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, 11 (1), 49-57

The Interaction Between the Helicase DHX33 and IPS-1 as a Novel Pathway to Sense Double-Stranded RNA and RNA Viruses in Myeloid Dendritic Cells

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The Interaction Between the Helicase DHX33 and IPS-1 as a Novel Pathway to Sense Double-Stranded RNA and RNA Viruses in Myeloid Dendritic Cells

Ying Liu et al. Cell Mol Immunol.

Abstract

In eukaryotes, there are at least 60 members of the DExD/H helicase family, many of which are able to sense viral nucleic acids. By screening all known family members, we identified the helicase DHX33 as a novel double-stranded RNA (dsRNA) sensor in myeloid dendritic cells (mDCs). The knockdown of DHX33 using small heteroduplex RNA (shRNA) blocked the ability of mDCs to produce type I interferon (IFN) in response to poly I:C and reovirus. The HELICc domain of DHX33 was shown to bind poly I:C. The interaction between DHX33 and IPS-1 is mediated by the HELICc region of DHX33 and the C-terminal domain of IPS-1 (also referred to MAVS and VISA). The inhibition of DHX33 expression by RNA interference blocked the poly I:C-induced activation of MAP kinases, NF-κB and IRF3. The interaction between the helicase DHX33 and IPS-1 was independent of RIG-I/MDA5 and may be a novel pathway for sensing poly I:C and RNA viruses in mDCs.

Figures

Figure 1
Figure 1
DHX33 senses both poly I:C and an RNA virus in D2SC mDCs. (a) IB showing the knockdown efficiency of shRNAs targeting the indicated proteins in D2SC cells. Scrambled shRNA served as a control (left most lane). Actin blots are shown as loading controls (lower panel). ELISA of IFN-α and IFN-β production by D2SC cells treated with the indicated shRNAs after stimulation with (b) 2.5 µg/ml long poly I:C, (c) 5 µg/ml short poly I:C or (d) reovirus. Cells were stimulated with nucleic acids, delivered by Lipofectamine 2000, for 16 h. Virus was added to the cells at a MOI of 10 for 2 h. Cells were collected, washed with PBS and cultured for an additional 16 h. (e) ELISA of TNF-α and IL-6 production by D2SC cells treated with the indicated shRNAs after 16 h of stimulation with 1 µg/ml LPS. N-STM, unstimulated D2SC cells treated with scrambled shRNA. Individual circles represent the value from each independent experiment. Bars represent the average values from at least three independent experiments. IB, immunoblot; IFN, interferon; LPS, lipopolysaccharide; MOI, multiplicity of infection; PBS, phosphate-buffered saline; shRNA, small heteroduplex RNA.
Figure 2
Figure 2
DHX33 knockdown in BMDCs abolishes their cytokine responses to poly I:C and reovirus. (a) QPCR showing the knockdown efficiency of shRNA targeting the indicated genes in BMDCs (left panel). IB showing the knockdown efficiency of shRNAs targeting the indicated proteins in BMDCs (right panel). Scrambled shRNA served as a control. ELISA of IFN-α and IFN-β production by BMDCs treated with the indicated shRNAs after stimulation with (b) 2.5 µg/ml long poly I:C, (c) 5 µg/ml short poly I:C or (d) reovirus at a MOI of 10. Cells were stimulated with nucleic acids, delivered by Lipofectamine 2000, for 16 h. Virus was added to the cells for 2 h. Cells were collected, washed with PBS and cultured for an additional 16 h. (e) ELISA of TNF-α and IL-6 production by BMDCs treated with the indicated shRNAs after 16 h of stimulation with 1 µg/ml LPS. N-STM, unstimulated D2SC cells treated with the scrambled shRNA. Individual circles represent the values from each independent experiment. Bars represent the average values from at least three independent experiments. BMDC, bone marrow-derived dendritic cell; IB, immunoblot; IFN, interferon; MOI, multiplicity of infection; LPS, lipopolysaccharide; PBS, phosphate-buffered saline; QPCR, quantitative PCR; shRNA, small heteroduplex RNA.
Figure 3
Figure 3
Recombinant DHX33 can rescue the defect in poly I:C-activated IFN production caused by DHX33 shRNA knockdown. (a) Immunoblot of endogenous DHX33 in D2SC mDCs (Control) and recombinant HA-DHX33 (sh+HA-X33) in D2SC mDCs in which endogenous DHX33 was selectively knocked down (shDHX33). (b) ELISA of IFN-β production by D2SC mDCs. IFN, interferon; mDC, myeloid dendritic cell; shRNA, small heteroduplex RNA.
Figure 4
Figure 4
DHX33 signaling is independent of MDA5 and RIG-I signaling. (a) IB showing the knockdown efficiency of shRNAs targeting the indicated proteins in RIG-I-deficient MEFs (left panel). QPCR showing the knockdown efficiency of shRNAs targeting the indicated genes in RIG-I-deficient MEFs (middle panel). Scrambled shRNA served as a control. ELISA of IFN-α production by RIG-I-deficient MEFs treated with the indicated shRNAs after 16 h of stimulation with 5 µg/ml short poly I:C delivered to the cells by Lipofectamine 2000 (right panel). (b) IB showing the knockdown efficiency of shRNAs targeting the indicated proteins in MDA5-deficient MEFs (left panel). QPCR analysis showing the knockdown efficiency of shRNAs targeting the indicated genes in MDA5-deficient MEFs (middle panel). Scrambled shRNA served as a control. ELISA of IFN-α production by MDA5-deficient MEFs treated with the indicated shRNAs after 16 h of stimulation with 2.5 µg/ml long poly I:C delivered to the cells by Lipofectamine 2000 (right panel). N-STM, unstimulated RIG-1- or MDA5-deficient MEFs treated with scrambled shRNA. The data represent the mean±s.d. of triplicate or quadruplicate measurements. Individual diamonds represent the values from each independent experiment. Bars represent the average values from at least three independent experiments. IB, immunoblot; IFN, interferon; MEF, mouse embryonic fibroblast; QPCR, quantitative PCR; shRNA, small heteroduplex RNA.
Figure 5
Figure 5
DHX33 interacts with both poly I:C and IPS-1. (a) IB of the indicated proteins precipitated with an anti-DHX33 antibody from whole-cell lysates of D2SC cells that were unstimulated (−) or stimulated with long poly I:C (+). An IgG antibody served as a control. (b) Top: schematic representation of DHX33 and its serial truncations. DExDc: Asp–Glu–Ala–Asp box motif; Helic C: helicase C-terminal domain. HA2: helicase-associated domain. DUF: domain of unknown function. Numbers denote amino-acid residues. Middle panel: immunoblot of purified HA-DHX33 truncated proteins using an anti-HA antibody (a–f). Bottom: immunoblot using an anti-HA antibody of proteins precipitated (IP) with an anti-Myc antibody from a mixture of an HA-DHX33 protein (a–f) individually incubated with a Myc-IPS-1 fusion protein. (c) Top: schematic representation of IPS-1 and its serial truncations. CARD: the caspase activation and recruitment domain. Numbers denote amino-acid residues. Middle panel: immunoblot using an anti-Myc antibody of purified Myc-IPS-1 truncated proteins (a–d). Bottom: immunoblot using an anti-Myc antibody of proteins precipitated (IP) from a mixture of a Myc-IPS-1 protein (a–d) individually incubated with an HA-DHX33 fusion protein. (d) Immunoblot using an anti-HA antibody of pulldown assays in which purified, serial truncations of HA-DHX33 (a–f) were individually incubated with biotinylated poly I:C, followed by the addition of NeutrAvidin beads. (e) Immunoblot using an anti-HA antibody of pulldown competition assay products in which 0.5, 5 or 50 µg/ml poly I:C or poly U was added to a mixture of HA-DHX33 plus biotinylated poly I:C, followed by the addition of NeutrAvidin beads. IB, immunoblot; IP, immunoprecipitation.
Figure 6
Figure 6
DHX33 is required for the activation of MAP kinases, NF-κB and IRF3 in mDCs. Immunoblot of the indicated proteins from the lysates of D2SC mDCs treated with a scrambled shRNA (control), a DHX33-targeting shRNA (DHX33) or an IPS-1-targeting shRNA (IPS-1) after stimulation for the indicated time with 2.5 µg/ml long poly I:C delivered by Lipofectamine 2000. β-Actin served as a loading control. mDC, myeloid dendritic cell; shRNA, small heteroduplex RNA.

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