DDX41 is required for cGAS-STING activation against DNA virus infection

Abstract

Upon binding double-stranded DNA (dsDNA), cyclic GMP-AMP synthase (cGAS) is activated and initiates the cGAS-stimulator of IFN genes (STING)-type I interferon pathway. DEAD-box helicase 41 (DDX41) is a DEAD-box helicase, and mutations in DDX41 cause myelodysplastic syndromes (MDSs) and acute myeloid leukemia (AML). Here, we show that DDX41-knockout (KO) cells have reduced type I interferon production after DNA virus infection. Unexpectedly, activations of cGAS and STING are affected in DDX41 KO cells, suggesting that DDX41 functions upstream of cGAS. The recombinant DDX41 protein exhibits ATP-dependent DNA-unwinding activity and ATP-independent strand-annealing activity. The MDS/AML-derived mutant R525H has reduced unwinding activity but retains normal strand-annealing activity and stimulates greater cGAS dinucleotide-synthesis activity than wild-type DDX41. Overexpression of R525H in either DDX41-deficient or -proficient cells results in higher type I interferon production. Our results have led to the hypothesis that DDX41 utilizes its unwinding and annealing activities to regulate the homeostasis of dsDNA and single-stranded DNA (ssDNA), which, in turn, regulates cGAS-STING activation.

Keywords: AML; CP: Immunology; CP: Molecular biology; DDX41; DNA virus; MDS; STING; acute myeloid leukemia; annealing; cGAS; myelodysplastic syndromes; unwinding.

Figure 1.. DDX41 knockout cells have reduced cytokine response to DNA stimulation

(A) Western blot assays of WT and DDX41 KO HeLa cell lines with the indicated antibodies. β-actin serves as a loading control. (B and C) qPCR analysis of the gene expression of IFN-β (B) and ISG56 (C) in WT and DDX41 KO HeLa cell lines after poly(dA:dT) treatment. GAPDH serves as an internal control, and time 0 was set as 1. (D and E) Western blot assays of proteins in the cGAS-STING pathway after poly(dA:dT) treatment in WT (D) and DDX41 KO (E) HeLa cell lines. (F–I) Quantification of the relative expression of cGAS (F), p-STING (G), p-TBK1 (H), and p-IRF3 (I). (J) Western blot assays of WT and DDX41 KO THP-1 cell lines with the indicated antibodies. (K) qPCR analysis of IFN-β gene expression in WT and DDX41 KO THP-1 macrophages after poly(dA:dT) treatment. (L) IFN-β protein detected by an ELISA kit (414,101, R&D Systems) in WT and DDX41 KO THP-1 macrophages after poly(dA:dT) treatment. (M) qPCR analysis of ISG56 gene expression in WT and DDX41 KO THP-1 macrophages after poly(dA:dT) treatment. (N) IRF-induced luciferase activity measured in WT and DDX41 KO THP-1 macrophages after poly(dA:dT) treatment. (O and P) Western blot assays of proteins in the cGAS-STING pathway in WT (O) and DDX41 KO (P) THP-1 macrophages after poly(dA:dT) treatment. (Q–T) Quantification of the relative expression of cGAS (Q), p-STING (R), p-TBK1 (S), and p-IRF3 (T). For (B), (C), (F)–(I), (K)–(N), and (Q)–(T): data represent the mean ± SEM of three independent experiments.

Figure 2.. DDX41 is required for the activation of cGAS and STING

(A) cGAMP production detected by an ELISA kit (Cayman Chemical) in WT and DDX41 KO THP-1 macrophages 6 h post mock or indicated DNA stimulation. Mock (no DNA) was set as 1. cGAS KO cells serve as a negative control. (B and C) cGAS oligomerization (B) and STING dimerization (C) status in WT and DDX41 KO THP-1 macrophages after mock or poly(dA:dT) stimulation (top). Denaturing condition and β-actin were used as loading controls (bottom). (D–F) Co-localization of STING with ERGIC (D), Golgi (E), and p-TBK1 (F) in WT and DDX41 KO THP-1 macrophages after mock or poly(dA:dT) stimulation (6 h). (G) Western blot assays of proteins in the cGAS-STING pathway after HSV-1 infection (MOI = 10, 6 hours post infection [h.p.i.) in BMDMs and BMDCs from the indicated mouse genotype. (H–J) cGAMP production (H), cGAS oligomerization (I), and STING dimerization (J) in BMDMs and BMDCs from the indicated mouse genotype after HSV-1 infection (MOI = 10, 6 h.p.i.). (K and L) Co-localization of STING with ERGIC (K) and Golgi (L) in BMDMs from the indicated mouse genotype after HSV-1 infection (MOI = 10, 6 h.p.i.). For (A) and (H): data represent the mean ± SEM of three independent experiments. ****p < 0.0001, ***p < 0.001, and **p < 0.01.

Figure 3.. Unwinding and annealing activities of DDX41 protein

(A) SDS-PAGE analysis of recombinant DDX41 proteins eluting from a Sephacryl S-300 HR column. DDX43 protein was loaded and used as a control. (B and C) Representative images of helicase reactions performed by incubating 0.5 nM of 3′- (B) or 5′-tailed (C) 13-bp duplex RNA substrate with increasing DDX41 protein concentration (0–300 nM) at 37°C for 15 min. NE, no enzyme; filled triangle, heat-denatured RNA substrate control; DDX43 protein (300 nM) was used as a control. (D–G) Representative images of helicase reactions performed by incubating 0.5 nM of forked (D), 5′-tailed (E), 3′-tailed (F), or blunt-ended (G) 20-bp duplex DNA substrate with increasing DDX41 protein concentration (0–300 nM) at 37°C for 15 min. (H and I) Representative images of helicase reactions performed by incubating 0.5 nM of forked 20-bp duplex DNA substrate at 37°C for 15 min with 150 nM DDX41 protein and different nucleoside-triphosphates (H) or with increasing concentration of ATP or ATP analogs (I). (J and K) Representative images of strand-annealing reactions performed by incubating 0.5 nM of two ssDNAs (one of them ³²P labeled) for 20- (J) and 30-bp (K) forked DNA substrate at room temperature for 30 min with increasing DDX41 protein (0–150 nM) with or without ATP (2 μM). NE + Com, no enzyme with complementary strand. (L) Quantitative analysis of DNA annealing by DDX41 protein in (K). Data represent the mean ± SEM of three independent experiments.

Figure 4.. DDX41 translocates from the nucleus to cytoplasm and interacts with cGAS after DNA stimuli

(A) Schematic representation of DDX41 constructs. Three nuclear localization signals (NLSs) are indicated in red (first one solid, second and third patterned), helicase core domain in yellow, and zinc finger domain (ZnF) in purple. The sequence of the first NLS is shown, and lysine 9 is underlined. (B) Subcellular localization of GFP-tagged DDX41 (top: full-length DDX41; bottom: NLS deletion) after poly(dA:dT) treatment in HeLa cells. (C) Subcellular localization of GFP-tagged DDX41 (full-length) after TSA treatment or mock in HeLa cells. (D) Subcellular localization of GFP-tagged DDX41 point mutants, K9A, K9Q, and K9R, in HeLa cells at 6 h post poly(dA:dT) treatment. (E–H) IFN-β (E) and ISG56 (F) gene expression, IFN-β protein (G), and luciferase activity (H) detected in WT and DDX41 KO THP-1 macrophages after the indicated DNA treatment. Mock, no DNA; dT₇₀; two random complementary strands: random 70-mer-T and random 70-mer-B; annealed random dsDNA: random-70mer-T and random-70mer-B annealed in vitro; two VACV complementary strands: VACV-70mer-T and VACV-70mer-B; annealed VACV dsDNA: VACV-70mer-T and VACV-70mer-B annealed in vitro. Mock was set as 1. (I) Luciferase activity detected in WT and DDX41 KO THP-1 macrophages 4 h after infection with HSV-1 and/or HSV-1 30-mer DNA. Mock (no DNA or virus) was set as 1. (J) Co-localization of two complementary DNA strands (VACV) in WT was greater than in DDX41 KO THP-1 macrophages at 4 h post transfection. Ten μg/mL DNA was used. (K) Co-localization of two non-complementary DNA strands in WT and DDX41 KO THP-1 macrophages at 1 h post transfection. Note: no signal was observed at 4 h post DNA transfection. (L) DDX41 co-localizes with FAM-labeled dsDNA (VACV) and less with ssDNA in THP-1 macrophages. Note: images of ssDNA were taken at 1 h post transfection; images of no DNA and dsDNA were taken at 4 h post transfection. FAM-ssDNA is VACV-70mer-T with F, and FAM-dsDNA is VACV-70mer-T with F and VACV-70mer-B annealed. (M) Western blot assays of DDX41 in the nuclear and cytosol fractions after THP-1 WT macrophages transfected with ssDNA or dsDNA. ssDNA is VACV-70mer-T, dsDNA is VACV-70mer-T and VACV-70mer-B annealed (the same for N and O). (N) Co-immunoprecipitation of DDX41 protein by cGAS antibody in THP-1 WT macrophages that were transfected with ssDNA or dsDNA and blotted with DDX41 (top) or cGAS antibody (bottom). Normal IgG was used as a control. (O) DDX41 co-localizes with cGAS after dsDNA transfection, but not ssDNA transfection, in THP-1 macrophages. (P–R) Representative images of PLA between DDX41 and dsDNA (P), ssDNA (Q), or DNA:RNA hybrid (R) in THP-1 macrophages, and their quantitative assays of PLA signal. 30 cells were counted in each PLA. (S) Co-localization of DDX41 (green) and BrdU (red, incorporated into HSV-1 genome DNA) in HeLa cells (top). Non-infection cells were used as a control (bottom). For (E)–(I) and (P)–(R): data represent the mean ± SEM of three independent experiments. ****p < 0.0001, ***p < 0.001, **p < 0.01. See Table S1 for related sequence.

Figure 5.. Biochemical characterization of DDX41 patient mutant R525H protein

(A) SDS-PAGE analysis of recombinant DDX41 proteins (WT and R525H) eluting from a Sephacryl S-300 HR column. (B) Circular-dichroism spectrum of DDX41-WT and mutant R525H proteins. The CD spectra for these proteins (1 mg/mL) were acquired using Chirascan plus (Applied Photophysics) in 1-mm cuvettes and recorded from 190 to 260 nm. The secondary-structure content was analyzed using DichroWeb (Miles et al., 2022) with the respective spectrum input. (C) A representative image of helicase reactions performed by incubating 0.5 nM of 30-bp forked duplex DNA substrate with increasing DDX41 protein concentration (0–300 nM) of WT and R525H. NE, no enzyme; filled triangle, heat-denatured DNA substrate control. (D) Quantitative analysis of helicase assays of DDX41 proteins in (C). (E and F) Representative images of strand-annealing reactions using 0.5 nM two ssDNAs (one of them ³²P labeled) for 30-bp DNA substrates and increasing DDX41 protein (0–150 nM) without (E) or with ATP (2 mM, F). NE + Com, no enzyme with complementary strand. (G) A representative EMSA image with increasing R525H or WT DDX41 protein (0–1.5 μM) binding with 0.5 nM 30-bp forked DNA substrate without (left) or with (right) ATP analog ATPλS (2 mM). (H) A representative image of ATP hydrolysis detected by TLC with DDX41 proteins (WT, R525H, and ATPase-dead E345A, 300 nM each) with M13 ssDNA effector (50 μM). (I) Quantitative assay for the results shown in (H). (J) A representative dot blot image of ATP bound by DDX41 proteins. (K) ATP binding by DDX41 proteins was determined by ATP agarose (AK-102, Jena Bioscience) and followed by western blot with an anti-DDX41 antibody (SC-166225, Santa Cruz). (L and M) TLC analysis of cGAS cyclic-dinucleotide synthesis under indicated conditions. Purified full-length human cGAS protein (2.5 μM), ATP (250 μM), GTP (250 μM), [α-³²P]-ATP (10 μCi), VACV tailed dsDNA (1 μM; Table S2), and increasing DDX41 protein (1.25, 2.5, 5, and 10 μM) (L). cGAS protein (2.5 μM) was incubated with substrate nucleotides (250 μM each), stimulatory DNA (VACV tailed dsDNA, 1 μM), and DDX41 or DDX43 protein (2.5 μM) as indicated (M). Reactions were terminated by treatment with alkaline phosphatase to remove free nucleotide triphosphate. For (D) and (I): data represent the mean ± SEM of three independent experiments.

Figure 6.. Patient mutant R525H has excessive cytokine response to DNA stimulation

(A) Western blot analysis of overexpression of vector, DDX41-WT, or DDX41-R525H in DDX41 KO THP-1 cells with the indicated antibodies. β-actin serves as a loading control. (B–F) qPCR analysis of the expression of IFN-β gene (B), cGAS oligomerization (C), cGAMP production (D), STING dimerization (E), and luciferase activity (F) in vector, DDX41-WT, or DDX41-R525H expressed in DDX41 KO THP-1 macrophages after poly(dA:dT) treatment. For (B), GAPDH serves as an internal control, and time 0 was set as 1; for C and E, denaturing condition and β-actin were used as loading controls; for D, mock was set as 1; for F, time 0 was set as 1. (G and H) Western blot analysis of WT THP-1 cells expressing vector (G and H), FLAG-tagged DDX41-WT or DDX41-R525H (G), and FLAG-tagged DDX43-WT or DDX43-D396A (H), with indicated antibodies. β-actin serves as a loading control. (I and J) qPCR analysis of the expression of IFN-β gene (I) and luciferase activity (J) in WT THP-1 macrophages expressing vector, DDX41-WT, DDX43-WT, DDX41-R525H, or DDX43-D396A after poly (dA:dT) treatment. GAPDH serves as an internal control and time 0 was set as 1. (K) Increased IFN-responsible genes occurring in R525H-expressing patient cells determined by GSEA (three patients with the R525H mutation were compared with 20 patients without the mutation). Normalized enrichment score, nominal p value, and false discovery rate (FDR) q value are indicated at the bottom. (L) A representative image of helicase analysis on a 30-bp forked duplex DNA using indicated DDX41 proteins. Helicase reactions were performed by incubating with increasing protein (0–300 nM) and 0.5 nM duplex DNA substrate at 37°C for 15 min. NE, no enzyme. The triangle indicates heat-denatured DNA substrate control. (M) A representative image of strand-annealing reactions using 0.5 nM two ssDNA (one labeled with ³²P) for forked 30-bp dsDNA substrates and DDX41 proteins (0–150 nM) without ATP. NE + Com, no enzyme with complementary strand added. (N) A model of DDX41’s role in the activation and inactivation of the cGAS-STING-type I IFN pathway. Upon DNA virus infection, DDX41 protein expression increases, and its strand-annealing activity dominates over its unwinding activity, and the produced dsDNA activates the cGAS-STING-IFN pathway. Once the virus is cleared, DDX41 expression reduces, and its unwinding activity dominates over its annealing activity, and the produced ssDNA inactivates the cGAS-STING-IFN pathway. Ac, acetylation. For simplicity, only cytosolic cGAS is shown. For (B), (D), (F), (I), and (J): data represent the mean ± SEM of three independent experiments. ns, not significant (p ≥ 0.05). ****p < 0.0001, ***p < 0.001, **p < 0.01, and *p < 0.05.