DEAD-box helicase gcDDX56 disrupts IRF3 nuclear import complex and promotes nuclear IRF3 degradation for enhancing...

DEAD-box helicases play multifaceted roles in antiviral immunity, acting as both viral sensors and regulators of interferon (IFN) signaling. However, the role of DDX56 in teleost antiviral immunity remains poorly characterized. This study investigates the role of grass carp DDX56 (gcDDX56) in regulating IFN responses during grass carp reovirus (GCRV) infection. Dual-luciferase reporter assays reveal that gcDDX56 potently suppresses GCRV-induced activation of IFN1/3 promoters and RLR signaling components (RIG-I, MDA5, MAVS, TBK1, IRF3/7). Mechanistically, gcDDX56 reduces IRF3 protein levels via autophagy-lysosome-mediated degradation, confirmed by cycloheximide (CHX) chase experiments and autophagy inhibitor rescue. Nuclear protein fractionation confirmed that gcDDX56 overexpression significantly reduced nuclear and cytoplasmic IRF3 protein levels during GCRV infection. Unlike mammalian DDX56, which directly interacts with IRF3, gcDDX56 does not bind gcIRF3. Instead, it sequesters importin β3 (gcKPNB3) in the nucleus, disrupting the gcIRF3-gcKPNB3 complex required for IRF3 nuclear import. Confocal microscopy validates nuclear co-localization of gcDDX56-gcKPNB3 and competitive binding with gcIRF3. Transcriptional profiling shows that gcDDX56 suppresses the expression of type I IFNs (ifn1, ifn3), IFN-stimulated genes (mx1, mx2), and RLR pathway components induced by gcIRF3-gcKPNB3. These findings establish a novel dual mechanism for gcDDX56: autophagy-dependent IRF3 degradation and gcKPNB3-mediated nuclear import blockade. This evolutionary divergence highlights teleost-specific adaptations in antiviral immunity, providing insights into GCRV pathogenesis and potential targets for aquaculture disease control.

DEAD-box helicases exhibit versatile cellular roles in antiviral defense, functioning both as sentinels detecting viral invaders and as regulators of immune signaling. The present study reveals that teleost fish have evolved unique strategies using a related protein called gcDDX56. Unlike previously studied DEAD-box helicases, we demonstrate for the first time that gcDDX56 hijacks an importin β's (gcKPNB3) dual functions (IRF3 transport/degradation) via domain-specific interaction, acting as a "switch" to tilt gcKPNB3 toward pro-viral degradation. This novel regulatory axis (gcDDX56-gcKPNB3-IRF3) reveals a "triple hit" mechanism (cytoplasmic degradation + nuclear clearance + import blockade) that maximizes IRF3 suppression, a strategy not reported for other viruses. Beyond advancing basic knowledge of vertebrate innate immunity (expanding DEAD-box helicase/importin β functional repertoires), these findings provide actionable targets (e.g., gcDDX56-Helicase C/gcKPNB3-KAP95 interface) for developing anti-GCRV therapies, addressing a pressing need in aquaculture to mitigate GCRV-induced losses.