Zika Virus NS3 Mimics a Cellular 14-3-3-Binding Motif to Antagonize RIG-I- and MDA5-Mediated Innate 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. 14-3-3 epsilon promotes the cytosolic-to-mitochondrial translocation of RIG-I, while 14-3-3 eta 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 14-3-3 epsilon and 14-3-3 eta 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-14-3-3 epsilon/eta interaction and to suppress antiviral signaling. Mutational perturbation of 14-3-3 epsilon/eta 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.