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NLRX1 promotes immediate IRF1-directed antiviral responses by limiting dsRNA-activated translational inhibition mediated by PKR

Nature Immunology volume 18, pages 1299–1309 (2017)Cite this article

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Abstract

NLRX1 is unique among the nucleotide-binding-domain and leucine-rich-repeat (NLR) proteins in its mitochondrial localization and ability to negatively regulate antiviral innate immunity dependent on the adaptors MAVS and STING. However, some studies have suggested a positive regulatory role for NLRX1 in inducing antiviral responses. We found that NLRX1 exerted opposing regulatory effects on viral activation of the transcription factors IRF1 and IRF3, which might potentially explain such contradictory results. Whereas NLRX1 suppressed MAVS-mediated activation of IRF3, it conversely facilitated virus-induced increases in IRF1 expression and thereby enhanced control of viral infection. NLRX1 had a minimal effect on the transcription of IRF1 mediated by the transcription factor NF-kB and regulated the abundance of IRF1 post-transcriptionally by preventing translational shutdown mediated by the double-stranded RNA (dsRNA)-activated kinase PKR and thereby allowed virus-induced increases in the abundance of IRF1 protein.

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Figure 1: NLRX1 is a positive regulator of innate immunity and an antiviral factor in hepatocytes.
Figure 2: Effect of NLRX1 deficiency on NF-κB signaling and the activation of IRF1 and IRF3 in SeV-infected PH5CH8 cells.
Figure 3: NLRX1–IRF1 signaling dominates the antiviral cytokine response in hepatocytes.
Figure 4: NLRX1 facilitates immediate IRF1 antiviral responses by promoting global protein synthesis in SeV-infected cells.
Figure 5: NLRX1 reduces the activation of PKR and subsequent inhibition of protein synthesis by competing for binding to viral RNA.

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Acknowledgements

We thank B. Glaunsinger (University of California, Berkeley) for the human IL6 3′ UTR expression vector, and L. Hensley, W. Lovell, M. Deng, and M. Chua for technical support. Supported by the US National Institutes of Health (U19-AI109965 to S.M.L., J.A.D. and J.P-Y.T.; R01-AI103083 to S.M.L.; R01-AI131685 to S.M.L. and J.K.W.; R01-AI103311 to N.J.M.; and R21-AI117575 to J.K.W.) and the University of North Carolina Cancer Research Fund (S.M.L. and J.P-Y.T.).

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Author notes

  1. Daisuke Yamane

    Present address: Department of Microbiology and Cell Biology, Tokyo Metropolitan Institute of Medical Science, Setagaya-ku, Tokyo, Japan

  2. Erik M Lenarcic and Daisuke Yamane: These authors contributed equally to this work.

Authors and Affiliations

  1. Lineberger Comprehensive Cancer Center, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Hui Feng, Erik M Lenarcic, Daisuke Yamane, Eliane Wauthier, Jinyao Mo, Haitao Guo, David R McGivern, Olga González-López, Ichiro Misumi, Lola M Reid, Jason K Whitmire, Jenny P-Y Ting, Joseph A Duncan, Nathaniel J Moorman & Stanley M Lemon

  2. Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Hui Feng, Daisuke Yamane, Jinyao Mo, David R McGivern, Olga González-López, Joseph A Duncan & Stanley M Lemon

  3. Department of Microbiology & Immunology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Erik M Lenarcic, Jason K Whitmire, Nathaniel J Moorman & Stanley M Lemon

  4. Department of Genetics, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Eliane Wauthier, Ichiro Misumi, Jason K Whitmire & Jenny P-Y Ting

  5. Department of Cell Biology and Physiology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA

    Haitao Guo & Lola M Reid

  6. Department of Pharmacology, The University of North Carolina, Chapel Hill, North Carolina, USA

    Joseph A Duncan

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Contributions

H.F. and S.M.L. designed the experiments, analyzed the data and wrote the manuscript; H.F., E.M.L., D.Y., D.R.M., O.G -L. and I.M. performed experiments; E.W., J.M., H.G., L.M.R., J.K.W., J.P -Y.T., J.A.D. and N.J.M. provided critical reagents and intellectual input; S.M.L. supervised the study; and all authors reviewed the manuscript.

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Correspondence to Stanley M Lemon.

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Integrated supplementary information

Supplementary Figure 1 The antiviral activity of NLRX1 results from positive regulation of cytokine responses.

(a) Kinetics of IL6 mRNA expression in NLRX1-deficient PH5CH8 cells infected with SeV. (b) Gaussia princeps luciferase (GLuc) analysis of HCV (JFH1-QL/GLuc) replication in NLRX1-T3 cells at 48 h post-transfection (hpt) with indicated treatment. NAC, N-acetyl-L-cysteine (3 mM); TUDCA, tauroursodeoxycholic acid (0.5 mM); 3-MA, 3-methyladenine (10 mM). Because basal levels of lipid peroxidation inhibit HCV replication, we used a lipid-insoluble antioxidant to avoid spurious increases in replication51. Viral RNA transfection and treatment with DAA or inhibitors were carried out as in Fig. 1b. (c) Transwell assay to assess the role of soluble cytokines in suppression of viral replication at 48 hpt. The upper wells were seeded with IRF1-deficient (indicator) PH5CH8 cells harboring a replicating HCV reporter virus genome (JFH1-QL/GLuc) that expresses GLuc, and placed in chambers in which the lower well was seeded with control versus NLRX1-deficient PH5CH8 cells stimulated by transfection with replicating HCV (JFH1-QL) RNA. (d) Replication of an HCV reporter virus assessed by GLuc expression at 48 hpt in control PH5CH8 cells versus NLRX1-T3 cells, both treated with ruxolitinib (3 μM, an inhibitor of JAK1 and JAK2), tofacitinib (1.5 μM, an inhibitor of JAK3), or no inhibitor (Null). (e,f) Impact of NLRX1 depletion on viral replication in RIG-I-deficient Huh-7.5 cells or MAVS-deficient PH5CH8 cells. Immunoblots of NLRX1 in Huh-7.5 cells tranduced with NLRX1-T3 sgRNA (e), or NLRX1-MAVS double-deficient PH5CH8 cells (f) are shown on the left. GLuc analysis of HCV replication at 72 hpt or qRT-PCR of intracellular HAV RNA at 72 hpi are shown on the right. Each symbol (throughout) represents an individual technical replicate. Data shown are representative of 2-3 independent experiments, each with 2-3 technical replicates, and are shown mean ± S.E.M. ns = nonsignificant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 by two way ANOVA (a,c) or t test (b,d-f).

Supplementary Figure 2 NLRX1 deficiency minimally impairs NF-κB signaling in virus-infected PH5CH8 cells.

(a,b) Impact of NLRX1 depletion on SeV-triggered (a) IFNB 1-Luc and (b) IRF3-responsive (4*PRDI/III)-Luc promoter activation in PH5CH8 cells. (c) Measurement of IL6 mRNA decay in SeV-infected NLRX1-T3 cells. Cells were infected with SeV for 2.5 h prior to the addition of actinomycin D (Act D, 10 μg/ml). Data are presented as percent mRNA remaining relative to the starting abundance determined by qRT-PCR (set as 100%). Data were fit to a one-phase decay model using the least-squares method. (d) Dual luciferase reporter analysis of IL6-3′UTR-Luc in PH5CH8 cells with or without NLRX1 overexpression. EV, empty vector. (e) (left) Immunoblots of p-IκBα, IκBα, p-RELA (S536) and RELA in SeV-infected NLRX1-deficient PH5CH8 cells. (right) Quantitative analysis of p-IκBα and p-RELA from three independent experiments. (f) Immunoblot confirmation of IRF3 and IRF1 depletion in PH5CH8 cells. (g) qRT-PCR quantitation of IL1B and IL6 mRNA responses after SeV infection of IRF3- and IRF1-deficient PH5CH8 cells. (h) ELISA assay for IL-6 protein abundance in IRF1- and IRF3-deficient cells infected with SeV. Unless otherwise indicated, assays were performed 3 h after infection with SeV. Each symbol represents an individual technical replicate (a,b,d,g,h) or experiment (e). Data are representative of 3 (a,b,d), 5 (c) or 2 (g,h) independent experiments, each with 3 technical replicates. Data are presented as mean ± S.E.M. Two-way ANOVA (a,b,e,g,h) and t test (d) were used for statistical analysis. ns = nonsignificant, *p < 0.05, **p < 0.01, *** P < 0.001, ****P < 0.0001.

Supplementary Figure 3 SeV infection results in MAVS and NF-κB signaling that leads to an immediate IRF1 mRNA response in PH5CH8 cells.

(a) Immunoblots showing SeV-induced IRF3 dimerization and IRF1 expression in MAVS-deficient or NLRX1-MAVS double-deficient PH5CH8 cells. (b) Immunoblots showing SeV-triggered IRF1 protein responses with (Act D) or without (Null) actinomycin D pretreatment (10 μg/ml, 30 min) in NLRX1-T3 cells. (c-f) IRF1 protein and IRF1 mRNA responses in IRF3-deficient (c), RELA-deficient (d), NFKB1-depleted (siRNA) (e) and RELA-NFKB1 double-deficient (f) PH5CH8 cells. Each symbol (c,d,f) represents an individual technical replicate. Immunoblots are representative of 2-3 independent experiments. qRT-PCR results are representative of 2 experiments, each with 2-3 technical replicates, and are presented as mean ± S.E.M. ns = nonsignificant, **p < 0.01, ****P < 0.0001 by t test.

Supplementary Figure 4 NLRX1 deficiency globally impairs protein synthesis in virus-infected cells.

(a) qRT-PCR analysis of the impact of NLRX1 deficiency on SeV-induced IRF3 mRNA response in PH5CH8 cells. (b) Immunoblots showing IRF1 protein stability in SeV-infected control and NLRX1-T3 cells. Cells were infected with SeV for 3 h followed by inhibition of nascent protein synthesis with cyclohexamide (CHX, 100 μg/ml). See also Fig. 4c in the main text. (c) Immunoblots of IRF1 expression in SeV-infected NLRX1-T3 cells with (Puro) or without (Null) puromycin (50 μg/ml) pretreatment. (d) Phosphoimager analysis of SDS-PAGE of lysates of cells pulsed with [35S]-Met/Cys from 2.5-3.0 h following SeV or mock infection. See also Fig. 4d in the main text. (e) Puromycin incorporation assays demonstrating cellular protein synthesis in mock- or SeV-infected control versus NLRX1-T2 cells. Cells were pulse-labeled with puromycin (10 μg/ml) for 10 min prior to harvest at indicated timepoints. (top panels) Immunoblot showing puromycin-labelled nascent proteins with infrared fluorescence intensity traces of puromycin immunoblots at 0 (blue) or 3 h (red) after infection. (bottom) Confocal microscopic images of pulse-labeled NLRX1-T2 cells stained with antibody specific for puromycin. Scale bar, 40 μm. (f) Similar puromycin incorporation assays in primary HFHs with partial RNAi-mediated depletion of NLRX1. (g) Puromycin incorporation assay in primary N lrx1−/− versus wild-type MEFs following SeV infection. The far right panel shows normalized infrared intensity values of immunoblots from 4 independent experiments. (h) Representative A254 traces from mock- and SeV-infected control and NLRX1-T3 cells showing a sharp reduction in 80S translationally-competent ribosomes with an increase in 40S subunits in infected NLRX1-T3 cells. Each symbol represents an individual technical replicate (a) or experiment (g). Data are representative of 3 (a,c,e,f), 4 (g) or 6 (b,d) independent experiments, and are presented as mean ± S.E.M. ns = nonsignificant, ***p < 0.001 by two-way ANOVA (a) and *p < 0.5 by t test (g).

Supplementary Figure 5 NLRX1 protects virus-induced IRF1 responses from PKR–eIF2α-mediated global translational shutdown.

(a) Immunoblots of p-eIF2α in NLRX1-deficient PH5CH8 cells 8 h after transfection of poly(I:C) or HAV RNA. (b) (left) Global protein synthesis visualized by puromycin incorporation in PKR-deficient and PKR-NLRX1 double-deficient PH5CH8 cells following SeV infection. Scale bar, 40 μm. (right) Quantitative analysis of 3 independent experiments, with an average of 110 cells evaluated for each condition. *p< 0.05 by two-way ANOVA. (c) Immunoprecipitation was performed to ascertain whether there is a physical association between endogenous NLRX1 and PKR. Ctrl, isotype control antibody. (d) Similar immunoprecipitation assay following overexpression of NLRX1 and PKR in NLRX1-T3 cells. Data are representative of 3 (a-c) and 2 (d) experiments. (e) Schematic showing why NLRX1 is required for optimal early innate immune responses to RNA virus infections in hepatocytes. RNA virus infection initiates RIG-I/MAVS-dependent signaling, leading to early (3 h) dimerization of constitutively expressed IRF3 and NF-κB-mediated IRF1 transcription. In addition to acting as a previously identified brake on MAVS/IRF3 signaling, NLRX1 dampens PKR-mediated translational shutdown by competing with PKR for binding to viral RNA, thereby preventing inhibition of IRF1 protein synthesis. Since IRF1 is dominant over IRF3 in controlling the early cytokine response to virus infection in hepatocytes, NLRX1 depletion results in reduced cytokine transcription.

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Feng, H., Lenarcic, E., Yamane, D. et al. NLRX1 promotes immediate IRF1-directed antiviral responses by limiting dsRNA-activated translational inhibition mediated by PKR. Nat Immunol 18, 1299–1309 (2017). https://doi.org/10.1038/ni.3853

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