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.

Keywords: DDX3X; DEAD-box helicase; E3 ligase; IFN; NS1; RLR signalling; TRIM25; antiviral immunity; influenza; ubiquitination.

Figure 1

TRIM25 interacts with DDX3X. (A) Anti-FLAG immunoprecipitation (IP) of whole-cell lysates (WCL) of HEK293T cells expressing FLAG-TRIM25 and HA-DDX3X, as well as FLAG-DDX3X and HA-TRIM25. Immunoblot (IB) analysis of IP with anti-FLAG and anti-HA antibodies and WCL with anti-FLAG, anti-HA and anti-actin antibodies. (B) Interaction between endogenous TRIM25 and endogenous DDX3X in HEK293T cells, with immunoblot analysis of WCL and anti-TRIM25 (top) or anti-DDX3X (bottom) IP. (C–E) Anti-FLAG IP of WCL of HEK293T cells overexpressing various recombinant proteins. IB analysis of IP with anti-FLAG and anti-HA antibodies and WCL with anti-FLAG, anti-HA and anti-actin antibodies. (C) FLAG-DDX3X together with HA-tagged wild-type TRIM25, TRIM25ΔRING or TRIM25ΔPRY-SPRY. In the schematic, R = RING, B = B-box, CC = coiled-coil. (D) FLAG-tagged wild-type DDX3X, DDX3X 1–517, DDX3X 1–414 and DDX3X NTE (residues 1–168) constructs together with HA-TRIM25. In the schematic, NTE = N-terminal extension and R = RecA-like domain. (E) FLAG-TRIM25 and HA-tagged wild-type DDX3X or DDX3XΔNTE.

Figure 2

TRIM25 modifies DDX3X with K63-linked polyubiquitin chains. (A) Immunoblot analysis of the abundance and ubiquitination (Ub) status of mCherry-DDX3X in HEK293T cells transfected with expression vector for mCherry-DDX3X or empty mCherry vector (control). (B) Immunoblot analysis of the abundance (top) and total ubiquitination (middle) of mCherry-DDX3X in HEK293T cells transfected with empty FLAG vector or expression vector for FLAG-TRIM25 or TRIM25ΔRING (bottom). (C) Immunoblot analysis of the abundance (first blot) and total ubiquitination (second blot) of mCherry-DDX3X in HEK293T cells transfected to co-express HA-ubiquitin and pSuper-shTRIM25 (TRIM25 KD) or pSuper-shScrambled (WT), assessed after immunoprecipitation with anti-mCherry antibody. (D) Immunoblots probed using anti-DDX3X (top) or anti-ubiquitin (bottom) antibody showing in vitro DDX3X ubiquitination reactions consisting of recombinant E1 (Ube1), E2 (UbcH5b), TRIM25, TRIM25ΔRING, ubiquitin (Ub), ATP and wild-type DDX3X(1–580). Arrowheads denote progressively ubiquitinated DDX3X. (E) TRIM25 enhanced the K63-linked polyubiquitination of DDX3X. HEK293T cells were transfected with expression plasmids for mCherry-DDX3X and FLAG-TRIM25 or empty FLAG vector, together with HA-tagged wild-type or K11_only, K48_only, or K63_only ubiquitin mutants. Whole-cell extracts were immunoprecipitated with anti-mCherry antibody-conjugated beads and probed with anti-HA antibody. (F) HEK293T cells transfected to express mCherry-DDX3X, HA-tagged K63_only ubiquitin and pSuper-shTRIM25 (TRIM25 siRNA) or pSuper-shScrambled (control siRNA) (500 ng). mCherry-tagged proteins were immunoprecipitated with anti-mCherry antibody-conjugated beads probed with anti-HA antibody. The empty FLAG vector (pcDNA3.1) used to subclone was transfected as the “–” for all data. All results are representative of two independent experiments.

Figure 3

TRIM25 ubiquitinates DDX3X at lysine residue 55. (A) DDX3X domain schematic showing location of di-glycine-modified DDX3X residues identified in this study and by others [25]. NTE = N-terminal extension. (B) Immunoblot analysis of DDX3X in vitro ubiquitination assay with recombinant E1, E2 (UbcH5b), ubiquitin (Ub) and ATP together with wild-type (WT) or lysine (K) > arginine (R) substituted DDX3X. Arrowheads denote progressively ubiquitinated DDX3X. (C) Immunoblot analysis (with anti-mCherry antibody) of the abundance (top) and total ubiquitination (anti-HA antibody, second blot) of mCherry-tagged WT and K55R substituted DDX3X in HEK293T cells transfected with empty FLAG vector or expression vector for FLAG-TRIM25 or TRIM25ΔRING, assessed after immunoprecipitation with anti-mCherry antibody.

Figure 4

TRIM25 and DDX3X cooperatively enhance IFNB1 promoter induction. Activity of firefly luciferase expressed under the control of the IFNB1 promoter (pGL3-IFN-β1) measured after 24 h of RLR signalling cascade activation with RIG-I 2CARD (A,C,E,G,I,J,K) or 18 h of poly(I:C) stimulation (B,D,F,H) in HEK293T cells transfected to express recombinant proteins as indicated. (A,B) Increasing amounts of HA-TRIM25 expression vector or control FLAG vector. (C,D) Increasing amounts of FLAG-DDX3X expression vector or control FLAG vector. (E,F) FLAG-DDX3X and HA-TRIM25. (G,H) FLAG-DDX3X and HA-TRIM25 in cells transfected with pSuper-shTRIM25 (TRIM25 KD) or pSuper-shScrambled (WT). (I) FLAG-tagged WT, K55R or K66R DDX3X and HA-TRIM25. (J) FLAG-tagged WT, K55R or K66R DDX3X and HA-tagged WT or ΔRING TRIM25. (K) FLAG-tagged WT, K55R or K66R DDX3X and HA-tagged WT or ΔPRY-SPRY TRIM25. Firefly luciferase results normalised to the activity of Renilla luciferase internal control. The empty vector pcDNA3.1 FLAG was transfected as the “–” for all data. All results are representative of three independent experiments. Representative anti-FLAG, anti-HA and anti-actin immunoblots are shown. Graphs show the mean ± SD of three replicates. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test and assessed based on the p value: NS p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, and **** p ≤ 0.0001.

Figure 5

Influenza A NS1 inhibits the TRIM25 catalysed ubiquitination of DDX3X. (A) Immunoblot probed using anti-DDX3X antibody showing in vitro DDX3X ubiquitination reactions consisting of recombinant Ube1, UbcH5b, TRIM25, DDX3X(1–580), ubiquitin, ATP and increasing concentrations of influenza A virus non-structural protein 1 (IAV–NS1) (NS1:TRIM25 molar ratio: 1:1, 3:1, 5:1), or, as a specificity control, E. coli maltose-binding protein (MBP) (MBP:TRIM25 molar ratio: 1:1, 3:1, 5:1). Arrowheads denote ubiquitinated DDX3X. (B) Immunoblot analysis (with anti-HA antibody) of the abundance (first blot) and total ubiquitination (second blot) of mCherry-DDX3X in HEK293T cells transfected with empty vector or expression vector for FLAG-TRIM25, HA-ubiquitin and increasing amounts of myc-NS1 (0–6 μg), assessed after immunoprecipitation with anti-mCherry antibody. (C) Activity of firefly luciferase expressed under the control of the IFNB1 promoter (pGL3-IFN-β1) measured after 24 h of RIG-I 2CARD stimulation in HEK293T cells expressing FLAG-DDX3X and HA-TRIM25 together with control vector (− NS1) or myc-NS1 (+ NS1). (D) Anti-FLAG IP of WCL of HEK293T cells overexpressing FLAG-DDX3X together with HA-TRIM25 and increasing amounts of myc-NS1 (0–5 μg). IP analysis with anti-FLAG or anti-HA antibodies and WCL with anti-FLAG, anti-HA and anti-actin antibodies. (E,F) Firefly luciferase activity expressed from the pGL3-IFN-β1 reporter plasmid after 24 h of RIG-I 2CARD stimulation in HEK293T cells expressing myc-NS1 together with increasing amounts of HA-TRIM25 (0–100 ng) (E) or FLAG-DDX3X (0–500 ng) (F). Firefly luciferase results normalised to the activity of Renilla luciferase internal control. The empty vector pcDNA3.1 FLAG was transfected as the “–” for all data. All results are representative of three independent experiments. Representative anti-FLAG, anti-HA, anti-myc and anti-actin immunoblots are shown. The graphs show the mean ± SD of three replicates. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test and assessed based on the p value: NS p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.