ZIKA VIRUS NS4B PROTEIN TARGETS TANK-BINDING KINASE 1 TO INHIBIT TYPE I INTERFERON PRODUCTION

MARIA BELEN SARRATEA; DANIELA REDOLFICONSTANZA AMERIO, LAURA IANNANTUONO LOPEZ, SOFÍA NOLI TRUANT; ANDRES SANCHEZ ALBERTI; EMILIO LUIS MALCHIODI

Congreso; Reunión Anual de Sociedades de Biociencias; 2021

Type I interferons (IFN I) play an essential role in antiviral innate immunity. During viralinfections, cytosolic nucleic acids can be sensed by intracellular pattern recognitionreceptors, triggering TANK-binding kinase 1 (TBK1)- interferon regulatory factor 3(IRF3) signaling axis to initiate IFN I transcription. However, many flavivirus use non-structural proteins to evade immune sensing favoring their survival. Here, we aimed tostudy the role of Zika virus (ZIKV) NS4b protein in the inhibition of IFN I induction and itsinteraction with host ligands.For this purpose, we performed transfection assays with a plasmid encodingrecombinant ZIKV NS4b or ZIKV NS4b C100S mutant. Using RAW-Lucia ISG cells, anIFN reporter cell-line, we showed that cells with ZIKV NS4b were able to reduceluciferase signals compared to empty vector. Interestingly, this reduction was abrogatedwith ZIKV NS4b C100S mutant (ANOVA+Tukey´s, p<0.05). Moreover, A549 cellstransfected with plasmid encoding ZIKV NS4b and stimulated with poly(I:C) secretedless IFN-β levels (ELISA) compared to control (ANOVA+Tukey´s, p<0.05).TBK1, a key component in IFN I production, has been proposed as a possible target ofNS4b. Using transfection assays in Hela cells, we showed that TBK1immunoprecipitated with ZIKV NS4b. Furthermore, we recombinantly produced N-terminal ZIKV NS4B in micelles and human TBK1. We performed Surface PlasmonResonance (SPR) assays to further characterize this interaction. SPR assays showedthat NS4b interacted with TBK1 with an equilibrium dissociation constant (KD) of 3.1 ±0.2 µM.Our results add evidence that ZIKV NS4b is involved in disrupting TBK1/IRF3 cascadeand the conserved residue C100 is important for this function. Besides, this is the firstreport of biophysical interaction between N-terminal ZIKV NS4b and TBK1. Altogether,the information gathered herein can be of substantial use in the rational design ofantiviral inhibitors.