Research ArticleIMMUNOLOGY

Broad and diverse mechanisms used by deubiquitinase family members in regulating the type I interferon signaling pathway during antiviral responses

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Science Advances  02 May 2018:
Vol. 4, no. 5, eaar2824
DOI: 10.1126/sciadv.aar2824
  • Fig. 1 A large number of DUBs are regulated by virus-induced type I IFN signaling at the transcriptional level.

    (A) Domain organization of DUB subfamilies [USPs, UCHs, Josephins, OTUs, and MPN(+)/JAMMs]. (B) DUB splice variants were mapped according to the UniProt database. Isoforms with incomplete catalytic domains or interaction domains were analyzed. (C) Schematic diagram illustrating the screening approach. (D) qRT-PCR analysis of the mRNA level of individual DUBs in THP-1 monocytes (left, lanes 1 to 6) or THP-1–derived macrophages (right, lanes 7 to 12) with VSV (MOI, 0.01) infection for the indicated time points. Data were normalized to the mRNA levels in untreated cells (0 hours) and were presented in a heat map. (E) qRT-PCR analysis of mRNA levels of individual DUBs in THP-1 monocytes (left, lanes 1 to 6) or THP-1–derived macrophages (right, lanes 7 to 12) treated with IFN-β (1000 U/ml) for the indicated time points. Data were normalized to the basal mRNA levels and were presented in a heat map. (F) qRT-PCR analysis of mRNA levels of the indicated DUBs in VSV-infected (MOI, 0.01) (lane 2), HSV-1–infected (MOI, 0.1) (lane 3), or IFN-β–treated (1000 U/ml) (lane 4) PBMCs. Data were normalized to the basal mRNA levels. (G) Distribution of different expression tendencies of DUBs between different cell types upon VSV infection or IFN-β stimulation. Each point represents the difference of a certain DUB expression among different cell types, plotted by the natural logarithm of expression difference ratio between THP-1 monocytes and THP-1–derived macrophages on the horizontal axis and the ratio between PBMCs and THP-1–derived macrophages on the vertical axis. Data are representative of three independent experiments (D to F).

  • Fig. 2 Identification of DUBs that modulate antiviral response.

    (A) Schematic overview of assay. 293T cells were transduced with lentiviruses expressing Cas9 and sgRNAs targeting individual DUBs. After puromycin selection, sg_DUB 293T cells were infected with VSV-eGFP (MOI, 0.01) for 18 hours. VSV-eGFP positive ratios were determined by FACS analysis, and the infection ratios were normalized to cells transduced with nontargeting control (NT) sgRNA. On the other hand, sg_DUB 293T cells were transfected with ISRE, IFN-β, or NF-κB luciferase reporters and then transfected with RIG-I (2CARD) or infected with SeV. The luciferase reporter induction was measured and normalized to NT sgRNA-expressing cells. (B) Demonstration of the hit candidate selection by Venn diagrams.

  • Fig. 3 DUB proteins act at different levels of type I IFN pathways.

    (A) Luciferase activity in 293T cells transfected with an IFN-β luciferase reporter, together with vectors for RIG-I (2CARD), MAVS, cGAS/STING, TBK1, and IRF3 (5D), along with an empty vector or with expression vectors for the indicated DUBs. Columns are colored by significance relative to EV (empty vector) control (t test); color scale is given in the figure. (B) Heat map summary of (A). Negative regulations are marked green, positive regulations are marked red, and nonsignificant (NS) changes are marked gray. (C) Summary of interactions between DUBs and key molecules in type I IFN signaling pathways. (D to I) Coimmunoprecipitation and immunoassay of extracts of A549 cells infected with or without SeV (MOI, 0.1) for 12 hours with the indicated antibodies. IgG, immunoglobulin G. (J and K) Coimmunoprecipitation and immunoassay of extracts of A549 cells infected with or without HSV-1 (MOI, 0.1) for 12 hours with the indicated antibodies. (L) Coimmunoprecipitation and immunoassay of extracts of A549 cells infected with or without SeV (MOI, 0.1) for 12 hours with the indicated antibodies. (M) Summary of multilayered regulations of DUBs, according to the combination of inhibitory function levels and endogenous protein interactions. Data are representative of three independent experiments [mean and SEM in (A)]. IP, immunoprecipitate; IB, immunoblot; WCL, whole cell lysate.

  • Fig. 4 DUBs regulate type I IFN signaling by altering ubiquitination in both catalytic activity–dependent and –independent manners.

    (A) Heat map summary of regulatory roles of DUBs on total ubiquitination of key molecules in the type I IFN signaling pathway. (B) Heat map summary of regulatory roles of DUBs on ubiquitination of key molecules with different linkages in the type I IFN signaling pathway. Data are obtained by band intensity analysis (IP-HA/IP-Flag) and are normalized to the control sample. (C) Luciferase activity in 293T cells transfected with an IFN-β luciferase reporter, together with vectors for RIG-I (2CARD), along with an empty vector or with expression vectors for the indicated WT DUB or catalytically inactive mutants. (D) Coimmunoprecipitation and immunoassay of extracts of 293T cells transfected with Flag-TRAF3, Myc-BRCC3, and Myc-BRCC3-H122Q with the indicated antibodies. (E) Coimmunoprecipitation and immunoassay of extracts of 293T cells transfected with Flag-RIG-I and HA-K11-Ub together with Myc-USP5 or Myc-USP5-C335A followed by SeV infection with the indicated antibodies. (F) Coimmunoprecipitation and immunoassay of extracts of 293T cells transfected with Flag-STING and HA-K27-Ub together with Myc-USP22 or Myc-USP22-C185A followed by HSV-1 infection with the indicated antibodies. (G) Coimmunoprecipitation and immunoassay of extracts of 293T cells transfected with Flag-RIG-I and HA-K48-Ub together with Myc-USP5 or Myc-USP5-C335A with the indicated antibodies. Data are representative of three independent experiments [mean and SEM in (C)]. NS, nonsignificant (P > 0.05); ***P < 0.001.

  • Fig. 5 USP22 removes K27-linked ubiquitination on STING through cooperation with USP13.

    (A) Luciferase activity in indicated sg_DUB 293T cells transfected with an IFN-β luciferase reporter, together with vectors for RIG-I (2CARD), along with an empty vector or with expression vectors for the indicated WT DUB or catalytically inactive mutants. (B) Coimmunoprecipitation and immunoassay of extracts of 293T cells transfected with Myc-USP13 and HA-USP22 with or without HSV-1 infection with the indicated antibodies. (C) Coimmunoprecipitation and immunoassay of extracts of 293T cells transfected with Flag-STING, Myc-USP13, and HA-USP22 with or without HSV-1 infection with the indicated antibodies. (D and E) sg_NT or sg_USP13 A549 cells were transfected with control siRNA or USP22 siRNA and then left uninfected of infected with HSV-1 for 18 hours. IFN-β mRNA levels (D) and HSV-1 (glycoprotein C mRNA) gRNA levels (E) were determined by qRT-PCR. Rel., relative. (F) Constructs of WT USP22, USP22 enzymatically inactive mutant (USP22-C185A), USP22 containing only the ZNF domain (USP22-ZNF), and USP22 containing only the UCH domain (USP22-UCH). (G) Coimmunoprecipitation and immunoassay of extracts of 293T cells transfected with Myc-STING, HA-USP13, and Flag-USP22-WT, USP22-C185A, USP22-ZNF, or USP22-UCH followed by HSV-1 infection with the indicated antibodies. (H) Coimmunoprecipitation and immunoassay of extracts of 293T cells transfected with Flag-STING and HA-K27-Ub together with Myc-USP22, USP22-C185A, USP22-ZNF, or USP22-UCH followed by HSV-1 infection with the indicated antibodies. Data are representative of three independent experiments [mean and SEM in (A), (D), and (E)]. NS, nonsignificant (P > 0.05); ***P < 0.001.

  • Fig. 6 USP5 inhibits type I IFN signaling and enhances viral replication through the promotion of STUB1-mediated RIG-I degradation.

    (A) Immunoblot analysis of extracts of A549 cells transfected with control siRNA or USP5 siRNA treated with CHX (100 μg/ml) for the indicated time after infection with VSV for 12 hours. (B) WT, RIG-I-KO 293T, or RIG-I-KO cells reconstituted with RIG-I were transfected with control siRNA or USP5 siRNA and then infected with VSV-eGFP for 18 hours. The mRNA level of IFN-β was determined by qRT-PCR. (C) Plaque titration of VSV-eGFP in the supernatant of the indicated cells transfected with control siRNA or USP5 siRNA followed by VSV-eGFP infection for 24 hours. pfu, plaque-forming units. (D) Coimmunoprecipitation and immunoassay of extracts of 293T cells transfected with Myc-USP5 and HA-STUB1 with or without SeV infection with the indicated antibodies. (E) Coimmunoprecipitation and immunoassay of extracts of 293T cells transfected with Flag-RIG-I, Myc-USP5, and HA-STUB1 treated with MG132 and infected with SeV with the indicated antibodies. (F) Immunoassay of extracts of sg_NT or sg_STUB1 293T cells transfected with Flag-RIG-I and Myc-USP5 followed by VSV infection for 9 hours. (G) qRT-PCR analysis of IFN-β mRNA levels in sg_NT or sg_STUB1 293T cells transfected with control siRNA or USP5 siRNA followed by VSV-eGFP infection for 18 hours. (H) Plaque titration of VSV-eGFP in the supernatant of the indicated cells transfected with control siRNA or USP5 siRNA followed by VSV-eGFP infection for 24 hours. (I) Constructs of WT USP5, USP5 enzymatically inactive mutant (USP5-C335A), and USP5 without the UBA domain (USP5-ΔUBA). (J) Coimmunoprecipitation and immunoassay of extracts of 293T cells transfected with Flag-RIG-I, HA-STUB1, and Myc-USP5-WT, USP5-C335A, or USP5-ΔUBA, followed by SeV infection and treatment with MG132 with the indicated antibodies. (K) Coimmunoprecipitation and immunoassay of extracts of 293T cells transfected with Flag-RIG-I, HA-K48-Ub, and Myc-USP5-WT, USP5-C335A, or USP5-ΔUBA, followed by SeV infection and treatment with MG132 with the indicated antibodies. (L) The six working models of DUBs in modulating type I IFNs. Data are representative of three independent experiments [mean and SEM in (B), (C), (G), and (H)]. NS, nonsignificant (P > 0.05); ***P < 0.001.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/5/eaar2824/DC1

    fig. S1. Knockdown efficiency of DUBs by sgRNAs.

    fig. S2. Identification of functional DUBs in antiviral response by FACS analysis and luciferase assay.

    fig. S3. Validation of identified DUB candidates in an overexpression system and by siRNA.

    fig. S4. DUBs interact with the key molecules in type I IFN signaling pathways and the induction of the indicated genes in sg_NT A549 and PBMCs after viral infection.

    fig. S5. Attenuated DUB expression in A549 cells and PBMCs affects virus-induced cytokine expression.

    fig. S6. Total ubiquitination and different linkages of ubiquitination of the key molecules in the type I IFN pathway are regulated by DUBs.

    fig. S7. USP5 inhibits type I IFN signaling and enhances viral replication through the promotion of STUB1-mediated RIG-I degradation.

    table S1. Relative mRNA expression of DUBs in THP-1 monocytes or macrophages after VSV infection or IFN-β treatment for the indicated time points and the relative mRNA expression of DUBs in PBMCs after VSV infection, HSV-1 infection, or IFN-β treatment.

    table S2. Summary of DUB expression patterns using different stimuli.

    table S3. List of sgRNA sequences, qRT-PCR primer sequences, and siRNA sequences used in this study.

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Knockdown efficiency of DUBs by sgRNAs.
    • fig. S2. Identification of functional DUBs in antiviral response by FACS analysis and luciferase assay.
    • fig. S3. Validation of identified DUB candidates in an overexpression system and by siRNA.
    • fig. S4. DUBs interact with the key molecules in type I IFN signaling pathways and the induction of the indicated genes in sg_NT A549 and PBMCs after viral infection.
    • fig. S5. Attenuated DUB expression in A549 cells and PBMCs affects virus-induced cytokine expression.
    • fig. S6. Total ubiquitination and different linkages of ubiquitination of the key molecules in the type I IFN pathway are regulated by DUBs.
    • fig. S7. USP5 inhibits type I IFN signaling and enhances viral replication through the promotion of STUB1-mediated RIG-I degradation.
    • Legends for tables S1 to S3

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    Other Supplementary Material for this manuscript includes the following:

    • table S1 (Microsoft Excel format). Relative mRNA expression of DUBs in THP-1 monocytes or macrophages after VSV infection or IFN-β treatment for the indicated time points and the relative mRNA expression of DUBs in PBMCs after VSV infection, HSV-1 infection, or IFN-β treatment.
    • table S2 (.pdf format). Summary of DUB expression patterns using different stimuli.
    • table S3 (Microsoft Excel format). List of sgRNA sequences, qRT-PCR primer sequences, and siRNA sequences used in this study.

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