Research ArticleVIROLOGY

PPM1A silences cytosolic RNA sensing and antiviral defense through direct dephosphorylation of MAVS and TBK1

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Science Advances  01 Jul 2016:
Vol. 2, no. 7, e1501889
DOI: 10.1126/sciadv.1501889
  • Fig. 1 PPM1A potently suppresses cytosolic RNA sensing and antiviral response.

    (A) In a dose-dependent manner, transfection of PPM1A elicited a marked suppression on IRF3-responsive IFN-β promoter (left) or 5xISRE promoter (right) in 293T cells, which was stimulated by expression of activated RIG-I (caRIG-I). The comparative level of PPM1A and caRIG-I was detected by immunoblotting (IB) (left). n = 4 experiments. *P < 0.001, compared with control, by Student’s t test. (B) PPM1A also strongly inhibited IRF3 activation, which was stimulated by either adaptor MAVS (left) or kinase TBK1 (right), in a dose-dependent manner. n = 3 experiments. *P < 0.001, compared with control, by Student’s t test. (C) Transfection of PPM1A D239N or R174G mutant that is catalytically defective, but not PPM1A wild type (WT), has a much weaker effect on caRIG-I–stimulated IRF3 activation. n = 3 experiments. *P < 0.001, compared with WT PPM1A, by Student’s t test. (D) caRIG-I–induced IRF3 activation, as revealed by anti-pIRF3 (Ser396) immunoblotting (top), was completely abolished by cotransfection of PPM1A WT, but not its phosphatase-defective forms. (E) Transfection of siRNA-targeting PPM1A (si-PPM1A) in HepG2 cells, which effectively depleted endogenous PPM1A expression (left), enhanced cytosolic RNA sensing in response to either caRIG-I stimulation or polyinosinic-polycytidylic acid [poly(I:C)] transfection (TpIC) (middle and right). n = 3 experiments. *P < 0.01, compared with control siRNA (si-Ctrl), by Student’s t test. (F) siRNA-mediated PPM1A depletion promoted SeV-induced nuclear translocation of endogenous IRF3, which was detected by immunofluorescence and microscopy. DAPI, 4′,6-diamidino-2-phenylindole. (G) Similar siRNA-mediated depletion of PPM1A in 293T cells, as displayed by immunoblotting of PPM1A (left), boosted VSV-induced IRF3 Ser396 phosphorylation (left) and enhanced antiviral response measured by virus-induced mRNA expression of antiviral proteins, including IFNB1, ISG15, and IFIT1 (right). gVSV, green fluorescent protein (GFP)–tagged VSV. (H) Antiviral response of control or Ppm1a−/− BMDMs against SeV infection was measured by mRNA induction at 12 hours post-infection (hpi) of various ISGs. Ppm1a−/− BMDMs exhibited stronger antiviral responses compared to control BMDMs. PPM1A expression was revealed by anti-PPM1A immunoblotting. n = 4 mice. *P < 0.01, compared with control, by Student’s t test. KO, knockout.

  • Fig. 2 PPM1A negatively regulates host antiviral defense in cells, mice, and zebrafish.

    (A) Ppm1a−/− 293T cells were generated by CRISPR/Cas9 strategy and infected with gVSV. An enhanced IRF3 activation induced by gVSV infection was detected by phospho-Ser396 immunoblotting when PPM1A was knocked out. Drastically boosted cellular viral resistance was observed by less amount of total GFP tags detected by immunoblotting (left) or by reduced virus-replicating (GFP+) cells (right). (B) siRNA-mediated depletion of PPM1A in 293T cells, as indicated in Fig. 1G, led to effectively reduced infection of gVSV. (C) Determination of gVSV loads in mouse organs by TCID50 (median tissue culture infectious dose) assay 12 hpi in Ppm1a−/− and WT mice, which were intravenously injected through the tail vein with gVSV. n = 6 mice for each group. *P < 0.01, compared with control Ppm1a+/+ group, by Student’s t test. (D) Survival of ~8-week-old Ppm1a−/− and WT mice given intravenous tail vein injection of gVSV [2 × 107 plaque-forming units (PFU)/g]. n = 6 mice for each group. P < 0.05, by paired Student’s t test. (E) Enhanced antiviral response was detected in Ppm1a−/− PBMCs by mRNA induction of antiviral proteins, including IFNB1, ISG15, and IFIT1 at 6 hpi of VSV injection in mice. n = 4 mice for each group. *P < 0.05, compared with control Ppm1a+/+ group, by Student’s t test. (F) 293T cells, which were previously transfected with MAVS in the absence or presence of PPM1A WT or phosphatase-defective mutant, were infected by gVSV. Visualized GFP represented cells that have active VSV replication. Restored numbers of virus-replicating (GFP+) cells indicated that overexpression of PPM1A impeded antiviral function of MAVS. (G) gVSV was microinjected into yolk of zebrafish embryos (1 × 103 PFU per embryo), which elicited a robust virus infection status and occurred strongly at brain but was also visible at muscle and gut tissues of fish. The infection was aggravated at 48 hpi and started to cause embryo death. (H) Zebrafish embryos were previously microinjected with MAVS or PPM1A mRNA to gain expression of proteins, as detected by immunoblotting (top). The survival rates of gVSV-infected zebrafish were recorded. A vulnerable phenotype of PPM1A- expressing embryos and a resistance phenotype of MAVS-expressing embryos to gVSV infection were observed upon VSV challenge. n = 150 embryos for each group. *P < 0.05, **P < 0.05, compared with sham group, by paired Student’s t test. PBS, phosphate-buffered saline.

  • Fig. 3 PPM1A is an inherent component and silencer of TBK1/IKKε complex.

    (A and B) Endogenous complex of PPM1A/TBK1 or PPM1A/IKKε was detected by coimmunoprecipitation using anti-TBK1 or anti-IKKε antibodies in 293T lysates and visualized by anti-PPM1A antibody. IgG, immunoglobulin G; IP, immunoprecipitation; WCL, whole-cell lysates. (C) Peptides of endogenous PPM1A were detected in abundance by mass spectrometry in TBK1-immunoprecipitated proteins by Flag tag, verifying that PPM1A was an endogenous component of TBK1 complex. LC-MS/MS, liquid chromatography–tandem mass spectrometry. (D) Domain mapping assays, performed by coimmunoprecipitation of Flag-tagged full-length (FL) PPM1A with serial truncations of TBK1 (left), revealed that the C terminus, that is, kinase domain of TBK1, was responsible and enough to recruit PPM1A (right). ULD, ubiquitin-like domain. (E) In vitro kinase assay was performed using separately purified IRF3 and TBK1, which was cotransfected with or without PPM1A (left), showing that TBK1 coexpressed with WT PPM1A lost its kinase ability to phosphorylate IRF3. SDS-PAGE, SDS–polyacrylamide gel electrophoresis. (F) siRNA-mediated depletion of PPM1A in 293T cells resulted in an enhanced activation of endogenous TBK1, in response to stimulations from activated RIG-I or STING. pTBK1, phosphorylated TBK1.

  • Fig. 4 PPM1A directly dephosphorylates MAVS and TBK1/IKKε.

    (A) In vitro phosphatase assays were performed by using purified PPM1A and separately isolated MAVS, which were previously coexpressed with TBK1 to gain phosphorylation modifications, as visualized by mobility shift (second lane). WT PPM1A, but not phosphatase SCP7 that was set as a negative control, removed phosphorylation on MAVS, similar to λPPase treatment. HA, hemagglutinin. (B) Similarly, coexpression of PPM1A with both MAVS and TBK1 in 293T cells led to disappearance of phospho-MAVS modifications, as monitored by mobility shift. (C) Coimmunoprecipitation with tagged MAVS and PPM1A showed that only minimal interaction between PPM1A and MAVS was detected, but their interaction was drastically enhanced in the presence of IKKε. (D) Endogenous complex of PPM1A/MAVS was detected upon VSV infection at 6 hpi by coimmunoprecipitation using anti-PPM1A antibody in 293T lysates and visualized by anti-MAVS antibody. (E) A significant portion of endogenous PPM1A (by anti-PPM1A antibody; green) was translocated to mitochondria (by MitoTracker staining; red) upon SeV infection at 6 hpi, captured, and visualized by immunofluorescence and the super-resolution microscopy. (F and G) In vitro phosphatase assays were performed by using separately purified TBK1 or IKKε with PPM1A WT or its phosphatase-defective mutant, showing that WT PPM1A eliminated phospho-Ser172 modification on TBK1 (F) or IKKε (G). (H) Phos-tag SDS-PAGE showed that PPM1A removed phosphorylation modifications on TBK1 to an extent similar to λPPase treatment (second panel).

  • Fig. 5 PPM1A and MAVS compete for antiviral signaling output.

    (A) Coimmunoprecipitation assay revealed that MAVS/IKKε association was disrupted in the presence of WT PPM1A, but not by its phosphatase-defective form. Note the faster mobility shift of IKKε and MAVS in the presence of WT PPM1A (third panel), indicating the dephosphorylation of both kinase and adaptor by PPM1A in cells. (B) Similarly, interaction between MAVS and TBK1 was diminished by WT PPM1A. (C) Interaction of TBK1 with IRF3 2SA mutant, which had an enhanced interaction to TBK1 and thereby used in analysis for TBK1/IRF3 interaction, was significantly improved by the siRNA-mediated depletion of PPM1A expression. (D) Coimmunoprecipitation assay with Myc-tagged TBK1 and Flag-tagged PPM1A revealed their strong association. The presence of cotransfected MAVS can lower TBK1 and PPM1A interaction. (E) Coimmunoprecipitation assay of Flag-tagged IRF3 with Myc-tagged MAVS, with or without PPM1A, showed that PPM1A enhanced IRF3/MAVS association, possibly by trapping IRF3 in an inactive MAVS.

  • Fig. 6 Model for PPM1A-guided silencing of cytosolic RNA sensing and antiviral defense.

    Phosphatase PPM1A is a natural component of TBK1/IKKε kinase complex, which is recruited by kinase domains of TBK1/IKKε to restrain cytosolic RNA sensing by directly dephosphorylating both MAVS and TBK1/IKKε, potentially serving as a threshold check of antiviral signaling. However, stronger activation from MAVS can release TBK1/IKKε from the grasp of PPM1A, leading to their activation and MAVS phosphorylation which led to IRF3 C-terminal phosphorylation and translocation to function as transcriptional factor.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/2/7/e1501889/DC1

    fig. S1. Reporter screen of RLR/IRF3 pathway by human Ser/Thr phosphatase cDNAs.

    fig. S2. Short hairpin RNA–mediated PPM1A depletion results in an enhanced antiviral signaling.

    fig. S3. Enhanced basal expression of ISGs in Ppm1a−/− BMDMs.

    fig. S4. Colocalization of TBK1 with endogenous PPM1A in cytosol.

    fig. S5. mRNA level of PPM1A is not significantly changed in response to virus infection.

    Oligo sequence of qPCR and CRISPR/Cas9

  • Supplementary Materials

    This PDF file includes:

    • fig. S1. Reporter screen of RLR/IRF3 pathway by human Ser/Thr phosphatase cDNAs.
    • fig. S2. Short hairpin RNA–mediated PPM1A depletion results in an enhanced antiviral signaling.
    • fig. S3. Enhanced basal expression of ISGs in Ppm1a−/− BMDMs.
    • fig. S4. Colocalization of TBK1 with endogenous PPM1A in cytosol.
    • fig. S5. mRNA level of PPM1A is not significantly changed in response to virus infection.
    • Oligo sequence of qPCR and CRISPR/Cas9

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