ZIKA VIRUS NS4B-C100S MUTANT PROTEIN WEAKLY BINDS TANK-BINDING KINASE AND ELICITS A STRONG ANTIVIRAL RESPONSE

BELEN, SARRATEA MARIA; REDOLFI, DANIELA; IANNANTUONO LÓPEZ, LAURA; NOLI TRUANT, SOFIA; GÓMEZ, GABRIELA ELENA ; DELFINO, JOSÉ MARÍA; MARIUZZA, ROY ; SANCHEZ ALBERTI, ANDRÉS; FERNÁNDEZ, MARISA MARIEL; EMILIO, MALCHIODI

Mar del Plata

Congreso; REUNIÓN CONJUNTA SAIC SAI & FAIC SAFIS 2022; 2022

SAIC SAI & FAIC SAFIS

Type I interferons (IFN-I) are key mediators in antiviral innate immunity. Nucleic acidsensing in the cytoplasm triggers TANK-binding kinase 1 (TBK1)- interferon regulatoryfactor 3 (IRF3) signaling axis and activate IFN-I transcription. Our previous reportsdemonstrated that the Zika virus (ZIKV) non-structural protein NS4B inhibits IFN-βproduction and directly binds TBK1. An attenuated ZIKV with NS4B C100S mutationshowed a potent induction of antiviral immune response in a mouse model. In this work,we evaluated the effect of NS4B C100S in the antiviral cellular response and study howit affects the interaction with host ligands.For this purpose, we performed transfection assays with plasmids encodingrecombinant ZIKV NS4B or NS4B-C100S mutant. We found that Hela cells transfectedwith NS4B-C100S secreted higher levels of IFN-β and IL-6 upon dsRNA analogstimulation (poly(I:C), compared to those transfected with wild type NS4B(ANOVA+Tukey´s, p<0.05).TBK1, an essential component in IFN-I production, acts as a ligand of ZIKV NS4B, asdemonstrated by immunoprecipitation. Here, we found that TBK1 alsoimmunoprecipitated with ZIKV NS4B-C100S mutant. To further study this interaction,we performed Surface Plasmon Resonance assays (SPR). Micelles of recombinantlyexpressed ZIKV NS4B-C100S were injected over a surface with immobilized TBK1.SPR showed that NS4B-C100S also interacts with TBK1, but with an equilibriumdissociation constant (KD) of 1.1±0.2 x10 -5 M, a 10 times lower affinity than TBK1-NS4Bwt binding (KD= 3.7±0.4 x10 -6 M).Our data suggest that the NS4B-C100S mutant is associated with a strongerinflammatory response and reduced capacity to bind TBK1. The results correlate withthe higher inflammatory and antiviral response reported for the mutated virus.Altogether, these results highlight the importance of the conserved C100 residue ofZIKV NS4B for its function and viral kinetic and may provide new insights for thedevelopment of antivirals.