we and Xia et al. [21
] did not observe the inhibitory effect of NS5 on TBK-1. This discrepancy may be due to differences in the used ZIKV strains, cell types, or expression constructs, as well as differences in overall experimental conditions. Indeed, Esser-Nobis et al. [48
] recently showed that infection with Zika viruses of different genetic lineages (Asian vs. African) led to differences in innate immune responses, resulting in differential IRF3 phosphorylation. In the above-mentioned studies, Wu et al. [20
] and Lin et al. [22
] used African ZIKV strain in their experiments, while Xia et al. [21
] used both African and Asian lineage viruses, which both showed NS5-related inhibition of IFN-β-promoter activation.
Different flaviviruses are known to have different functions for the same/similar proteins. Flavivirus NS5 proteins have two active domains: A methyltransferase and an RNA-dependent RNA polymerase domain. Even though these biological functions are similar amongst flaviviruses, NS5 sequences of flaviviruses show up to 45% differences at amino acid level [17
] allowing these proteins to have variable biological functions. For instance, DENV NS5 induces STAT2 degradation via E3 ubiquitin ligase [49
], WNV NS5 inhibits IFNAR1 expression [50
] and STAT1 phosphorylation [51
], YFV NS5 binds to STAT2 and prevents its binding to ISRE sites [52
], and ZIKV NS5 binds to STAT2 and induces its proteasomal degradation [17
]. ZIKV NS5 also inhibits STAT1 phosphorylation which leads to reduced IFN-induced ISRE activation [15
]. Even minor changes in amino acid sequences have been shown to impact these mechanisms. For example, a single amino acid substitution in ZIKV NS1 leads to a protein variant that targets the RIG-I pathway by inhibiting TBK-1 phosphorylation [21
]. In addition, a single amino acid substitution in WNV NS2A protein leads to an attenuated virus phenotype with an inability to inhibit IFN-α/β production [53
]. In our study, the NS genes originate from a highly pathogenic Asian lineage ZIKV strain isolated from fetal brain. Whether there was a selection towards a more fit/aggressive virus type with an exceptional ability to inhibit innate immune responses remains to be studied.
In our study, we found a direct interaction between ZIKV NS5 and IKKε. In addition, NS5 seemed to reduce the expression and phosphorylation of IKKε. Importantly, with higher expression levels of IKKε, the inhibitory effect of NS5 on IFN-λ1 promoter activation was abolished, suggesting that the stoichiometry of NS5 and IKKε has an impact on the inhibitory activity of NS5. Other viral proteins are also known to interfere with the functions of IKKε. For example, DENV NS2B/NS3 protein complex interacts with IKKε and blocks its kinase activity [54
], MERS-CoV ORF4b protein inhibits the formation of MAVS and IKKε complexes [55
], and arenavirus NP binds to and colocalizes with IKKε, preventing IKKε autophosphorylation and IKKε-induced IRF3 phosphorylation [56
]. It remains to be studied whether the inhibitory effect of ZIKV NS5 on IFN gene expression is due to direct binding of NS5 with IKKε or whether NS5 targets some scaffold/modulating proteins on the IKKε signalosome.
ZIKV has a huge potential to efficiently spread among naïve human populations through Aedes species and possibly other mosquito vectors. This global threat and newly-discovered disease burden of ZIKV emphasizes the need to systematically study the role of individual ZIKV proteins in virus–host cell interactions. ZIKV NS5 is a crucial protein for the life cycle of the virus. Importantly, the ZIKV NS5 structure has already been resolved [41
] facilitating the development of small molecular inhibitors that could be used as antiviral substances [58
]. In this study, we describe a new phenomenon of how ZIKV NS5 protein interferes with the host innate immune system providing potentially novel targets for rational drug design.