Subcellular trafficking of host RNA helicase DDX3X modulates innate antiviral signalling and parainfluenza virus immunity

Heaton SM, Atkinson SA, Jans DA, Sweeney MN and Borg NA

Infection & Immunity Program, Biomedicine Discovery Institute, Department of Biochemistry & Molecular Biology, Monash University, Clayton, 3800, Australia.

DEAD-box RNA helicase 3, X-linked (DDX3X) multiply regulates the retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) antiviral signalling cascade. Accordingly, manipulating DDX3X is crucial to the replication strategy of a growing list of evolutionarily diverse pathogens including influenza A virus, HBV, HCV, dengue virus and HIV-1. To fulfil these host- and pathogen-directed roles, DDX3X localises to various subcellular compartments. Interestingly, nuclear export of DDX3X was previously attributed to an exportin-1/CRM1-dependent mechanism that, uniquely, required neither a nuclear export signal (NES) nor the nucleocytosolic Ran-GTP/GDP gradient. We examined the DDX3X-exportin-1 interaction by analytical ultracentrifugation and confocal microscopy. We probed its role in immune signalling and human parainfluenza-3 (hPIV-3) replication in human lung A549 cells using plaque assays, luciferase gene reporter assays, NanoString transcriptome analysis, FACS and ELISA, then dissected its mechanism of immune regulation by constitutively activating RLR signalling proteins or treating with poly(I:C), LPS or IFN-β. Contrasting the current model, we find the region bound by exportin-1 bears no primary sequence homology to any recognised protein domain in evolution. We show exportin-1 is the bulk nuclear export receptor for DDX3X, which is specifically impaired by treatment with exportin-1 inhibitors, DDX3X point mutagenesis or removal of Ran-GTP. In response to viral RNA exposure during hPIV-3 infection, DDX3X accumulates inside the nucleus to support IFN-β expression. However, ectopic nuclear DDX3X impairs RLR signalling, deregulates dozens of genes relevant to immunity and fails to suppress hPIV-3 replication. Our results redefine the molecular mechanism of DDX3X nuclear export and reveal new modes of immune regulation.