Research ArticleIMMUNOLOGY

Control of antiviral innate immune response by protein geranylgeranylation

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Science Advances  29 May 2019:
Vol. 5, no. 5, eaav7999
DOI: 10.1126/sciadv.aav7999
  • Fig. 1 Protein geranylgeranylation negatively regulates RLR-mediated antiviral innate immune response.

    (A to C) Quantitative reverse transcription polymerase chain reaction (RT-PCR) analysis of cytokine genes or SeV-P gene transcript abundance from Pggt1b+/+ Lyz2-Cre and Pggt1bfl/fl Lyz2-Cre BMDMs after transfection of mock or RIG-I ligands [poly(I:C) LMW and 5′ppp-dsRNA (1 μg/ml)], Sting ligand 2′3′-cGAMP (8 μg/ml), and cGAS ligand ISD (1 μg/ml) with Lipofectamine for 4 hours (A and B), or after infection with SeV (50 HA (Hemagglutinin) units/ml in all succeeding experiments) for 4 hours (C); fold, fold change relative to wild-type negative control throughout. (D) Enzyme-linked immunosorbent assay (ELISA) measurement of interferon-β (IFN-β) and interleukin-6 (IL-6) in supernatants of Pggt1b+/+ Lyz2-Cre and Pggt1bfl/fl Lyz2-Cre BMDMs infected with SeV for 24 hours. (E and F) Immunoblot analysis of phosphorylated (p-) or total IRF3, TBK1, IKK, and Pggt1b or β-actin (loading control throughout) in Pggt1b+/+ Lyz2-Cre and Pggt1bfl/fl Lyz2-Cre BMDMs transfected with poly(I:C) LMW (E) or infected with SeV (F). Numbers below lanes indicate densitometry of phosphorylated IRF3, TBK1, or IKK relative to that of total IRF3, TBK1, or IKK, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 [two-way analysis of variance (ANOVA)]. ns, not significant. Data are representative of three independent experiments with three biological replicates.

  • Fig. 2 MAM-localized Rac1 inhibits RLR signaling in a protein geranylgeranylation–dependent manner.

    (A to C) Quantitative RT-PCR analysis of transcript abundance of Ifnb1, Il6, Rac1, and Rac2 in wild-type (WT), Rac1−/−, or Rac2−/− iBMDMs 4 hours after SeV infection. (D) Immunoblot of phospho-IRF3 and phospho-IKK in cell lysates of wild-type or Rac1−/− iBMDMs following SeV infection. Numbers below lanes indicate densitometry of phosphorylated IRF3 or IKK relative to that of total IRF3 or IKK, respectively. (E to H) Quantitative RT-PCR analysis of transcript abundance of Ifnb1, Il6, and SeV-P (E to G) and immunoblot of Rac1 (H) in Rac1−/− iBMDMs reconstituted with wild-type, C189S, G12V, or G12V/C189S of Rac1 4 hours after SeV infection. (I and J) Confocal microscopy of immunofluorescent staining of Rac1 (green) and SIGMA1R (red) in Pggt1b+/+ Lyz2-Cre and Pggt1bfl/fl Lyz2-Cre BMDMs 4 hours after SeV infection. Cells were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Scale bars, 10 μm (I). See each single channel in fig. S3J. The percentage of colocalization between Rac1 and SIGMA1R was calculated using the ImageJ software from at least 50 cells randomly selected from the immunostaining slides (J). (K to N) Representative PLA images of Pggt1b+/+ Lyz2-Cre and Pggt1bfl/fl Lyz2-Cre BMDMs (K) or Rac1−/− iBMDMs reconstituted with wild-type, G12V, or C189S of Rac1 (M) 4 hours after SeV infection. The cells were probed with Rac1 and SIGMA1R antibodies. Red spots indicate the association of Rac1 with SIGMA1R. (K and M) Cells were counterstained with DAPI (blue). Scale bars, 10 μm. (L and N) Quantitation of PLA spots per cell in (K) and (M) (n = 20 cells). (O) Schematic display of the C-terminal amino acid sequence of Rac1 highlighting the C178 for palmitoylation and C189 for geranylgeranylation sites. (P) Immunoblots of Rac1 after S-palmitoylated protein assay (acyl-RAC; see Materials and Methods) of captured proteins (Pulldown) and inputs from Pggt1b+/+ Lyz2-Cre and Pggt1bfl/fl Lyz2-Cre BMDM cell lysate following infection with or without SeV for 4 hours. (Q) Quantitative RT-PCR of Ifnb1 and Il6 abundance in MEF cells stably expressing wild-type and C178S mutant forms of Rac1 after transfection with poly(I:C) LMW. **P < 0.01, ****P < 0.0001 (two-way ANOVA). Data are representative of three independent experiments.

  • Fig. 3 Rac1 directly engages MAVS signalosome and inhibits MAVS signaling in a protein geranylgeranylation–dependent manner.

    (A) Immunoblots (IB) of MAVS and TRADD in anti-Rac1 immunoprecipitates (IP) from the mitochondrial fraction of BMDMs infected with or without SeV. IgG, immunoglobulin G; VDAC, voltage-dependent anion channel. (B and C) Confocal microscopy of immunofluorescent staining of Rac1 (green) and MAVS (red) in BMDMs 4 hours after infection with SeV. Scale bars, 10 μm (B). See each single channel in fig. S5A. The percentage of colocalization of Rac1 and MAVS was calculated using ImageJ (C). (D to G) Representative PLA confocal images for Rac1 and MAVS in Pggt1b+/+ Lyz2-Cre and Pggt1bfl/fl Lyz2-Cre BMDMs (D) or Rac1−/− iBMDMs reconstituted with wild-type, G12V, or C189S of Rac1 (F) 4 hours after SeV infection. Cells were counterstained with DAPI (blue). Scale bars, 10 μm (D and F). (E and G) Quantification of PLA spots per cell in (D) and (F) (n = 20 cells). (H) Luciferase activity in 293T cell lysate 24 hours after transfection with an IFN-β luciferase reporter, HA-MAVS together with wild-type, G12V, or C189S Rac1 (top). Immunoblots of HA-MAVS and Rac1 in whole-cell lysates (WCL) of transfected 293T cells (bottom). (I) Immunoblot of crude mitochondrial and cytoplasm fractions from 293T cells cotransfected with Myc-MAVS and Flag-Rac1 wild-type, G12V, or C189S mutants with indicated antibodies. (J) Schematic of MAVS (wild-type) and its truncation mutants (top), and coimmunoprecipitation analysis of the interaction of Flag-Rac1 with Myc-MAVS or its truncation mutants in 293T cells (bottom). **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way ANOVA). Data are representative of three independent experiments.

  • Fig. 4 MAM-localized Rac1 inhibits Trim31-MAVS interaction and subsequent MAVS Lys63-linked ubiquitination, aggregation, and activation upon RNA virus infection.

    (A) Immunoblot of MAVS and Pggt1b in the RIPA-soluble fractions and WCL of BMDMs infected with SeV. (B) Densitometry of MAVS relative to that of β-actin in the RIPA-soluble fractions and WCL of (A). (C) Anti-MAVS immunoblot of crude mitochondrial extracts of BMDMs infected with SeV for 4 hours and resolved by SDD-AGE (top) or total cell lysate of BMDMs resolved by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) (bottom). (D) Pggt1b+/+ Lyz2-Cre and Pggt1bfl/fl Lyz2-Cre BMDMs infected with SeV for 4 hours were lysed, boiled, and sheared by sonication. After centrifugation, supernatants were immunoprecipitated with an anti-MAVS antibody and probed with pan-ubiquitin, K63-, or K48-ubiquitin–specific antibodies. Numbers below lanes indicate densitometry relative to β-actin. (E and F) PLA confocal images for MAVS and TRIM31 (red) in BMDMs 4 hours after SeV infection. Cells were counterstained with DAPI (blue). Scale bars, 10 μm (E); (F) quantification of PLA foci per cell in (E) (n = 20 cells), *P < 0.05, ****P < 0.0001 (two-way ANOVA). (G) Immunoblot of total cell lysate (bottom) or anti-Myc immunoprecipitates (top) of 293T cells transfected with Myc-tagged Mavs and Flag-tagged Trim31 together with the wild-type, G12V, or C189S mutant form of Rac1. (H) Immunoblot of total cell lysate (bottom) or anti-Myc immunoprecipitates (top) of 293T cells transfected with Myc-tagged Mavs, Flag-tagged Trim31, and HA-tagged K63-ubiqutin together with the wild-type, G12V, or C189S mutant form of Rac1. Data are representative of three independent biological experiments.

  • Fig. 5 MAM-localized Rac1 facilitates the recruitment of caspase-8 and cFLIPL to MAVS signalosome and promotes the cleavage of Ripk1.

    (A) Immunoblot with anti-Ripk1 of cell lysate from BMDMs at different time points after SeV infection and the densitometry of cleaved 37KD Ripk1 fragment relative to that of β-actin in the WCL. (B and C) Representative PLA confocal images for MAVS and TRADD and Casp-8 in Pggt1b+/+ Lyz2-Cre and Pggt1bfl/fl Lyz2-Cre BMDMs (B) or MAVS and TRADD, FADD, Casp-8, and cFLIPL in wild-type (Rac1+/+) and Rac1−/− iBMDMs (C) 4 hours after SeV infection. Cells were counterstained with DAPI (blue). Scale bars, 10 μm. Quantification of PLA spots per cell is shown in the right panels of (B) and (C) (n = 20 cells). (D) Immunoblot of input (middle and bottom) or anti-Myc immunoprecipitates (top) in mitochondrial fractions of 293T cells transfected with Myc-tagged TRADD and Flag-tagged Casp-8 together with the wild-type, G12V, or C189S mutant form of Rac1. (E) Immunoblot of anti–caspase-8 immunoprecipitates and input from mitochondrial (mito) or cytosolic (cyto) fractions of BMDMs 4 hours after SeV infection. (F) Immunoblot of input (bottom) or anti-Myc immunoprecipitates (top) in mitochondrial (left) or cytosolic (right) fractions of 293T cells transfected with Myc-tagged caspase-8 and Flag-tagged cFLIPL together with the wild-type, G12V, or C189S mutant form of Rac1. **P < 0.01, ***P < 0.001, ****P < 0.0001 (two-way ANOVA). Data are representative of three independent experiments.

  • Fig. 6 Myeloid Pggt1b deficiency better protects mice from lethal PR8 challenge.

    (A and B) Quantitative RT-PCR analysis of Ifnb1, Ifna4, Il6, and Tnf in murine alveolar macrophages (mAMs) 9 hours after acute infection with influenza A virus (PR8, 1 MOI). (C and D) Fluorescence microscopy of mAMs 24 hours after infection with a PR8-mNeon strain of (0.01 MOI) containing TPCK-trypsin (1 μg/ml) (C) and percentage of mNeon-positive cells (D). (E and F) Survival curves (E) and body weights (F) of Pggt1b+/+Lyz2-Cre or Pggt1bfl/f Lyz2-Cre mice infected with 50 plaque-forming units (PFU) PR8 (n = 16); results are pooled together from two independent biological experiments. (G to I) Viral titer (G), IFN-β (H), and IL-6 (I) levels in lung homogenates from mice 3 days after infection with 50 PFU of PR8 (n = 8 per group). (J) Quantitative RT-PCR analysis of IFNB1 and IFNA1 in mock or 10 μM simvastatin-treated human lung alveolar macrophages (hAMs) 9 hours after infection with 1 MOI PR8. DMSO, dimethyl sulfoxide. (K) Quantitative RT-PCR of Ifnb1 and Il6 in mouse lung alveolar macrophages (mAMs) pretreated with 10 μM simvastatin overnight followed by PR8 (1 MOI) infection for 9 hours. (L) Quantitative RT-PCR of IAV viral genes M1 and NS1 in mAMs pretreated with simvastatin overnight followed by infection with PR8 (0.01 MOI) containing TPCK-trypsin (1 μg/ml) for 24 hours. **P <0.01, ***P <0.001, ****P <0.0001 (two-way ANOVA). Data are representative of three independent biological experiments.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/5/eaav7999/DC1

    Fig. S1. Protein geranylgeranylation has no effect on DNA virus–induced antiviral innate immune response.

    Fig. S2. Rac1 but not RhoA or Cdc42 suppresses MAVS signaling in a protein geranylgeranylation–dependent manner.

    Fig. S3. Protein geranylgeranylation targets Rac1 to MERC ER membranes upon RLR engagement.

    Fig. S4. Chaperon but not the signaling function of SIGMA1R and Rac1 palmitoylation is important for anchoring Rac1 to MERC ER membranes.

    Fig. S5. Rac1 directly engages MAVS signalosome and inhibits MAVS signaling in a protein geranylgeranylation–dependent manner.

    Fig. S6. MERC-localized Rac1 inhibits Trim31-MAVS interaction and subsequent MAVS Lys63-linked ubiquitination, aggregation, and activation upon RNA virus infection.

    Fig. S7. MERC-localized Rac1 promotes cleavage of Ripk1 and the association of TANK with ubiquitinated Ripk1.

    Fig. S8. Pggt1b deficiency and Statin pretreatment in BMDMs enhance antiviral innate immune response after influenza A virus PR8 challenge.

    Fig. S9. Graphic summary.

    Table S1. Primers for quantitative RT-PCR.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Protein geranylgeranylation has no effect on DNA virus–induced antiviral innate immune response.
    • Fig. S2. Rac1 but not RhoA or Cdc42 suppresses MAVS signaling in a protein geranylgeranylation–dependent manner.
    • Fig. S3. Protein geranylgeranylation targets Rac1 to MERC ER membranes upon RLR engagement.
    • Fig. S4. Chaperon but not the signaling function of SIGMA1R and Rac1 palmitoylation is important for anchoring Rac1 to MERC ER membranes.
    • Fig. S5. Rac1 directly engages MAVS signalosome and inhibits MAVS signaling in a protein geranylgeranylation–dependent manner.
    • Fig. S6. MERC-localized Rac1 inhibits Trim31-MAVS interaction and subsequent MAVS Lys63-linked ubiquitination, aggregation, and activation upon RNA virus infection.
    • Fig. S7. MERC-localized Rac1 promotes cleavage of Ripk1 and the association of TANK with ubiquitinated Ripk1.
    • Fig. S8. Pggt1b deficiency and Statin pretreatment in BMDMs enhance antiviral innate immune response after influenza A virus PR8 challenge.
    • Fig. S9. Graphic summary.
    • Table S1. Primers for quantitative RT-PCR.

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