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

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.

The multifaceted roles of NLRP3-modulating proteins in virus infection

“…DDX3X, which is encoded on the X-chromosome, has a role to play in multiple biological functions including innate immunity and the cellular stress response. We and others have previously shown that DDX3X plays a prominent role in enhancing the RLR-mediated type I interferon (IFN) antiviral response by associating with various RLR signaling molecules including TBK1 (TANK-binding kinase I) and IKKε (I-kappa-B kinase-ε) and direct interaction with the IFNB1 promoter ( 39 – 43 ) ( Figure 1 ). DDX3X also participates in the formation of cytoplasmic stress granules, membraneless cytosolic bodies that form during the cell stress response, as well as during viral infection ( 44 , 45 ).…”

“…The determination of the DDX3X AKT-phosphorylation site/s would validate these results and potentially highlight a unique DDX3X-targeting site. Likewise, other post-translational modifiers of DDX3X, including tripartite motif protein 25 (TRIM25) ( 39 ) and TANK binding kinase 1 (TBK1) ( 43 ), may influence NLRP3 inflammasome activation but these remain to be explored.…”

“…During IAV infection DDX3X regulates stress granule formation, the type I IFN response and NLRP3 inflammasome activation (Table 1) (56,71). On the other hand, the IAV NS1 protein, which interacts with DDX3X in an RNA-independent manner, inhibits both stress-granule formation and the type I IFN response, however whether NS1 promotes or inhibits NLRP3 inflammasome activation is contentious (39,56,(71)(72)(73)(74)(75). Despite the influence of NS1, DDX3X protects IAV-infected mice against severe lung pathology and viral spread, thus aiding their survival (Table 1) (56).…”

Regulation of Tripartite Motif-Containing Proteins on Immune Response and Viral Evasion

“…Recently, it was found that TRIM25 enhances the antiviral activity of zinc finger antiviral protein (ZAP) with both ubiquitin ligase activity and multimerization ( Li et al, 2017 ; Zheng et al, 2017 ). In addition, TRIM25 ubiquitinates DDX3X (a critical component of TBK1) at K55, and TRIM25 and DDX3X cooperatively enhance type I IFN induction following RIG-I activation ( Soulat et al, 2008 ; Atkinson et al, 2021 ).…”

“…Moreover, the N-terminal domain of NS1 encoded by IBV is responsible for interaction with TRIM25, and this interaction blocks the Lys63-linked ubiquitination of RIG-I ( Jiang et al, 2016 ). Recently, it was found that NS1 disrupts the TRIM25:DDX3X interaction, abrogating both TRIM25-mediated ubiquitination of DDX3X and cooperative activation of the IFNB1 promoter ( Atkinson et al, 2021 ).…”