Research ArticleIMMUNOLOGY

NDR2 promotes the antiviral immune response via facilitating TRIM25-mediated RIG-I activation in macrophages

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Science Advances  06 Feb 2019:
Vol. 5, no. 2, eaav0163
DOI: 10.1126/sciadv.aav0163
  • Fig. 1 NDR2 facilitates RNA virus–induced IFN-β, IL-6, and TNF-α production via a kinase activity–independent mechanism in macrophages.

    (A and B) IFN-β, IL-6, and TNF-α expression in NDR2f/f and Lysm+NDR2f/f PMs infected with VSV in the indicated times was detected by enzyme-linked immunosorbent assay (ELISA) (A) and real-time polymerase chain reaction (PCR) analysis (B). (C) Real-time PCR analysis of IFN-β, IL-6, and TNF-α transcripts in NDR2f/f and Lysm+NDR2f/f PMs treated with phosphate-buffered saline (PBS) or with the infection of H1N1, SeV, RSV, EMCV, HSV-1, or MHV68 for 8 hours. (D) Fluorescence-activated cell sorting (FACS) analysis of enhanced green fluorescent protein (eGFP) fluorescence intensity in NDR2f/f and Lysm+NDR2f/f PMs infected with VSV-eGFP. (E) VSV-eGFP titers by TCID50 assay in supernatants of NDR2f/f and Lysm+NDR2f/f PMs infected with VSV-eGFP for 6 hours. (F) NDR2f/f and Lysm+NDR2f/f PMs were transfected with empty vector (mock), NDR2, or its kinase-inactive mutants overexpressing plasmids for 36 hours and then infected with VSV, followed by real-time PCR analysis of IFN-β, IL-6, and TNF-α expression. AA, K282A/T442A. (G) FACS analysis of RAW264.7 cells stably overexpressing NDR2 or its kinase-inactive mutants infected with VSV-eGFP. Data are means ± SD and are representative of three independent experiments. Student’s t test was used for statistical calculation. ns, no significance. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 2 NDR2-deficient mice are more vulnerable to RNA viral infection.

    (A) ELISA of IFN-β, IL-6, and TNF-α in serum obtained from 8-week-old male NDR2f/f and Lysm+NDR2f/f mice (n = 6 per group) 12 hours after intraperitoneal infection with VSV [1 × 107 plaque-forming units (PFU) g−1]. (B) VSV titers by TCID50 assay in spleens, lungs, and livers from mice in (A). (C) Pathology of NDR2f/f and Lysm+NDR2f/f mice in response to VSV infection. Hematoxylin and eosin staining of lung sections from mice in (A). Scale bars, 200 μm (for 4×) and 50 μm (for 20×). (D) Neutrophil (CD11b+Gr-1+) infiltration in murine bronchoalveolar lavage fluid (BALF) from mice treated with intraperitoneal injections of PBS (n = 3) or VSV (1 × 107 PFU g−1 and n = 3) was assessed 12 hours after injection. (E) Survival of 8-week-old male NDR2f/f and Lysm+NDR2f/f mice administered VSV (1 × 108 PFU g−1) via tail intravenous injection (n = 8 per group; Wilcoxon test). (F) Survival curve for 8-week-old male NDR2f/f (n = 8) and Lysm+NDR2f/f (n = 9) mice infected with H1N1 virus (2 × 103 PFU per mouse) by intranasal inoculation (Wilcoxon test). (G) Real-time PCR analysis of flu-M mRNA of lungs from 8-week-old male NDR2f/f and Lysm+NDR2f/f mice 4 days after intranasal inoculation with H1N1 virus (2 × 103 PFU per mouse) (n = 6 per group). Data are means ± SD and are representative of three independent experiments. Student’s t test was used for statistical calculation. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 3 NDR2 specifically promotes RIG-I pathway activation in a kinase-independent manner.

    (A and B) NDR2f/f and Lysm+NDR2f/f PMs were infected with VSV (A) or SeV (B) for the indicated times, and then, immunoblotting analysis was performed to detect the total and phosphorylated (p-) TBK1, IRF3, P65, ERK1/2, JNK1/2, and P38. (C) BMDMs were transfected with NDR2-specific or scrambled small interfering RNA (siRNA), followed by VSV infection for the indicated periods, and then, immunoblotting analysis was performed to detect the total and phosphorylated TBK1, IRF3, P65, ERK1/2, JNK1/2, and P38. (D) NDR2f/f and Lysm+NDR2f/f PMs were transfected with LMW poly(I:C) (1 μg ml−1) for the indicated times, and then, immunoblotting analysis was performed to detect the total and phosphorylated TBK1, IRF3, P65, ERK1/2, JNK1/2, and P38. (E) NDR2f/f and Lysm+NDR2f/f PMs were transfected with vectors expressing NDR2 or its kinase-inactive mutants for 36 hours, followed by infection with VSV to detect the total and phosphorylated TBK1, IRF3, P65, ERK1/2, JNK1/2, and P38 by immunoblotting. (F) Human embryonic kidney (HEK) 293 cells were transfected with RIG-I CARDs (caspase activation and recruitment domains), MDA5 CARDs, MAVS, TBK1, and IKKi, along with IFN-β reporter plasmid and NDR2 or its kinase-inactive mutant vectors for 48 hours, and luciferase activity was analyzed. Data are means ± SD and are representative of three independent experiments. Student’s t test was used for statistical calculation. **P < 0.01 and ***P < 0.001.

  • Fig. 4 NDR2 interacts with RIG-I and promotes RIG-I K63-linked polyubiquitination.

    (A) Immunoblot analysis of extracts of RAW264.7 macrophages stably overexpressing Flag-NDR2 and infected with VSV for the indicated times was performed, and then, whole-cell lysates were immunoprecipitated with anti-Flag mouse magnetic (M2) beads, followed by immunoblotting with the indicated antibodies. (B) Flag-tagged RIG-I, MAVS, TBK1, or IKKi was coexpressed with Myc-NDR2 in HEK293 cells, followed by IP with anti-Flag M2 beads, followed by immunoblotting with anti-Flag or anti-Myc antibodies. (C) Lysates from murine PMs infected with VSV for the indicated time periods were subjected to IP with anti–RIG-I antibody, followed by Western blot analysis with the indicated antibodies. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (D) Flag-tagged full-length NDR2(1–464), dN(90–464), dC(1–383), dNC(90–383), or dS-TKc truncation mutants were coexpressed with Myc–RIG-I in HEK293 cells, and whole-cell lysates were immunoprecipitated with anti-Flag M2 beads, followed by immunoblotting with anti-Flag or anti-Myc antibodies. (E) Flag-tagged full-length RIG-I(1–926), 2CARD(1–233), Heli(234–734), CTD(735–926), or dCARD(234–926) truncation mutants were coexpressed with Myc-NDR2 in HEK293 cells, and whole-cell lysates were immunoprecipitated with anti-Flag M2 beads, followed by immunoblotting with anti-Flag or anti-Myc antibodies. (F) Immunoblot analysis of the polyubiquitination of RIG-I in HEK293 cells cotransfected with Flag–RIG-I, hemagglutinin (HA)–ubiquitin (HA-ub), and increasing concentrations (wedge) of vectors for the Myc-NDR2 constructs, followed by denaturation-IP with anti-Flag M2 beads and immunoblot analysis with an anti-HA antibody. (G) Immunoblot analysis of the polyubiquitination of RIG-I in HEK293 cells cotransfected with Flag–RIG-I, HA-ubiquitin, and Myc-NDR2 or its kinase-inactive mutant constructs, followed by denaturation-IP with anti-Flag M2 beads and immunoblot analysis with anti-HA antibodies. (H) Immunoblot analysis of the ubiquitination of RIG-I in HEK293 cells cotransfected with Flag–RIG-I, Myc-NDR2, and HA-ub or the mutant ubiquitin constructs, followed by denaturation-IP with anti-Flag M2 beads and immunoblot analysis with anti-HA antibodies. (I) NDR2f/f and Lysm+NDR2f/f BMDMs were infected with or without VSV for the indicated periods. Cell lysates were immunoprecipitated with anti–RIG-I, and the immunoprecipitates were analyzed by immunoblots with the indicated antibodies.

  • Fig. 5 NDR2 enhances TRIM25-mediated RIG-I K63-linked ubiquitination via facilitating recruitment of TRIM25 to RIG-I.

    (A) Flag-tagged Riplet, TRIM4, or TRIM25 were coexpressed with Myc-NDR2 in HEK293 cells, and then, whole-cell lysates were immunoprecipitated with anti-Flag M2 beads, followed by immunoblotting with anti-Flag or anti-Myc antibodies. (B) Recombinant Myc-tagged NDR2 proteins were mixed with Flag-tagged GFP-, TRIM25-, or RIG-I–purified proteins. The proteins were then pulled down with anti-Flag M2 beads, followed by immunoblotting with anti-Flag or anti-Myc antibodies. (C) Lysates from HEK293 cells infected with SeV for the indicated time were immunoprecipitated with anti-TRIM25 antibodies, followed by Western blot analysis with the indicated antibodies. IgG, immunoglobulin G. (D) IP and immunoblot analysis of HEK293 cells transfected with the indicated combinations of plasmids encoding HA-NDR2, Flag–RIG-I, and Myc-TRIM25. Then, whole-cell lysates were immunoprecipitated with anti-Flag M2 beads, followed by immunoblotting with anti-Flag, anti-Myc, or anti-HA antibodies. (E) Recombinant RIG-I, TRIM25, NDR2, NDR2 dN, NDR2 dSTKc, and ubiquitination-associated proteins were mixed in adenosine 5′-triphosphate buffer and incubated at 30°C for 2 hours. Then, reaction aliquots were quenched and evaluated by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting. (F) Immunoblot analysis of the polyubiquitination of RIG-I in WT and TRIM25-deficient L929 cells transfected with empty vector or Flag-NDR2 plasmids. Thirty-six hours later, the cells were infected with VSV for the indicated times, followed by denaturation-IP with anti–RIG-I and immunoblot analysis with the indicated antibodies. (G) Real-time PCR analysis of IFN-β, IL-6, and TNF-α mRNA in WT and TRIM25-deficient L929 cells transfected with plasmids encoding Flag-NDR2 or empty vector for 24 hours, followed by VSV infection for the indicated periods. Data are means ± SD and are representative of three independent experiments. Student’s t test was used for statistical calculation. *P < 0.05, **P < 0.01, and ***P < 0.001.

  • Fig. 6 Viral infection dampens NDR2 expression in an IFN/STAT1-dependent manner.

    (A) NDR2 expression in PBMCs from RSV-infected patients (n = 77) and healthy controls (n = 40) was analyzed by real-time PCR analysis. (B and C) NDR2 expression in PMs infected with RNA viruses (VSV, H1N1, SeV, and RSV) or DNA viruses (HSV-1 and MHV68) for the indicated periods was detected by real-time PCR (B) and immunoblotting (C). STAT1 expression was detected to ensure that the antiviral immune response was triggered in the virus-infected cells. (D and E) NDR2 expression in PMs stimulated with murine recombinant IFN-β (100 IU ml−1) (D) or in Thp1 cells stimulated with human recombinant IFN-α (100 IU ml−1) (E) for the indicated times was detected by immunoblotting. (F) WT, IFNαR−/−, or STAT1−/− immortalized BMDMs were infected with VSV for the indicated periods, followed by immunoblotting analysis of NDR2 expression. (G and H) H3K27me3 (G) and H3K4me3 (H) modifications were detected by chromatin IP (ChIP)–quantitative PCR on NDR2 gene promoter loci in the lysates of Thp1 cells infected with VSV for 8 hours or with IFN-α (100 IU ml−1) for 4 hours. Data are means ± SD and are representative of three independent experiments. Student’s t test was used for statistical calculation. *P < 0.05, **P < 0.01, and ***P < 0.001.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/2/eaav0163/DC1

    Fig. S1. NDR2 specifically enhances RNA virus–induced IFN-β, IL-6, and TNF-α production in macrophages in a kinase-independent manner.

    Fig. S2. NDR2 protects mice from RNA virus infections.

    Fig. S3. NDR2 enhances RIG-I pathway activation in a kinase-independent manner.

    Fig. S4. Schematic structure of NDR2 and TRIM25 and their derivatives.

    Fig. S5. NDR2 promotes TRIM25-mediated K63-linked polyubiquitination of RIG-I.

    Fig. S6. Virus infections inhibit NDR2 expression via STAT1-dependent mechanism.

    Table S1. List of antibody information.

    Table S2. List of primers for qPCR.

    Table S3. List of gRNAs for CRISPR-CAS9.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. NDR2 specifically enhances RNA virus–induced IFN-β, IL-6, and TNF-α production in macrophages in a kinase-independent manner.
    • Fig. S2. NDR2 protects mice from RNA virus infections.
    • Fig. S3. NDR2 enhances RIG-I pathway activation in a kinase-independent manner.
    • Fig. S4. Schematic structure of NDR2 and TRIM25 and their derivatives.
    • Fig. S5. NDR2 promotes TRIM25-mediated K63-linked polyubiquitination of RIG-I.
    • Fig. S6. Virus infections inhibit NDR2 expression via STAT1-dependent mechanism.
    • Table S1. List of antibody information.
    • Table S2. List of primers for qPCR.
    • Table S3. List of gRNAs for CRISPR-CAS9.

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