Expression of DDX60 mRNA. (A and B) Mouse BM-DCs were stimulated with poly(I·C) (A) or infected with MV in the presence of anti-IFN-AR antibody (B). Total RNA was extracted from the cells, and microarray analysis was performed. The heat maps in the left column show the expression profiles of the genes encoding the helicase domain. The heat maps in the right column show the genes encoding the helicase domain whose expression levels changed more than 4-fold. (C) Northern blot of human DDX60 mRNAs in specified tissues. Northern blots of human tissues were probed with DDX60 cDNA. (D and E) Mouse BM-DCs, HeLa cells, or RAW 264.7 cells were stimulated with 50 μg of poly(I·C)/ml (D), 800 U of IFN-β/ml (E), 100 U of TNF-α/ml (E), or 1,000 U of IL-1β/ml (E). Expression of DDX60, RIG-I, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), and β-actin mRNA was examined by RT-PCR. (F and G) HEK293 cells, RAW 264.7 cells, or HeLa cells were infected with VSV at an MOI of 1 (F) or PV at an MOI of 0.1 (G). Expression of DDX60, IFN-β, β-actin, and/or GAPDH was examined by RT-PCR (F) or RT-qPCR (G). (H) PV was injected intraperitoneally (i.p.) into PVR-transgenic mice susceptible to PV. Tissue RNA extraction was performed before or 3 days after infection, and RT-PCR was carried out on these samples.

Intracellular localization of DDX60. (A and B) HA-tagged DDX60 expression vector was transfected into HeLa cells, and transfected cells were stimulated with 50 μg/ml poly(I·C) (A) or infected with VSV at an MOI of 10 (B). The cells were fixed and stained with anti-HA antibodies and observed using confocal microscopy. DIC, differential interference contrast; pAb, polyclonal antibody. (C to E) HA-tagged DDX60 was transfected into HeLa cells together with FLAG-tagged EXOSC1 (C), EXOSC4 (D), or EXOSC5 (E). Transfected cells were fixed and stained with anti-HA and anti-FLAG antibodies and observed using confocal microscopy. mAb, monoclonal antibody. (F to I) HEK293 cells stably expressing DDX60-HA were infected with VSV or SeV. DDX60-HA was stained with anti-HA antibody. The ER, mitochondria, early endosome, and autophagosome were stained with calnexin (F), Mitotracker Red (G), anti-EEA1 antibody (H), and anti-LC3 antibody (I).

Antiviral activity of DDX60. (A and B) HeLa cells (A) or the cells stably expressing shRNA for EXOSC5 or GFP (B) were transiently transfected with empty, DDX60-expressing, and/or RIG-I-expressing vectors. After 24 h, cells were infected with VSV at an MOI of 0.1 for 30 h. Cells were fixed and stained with crystal violet. (C and D) HEK293 clones stably expressing DDX60 or RIG-I (C) and HeLa cells subjected to mock treatment or transiently expressing DDX60 (D) were infected with VSV at an MOI of 0.1 for 24 h (C) or 48 h (D). Viral titers of culture media were measured by plaque assay. (E) Empty or DDX60-expressing vectors were transfected into HeLa cells, and 24 h after transfection, cells were infected with PV at an MOI of 0.1 for 26 h. Viral titers of culture media were measured by a plaque assay. (F) DDX60-expressing vector was transfected into HeLa clones stably expressing shRNA for GFP or DDX60, and the protein results were observed by Western blotting. (G) Control or DDX60 knockdown cells were infected with VSV at an MOI of 0.1 for 12 h, and viral titers of the culture media were measured by a plaque assay. (H) Control and DDX60 knockdown cells were infected with VSV at the indicated MOI for 12 h. The cells were fixed and stained with crystal violet. (I and J) Empty or DDX60-expressing vectors were transfected into HeLa cells with negative-control siRNA or siRNA for IPS-1. The cells were infected with VSV at an MOI of 1. After 24 h, cells were stained with crystal violet (I), and the viral titers of culture media were determined by a plaque assay (J).

Binding of the DDX60 helicase domain to viral RNA. (A) Schematic diagram showing the helicase region of DDX60 used for the following gel shift assay. (B) A His-tagged DDX60 helicase fragment was expressed in E. coli and purified using Ni-NTA resin. Purified products were analyzed by SDS-PAGE and stained with CBB. Lane A represents the nonabsorbed fraction, lane B represents the wash fraction, and lanes 1 to 10 represent the eluted fractions. The lane 3 fraction contains DDX60 protein. (C to F) Purified DDX60 and DDX6 fragments were incubated with in vitro-synthesized VSV ssRNA (C), dsRNA (D), dsRNA treated with calf intestinal alkaline phosphatase (CIAP) (E), or dsDNA (F), and the products were analyzed using agarose gel. The gel was stained with ethidium bromide.

DDX60 increases the association of RIG-I to short synthetic dsRNA. DDX60- and RIG-I-expressing vectors were transfected into HEK293FT cells. At 24 h later, cell lysate was prepared. The lysate was incubated with or without biotin-conjugated dsRNA, and the dsRNA was recovered using streptavidin Sepharose beads. The recovered fraction was analyzed by Western blotting.

Effects of DDX60 knockdown on antiviral responses. (A to C) Control or DDX60 knockdown cells were stimulated with human IFN-β (A) or infected with VSV at an MOI of 1 (B) or 0.1 (C), and expression of IFIT1 (A) and IFN-β (B and C) mRNA was examined by RT-qPCR. (D) Control or DDX60 knockdown HEK293 cells were infected with VSV at an MOI of 1, and cell lysates were prepared at the indicated times. Lysates were analyzed by native PAGE, and Western blot analysis was performed with anti-IRF-3 antibody. (E to G) Control or DDX60 knockdown HeLa cells were infected with HSV-1, and total RNA was extracted at the indicated times. RT-qPCR was performed to examine expression of IFN-β (E), IP10 (F), and DDX60 (G).

The phylogenetic tree of DEXD/H box RNA helicase. (A) Schematic diagram of DDX60. DDX60 encodes a peptide of 1,712 amino acids (aa) that contains a DEXD/H box (DEXDc; aa 760 to 956) and HELICc (aa 1247 to 1330). (B) The phylogenetic tree of DEXD/H box RNA heliases. Ce, C. elegans. The bootstrap probabilities and genetic distances are shown in red and black, respectively.

Interactions between DDX60 and RNA exosome components. (A and B) HA-tagged DDX60 and FLAG-tagged EXOSC1 (A) and EXOSC4 (B) expression vector was transfected into HEK293FT cells. After 24 h, cell lysates were prepared and immunoprecipitation (IP) was performed with anti-FLAG antibody. The samples were analyzed by SDS-PAGE and detected by Western blotting (WB) using anti-HA or anti-FLAG antibodies. WCE, whole-cell extract (WCE). (C) HA-tagged DDX60 partial fragments and/or FLAG-tagged EXOSC4 expression vectors were transfected into HEK293FT cells, and immunoprecipitation was carried out as described for panel A in the presence of RNase A. (D) HA-tagged DDX60 fragment-expressing vectors were transfected into HeLa cells. The cells were stained with anti-HA antibody and DAPI and then observed by confocal microscopy.

Antiviral activity of RNA exosome. (A and B) HeLa cells were transfected with expression vectors containing EXOSC1, EXOSC4, and EXOSC5, and 24 h later, the transfected cells were infected with VSV at an MOI of 1 or 0.5. One day after infection, the cells were fixed and stained with crystal violet (A). Viral titers of culture media after 76 h were measured by a plaque assay (B). (C) Expression of EXOSC4 and EXOSC5 in stable HeLa clones, which express shRNA for EXOSC4, EXOSC5, or GFP, was examined by RT-PCR (upper panel), RT-qPCR (middle panel), and Western blotting (lower panel). The amounts of EXOSC4 and EXOSC5 cDNA in each sample were normalized by dividing by the amount of GAPDH. (D and E) Cell growth rates of stable HeLa clones, which express shRNA for EXOSC4, EXOSC5, DDX60, or GFP, were determined (D). The cells were infected with VSV at an MOI of 0.1 for 48 h, and viral titers of culture media were determined by a plaque assay (E). (F) FLAG-tagged EXOSC4- and HA-tagged DDX60-expressing vectors were transfected into HEK293FT cells. After VSV or mock infection, the immunoprecipitation was performed with anti-HA antibody.

Association of DDX60 with RLRs. (A to C) Vectors expressing HA-tagged DDX60 were transfected into HEK293FT cells with FLAG-tagged LGP2, RIG-I, MDA5, IPS-1, IKK-ε, and/or Ubc13, and cell lysates were prepared. The lysates were treated with RNase III (B) or RNase A (C). Immunoprecipitation was carried out with anti-FLAG antibody, and the precipitates (IP) and 10% of whole-cell extract (WCE) were analyzed using SDS-PAGE. Proteins were stained by Western blotting using anti-HA or anti-FLAG antibody. (D and E) HEK293FT cells were transfected with empty or HA-tagged DDX60-expressing vectors, and cells were stimulated with dsRNA or infected with VSV. Cell lysates were prepared at the indicated times, and immunoprecipitation was performed with anti-HA antibody. The precipitates were analyzed using SDS-PAGE, and Western blotting was carried out using anti-HA and anti-HMGB1 (D) or anti-RIG-I (E) antibodies. (F and G) Vectors expressing HA-tagged DDX60 and FLAG-tagged RIG-I (F) or MDA5 (G) were transfected into HeLa cells. After 24 h, cells were fixed and stained with anti-HA or anti-FLAG antibody and then observed using confocal microscopy. (H) The upper panel shows a schematic diagram of RIG-I partial fragments. The lower panel shows results of an immunoprecipitation assay performed as described for panel A. DDX60 was found to bind to the RIG-IC region.

DDX60 promotes RIG-I- or MDA5-mediated signaling. (A to C) Activation of the IFN-β promoter was examined using a reporter gene assay and p125luc plasmid. Vectors expressing RIG-I (A), MDA5 (B), DDX6 (C), and the wild type (WT) or DDX60-K791A (C) were transfected into HEK293 cells together with the reporter plasmid and Renilla luciferase plasmid (internal control). After 24 h, the cells were left unstimulated or stimulated with poly(I·C) by the use of DEAE-dextran for 4 h. Cell lysates were prepared, and luciferase activity was measured. (D) Control or DDX60 knockdown HEK293 cells were transfected with the p125luc reporter, Renilla luciferase plasmid, and/or in vitro-synthesized VSV dsRNA. After 24 h, cell lysates were prepared and luciferase activity was measured. (E and F) siRNA for DDX60 or control siRNA was transfected into HEK293 cells. The cells were left unstimulated or stimulated with poly(I:C), and expression of IFN-β and DDX60 mRNA was measured by RT-qPCR. Expression values were normalized using GAPDH. (G) siRNA for DDX60 or the control was transfected into HEK293 cells together with DDX60-expressing vector. The DDX60 protein was observed by Western blotting. (H) Vectors expressing TICAM-1 and/or DDX60 were transfected into HEK293 cells together with the p125luc reporter and Renilla luciferase plasmids. After 24 h, the cell lysates were prepared and luciferase activities were measured. (I) Vectors expressing TLR3 and/or DDX60 were transfected into HEK293 cells together with the p125luc reporter and Renilla luciferase plasmids. After 24 h, the cells were left unstimulated or stimulated with poly(I:C) for 4 h, the cell lysates were prepared, and luciferase activity was measured. (J and K) HeLa cells expressing shRNA for DDX60 (J) or EXOSC4 (K) were stimulated with 50 μg/ml of poly(I·C) (no transfection) (J) or dsRNA (transfection) (K). RT-qPCR was performed to measure IFN-β mRNA expression. (L) HeLa cells expressing shRNA for GFP, EXOSC4, or EXOSC5 were infected with VSV at an MOI of 1. Levels of induction of IFN-β mRNA were calculated as described for panel J. (M and N) Empty or IPS-1-expressing vector (M) and RIG-I CARD-, MDA5-, or TBK1-expressing vector (N) were transfected into control or DDX60 knockdown HEK293 cells together with p125luc reporter and Renilla luciferase plasmids. After 24 h, cell lysates were prepared and luciferase activity was measured. (O) shRNA for DDX60 did not inhibit the signaling from TLR3 (H to J). Although DDX60 promotes RLR-dependent signaling (A to E), shRNA for DDX60 did not reduce the signaling induced by RIG-I CARD, MDA5, IPS-1, or TBK1 overexpression (M and N). These data suggest that shRNA suppresses signaling upstream of RIG-I and MDA5.

Knockdown of DDX60 decreases expression of type I IFN during viral infection in HeLa cells. (A to L) Control cells or HeLa cells from a stable cell line expressing shRNA for DDX60 were infected with VSV (A to D), PV (E to H), or SeV (I to L). Total RNA was extracted at the indicated times. RT-qPCR was performed to measure expression of IFN-β (A, E, and I), IFIT1 (B, F, and J), IP10 (C, G, and K), and DDX60 (D, H, and L). The expression level of each sample was normalized to GAPDH expression.