TRIM25 and DEAD-Box RNA Helicase DDX3X Cooperate to Regulate RIG-I-Mediated Antiviral Immunity.

Sarah C Atkinson,Michelle D Audsley,Steven M Heaton,Oded Kleifeld,Natalie A Borg

doi:10.3390/ijms22169094

Abstract

The cytoplasmic retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs) initiate interferon (IFN) production and antiviral gene expression in response to RNA virus infection. Consequently, RLR signalling is tightly regulated by both host and viral factors. Tripartite motif protein 25 (TRIM25) is an E3 ligase that ubiquitinates multiple substrates within the RLR signalling cascade, playing both ubiquitination-dependent and -independent roles in RIG-I-mediated IFN induction. However, additional regulatory roles are emerging. Here, we show a novel interaction between TRIM25 and another protein in the RLR pathway that is essential for type I IFN induction, DEAD-box helicase 3X (DDX3X). In vitro assays and knockdown studies reveal that TRIM25 ubiquitinates DDX3X at lysine 55 (K55) and that TRIM25 and DDX3X cooperatively enhance IFNB1 induction following RIG-I activation, but the latter is independent of TRIM25’s catalytic activity. Furthermore, we found that the influenza A virus non-structural protein 1 (NS1) disrupts the TRIM25:DDX3X interaction, abrogating both TRIM25-mediated ubiquitination of DDX3X and cooperative activation of the IFNB1 promoter. Thus, our results reveal a new interplay between two RLR-host proteins that cooperatively enhance IFN-β production. We also uncover a new and further mechanism by which influenza A virus NS1 suppresses host antiviral defence.

Highlights

The retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), which include RIG-I, sense virally-derived nucleic acids in the cytosol to initiate an antiviral immune response [1,2]
In addition to tripartite motif 25 (TRIM25) peptides, we identified a substantial enrichment of peptides from another protein with roles in the RLR-signalling cascade, DEADbox helicase 3X (DDX3X), and significantly these peptides covered ~17% of the DDX3X sequence (Supplementary Table S1)
Given the unexpected result that the DDX3X(K55R) mutant, which is no longer ubiquitinated by TRIM25, caused an increase in IFNB1 gene induction in the presence of TRIM25, we further investigated the association of TRIM25 catalytic activity and DDX3X binding with IFNB1 gene induction

Summary

Introduction

The retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), which include RIG-I ( termed DDX58), sense virally-derived nucleic acids in the cytosol to initiate an antiviral immune response [1,2]. RIG-I binds MAVS (mitochondrial antiviral signalling protein; termed IPS-1), an adaptor protein which aggregates along the mitochondrial outer membrane and converges downstream elements of the RLR signalling apparatus, including tumour necrosis factor receptor-associated factor-3 (TRAF3), inhibitor of NF-κB kinase subunit ε (IKKε), TANK-binding kinase 1 (TBK1), and DDX3X (DEAD-box helicase 3, X-linked) [3,4,5,6] These signals converge at interferon regulatory factors (IRFs; IRF3/7), which drive the expression of type I interferon (IFN-I; namely IFN-α and IFN-β) and type III interferon (IFN-III; namely IFN-λ) and in turn modulate the expression of hundreds of antiviral genes in infected cells, neighbouring tissues and systemically. In addition to its RING-dependent functions, TRIM25 performs RING-independent antiviral functions that are independent of RIG-I [20,21,22]

Methods

Results

Conclusion