The RNA helicase DDX39A plays an important role in the RNA splicing/export process. In our study, human DDX39A facilitated RNA virus escape from innate immunity to promote virus proliferation by trapping TRAF3, TRAF6, and MAVS mRNAs in the HEK293T cell nucleus. DDX39A was a target for SUMOylation. SUMO1, 2, and 3 modifications were found on immunoprecipitated DDX39A. However, only the SUMO1 modification decreased in vesicular stomatitis virus–infected HEK293T cells. Further studies have found that viral infection reduced SUMO1 modification of DDX39A and enhanced its ability to bind innate immunity–associated mRNAs by regulating the abundance of RanBP2 with SUMO1 E3 ligase activity. RanBP2 acted as an E3 SUMO ligase of DDX39A, which enhanced SUMO1 modification of DDX39A and attenuated its ability to bind RNA. This work described that specific mRNAs encoding antiviral signaling components were bound and sequestered in the nucleus by DDX39A to limit their expression, which proposed a new protein SUMOylation model to regulate innate immunity in viral infection.

The innate immune system can sense viral infection through pattern recognition receptors to initiate host innate immune responses to eliminate pathogens (1, 2). Upon viral infection, RNA viral nucleic acids are recognized by cytosolic sensors, retinoic acid–inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) protein (3). Both RIG-I and MDA5 expose the N-terminal CARD to bind the mitochondrial adaptor protein MAVS, which leads to activation of IκB kinase-γ (IKKγ) or downstream signaling of TRAF family member–associated NF-κB activator (TANK)-binding kinase 1 (TBK1), then phosphorylation of IFN regulatory factor (IRF) 3 forms a dimer and releases NF-κB/P65 from IκBα, which translocates into the nucleus to induce IFN-β production (35).

DEAD-box (DDX) RNA helicases, characterized by the conserved motif Asp-Glu-Ala-Asp (DEAD), comprise the largest superfamily 2 helicase, which plays an important role in antiviral immune response (68). Studies have reported that some members of the family can participate in the fight against viral infections. The members of the DDX family exert antiviral effects by recognizing viral dsRNA or regulating downstream signaling pathways. DDX58 (RIG-I) recognizes viral RNA and mediates induction of the NF-κB signaling pathway and IFN-α/β production (9). As a bridge adapter for IKKε and IRF3, DDX3 enhances IKKε autophosphorylation and activation to facilitate the induction of type I IFNs (10). In addition, DDX24 hijacks the adaptor proteins FADD and RIP1 to inhibit RNA-mediated innate immune signaling (11).

DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 39A (DDX39A), also known as URH49 and BAT1, was initially identified as an essential splicing factor dependent on U2AF65-based pre-mRNA recruitment (12). As a member of the nucleic acid–binding protein DDX RNA helicases family, DDX39A plays an important role in pre-mRNA splicing (13). In addition, excess DDX39A potently inhibits mRNA export and the recruitment of Aly to spliced mRNP for export-oriented mRNA (14). DDX39A also acts in cytoplasmic mRNA localization and maintenance of genome integrity as well as mitosis and cytokinesis (1517). During influenza A virus infection, DDX39A plays a role in regulating nucleus-cytoplasm transport of antiviral MxA (18). Influenza virus nuclear protein uses DDX39A to enhance viral RNA synthesis (19). DDX39A is essential for efficient Kaposi sarcoma–associated herpesvirus ORF57 expression (20). However, how DDX39A functions in the RNA virus-mediated type I IFN pathway and which immune-related RNAs are bound by DDX39A still be investigated.

In our research, we demonstrated that DDX39A negatively regulated type I IFN production and promoted virus proliferation by binding to innate immunity–associated RNAs (TRAF3, TRAF6, and MAVS). DDX39A can be used as a substrate for the small ubiquitin-related modifier (SUMO) modification. Interestingly, the SUMOylation of DDX39A was regulated during viral infection, which in turn affected the ability of DDX39A to bind RNA. Mechanistically, RNA viral infection downregulated the expression of RanBP2, an E3 SUMO ligase of DDX39A, which inhibited the SUMO1 modification of DDX39A and thus promoted the binding of DDX39A to RNA. Enhanced binding of DDX39A to innate immune–related RNAs inhibited the nuclear export of these RNAs, thereby inhibiting the expression of corresponding proteins (TRAF3, TRAF6, and MAVS). Our results provide a novel model on posttranslational modification protein interacted with mRNA to regulate innate immunity.

HEK293T, Vero, and HeLa cells were obtained from American Type Culture Collection and cultured in DMEM (Life Technologies) with 10% (v/v) FBS (Biological Industries) supplemented with an antibiotic–antimycotic mixture of 100 mg/ml streptomycin and 100 IU/ml penicillin. The cells were maintained in a humidified 5% CO2 incubator at 37°C.

Transfection of DDX39A plasmid was performed using jetPEI reagent (Polytransfection, Illkirch, France) at a final concentration of 1 μg/ml for HEK293T cells or and 0.5 μg/ml for HeLa cells, according to the manufacturer protocol.

Abs against DDX39A (Y404360), p65 (Y021012), Flag-tag (G188), MAVS (Y401304), and SUMO1 (Y400655) were purchased from Applied Biological Materials (Vancouver, Canada). Abs against p-TBK1 (ab109272), TBK1 (ab40676), and p-IRF3 (ab76493) were purchased from Abcam (Cambridge, MA). Abs against HA-tag (3724), Myc-tag (2278), ubiquitin (3936), TRAF1 (4715), TRAF3 (61095), TBK1 (3504), and SUMO-2/3 (4971) were from Cell Signaling Technology (Danvers, MA). Abs against RanBP2 (sc-74518), IRF3 (sc-33641), TRAF6 (sc-8409), and histone H3 (sc-56616) were from Santa Cruz Biotechnology (Santa Cruz, CA). Abs to β-actin (HC201) and HRP-labeled secondary Abs were purchased from Transgen (Beijing, China) and Sanjian (Tian, China), respectively.

Anti-HA-tag mouse mAb agarose-conjugated (M20013S), anti-DYKDDDDK-tag (Flag) mouse mAb agarose-conjugated (M20018M), and anti-Myc-Tag mouse mAb agarose-conjugated (M20012S) were purchased from Abmart (Shanghai, China).

Vesicular stomatitis virus (VSV) was proliferated and amplified in monolayer Vero cells. Senda virus (SeV) and encephalomyocarditis virus (EMCV) was obtained from American Type Culture Collection. Cells were infected with VSV (multiplicity of infection [MOI] = 1), EMCV (MOI = 5), and SeV (MOI = 5) for the indicated hours, according to the experimental requirements.

DDX39A-deficient HEK293T cells were generated by using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology. The single guide RNA (sgRNA)–targeting DDX39A were cloned into a Cas9-expressing lentiviral transfer vector (lentiCRISPRv2) according to the methods of the Feng Zhang laboratory (21). Briefly, lentiCRISPRv2 was digested with BsmBI and ligated with annealed oligonucleotides (DDX39A-sgRNA). The lentiCRISPRv2 plasmid was cotransfected into HEK293T cells with packaging vectors pMD2G and psPAX2 using Lipofectamine 3000 (Invitrogen, Carlsbad, CA). Two days after transfection, the viral supernatant was harvested and stored at −80°C. HEK293T cells were incubated with virus supernatant containing 8 μg/ml polybrene for 48 h and selected with 1.5 μg/ml puromycin to generate a stable cell line. The oligonucleotides for sgRNAs are as follows: A, 5′-CACCGCAGCAGATTGAGCCTGTCAA-3′; B, 5′-AAACTTGACAGGCTCAATCTGCTGC-3′.

The DDX39A and RanBP2 cDNA were amplified from a human NK cell cDNA library. The specific primer pairs (Table I) with a common sequence of vectors were used to amplify the DDX39A and RanBP2 mRNA and ligated to pFlag-CMV2, pMyc-CMV2, pHA-CMV2, and pEGFP vectors using a One Step Cloning Kit (Vazyme, Nanjing, China). DDX39A point mutation primers (Table II) were generated by use of the Web-based QuickChange Primer Design Program. DDX39A point mutations were generated by standard molecular biology techniques.

For immunoblot analysis, transfected or virus-infected cells were washed twice with cold PBS and lysed in 100 μl of lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% Triton X-100, 10% glycerol, 1 mM EDTA) supplemented with a proteinase inhibitor (20 nM PMSF [Sigma-Aldrich] or 2.5 mM deSUMOylation protease inhibitor N-ethylmaleimide [Sigma-Aldrich]). The protein concentrations in the extracts were measured and boiled in buffer for 10 min. Equal amounts of extracts were separated with 12% SDS-PAGE and transferred to the methanol-activated PVDF membrane (Millipore). Membranes were blocked in TBST/5% nonfat dry milk for 1 h and incubated with an Ab against DDX39A (1:1000), RanBP2 (1:500), β-actin (1:5000) or labeled Abs (1:3000) for overnight at 4°C, followed by washing and incubation with HRP-conjugated secondary Abs for 1 h at room temperature. Immunodetection was completed using Pierce ECL Western Blotting Substrate (Thermo Fisher Scientific).

HEK293T cells were transfected with Flag–DDX39A, Flag–GFP or Flag truncations of DDX39A for 24 h. Each cell pellet was washed twice with ice-cold PBS and lysed in 400 μl lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% Triton X-100, 10% glycerol, 1 mM EDTA) supplemented with 20 nM PMSF and 40 U RNase Inhibitor (Invitrogen). The anti-DYKDDDDK-tag (Flag) agarose-conjugated beads were washed three times with 1 ml of lysis buffer. Three hundred microliters of each sample was incubated with anti-Flag agarose beads for 6 h at 4°C, and 30 μl of each sample was kept as input (10%). The beads were washed sequentially in high-salt lysis buffer (adjusted to 500 mM NaCl) and lysis buffer. The RNA content of the resulting eluate and input was extracted with TRIzol reagent (Takara Bio, Beijing, China). For RNA immunoprecipitation (RIP) sequencing (RIP-seq), purified immunoprecipitated RNA was sent to Genewiz (Suzhou, China) for high-throughput RNA sequencing based on the Illumina HiSeq platform. For RIP–quantitative RT-PCR (qRT-PCR), the primers are shown in Table III.

HEK293T cells were transfected with small interfering RNA (siRNA) 1 (siDDX39A-1) or siRNA2 (siDDX39A-2) at a final concentration of 50 nmol/l using Lipofectamine 3000 (Invitrogen). All siRNAs are synthesized by GenePharma (Shanghai, China) (Table IV). The knockdown efficiency was confirmed by qRT-PCR and Western blotting.

The concentrations of IFN-β in culture supernatants were detected by ELISA Kits (R&D Systems) according to the manufacturer’s instructions. In addition, HEK293T cells were infected with a virus or transfected with siRNA or plasmid(s). Cells were harvested at the indicated times, and total RNA was extracted with TRIzol reagent. First-strand cDNA was synthesized from HEK293T cells or RIP-purified RNAs using a First-Strand Synthesis System (Transgen). The relative gene expression was analyzed by qRT-PCR with TransStart Tip Green qPCR SuperMix (Transgen). The comparative cycle threshold method was used to calculate the relative gene expression using a 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA). The mRNA level of the endogenous gene β-actin in each sample was used as a positive control; all data presented were relatively quantitative and analyzed using GraphPad Prism 6.0 software. The primer pairs used for quantitative real-time PCR are listed in Table III.

The procedure is based on a previously described report (22). Briefly, HEK293T cells were transfected with the Flag-DDX39A plasmid and then infected with VSV. At 24 h postinfection, cytoplasmic and nuclear proteins were extracted using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime Institute of Biotechnology, Shanghai, China). The abundance of p65 in the nucleus and cytoplasmic was detected by Western blotting, respectively.

HEK293T cells were transfected with related expression plasmid or siRNA. At 24 h posttransfection, cells were washed twice with cold PBS and resuspended in lysis buffer (10 mM Tris-HCl, 140 mM NaCl, 1.5 mM MgCl2, 10 mM EDTA, 0.5% NP-40, and 40 U/ml RNasin [pH 7.4]) for 20 min. The supernatants as the cytoplasmic fraction of RNA were collected after centrifugation at 12,000 × g for 10 min. The resuspended nuclear pellets were washed twice with lysis buffer as a nuclear fraction. Next, cytoplasmic and nuclear RNA were extracted by TRIzol reagent. The purified RNAs were synthesized into first-strand cDNA using a First-Strand Synthesis System. After RT-PCR using specific primers (Table III), mRNA levels of relevant transcription factors were detected by 2% agarose gels.

HeLa cells were seeded in 12-well plates and grown until the cell density was ∼30–40%. After 24 h, the cells were transfected with plasmid expressing DDX39A, SUMO1/2/3, RanBP2, or empty vector (0.5 μg). At 18 h posttransfection, the cells were washed by PBS and fixed with 4% paraformaldehyde for 15 min and then permeabilized by 0.3% Triton X-100 in PBS for 10 min at room temperature. Cells were then blocked in 1% BSA in PBS for 30 min. Next, cells were incubated with primary Abs (anti-Flag, anti-Myc, anti-HA; diluted at 1:200) or anti-RanBp2 Ab (diluted at 1:200) in 3% BSA in PBS at 4°C overnight. Next, cells were washed by PBST buffer (1× PBS and 0.1% Tween 20) and incubated with secondary Abs (FITC-conjugated anti-mouse IgG or PE-conjugated anti-rabbit IgG) (diluted at 1:200) for 1 h. Finally, cells were washed and stained with DAPI. Imaging of the cells was carried out using an Olympus confocal microscope. Images were taken at ×100 magnification.

The procedure is based on a previously described report (23). Briefly, HEK293T cells (1 × 105) were seeded into 24-well plates until the cell density was ∼70–80%. The cells were transfected with 50 ng of luciferase reporter (IFN-β–Luc) and 50 ng of internal control (LacZ) and the indicated appropriate control or expression plasmid (s) or siRNA. At 24 h posttransfection, the lysed samples were prepared, and the luciferase activity was measured using a multimode microplate reader (Promega) according to the manufacturer’s recommendations.

The virus titer was determined using the half maximal tissue-culture infectious dose (TCID50) method. Vero cells (1 × 104 cells per well) were seeded in 96-well plates. After 24 h, Vero monolayer cells were inoculated with 100 μl of 10-fold serially diluted (10−2–10−6) viral suspension in 2% DMEM for 1 h at 37°C. In addition, normal cells treated with PBS were used as controls. Cells were incubated at 37°C for 7 d, and TCID50 was calculated using the Reed–Muench method (24).

Data were subjected to one-way ANOVA and expressed as mean ± SEM. Pairwise multiple comparison was conducted to determine which group differed by two-way ANOVA followed by Bonferroni posttests using Prism 6.0 (GraphPad Software). Results were considered statistically significant if p < 0.05.

DDX39A is an essential mRNA export factor implicated in multiple steps in RNA metabolism (2527). We found that DDX39A was highly conserved between mammals and amphibians, with the conserved motifs of the DDX family. According to residues that interact with RNA in the DDX family consensus sequence (28, 29), the predicted residues of DDX39A interacting with RNA were highlighted in red (Supplemental Fig. 1A). Next, Flag-DDX39A RIP and RIP-seq analysis were performed to search for RNAs related to antiviral immunity. An overview of the DDX39A-associated RNA transcript expression profiles is summarized in Supplemental Table I. We found that the mRNA expression profiles revealed DDX39A preferentially bound to antiviral transcripts (TRAF3, TRAF6, MAVS) in the RNA virus-mediated IFN signaling pathway (Fig. 1A). To control nonspecific binding to Flag-tagged recombinant DDX39A, a Flag-tagged DDX39AΔRna mutant with all predicted RNA binding sites mutated (Supplemental Fig. 1B) and Flag-tagged GFP were used for parallel reactions. The DDX39A mRNA complexes were purified by immunoprecipitation from cell extracts (Fig. 1B) and innate immunity–associated RNAs were analyzed by qRT-PCR. The results further confirmed that specific mRNAs (TRAF3, TRAF6, MAVS) encoding antiviral signaling components bind to DDX39A (Fig. 1D–F), whereas other RNAs (TRAF1, TBK1, NF-κB) did not specifically bind to DDX39A compared with the control (Fig. 1C, 1G, 1H). In addition, we verified the difference in RNA-binding capacity of DDX39A in HEK293T cells infected with or without VSV (Supplemental Fig. 2). Notably, viral infection specifically enhanced the binding of DDX39A to TRAF3, TRAF6, and MAVS transcripts, indicating DDX39A might be involved in antiviral innate immune response.

FIGURE 1.

RIP assay and RNAs analyses by qRT-PCR to detect the enrichment of different innate immune signaling pathway factor mRNAs in the DDX39A-RNA complex. (A) The heat map generated from RIP-seq data showing expression of the immune-associated transcripts bound to DDX39A. These genes (TRAF3, TRAF6, MAVS) are highlighted with the red stars, indicating that there is a change >2.0-fold in DDX39A and control. (B) Detection of the DDX39A-RNA complex precipitated with anti-FLAG agarose beads by Western blotting. (CH) Immunoprecipitation of DDX39A-related RNA was purified and subjected to qRT-PCR, and nontarget-associated RNA was isolated from GFP and DDX39AΔRna (Mut) with all RNA binding site–mutated RIP reactions performed in parallel as nonspecific binding. Data are representative of three independent experiments. Data are mean ± SEM of triplicate samples in (C)–(H). ***p < 0.001, ****p < 0.0001. ns, no significance.

FIGURE 1.

RIP assay and RNAs analyses by qRT-PCR to detect the enrichment of different innate immune signaling pathway factor mRNAs in the DDX39A-RNA complex. (A) The heat map generated from RIP-seq data showing expression of the immune-associated transcripts bound to DDX39A. These genes (TRAF3, TRAF6, MAVS) are highlighted with the red stars, indicating that there is a change >2.0-fold in DDX39A and control. (B) Detection of the DDX39A-RNA complex precipitated with anti-FLAG agarose beads by Western blotting. (CH) Immunoprecipitation of DDX39A-related RNA was purified and subjected to qRT-PCR, and nontarget-associated RNA was isolated from GFP and DDX39AΔRna (Mut) with all RNA binding site–mutated RIP reactions performed in parallel as nonspecific binding. Data are representative of three independent experiments. Data are mean ± SEM of triplicate samples in (C)–(H). ***p < 0.001, ****p < 0.0001. ns, no significance.

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Table I.
Primers used for PCR amplification
Genes NameGenBank NumberSequence of Primer(5′-3′)
pCMV-DDX39A NM_005804.3 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGATGGCAGAACAGGATGTGG-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTACCGGCTCTGCTCGATGT-3′ 
pEGFP-DDX39A NM_005804.3 Forward: 5′-GTACCGCGGGCCCGGGATCCATGGCAGAACAGGATGTGG-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTACCGGCTCTGCTCGATGT-3′ 
pCMV-RanBP2(D1) NM_006267.5 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGAGGCGCAGCAAGGCTGACG-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTAGTTTATTTCATGCTGAACTAGAA-3′ 
pCMV-RanBP2(D2) NM_006267.5 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGACTCTAAGAGCCCAGG-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTAGGCTTTTAAAATGCTCTGGGC-3′ 
pCMV-RanBP2(D3) NM_006267.5 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGCCAGGAACAAATGTAGCCATGG-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTGTATTAGAAGAACCCTGAAACAT-3′ 
pCMV-RanBP2(D4) NM_006267.5 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGCATAAACCTATTGCAGAAGCTC-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTAGCTTGACCGAGAAACATGAGGT-3′ 
pCMV-RanBP2(D5) NM_006267.5 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGTTAGCCAGTGATTTCTCTGATGG-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTATATCTGTCCACATTCTGTGATAG-3′ 
pCMV-RanBP2(D6) NM_006267.5 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGTTAGCCAGTGATTTCTCTGATGGT-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTATGTCTGAGATTTTTGAGCTTCCTG-3′ 
pCMV-GFP NM_006267.5 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGGTGAGCAAGGGCGAGGAGC-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTACTTGTACAGCTCGTCCATGCC-3′ 
pCMV-DDX39a (1–258) NM_005804.3 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGATGGCAGAACAGGATGTGG-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTACAGCGTGAGCTTGGT-3′ 
pCMV-DDX39a (75–427) NM_005804.3 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGATTCCCCAGGCCATC-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTACCGGCTCTGCTCGATGT-3′ 
pCMV-DDX39a (260–427) NM_005804.3 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGGGCCTGCAGCAGTAC-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTACCGGCTCTGCTCGATGT-3′ 
Genes NameGenBank NumberSequence of Primer(5′-3′)
pCMV-DDX39A NM_005804.3 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGATGGCAGAACAGGATGTGG-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTACCGGCTCTGCTCGATGT-3′ 
pEGFP-DDX39A NM_005804.3 Forward: 5′-GTACCGCGGGCCCGGGATCCATGGCAGAACAGGATGTGG-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTACCGGCTCTGCTCGATGT-3′ 
pCMV-RanBP2(D1) NM_006267.5 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGAGGCGCAGCAAGGCTGACG-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTAGTTTATTTCATGCTGAACTAGAA-3′ 
pCMV-RanBP2(D2) NM_006267.5 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGACTCTAAGAGCCCAGG-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTAGGCTTTTAAAATGCTCTGGGC-3′ 
pCMV-RanBP2(D3) NM_006267.5 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGCCAGGAACAAATGTAGCCATGG-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTGTATTAGAAGAACCCTGAAACAT-3′ 
pCMV-RanBP2(D4) NM_006267.5 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGCATAAACCTATTGCAGAAGCTC-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTAGCTTGACCGAGAAACATGAGGT-3′ 
pCMV-RanBP2(D5) NM_006267.5 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGTTAGCCAGTGATTTCTCTGATGG-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTATATCTGTCCACATTCTGTGATAG-3′ 
pCMV-RanBP2(D6) NM_006267.5 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGTTAGCCAGTGATTTCTCTGATGGT-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTATGTCTGAGATTTTTGAGCTTCCTG-3′ 
pCMV-GFP NM_006267.5 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGGTGAGCAAGGGCGAGGAGC-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTACTTGTACAGCTCGTCCATGCC-3′ 
pCMV-DDX39a (1–258) NM_005804.3 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGATGGCAGAACAGGATGTGG-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTACAGCGTGAGCTTGGT-3′ 
pCMV-DDX39a (75–427) NM_005804.3 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGATTCCCCAGGCCATC-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTACCGGCTCTGCTCGATGT-3′ 
pCMV-DDX39a (260–427) NM_005804.3 Forward: 5′-CCAGTCGACTCTAGAGGATCCATGGGCCTGCAGCAGTAC-3′ 
Reverse: 5′-CAGGGATGCCACCCGGGATCCCTACCGGCTCTGCTCGATGT-3′ 
Table II.
Primers used for DDX39A mutants
MutantsSequence of Primer(5′-3′)
H120L/R122K Forward: 5′-GGTCATGTGCCTCACGAAGGAGCTGGCCTTCCAAATCGGCAAGGAATATG-3′ 
Reverse: 5′-AGGCCAGCTCCCTCGTGTGGCACATGACCAGGACCGTCACCTGTCCGT-3′ 
K199R Forward: 5′-CGAGTGTGACAGGATGCTGGAGCAGCTGGACATGCGGCGGGAT-3′ 
Reverse: 5′-TGCTCCAGCATCCTGTCACACTCGTCCAGCACAAAGTGCTTCA-3′ 
K294R/V296L Forward: 5′-TCTTCGTCAGGTCATTGCAGCGCTGCATGGCCCTGGCCCAGC-3′ 
Reverse: 5′-AGCGCTGCAATGACCTGACGAAGATTATCACCTGGTTAAACTC-3′ 
T171S/G173A/R174K/A176G Forward: 5′-TCCCCGGCCAAGATCCTGGGGCTCGTGCGGAATAGGAGCTTCA-3′ 
T171S/G173A/R174K/A176G Reverse: 5′-GCCCCAGGATCTTGGCCGGGGACCCCACCACGACATGGGGACA-3′ 
T343S Forward: 5′-TGGTGGCCAGCAATCTGTTTGGCCGGGGGATGGACAT-3′ 
Reverse: 5′-AACAGATTGCTGGCCACCAGGATCCGCCGCTGGAAA-3′ 
K154/155R Forward: 5′-TCTCTCCATCCCGCCGGATGAAGAAGTGTTGAAGAAGAACTGTCCC-3′ 
Reverse: 5′-CTTCTTCATCCGGCGGGATGGAGAGACCACCGAAGAACACAGACAC-3′ 
K52R Forward: 5′-TTCTGCTGCCGCCGGAGCTCCTGCGGGCCATCGTGGACTG-3′ 
Reverse: 5′-AGCTCCGGCGGCAGCAGAAAGTCCCGGAAGCCAGAGCTG-3′ 
MutantsSequence of Primer(5′-3′)
H120L/R122K Forward: 5′-GGTCATGTGCCTCACGAAGGAGCTGGCCTTCCAAATCGGCAAGGAATATG-3′ 
Reverse: 5′-AGGCCAGCTCCCTCGTGTGGCACATGACCAGGACCGTCACCTGTCCGT-3′ 
K199R Forward: 5′-CGAGTGTGACAGGATGCTGGAGCAGCTGGACATGCGGCGGGAT-3′ 
Reverse: 5′-TGCTCCAGCATCCTGTCACACTCGTCCAGCACAAAGTGCTTCA-3′ 
K294R/V296L Forward: 5′-TCTTCGTCAGGTCATTGCAGCGCTGCATGGCCCTGGCCCAGC-3′ 
Reverse: 5′-AGCGCTGCAATGACCTGACGAAGATTATCACCTGGTTAAACTC-3′ 
T171S/G173A/R174K/A176G Forward: 5′-TCCCCGGCCAAGATCCTGGGGCTCGTGCGGAATAGGAGCTTCA-3′ 
T171S/G173A/R174K/A176G Reverse: 5′-GCCCCAGGATCTTGGCCGGGGACCCCACCACGACATGGGGACA-3′ 
T343S Forward: 5′-TGGTGGCCAGCAATCTGTTTGGCCGGGGGATGGACAT-3′ 
Reverse: 5′-AACAGATTGCTGGCCACCAGGATCCGCCGCTGGAAA-3′ 
K154/155R Forward: 5′-TCTCTCCATCCCGCCGGATGAAGAAGTGTTGAAGAAGAACTGTCCC-3′ 
Reverse: 5′-CTTCTTCATCCGGCGGGATGGAGAGACCACCGAAGAACACAGACAC-3′ 
K52R Forward: 5′-TTCTGCTGCCGCCGGAGCTCCTGCGGGCCATCGTGGACTG-3′ 
Reverse: 5′-AGCTCCGGCGGCAGCAGAAAGTCCCGGAAGCCAGAGCTG-3′ 
Table III.
Primers used for qRT-PCR amplification
GenesSequence of Primer(5′-3′)
DDX39A Forward: 5′-GCAGATTGAGCCTGTCAACG-3′ 
Reverse: 5′-AGACCACCGAAGAACACAGAC-3′ 
RanBP2 Forward: 5′-AAGCCAGTGCTACCAAATGTATTG-3′ 
Reverse: 5′-AATTCTGTATTAGAAGAACCCTGAAAC-3′ 
EMCV Forward: 5′-TGCAGTGGTTGCTCCCCTGA-3′ 
Reverse: 5′-TGACCGGAATGGGCGACTGT-3′ 
SeV Forward: 5′-GCTGCCGACAAGGTGAGAGC-3′ 
Reverse: 5′-GCCCGCCATGCCTCTCTCTA-3′ 
VSV Forward: 5′-GAGGAGTCACCTGGACAATCACT-3′ 
Reverse: 5′-TGCAAGGAAAGCATTGAACAA-3′ 
β-actin Forward: 5′-GGCGGCACCACCATGTACCC-3′ 
Reverse: 5′-AGGGGCCGGACTCGTCATACT-3′ 
Rnu6 Forward: 5′-GTGCTCGCTTCGGCAGCA-3′ 
Reverse: 5′-AATATGGAACGCTTCACGAAT-3′ 
Gapdh Forward: 5′-GGAGCGAGATCCCTCCAAAAT-3′ 
Reverse: 5′-GGCTGTTGTCATACTTCTCATGG-3′ 
IFN-β Forward: 5′-AGGACAGGATGAACTTTGAC-3′ 
Reverse: 5′-TGATAGACATTAGCCAGGAG-3′ 
ISG15 Forward: 5′-CGCAGATCACCCAGAAGATCG-3′ 
Reverse: 5′-TTCGTCGCATTTGTCCACCA-3′ 
TRAF1 Forward: 5′-ACACTCCACCGGAAGCTC-3′ 
Reverse: 5′-TGTCTGGGTGACCTCAAGGA-3′ 
TRAF3 Forward: 5′-CTCACAAGTGCAGCGTCCAG-3′ 
Reverse: 5′-GCTCCACTCCTTCAGCAGGTT-3′ 
TRAF6 Forward: 5′-GCGCACTAGAACGAGCAAG-3′ 
Reverse: 5′-GGCAGTTCCACCCACACTAT-3′ 
TBK1 Forward: 5′-CGGAGACCCGGCTGGTATAA-3′ 
Reverse: 5′-ATCCACTGGACGAAGGAAGC-3′ 
MAVS Forward: 5′-AAGAGACCAGGGACCTCGGA-3′ 
Reverse: 5′-ACAGGCATGGGGTAACTTGG-3′ 
NF-κB Forward: 5′-GCCAACAGATGGCCCATACC-3′ 
Reverse: 5′-TGCTGGTCCCACATAGTTGC-3′ 
GenesSequence of Primer(5′-3′)
DDX39A Forward: 5′-GCAGATTGAGCCTGTCAACG-3′ 
Reverse: 5′-AGACCACCGAAGAACACAGAC-3′ 
RanBP2 Forward: 5′-AAGCCAGTGCTACCAAATGTATTG-3′ 
Reverse: 5′-AATTCTGTATTAGAAGAACCCTGAAAC-3′ 
EMCV Forward: 5′-TGCAGTGGTTGCTCCCCTGA-3′ 
Reverse: 5′-TGACCGGAATGGGCGACTGT-3′ 
SeV Forward: 5′-GCTGCCGACAAGGTGAGAGC-3′ 
Reverse: 5′-GCCCGCCATGCCTCTCTCTA-3′ 
VSV Forward: 5′-GAGGAGTCACCTGGACAATCACT-3′ 
Reverse: 5′-TGCAAGGAAAGCATTGAACAA-3′ 
β-actin Forward: 5′-GGCGGCACCACCATGTACCC-3′ 
Reverse: 5′-AGGGGCCGGACTCGTCATACT-3′ 
Rnu6 Forward: 5′-GTGCTCGCTTCGGCAGCA-3′ 
Reverse: 5′-AATATGGAACGCTTCACGAAT-3′ 
Gapdh Forward: 5′-GGAGCGAGATCCCTCCAAAAT-3′ 
Reverse: 5′-GGCTGTTGTCATACTTCTCATGG-3′ 
IFN-β Forward: 5′-AGGACAGGATGAACTTTGAC-3′ 
Reverse: 5′-TGATAGACATTAGCCAGGAG-3′ 
ISG15 Forward: 5′-CGCAGATCACCCAGAAGATCG-3′ 
Reverse: 5′-TTCGTCGCATTTGTCCACCA-3′ 
TRAF1 Forward: 5′-ACACTCCACCGGAAGCTC-3′ 
Reverse: 5′-TGTCTGGGTGACCTCAAGGA-3′ 
TRAF3 Forward: 5′-CTCACAAGTGCAGCGTCCAG-3′ 
Reverse: 5′-GCTCCACTCCTTCAGCAGGTT-3′ 
TRAF6 Forward: 5′-GCGCACTAGAACGAGCAAG-3′ 
Reverse: 5′-GGCAGTTCCACCCACACTAT-3′ 
TBK1 Forward: 5′-CGGAGACCCGGCTGGTATAA-3′ 
Reverse: 5′-ATCCACTGGACGAAGGAAGC-3′ 
MAVS Forward: 5′-AAGAGACCAGGGACCTCGGA-3′ 
Reverse: 5′-ACAGGCATGGGGTAACTTGG-3′ 
NF-κB Forward: 5′-GCCAACAGATGGCCCATACC-3′ 
Reverse: 5′-TGCTGGTCCCACATAGTTGC-3′ 
Table IV.
Primers used in the small RNA interfering assay
Primer NamePrimer Sequence (5′-′′)
Negative control Forward: 5′-UUCUCCGAACGUGUCACGUTT-3′ 
Reverse: 5′-ACGUGACACGUUCGGAGAATT-3′ 
siDDX39A-1 Forward: 5′-GCAGCAGUACUACGUCAAATT-3′ 
Reverse: 5′-UUUGACGUAGUACUGCUGCTT-3′ 
siDDX39A-2 Forward: 5′-GCACCAAAGGCCUAGCCAUTT-3′ 
Reverse: 5′-AUGGCUAGGCCUUUGGUGCTT-3′ 
siRanBP2-1 Forward: 5′-GCGCGAAAUUGUUUCGUUUTT-3′ 
Reverse: 5′-AAACGAAACAAUUUCGCGCTT-3′ 
siRanBP2-2 Forward: 5′-CCUCUAAACCAACUCAUAATT-3′ 
Reverse: 5′-UUAUGAGUUGGUUUAGAGGTT-3′ 
Primer NamePrimer Sequence (5′-′′)
Negative control Forward: 5′-UUCUCCGAACGUGUCACGUTT-3′ 
Reverse: 5′-ACGUGACACGUUCGGAGAATT-3′ 
siDDX39A-1 Forward: 5′-GCAGCAGUACUACGUCAAATT-3′ 
Reverse: 5′-UUUGACGUAGUACUGCUGCTT-3′ 
siDDX39A-2 Forward: 5′-GCACCAAAGGCCUAGCCAUTT-3′ 
Reverse: 5′-AUGGCUAGGCCUUUGGUGCTT-3′ 
siRanBP2-1 Forward: 5′-GCGCGAAAUUGUUUCGUUUTT-3′ 
Reverse: 5′-AAACGAAACAAUUUCGCGCTT-3′ 
siRanBP2-2 Forward: 5′-CCUCUAAACCAACUCAUAATT-3′ 
Reverse: 5′-UUAUGAGUUGGUUUAGAGGTT-3′ 

The host innate immune response, predominantly type I IFN (IFN-α and IFN-β), serves as the first line of defense and plays a role in the nonspecific immune system (30, 31). Next, the function of DDX39A in the IFN signaling pathway induced by RNA virus (VSV, SeV, EMCV) was examined. The results demonstrated that the overexpression of DDX39A significantly inhibited the production of IFN-β during viral infection (Fig. 2A, Supplemental Fig. 3B). When the expression of DDX39A was dramatically silenced by siRNA, the mRNA level of IFN-β was increased (Fig. 2B, 2C). The DDX39A gene knockout (KO) HEK293T cell lines were then constructed (Supplemental Fig. 3A). Depletion of DDX39A significantly promoted VSV-induced IFN-β (Fig. 2D, Supplemental Fig. 3C) and IGS15 production (Fig. 2E) compared with wild-type (WT) controls. Furthermore, overexpression of DDX39A in DDX39A-deficient cells inhibited VSV-induced IFN-β and Interferon-stimulated gene (ISG)15 expression (Fig. 2F). IFN expression is tightly regulated by p-IRF3 and NF-κB/p65 nuclear translocation. Our study found DDX39A KO promoted VSV-induced expression of p-IRF3 (Fig. 2G). We then examined the p65 nuclear and cytoplasmic levels by Western blot analysis after fractionation experiments. P65 in the nucleus was significantly reduced, but whole cell extracts were not significantly changed when DDX39A was overexpressed in VSV-infected cells (Fig. 2H). The luciferase reporter system further confirmed that DDX39A can inhibit RIG-I– and MAVS-activated IFN-β promoter activity but did not inhibit TBK1 and IRF3-induced IFN-β activation (Supplemental Fig. 3D–K). Collectively, these results suggested DDX39A was an antagonist of IFN and negatively regulated type I IFN production.

FIGURE 2.

DDX39A is an antagonist of IFN. (A) HEK293T cells were transfected with DDX39A and vector expression plasmid for 24 h, followed by infection with SeV, EMCV, or VSV for 12 h or left uninfected. The IFN-β mRNA was analyzed by qRT-PCR. (B) Negative control (NC) and siRNA1 (siDDX39A-1) or siRNA2 (siDDX39A-2) targeting DDX39A were transfected into HEK293T cells for 24 h. Detection of siRNA interference by DDX39A polyclonal immunoblotting and qRT-PCR. (C) qRT-PCR analysis of IFN-β mRNA in SeV-infected HEK293T cells after transfection of siRNA1 or siRNA2 for 24 h. (D, E, and G) DDX39A WT and KO cells were infected with VSV for the times indicated. The IFN-β (D) and ISG15 (E) mRNA level were detected by qRT-PCR, and IRF3 and p-IRF3 were detected by Western blotting (G). (F) DDX39A WT and KO cells were transfected with vector or/and Flag-DDX39A plasmid for 24 h and then infected with VSV. The IFN-β and ISG15 mRNA levels were analyzed by qRT-PCR at 12 h postinfection. (H) HEK293T cells were transfected with Flag-DDX39A or empty vector for 24 h and followed by infection with VSV 12 h later.Total cell lysate and nuclear and cytoplasmic fractions were then analyzed by Western blotting with anti-p65, –β-actin, -HIST3H3, and anti-Flag Abs. HIST3H3 and β-actin were used as internal controls for nuclear protein and cytoplasmic protein loading, respectively. Data are representative of three independent experiments. Data are mean ± SEM of triplicate samples in (A)–(F). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, no significance.

FIGURE 2.

DDX39A is an antagonist of IFN. (A) HEK293T cells were transfected with DDX39A and vector expression plasmid for 24 h, followed by infection with SeV, EMCV, or VSV for 12 h or left uninfected. The IFN-β mRNA was analyzed by qRT-PCR. (B) Negative control (NC) and siRNA1 (siDDX39A-1) or siRNA2 (siDDX39A-2) targeting DDX39A were transfected into HEK293T cells for 24 h. Detection of siRNA interference by DDX39A polyclonal immunoblotting and qRT-PCR. (C) qRT-PCR analysis of IFN-β mRNA in SeV-infected HEK293T cells after transfection of siRNA1 or siRNA2 for 24 h. (D, E, and G) DDX39A WT and KO cells were infected with VSV for the times indicated. The IFN-β (D) and ISG15 (E) mRNA level were detected by qRT-PCR, and IRF3 and p-IRF3 were detected by Western blotting (G). (F) DDX39A WT and KO cells were transfected with vector or/and Flag-DDX39A plasmid for 24 h and then infected with VSV. The IFN-β and ISG15 mRNA levels were analyzed by qRT-PCR at 12 h postinfection. (H) HEK293T cells were transfected with Flag-DDX39A or empty vector for 24 h and followed by infection with VSV 12 h later.Total cell lysate and nuclear and cytoplasmic fractions were then analyzed by Western blotting with anti-p65, –β-actin, -HIST3H3, and anti-Flag Abs. HIST3H3 and β-actin were used as internal controls for nuclear protein and cytoplasmic protein loading, respectively. Data are representative of three independent experiments. Data are mean ± SEM of triplicate samples in (A)–(F). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, no significance.

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Depletion of DDX39A in HEK293T cells promotes IFN-β production; however, the molecular mechanism whereby DDX39A regulates IFN-β production remains unclear. Compared with the control, overexpression of DDX39A reduced TRAF3, TRAF6, and MAVS expression in HEK293T cells (Fig. 3A), whereas VSV infection further inhibited the expression of these transcription factors (Fig. 3B). Consistently, knockdown of DDX39A markedly promoted the expression of these transcription factors (Fig. 3C). Moreover, DDX39A was overexpressed in KO cells and restored the inhibitory effects on the expression of these transcription factors, and VSV infection further enhanced the effect (Fig. 3D). Fractionation experiment results showed that the total mRNA levels of these transcription factors were not influenced by DDX39A (Fig. 3E, 3F). However, the nuclear abundance of TRAF3, TRAF6, and MAVS transcripts were higher in HEK293T cells overexpressing DDX39A (Fig. 3E) and were reduced after knockdown of endogenous DDX39A in HEK293T cells (Fig. 3F). Taken together, our data demonstrated that DDX39A increased the abundance of these antiviral transcripts in the nucleus, thereby inhibiting the protein expression of antiviral molecules TRAF3, TRAF6, and MAVS.

FIGURE 3.

Expression of antiviral transcripts and distribution of nuclei in the presence of DDX39A. (A) Western blot analysis of TRAF3, TRAF6, MAVS, and TBK1 in HEK293T cells transfected with vector or various doses of Flag-DDX39A plasmid (1.0 or 2.0 μg/ml). (B) Western blot analysis of TRAF3, TRAF6, MAVS, and TBK1 in HEK293T cells transfected with Flag-DDX39A or vector upon or without VSV infection for 12 h. (C) Western blot analysis of TRAF3, TRAF6, MAVS, and TBK1 in DDX39A WT and KO cells infected with VSV at indicated time. (D) Western blot analysis of TRAF3, TRAF6, MAVS, and TBK1 in DDX39A KO cells transfected vector or Flag-DDX39A with or without VSV infection for 12 h. (E and F) RT-PCR analysis of the nuclear and cytoplasmic fractions of TRAF3, TRAF6, and MAVS in HEK293T cells transfected with Flag-DDX39A (E) or siDDX39A (F). The loading of cytoplasmic and nuclei mRNA was confirmed by Gapdh and Rnu6, respectively. Data are representative of three independent experiments.

FIGURE 3.

Expression of antiviral transcripts and distribution of nuclei in the presence of DDX39A. (A) Western blot analysis of TRAF3, TRAF6, MAVS, and TBK1 in HEK293T cells transfected with vector or various doses of Flag-DDX39A plasmid (1.0 or 2.0 μg/ml). (B) Western blot analysis of TRAF3, TRAF6, MAVS, and TBK1 in HEK293T cells transfected with Flag-DDX39A or vector upon or without VSV infection for 12 h. (C) Western blot analysis of TRAF3, TRAF6, MAVS, and TBK1 in DDX39A WT and KO cells infected with VSV at indicated time. (D) Western blot analysis of TRAF3, TRAF6, MAVS, and TBK1 in DDX39A KO cells transfected vector or Flag-DDX39A with or without VSV infection for 12 h. (E and F) RT-PCR analysis of the nuclear and cytoplasmic fractions of TRAF3, TRAF6, and MAVS in HEK293T cells transfected with Flag-DDX39A (E) or siDDX39A (F). The loading of cytoplasmic and nuclei mRNA was confirmed by Gapdh and Rnu6, respectively. Data are representative of three independent experiments.

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Because of the different roles of host RNA-binding proteins in RNA virus replication, we investigated the effect of DDX39A on RNA virus proliferation. The mRNA levels of SeV, EMCV, and VSV and the VSV virus titer were significantly higher in cells overexpressing DDX39A (Fig. 4A–D). The VSVΔM51-GFP was used to further substantiate the presence of biologically active IFN. As shown in Fig. 4E, DDX39A promoted VSV proliferation. Consistently, knockdown of DDX39A reduced the SeV, EMCV, and VSV virus replication (Fig. 4F–H). Furthermore, the VSV virus titer exhibited a downward trend in DDX39A siRNA-transfected cells (Fig. 4I). In line with that, the proliferation of RNA viruses was significantly inhibited in DDX39A KO cells (Fig. 4J–L). Taken together, these data provided more evidence that DDX39A promoted proliferation of multiple RNA viruses.

FIGURE 4.

DDX39A contributes to RNA virus proliferation. (AC) qRT-PCR analysis of SeV, EMCV, and VSV mRNA in HEK293T cells transfected with Flag-DDX39A or vector upon viral infection at indicated time. (D and I) VSV was inoculated after transfection with Flag-DDX39A (D) or siDDX39A-1 (I) and then the virus load was detected by TCID50. (E) HEK293T were transfected with Flag-DDX39A and then infected with 0.1 MOI VSVΔM51-GFP for 12 h; immunofluorescence microscopy imaging detected the proliferation of VSV. Original magnification ×100. (F and G) Analysis of virus levels by qRT-PCR detection of SeV (F) or EMCV (G) or VSV (H) mRNA in HEK293T cells transfected with siDDX39A-1 or siNC. (JL) qRT-PCR analysis of SeV, EMCV, and VSV mRNA in DDX39A WT and KO HEK293T cells infected with different RNA virus at indicated time. Data are from three independent experiments [(A–D) and (F–L), mean ± SEM] or one representative experiment of three independent experiments with similar results (D and L). *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 4.

DDX39A contributes to RNA virus proliferation. (AC) qRT-PCR analysis of SeV, EMCV, and VSV mRNA in HEK293T cells transfected with Flag-DDX39A or vector upon viral infection at indicated time. (D and I) VSV was inoculated after transfection with Flag-DDX39A (D) or siDDX39A-1 (I) and then the virus load was detected by TCID50. (E) HEK293T were transfected with Flag-DDX39A and then infected with 0.1 MOI VSVΔM51-GFP for 12 h; immunofluorescence microscopy imaging detected the proliferation of VSV. Original magnification ×100. (F and G) Analysis of virus levels by qRT-PCR detection of SeV (F) or EMCV (G) or VSV (H) mRNA in HEK293T cells transfected with siDDX39A-1 or siNC. (JL) qRT-PCR analysis of SeV, EMCV, and VSV mRNA in DDX39A WT and KO HEK293T cells infected with different RNA virus at indicated time. Data are from three independent experiments [(A–D) and (F–L), mean ± SEM] or one representative experiment of three independent experiments with similar results (D and L). *p < 0.05, **p < 0.01, ***p < 0.001.

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The virus enhances RNA-binding activity of DDX39A, which may be associated with viral effects on protein expression or posttranslational modification. However, the results showed that DDX39A mRNA and protein levels did not change in SeV and VSV infection (Fig. 5A–C). SUMOylation is a common posttranslational modification that has been confirmed in some RNA-binding proteins (3234). DDX39A expression is detected by Western blotting using anti-Flag Ab or SUMO1 or SUMO2/3. A prominent band of SUMO-modified DDX39A appeared in the Western blot assay. We also found that the upper band belonged to SUMOylated DDX39A. Coimmunoprecipitation analysis revealed DDX39A was a target for modification by SUMO1 or SUMO2/3. Excitingly, compared with the control, SUMO1 modification of DDX39A was reduced in VSV-infected HEK293T cells (Fig. 5D). VSV infection reduced the SUMO1 modification of DDX39A but not SUMO2 or SUMO3 when Myc-SUMO (SUMO1, SUMO2, SUMO3) and Flag-DDX39A were cotransfected in HEK293T cells (Fig. 5E). Immunofluorescence of both Myc-SUMO (SUMO1, SUMO2, SUMO3) and Flag-DDX39A showed that DDX39A and SUMO colocalized in the nucleus (Fig. 5F). Collectively, these results suggest DDX39A was posttranslationally modified by SUMOylation. Viral infection downregulated SUMO1 modification of DDX39A protein.

FIGURE 5.

Effect of viral infection on posttranslational SUMO modification of DDX39A. (AC) HEK293T cells were inoculated without or with VSV or SeV at the indicated times. The mRNA and protein levels of DDX39A were analyzed by qRT-PCR (A and B) and Western blot (C), respectively. (D) HEK293T cells were transfected with Flag-DDX39A, then inoculated with or without VSV. Immunoprecipitation and immunoblot analysis of DDX39A SUMO modification using SUMO1 Ab or SUMO2/3 Ab. (E) Immunoprecipitation and immunoblot analysis of DDX39A SUMO modification in HEK293T cells cotransfected with Flag-DDX39A and Myc-SUMO (SUMO1, SUMO2, or SUMO3) upon VSV infection for 12 h. (F) Colocalization of Flag-DDX39A with Myc-SUMO (SUMO1 or SUMO2 or SUMO3) transfected in HeLa cells. Nuclei were stained with DAPI (blue). Scale bar, 7 μm. Data are from three independent experiments [(A and B), mean ± SEM] or one representative experiment of three independent experiments with similar results.

FIGURE 5.

Effect of viral infection on posttranslational SUMO modification of DDX39A. (AC) HEK293T cells were inoculated without or with VSV or SeV at the indicated times. The mRNA and protein levels of DDX39A were analyzed by qRT-PCR (A and B) and Western blot (C), respectively. (D) HEK293T cells were transfected with Flag-DDX39A, then inoculated with or without VSV. Immunoprecipitation and immunoblot analysis of DDX39A SUMO modification using SUMO1 Ab or SUMO2/3 Ab. (E) Immunoprecipitation and immunoblot analysis of DDX39A SUMO modification in HEK293T cells cotransfected with Flag-DDX39A and Myc-SUMO (SUMO1, SUMO2, or SUMO3) upon VSV infection for 12 h. (F) Colocalization of Flag-DDX39A with Myc-SUMO (SUMO1 or SUMO2 or SUMO3) transfected in HeLa cells. Nuclei were stained with DAPI (blue). Scale bar, 7 μm. Data are from three independent experiments [(A and B), mean ± SEM] or one representative experiment of three independent experiments with similar results.

Close modal

Our RIP studies revealed that the DDX39A protein binds to multiple innate immune–associated RNAs, raising the possibility that SUMO1 modification may modulate the RNA-binding activity of DDX39A. SUMO1-modified DDX39A levels increased when SUMO1 and DDX39A were coexpressed in HEK293T cells (Fig. 6A). Furthermore, three SUMOylation sites of DDX39A (K52, K154, K155) were predicted using the SUMOylation site prediction program (35). Mutation of lysine 52, 154, and 155 to arginine suppressed SUMOylation of DDX39A to a certain extent (Fig. 6B). We found that coexpression of a SUMO1 inhibited the ability of DDX39A to bind the specified mRNAs (TRAF3, TRAF6, MAVS) but did not inhibit the binding of TBK1 (Fig. 6C–F). To test whether SUMO1 modification affected the binding of DDX39A to mRNAs in cells, Flag-tagged DDX39A wild or mutant expression vectors were transiently transfected into HEK293T cells to perform RIP. Compared with WT DDX39A, SUMOylation-incompetent DDX39AK52/154/155R enhanced the binding of TRAF3, TRAF6, and MAVS, but did not enhance the binding of TBK1 (Fig. 6G–J). Our results opened up the possibility that SUMO1 modification of DDX39A protein affected its ability to bind these antiviral RNA transcripts.

FIGURE 6.

Posttranslational modification of DDX39A protein affects its ability to bind RNA. (A and B) Immunoprecipitation and immunoblot analysis of DDX39A SUMO modification in HEK293T cells transfected with Flag-DDX39A (WT) (A) or Flag-DDX39A mutant (B) together with Myc-SUMO1(3.0 or 6.0 μg). (CF) RIP–qRT-PCR analysis of TRAF3, TRAF6, MAVS, and TBK1 in HEK293T cells transfected with Flag-DDX39A WT together with Myc-SUMO1. (GJ) RIP–qRT-PCR analysis of TRAF3, TRAF6, MAVS, and TBK1 in HEK293T cells transfected with Flag-DDX39A WT or mutant. Data are from three independent experiments (mean ± SEM) or one representative experiment of three independent experiments with similar results. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

FIGURE 6.

Posttranslational modification of DDX39A protein affects its ability to bind RNA. (A and B) Immunoprecipitation and immunoblot analysis of DDX39A SUMO modification in HEK293T cells transfected with Flag-DDX39A (WT) (A) or Flag-DDX39A mutant (B) together with Myc-SUMO1(3.0 or 6.0 μg). (CF) RIP–qRT-PCR analysis of TRAF3, TRAF6, MAVS, and TBK1 in HEK293T cells transfected with Flag-DDX39A WT together with Myc-SUMO1. (GJ) RIP–qRT-PCR analysis of TRAF3, TRAF6, MAVS, and TBK1 in HEK293T cells transfected with Flag-DDX39A WT or mutant. Data are from three independent experiments (mean ± SEM) or one representative experiment of three independent experiments with similar results. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Close modal

To investigate the molecular mechanisms of regulation of posttranslational modification of DDX39A protein, Flag-tagged DDX39A was immunoprecipitated, and the associated proteins were analyzed using mass spectrometry. E3 SUMO ligase RanBP2 peptide was identified in the Flag-DDX39A–immunoprecipitated complex (Fig. 7A). The interactions between DDX39A and endogenous RanBP2 were then confirmed by coimmunoprecipitation (Fig. 7B). Analysis showed that DDX39A consisted of a Q motif, DEADc domain, and HELICc region (Fig. 7C). The immunoprecipitation results indicated the key to the interaction between DDX39A and RanBP2 was the DEADc domain (Fig. 7D). Subsequently, we mapped the domains of RanBP2 and constructed the corresponding truncated segments (Fig. 7E). In immunoprecipitation assays, RanBP2-D1 and RanBP2-D5 both bound to DDX39A, indicating DDX39A interacts with RanBP2 via its N-terminal region or C-terminal region (Fig. 7F). An indirect immunofluorescence assay revealed that DDX39A and RanBP2-D1 or -D5 but not RanBP2-D6 were colocalized in HeLa cells (Fig. 7G). RanBP2 has SUMO1 E3 ligase activity, which strongly enhances SUMO1 transfer from Ubc9 to the SUMO1 target protein (36, 37). The colocalization of endogenous RanBP2, Myc-SUMO1, and GFP-DDX39A was confirmed by immunofluorescence (Fig. 7H). A fragment of RanBP2 (RanBP2ΔFG) (aa 2553–2838) previously shown to contain E3 ligase activity was sufficient to induce SUMO1 modification (36, 38, 39). RanBP2-D5 overlapped with its E3 ligase domain and also includes the RanBP2ΔFG fragment. In cell SUMO assays, SUMO1-modified DDX39A levels were significantly enhanced when overexpressing RanBP2-D5 (Fig. 7I). In line with that, SUMO1 modification of DDX39A was found to be reduced with downregulation of endogenous RanBP2 expression (Fig. 7J). However, we found that RanBP2-D5 also increased the SUMOylation of the DDX39A mutants to some extent, indicating that there were still undiscovered SUMO sites in DDX39A (Fig. 7K). Collectively, these data reflected that RanBP2, a SUMO1 E3 ligase associated with DDX39A, enhanced the SUMO1 modification of DDX39A.

FIGURE 7.

RanBP2 interacts with DDX39A and as its E3 sumo ligase. (A) HEK293T cells were transfected with Flag-tagged DDX39A plasmids for 24 h, cells were lysed, and cellular proteins were immunopurified with anti-FLAG affinity beads. The DDX39A-associated complex was resolved on SDS-PAGE and silver-stained, followed by mass spectrometry analysis. (B) Detection of the interaction between endogenous RanBP2 and Flag-DDX39A by coimmunoprecipitation. (C) Schematic of DDX39A domains. (D) The interaction between endogenous RanBP2 and truncated DDX39A was detected by coimmunoprecipitation. (E) Mapping of RanBP2 (WT) and internal deletion mutants. (F) Coimmunoprecipitation analysis of the interaction of DDX39A and truncated RanBP2. (G) Immunofluorescence was used to detect the interaction between GFP-DDX39A and Flag-RanBP2 (D1 or D5 or D6) in HeLa cells. (H) Immunofluorescence analysis of GFP-DDX39A and Myc-SUMO1 and endogenous RanBP2 in HeLa cells. Scale bars, 7 μm. (I and J) HEK293T cells were cotransfected with Myc-SUMO1 and HA-DDX39A, followed with Flag-RanBP2 (I) or siRanBP2 (J). Flag-DDX39A was immunoprecipitated, and SUMOylated DDX39A was detected by Western blotting using anti-Myc Ab. (K) HEK293T cells were cotransfected with HA-RanBP2 D5 and different Flag-DDX39A mutants; SUMOylated DDX39A mutants in the immunoprecipitation were detected by immunoblotting with SUMO1.

FIGURE 7.

RanBP2 interacts with DDX39A and as its E3 sumo ligase. (A) HEK293T cells were transfected with Flag-tagged DDX39A plasmids for 24 h, cells were lysed, and cellular proteins were immunopurified with anti-FLAG affinity beads. The DDX39A-associated complex was resolved on SDS-PAGE and silver-stained, followed by mass spectrometry analysis. (B) Detection of the interaction between endogenous RanBP2 and Flag-DDX39A by coimmunoprecipitation. (C) Schematic of DDX39A domains. (D) The interaction between endogenous RanBP2 and truncated DDX39A was detected by coimmunoprecipitation. (E) Mapping of RanBP2 (WT) and internal deletion mutants. (F) Coimmunoprecipitation analysis of the interaction of DDX39A and truncated RanBP2. (G) Immunofluorescence was used to detect the interaction between GFP-DDX39A and Flag-RanBP2 (D1 or D5 or D6) in HeLa cells. (H) Immunofluorescence analysis of GFP-DDX39A and Myc-SUMO1 and endogenous RanBP2 in HeLa cells. Scale bars, 7 μm. (I and J) HEK293T cells were cotransfected with Myc-SUMO1 and HA-DDX39A, followed with Flag-RanBP2 (I) or siRanBP2 (J). Flag-DDX39A was immunoprecipitated, and SUMOylated DDX39A was detected by Western blotting using anti-Myc Ab. (K) HEK293T cells were cotransfected with HA-RanBP2 D5 and different Flag-DDX39A mutants; SUMOylated DDX39A mutants in the immunoprecipitation were detected by immunoblotting with SUMO1.

Close modal

Next, the dynamic expression of RanBP2 in virus-infected or mock HEK293T cells was detected by qRT-PCR. The results showed that the mRNA levels of RanBP2 in the virus-infected HEK293T cells were significantly downregulated (Fig. 8A, 8B). Further Western blot analysis revealed that RNA virus infection downregulated RanBP2 but had no effect on DDX39A (Fig. 8C, 8D). Previous experiments have verified viral infection affects SUMO1 modification of DDX39A protein, resulting in a change in the ability to bind RNA. RanBP2 has been identified as a SUMO1 E3 ligase that specifically targets DDX39A in our study. We hypothesized that RanBP2-mediated DDX39A SUMOylation affected the ability of DDX39A to bind RNA. To further verify our hypothesis, the role of RanBP2 on the binding ability of DDX39A to RNA was investigated. The results showed DDX39A enhanced the ability to bind TRAF3, TRAF6, and MAVS in RanBP2 knockdown HEK293T cells (Fig. 8E–G). RanBP2-D5 effectively reduced the binding of antiviral transcripts toward DDX39A (Fig. 8H–J). RanBP2-D5 inhibits nuclear abundance of TRAF3, TRAF6, and MAVS transcripts in HEK293T cells overexpressing DDX39A (Fig. 8K). In addition, overexpression of RanBP2-D5 enhanced N-terminal CARD-like domains of RIG-I–mediated IFN induction and reversed the inhibitory effect of DDX39A (Fig. 8L). Collectively, these data reflected RanBP2 plays an important role in regulating DDX39A binding to TRAF3, TRAF6, and MAVS (Fig. 9).

FIGURE 8.

Effect of RanBP2 on the ability of DDX39A to bind RNA. (A and B) qRT-PCR analysis of RanBP2 in HEK293T cells inoculated without or with VSV or SeV at the indicated times. (C and D) Immunoblot analysis of RanBP2 and DDX39A expression in HEK293T cells inoculated without or with SeV (C) or VSV (D) at the indicated times. (EG) RIP–qRT-PCR analysis of TRAF3, TRAF6, and MAVS in HEK293T cells overexpressing Flag-DDX39A together with siRanBP2 or siNC. (HJ) RIP–qRT-PCR analysis of TRAF3, TRAF6, and MAVS in HEK293T cells overexpressing Flag-DDX39A together with HA-RanBP2-D5. (K) RT-PCR analysis of the nuclear and cytoplasmic fractions of TRAF3, TRAF6, and MAVS in HEK293T cells cotransfected with HA-DDX39A and Flag-RanBP2-D5. The loading of cytoplasmic and nuclei mRNA was confirmed by Gapdh and Rnu6, respectively. (L) IFN-β promoter activity was analyzed by luciferase assays in HEK293T cells cotransfected with Myc-DDX39A and Flag-RIGI-N, together with IFN-β reporter as well as HA-RanBP2-D5. Data are from three independent experiments [(A and B) and (E–J), mean ± SEM) or one representative experiment of three independent experiments with similar results. *p < 0.05, **p < 0.01, ***p < 0.001.

FIGURE 8.

Effect of RanBP2 on the ability of DDX39A to bind RNA. (A and B) qRT-PCR analysis of RanBP2 in HEK293T cells inoculated without or with VSV or SeV at the indicated times. (C and D) Immunoblot analysis of RanBP2 and DDX39A expression in HEK293T cells inoculated without or with SeV (C) or VSV (D) at the indicated times. (EG) RIP–qRT-PCR analysis of TRAF3, TRAF6, and MAVS in HEK293T cells overexpressing Flag-DDX39A together with siRanBP2 or siNC. (HJ) RIP–qRT-PCR analysis of TRAF3, TRAF6, and MAVS in HEK293T cells overexpressing Flag-DDX39A together with HA-RanBP2-D5. (K) RT-PCR analysis of the nuclear and cytoplasmic fractions of TRAF3, TRAF6, and MAVS in HEK293T cells cotransfected with HA-DDX39A and Flag-RanBP2-D5. The loading of cytoplasmic and nuclei mRNA was confirmed by Gapdh and Rnu6, respectively. (L) IFN-β promoter activity was analyzed by luciferase assays in HEK293T cells cotransfected with Myc-DDX39A and Flag-RIGI-N, together with IFN-β reporter as well as HA-RanBP2-D5. Data are from three independent experiments [(A and B) and (E–J), mean ± SEM) or one representative experiment of three independent experiments with similar results. *p < 0.05, **p < 0.01, ***p < 0.001.

Close modal
FIGURE 9.

Proposed model for the regulation of SUMOylation of DDX39A by the virus affects the binding ability to RNA and regulates type I IFN.

FIGURE 9.

Proposed model for the regulation of SUMOylation of DDX39A by the virus affects the binding ability to RNA and regulates type I IFN.

Close modal

DDX39A is a conserved helicase involved in alteration of RNA secondary structure (40), most notably for its role in regulation of RNA processing events, especially splicing, and mRNA export (41, 42). In our study, DDX39A was identified as a SUMO substrate, and its binding to RNA activity was regulated by the SUMO mechanism. SUMO, as a posttranslational modifier, plays a crucial regulatory role in a number of cellular processes, including regulating mRNA export, RNA editing, and RNA binding (43, 44). It has been identified that a significant proportion (17%) of SUMO-modified proteins are involved in RNA-related processes in yeast (45). SUMOylation of ADAR1 in vitro alters its activity and function and resulted in inhibition of RNA-editing activity (46). K237 of the hnRNP C protein was modified by SUMO, and SUMOylation inhibited the RNA-binding ability of hnRNP C (34). Moreover, the modification of La by SUMO plays a role in regulating RNA binding and the conformational changes in La more favorable for RNA binding (47).

Three SUMOylation sites of DDX39A (K52, K154, K155) were predicted according to one consensus motif (ψKxD/E) and two nonconsensus SUMO motifs for SUMOylation at DDX39A. Three predicted SUMOylation sites were mutated in DDX39A, and the effect on SUMO1 modification was analyzed. Interestingly, SUMO1 modification of DDX39A was significantly inhibited but did not completely disappear after simultaneous mutagenesis at the three SUMOylation sites, suggesting that there are undiscovered SUMOylation sites in DDX39A. In addition, the regulation of SUMO1 modification alters the interface or spatial structure of the DDX39A protein, which may result in the blockage of DDX39A binding to RNA, thereby displaying a significantly lower affinity of DDX39A for TRAF3, TRAF6, and MAVS. However, we cannot at this time exclude the possibility that these antiviral transcripts could be binding to DDX39A via some other components of the complex. Our finding raised the possibility that RNA-binding activity of DDX39A may be spatially controlled through its posttranslational modification.

A previous study showed DDX family controls RNA metabolism to regulate the antiviral innate immune response. DDX46 recruits ALKBH5 to erase m6A modification on DDX46-bound antiviral transcripts MAVS, TRAF3, and TRAF6 to entrap these antiviral transcripts in the nucleus to inhibit IFN-β production. DDX46 bound MAVS, TRAF3, and TRAF6 transcripts via their conserved CCGGUU element (48). These are the same mRNAs that this study shows preferentially bind to DDX39A. However, the relative contributions of these two DDX family members to regulating the innate response and how DDX39A selectively binds to mRNA from innate immune molecules needed further clarification.

DDX39A was shown to be a target for SUMOylation in our study. DDX39A has SUMO1, 2, and 3 modifications. However, only the SUMO1 modifications decreased in VSV-infected cells. RanBP2 acted as an E3 SUMO1 ligase of DDX39A. Virus infection decreased RanBP2 levels, resulting in a reduction in DDX39A SUMO1 modification. Attenuation of SUMO1 modification enhanced the ability of DDX39A to bind to antiviral transcripts (TRAF3, TRAF6, MAVS). However, whether SUMO1 modification can affect the ability of DDX39A to bind to other targeted mRNAs requires further exploration. Based on our findings, our model for the regulation of SUMO1 modification of DDX39A, which impacted the binding capacity and nuclear export to RNA and regulates type I IFN, is presented in Fig. 9. We verified the difference in RNA-binding capacity of DDX39A under SUMOylation. Attenuation of SUMO1 modification enhanced the ability of DDX39A to bind to antiviral transcripts (TRAF3, TRAF6, MAVS), leading to increased retention of these antiviral transcripts in the nucleus, thereby restricting the protein expressions and preventing downstream signaling in the antiviral innate response. Viral infection downregulated the abundance of RanBP2, resulting in reduced SUMO1 modification of DDX39A. Our study has demonstrated that the mechanism exploited by DDX39A for inhibiting type I IFN production to regulate innate immunity is its preferential binding to antiviral transcripts and its impact on mRNA export, which is additionally regulated by SUMO1-mediated modification of DDX39A in viral infection.

This work was supported by the National Key Research and Development Program of China (2018YFD0500500) and the National Natural Science Foundation of China (31272540).

The online version of this article contains supplemental material.

Abbreviations used in this article:

CRISPR

clustered regularly interspaced short palindromic repeats

DDX

DEAD-box

DDX39A

DEAD/H box polypeptide 39A

EMCV

encephalomyocarditis virus

IRF

IFN regulatory factor

KO

knockout

MOI

multiplicity of infection

qRT-PCR

quantitative RT-PCR

RIG-I

retinoic acid–inducible gene I

RIP

RNA immunoprecipitation

RIP-seq

RIP sequencing

SeV

Senda virus

sgRNA

single guide RNA

siRNA

small interfering RNA

SUMO

small ubiquitin-related modifier

TBK1

TANK-binding kinase 1

TCID50

half maximal tissue-culture infectious dose

VSV

vesicular stomatitis virus

WT

wild-type.

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The authors have no financial conflicts of interest.

Supplementary data