Zika virus (ZIKV) is a member of the Flaviviridae family and causes congenital microcephaly and Guillain–Barré syndrome. Currently, there is a lack of approved vaccines or therapies against ZIKV infection. In this study, we profile vRNA‒host protein interactomes at ZIKV stem‒loop B (SLB) and reveal that interleukin enhancer binding factor 3 (ILF3) and DEAH-box helicase 9 (DHX9) form positive regulators of antiviral RNA inference in undifferentiated human neuroblastoma cells and induced pluripotent stem cell-derived human neural stem cells (iPSC–NSCs). Functionally, ablation of ILF3 in brain organoids and Nestin-Cre ILF3 cKO foetal mice significantly enhance ZIKV replication and aggravated ZIKV-induced microcephalic phenotypes. Mechanistically, ILF3/DHX9 enhance DICER processing of ZIKV vRNA-derived siRNAs (vsiR-1 and vsiR-2) to exert anti-flavivirus activity. VsiR-1 strongly inhibits ZIKV NS5 polymerase activity and RNA translation. Treatment with the vsiR-1 mimic inhibits ZIKV replication in vitro and in vivo and protected mice from ZIKV-induced microcephaly. Overall, we propose a novel therapeutic strategy to combat flavivirus infection.
ZIKV has a single-stranded, positive-sense RNA genome ~10.8 kb in length that contains two flanking noncoding regions (UTRs) and a single long open reading frame (ORF) encoding a polyprotein: 5′-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-3′. ZIKV circulates as two genetic lineages, African and Asian. The ancestral African lineage (MR766) is maintained in a sylvatic transmission cycle and infects few human hosts<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 1" title="Haddow,