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. 2021 Dec 6;13(12):2448.
doi: 10.3390/v13122448.

INMI1 Zika Virus NS4B Antagonizes the Interferon Signaling by Suppressing STAT1 Phosphorylation

Elisa Fanunza  1 , Nicole Grandi  1 , Marina Quartu  2 , Fabrizio Carletti  3 , Laura Ermellino  1 , Jessica Milia  1 , Angela Corona  1 , Maria Rosaria Capobianchi  3 , Giuseppe Ippolito  3 , Enzo Tramontano  1
Affiliations

INMI1 Zika Virus NS4B Antagonizes the Interferon Signaling by Suppressing STAT1 Phosphorylation

Elisa Fanunza et al. Viruses. .

Abstract

The evasion of the Interferon response has important implications in Zika virus (ZIKV) disease. Mutations in ZIKV viral protein NS4B, associated with modulation of the interferon (IFN) system, have been linked to increased pathogenicity in animal models. In this study, we unravel ZIKV NS4B as antagonist of the IFN signaling cascade. Firstly, we reported the genomic characterization of NS4B isolated from a strain of the 2016 outbreak, ZIKV Brazil/2016/INMI1, and we predicted its membrane topology. Secondly, we analyzed its phylogenetic correlation with other flaviviruses, finding a high similarity with dengue virus 2 (DEN2) strains; in particular, the highest conservation was found when NS4B was aligned with the IFN inhibitory domain of DEN2 NS4B. Hence, we asked whether ZIKV NS4B was also able to inhibit the IFN signaling cascade, as reported for DEN2 NS4B. Our results showed that ZIKV NS4B was able to strongly inhibit the IFN stimulated response element and the IFN-γ-activated site transcription, blocking IFN-I/-II responses. mRNA expression levels of the IFN stimulated genes ISG15 and OAS1 were also strongly reduced in presence of NS4B. We found that the viral protein was acting by suppressing the STAT1 phosphorylation and consequently blocking the nuclear transport of both STAT1 and STAT2.

Keywords: NS4B; STAT1; STAT2; Zika virus; innate immunity; interferon; interferon evasion; phosphorylation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
ZIKV INMI1 NS4B expression and prediction of membrane topology. Protein expression level was verified by (a) SDS-Page and (b) immunofluorescence; scale bar, 10 μm; (c) schematic representation of ZIKV INMI1 NS4B transmembrane segments predicted by HMMTOP, DAS, TMHMM, TMpred, TOPCONS and Split; the blue boxes represent predicted transmembrane segments (TMS); the positions of the first and last amino acid of TMS are indicated; (d) consensus model of ZIKV INMI1 NS4B topology.
Figure 2
Figure 2
Phylogenic analysis of NS4B. (a) Phylogenetic tree of the amino acid sequence of NS4B protein of ZIKV INMI1 and of other representative flaviviruses. The highly supported cluster formed by the Spondweni (SPOV) group of mosquito-borne flaviviruses, including INMI1 KU991811 (green triangle) and other ZIKV strains, is highlighted with green branches; (b) amino acid identity matrix of NS4B77-125; (c) amino acid alignment of NS4B77-125 of INMI1 KU991811 and DEN2 isolates; (d) amino acid alignment of INMI1 KU991811 NS4B and WNV, KUNV and WNV9 whole NS4B; residues important for IFN modulation in WNV are highlighted in red; (*) identical nucleotides, (:) conserved substitution, (.) semi-conserved substitution.
Figure 3
Figure 3
ZIKV INMI1 NS4B inhibits IFN production and signaling. (a) Percentage of pIFNβ-luc expression in HEK293T cells transfected with increasing concentrations (30 ng/well and 60 ng/well) of empty vector (EV), ZIKV NS2A or NS4B after stimulation with IAV vRNA; percentage of pISRE-luc expression in (b) HEK293T cells and (c) Vero cells transfected with increasing concentrations (30 ng/well and 60 ng/well) of EV, ZIKV NS2A or NS4B and stimulated with IFN-α; (d) percentage of pGAS-luc expression in HEK293T cells transfected with increasing concentrations of EV, ZIKV NS2A or NS4B after stimulation with IFN-γ; (e) percentage of NF-κB expression in HEK293T cells transfected with increasing concentrations of ZIKV NS4B (7.5–15–30–60 ng/well) after stimulation with TNF-α; (f) ISG15 and (g) OAS1 transcript levels in HEK293T cells transfected with EV, ZIKV NS2A or NS4B in presence of IFN-α. (a–e) Results are shown as percentage of (a) pIFN-β-luc; (b,c) pISRE-luc; (d) pGAS-luc and (e) pNFκB-luc activation in NS2A or NS4B transfected cells over empty vector (EV) transfected controls (100%). (a–g) Significance calculated using a two-tailed unpaired Student’s t-test, * p< 0.05, ** p <  0.01, *** p <  0.001, **** p <  0.0001. Error bars indicate the mean ± SD (data from at least 3 independent experiments).
Figure 4
Figure 4
ZIKV INMI1 NS4B inhibits STAT1 phosphorylation. (a) Inhibition of pISRE-luc expression in HEK293T cells transfected with increasing concentrations of ZIKV NS2A (red line), NS4B (green line) or the combination of the two plasmids (blue line), and stimulated with IFN-α (10 ng/mL). Results are shown as percentages of pISRE-luc activation in NS2A or NS4B transfected cells over empty vector (EV) transfected controls; (b) the isobologram visualizes the synergistic effect of the combination of ZIKV NS2A and NS4B at their respective EC50; (c) immunofluorescence of HEK293T transfected for 24 h and 36 h with ZIKV NS2A (row 1 and 3) and NS4B (row 2 and 4) and stimulated with IFN-α; FLAG (green) and P-STAT1 (red) signals are detected; nuclei (blue) are stained with Hoechst; scale bar, 10 μm; (d) quantification was performed counting positive cells for nuclear P-STAT1 (red signal) in EV, NS2A and NS4B transfected cells, 6–10 fields per each condition, from a total of 3 biologically independent experiments, were analyzed; cells count was performed using Cell Counter plugin of the image analysis program ImageJ, **** p <  0.0001 as obtained by unpaired t test. (e) Western blot of HEK293T transfected with empty vector (EV) (lane 1–2) or ZIKV NS4B (lane 3–4) stimulated (+) (lanes 2–4) or not (−) (lanes 1–3) with IFN-α; membranes were stained for P-STAT1, STAT1-tot and GAPDH antibodies; (f) Western blot of HEK293T transfected with empty vector (EV) (lane 1–2) or ZIKV NS4B (lane 3–4); cells were treated (+) (lanes 2–4) or not (−) (lanes 1–3) with MG132; blot membranes were stained for STAT1-tot and GAPDH antibodies.
Figure 5
Figure 5
ZIKV INMI1 NS4B blocks STAT2 nuclear transport. (a) Immunofluorescence of HEK293T cells transfected with empty vector (EV) or ZIKV NS2A (row 3) stimulated with IFN-α. FLAG (green) and P-STAT2 (red) signals were detected; nuclei (blue) were stained with Hoechst; scale bar, 10 μm; (b) quantification was performed counting positive cells for nuclear P-STAT1 (red signal) in EV and NS4B transfected cells; 6–10 fields per each condition, from a total of 3 biologically independent experiments, were analyzed; cells count was performed using Cell Counter plugin of ImageJ. **** p <  0.0001 values are reported, as obtained by unpaired t test.
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