Research ArticleImmunology

IRE1α promotes viral infection by conferring resistance to apoptosis

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Science Signaling  06 Jun 2017:
Vol. 10, Issue 482, eaai7814
DOI: 10.1126/scisignal.aai7814
  • Fig. 1 Xbp1 deficiency enhances the susceptibility of MEFs to HSV and VSV.

    (A to F) WT and Xbp1−/− MEFs were infected with VSV-GFP at a multiplicity of infection (MOI) of 1 [(A) to (C)] or with HSV-1–GFP at an MOI of 10 [(D) to (F)]. Twenty-four hours later, the extent of infection was determined by measuring the relative abundance of GFP by flow cytometry. Data are from one experiment representative of three independent experiments (A and D). The mean fluorescence intensity (MFI) of GFP in the indicated cells was then determined. Data are means ± SD of three independent experiments (B and E). Viral titers in the cell culture medium were measured by plaque assay at 48 (C) and 72 (F) hours after infection. PFU, plaque-forming units. Data are means ± SD of three independent experiments (C and F). *P < 0.05; ***P < 0.001 compared to WT, unpaired t test.

  • Fig. 2 Xbp1-deficient cells are resistant to cell death during infection with VSV and HSV.

    (A to D) WT and Xbp1−/− MEFs were left uninfected (mock) or were infected with VSV-GFP [(A) and (B)] or HSV-1–GFP [(C) and (D)] for 24 hours. Cell death was then assessed with a membrane-impermeant, amine-reactive fluorescent dye, which was measured by flow cytometry. Data are from one experiment and are representative of three experiments (A and C). The percentages of dead cells were then determined. Data are means ± SD of three independent experiments (B and D). (E and F) BMDMs were cultured from Xbp1flox/flox ESR Cre+ (Xbp1Δ) or Cre littermate (WT) mice in the presence of tamoxifen. Cells were infected with VSV-GFP at the indicated MOI for 24 hours. Viability was then assessed by measuring 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reduction. Data are means ± SD of three replicates and are representative of three experiments (E and F). **P < 0.01 compared to WT, unpaired t test.

  • Fig. 3 Apoptosis induced by VSV and HSV limits viral infection.

    (A to B) WT and Xbp1−/− MEFs were left uninfected (mock) or were infected with VSV-GFP (A) or HSV-1–GFP (B) for 24 hours. Cells were then stained with an antibody specific for active (cleaved) caspase-3, which was measured by flow cytometry. Data are from one experiment and are representative of three experiments. (C) BMDMs were cultured from Xbp1flox/flox ESR Cre+ (Xbp1Δ) or Cre littermate (WT) mice in the presence of tamoxifen. Cells were infected with VSV-GFP at the indicated MOI for 7 hours. Caspase-3 activity was then assessed by measuring fluorometric substrate cleavage and is shown relative to that in uninfected cells. Data are means ± SD of three replicates and are representative of three experiments. (D to F) MEFs were infected in the presence of zVAD to inhibit caspase activity. Twenty-four hours after infection, cell death was assessed with a membrane-impermeant, amine-reactive fluorescent dye, which was measured by flow cytometry. The extent of infection was determined by measuring the relative abundance of GFP by flow cytometry. Data are from one experiment and are representative of three independent experiments. *P < 0.01 compared to WT, unpaired t test.

  • Fig. 4 Xbp1-deficient cells are resistant to the intrinsic pathway of apoptosis.

    (A and B) WT and Xbp1−/− MEFs were treated with staurosporine (sts), gliotoxin (glio), TNF and low-dose cycloheximide (TNF), Fas antibody and low-dose cycloheximide (Fas), and tunicamycin (TM) to induce the UPR, or high-dose cycloheximide (CHX). Twenty-four hours later, viability was assessed by measuring MTS reduction (A). Seven hours after treatment, caspase-3 activity was assessed by measuring fluorometric substrate cleavage and is shown relative to WT cells (B). Data are means ± SD of three replicates and are representative of three experiments. (C) BMDMs were cultured from Xbp1flox/flox ESR Cre+ (Xbp1Δ) or Cre littermate (WT) mice in the presence of tamoxifen and treated with the indicated inducers of cell death as described in (A). Twenty-four hours later, viability was assessed by measuring MTS reduction. Data are means ± SD of three replicates and are representative of three experiments. *P < 0.05; **P < 0.001 compared to WT, unpaired t test.

  • Fig. 5 The resistance of Xbp1-deficient cells to apoptosis results from the activation of IRE1α.

    (A to F) Analysis of the effects of IRE1α knockdown on cell death. (A, E, and F) WT and Xbp1−/− MEFs were transfected with siRNA targeting Ire1α or control siRNA (ctrl siRNA). Cells were then left uninfected (mock) or infected with VSV-GFP for 24 hours. Cell death was then assessed with a membrane-impermeant, amine-reactive fluorescent dye, and the relative abundance of GFP was measured by flow cytometry. Data are from one experiment and are representative of three (A) or two (E and F) independent experiments. (B and C) The indicated siRNA-transfected MEFs were left untreated (mock) or treated with staurosporine. Seven hours later, caspase-3 activity was assessed by measuring fluorometric substrate cleavage and is shown relative to that in untreated WT cells (B). Twenty-four hours after treatment, viability was assessed by measuring MTS reduction (C). (D) BMDMs were cultured from Xbp1flox/flox ESR Cre+ (Xbp1Δ) or Cre littermate (WT) mice in the presence of tamoxifen and the IRE1α inhibitor 4μ8C. Cells were then infected with VSV-GFP at an MOI of 2 or were treated with staurosporine. Viability was assessed 24 hours later by measuring MTS reduction. Data are means ± SD of three replicates and are representative of three experiments (B, C, and D). *P < 0.01; **P < 0.001, unpaired t test.

  • Fig. 6 IRE1α mediates reduction in proapoptotic miR-125a.

    (A) BMDMs were cultured from Xbp1flox/flox ESR Cre+ (Xbp1Δ) or Cre littermate (WT) mice in the presence of tamoxifen. Volcano plot demonstrating the distribution of miRNAs between WT and Xbp1Δ BMDMs measured using the NanoString nCounter assay. Data are from one experiment with quadruplicate samples. (B) BMDMs were cultured from Xbp1flox/flox ESR Cre+ (Xbp1Δ), Xbp1flox/flox Ern1 flox/flox ESR Cre+ (Xbp1Δ Ire1αΔ), or Cre littermate (WT) mice in the presence of tamoxifen. The relative abundances of Bcl-xL, Mcl-1, and β-actin in the cell lysates were determined by Western blotting and densitometry. The ratio of Bcl-xL or Mcl-1 to β-actin is shown, normalized to that in WT cells. Data are means ± SD of three independent experiments. a.u., arbitrary units. (C and D) WT and Xbp1−/− MEFs were transfected with negative control miRNA mimetic (miR-ctrl) or a miR-125a mimetic. Cells were left untreated (mock) or treated with staurosporine. Seven hours later, caspase-3 activity was assessed by measuring fluorometric substrate cleavage and is shown relative to that in untreated WT cells (C). Twenty-four hours after treatment, viability was assessed by measuring MTS reduction (D). Data are means ± SD of three replicates and are representative of two experiments. (E) The indicated miRNA-transfected MEFs were infected with VSV-GFP for 24 hours. Cell death was then assessed with a membrane-impermeant, amine-reactive fluorescent dye, which was measured by flow cytometry. The extent of infection was determined by measuring the relative abundance of GFP by flow cytometry. Data are from one experiment and are representative of two independent experiments. *P < 0.01; **P < 0.001, unpaired t test.

  • Fig. 7 IRE1α-mediated apoptosis resistance induced by HCV NS4B.

    (A) HeLa cells were transfected with constructs expressing GFP alone (vector) or together with HCV NS4B. RNA was isolated 72 hours later. XBP1 mRNA maturation from the unspliced (u) to spliced (s) form was analyzed by reverse transcription PCR (RT-PCR). Data are from one experiment and are representative of two independent experiments. (B and C) Transfected cells were treated with IRE1α inhibitor 2 for 24 hours, and staurosporine was then added. Twenty hours later, cell death was assessed with a membrane-impermeant, amine-reactive fluorescent dye, which was measured by flow cytometry. Data are from one experiment and are representative of three independent experiments (B). The percentage of dead cells among transfected GFP-positive cells was calculated (C). Data are means ± SD of three independent experiments. (D and E) Huh-7.5 cells were infected with trans-packaged HCV encoding luciferase. Forty-eight hours later, viability was determined by measuring cellular adenosine triphosphate (D). Secretion of virally encoded luciferase was measured 24 and 48 hours after infection (E). Data are means ± SD of three replicates and are representative of two experiments. (F and G) RNA was isolated from the liver tissue of HCV-infected patients (n = 11) and HCV-negative controls (n = 6). Expression of spliced XBP1 and miR-125a (relative to an internal control) was determined by quantitative RT-PCR. Data are means ± SEM. *P < 0.05; **P < 0.01, unpaired t test. ***P < 0.01, Mann-Whitney test.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/482/eaai7814/DC1

    Fig. S1. Increased IFN and ISG expression during VSV infection of Xbp1-deficient MEFs.

    Fig. S2. Knockdown of Xbp1 with siRNA mimics Xbp1 deficiency, and reconstitution with Xbp1 reverses resistance to cell death.

    Fig. S3. Xbp1 deficiency does not increase susceptibility to a noncytotoxic virus and mimics BCL2 overexpression.

    Fig. S4. Resistance to virally induced apoptosis in Xpb1-deficient cells is independent of Beclin 1.

    Fig. S5. Death of VSV- and HSV-infected cells does not require Chop.

    Fig. S6. VSV infection does not activate the UPR.

    Fig. S7. The resistance of Xbp1-deficient cells to apoptosis results from the activation of IRE1α.

    Fig. S8. The miRNA miR-125a regulates resistance to apoptosis.

  • Supplementary Materials for:

    IRE1α promotes viral infection by conferring resistance to apoptosis

    Susan L. Fink,* Teshika R. Jayewickreme, Ryan D. Molony, Takao Iwawaki, Charles S. Landis, Brett D. Lindenbach, Akiko Iwasaki*

    *Corresponding author. Email: akiko.iwasaki{at}yale.edu (A.I.); sfink{at}uw.edu (S.L.F.)

    This PDF file includes:

    • Fig. S1. Increased IFN and ISG expression during VSV infection of Xbp1-deficient MEFs.
    • Fig. S2. Knockdown of Xbp1 with siRNA mimics Xbp1 deficiency, and reconstitution with Xbp1 reverses resistance to cell death.
    • Fig. S3. Xbp1 deficiency does not increase susceptibility to a noncytotoxic virus and mimics BCL2 overexpression.
    • Fig. S4. Resistance to virally induced apoptosis in Xbp1-deficient cells is independent of Beclin 1.
    • Fig. S5. Death of VSV- and HSV-infected cells does not require Chop.
    • Fig. S6. VSV infection does not activate the UPR.
    • Fig. S7. The resistance of Xbp1-deficient cells to apoptosis results from the activation of IRE1α.
    • Fig. S8. The miRNA miR-125a regulates resistance to apoptosis.

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    Citation: S. L. Fink, T. R. Jayewickreme, R. D. Molony, T. Iwawaki, C. S. Landis, B. D. Lindenbach, A. Iwasaki, IRE1α promotes viral infection by conferring resistance to apoptosis. Sci. Signal. 10, eaai7814 (2017).

    © 2017 American Association for the Advancement of Science

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