Research ArticleANTIVIRAL IMMUNITY

The host microRNA miR-301a blocks the IRF1-mediated neuronal innate immune response to Japanese encephalitis virus infection

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Sci. Signal.  14 Feb 2017:
Vol. 10, Issue 466, pp. eaaf5185
DOI: 10.1126/scisignal.aaf5185
  • Fig. 1 miR-301a expression is induced early in JEV-infected neuronal cells.

    (A) HT22 cells were left uninfected (MI) or were infected with JEV at the indicated multiplicity of infections (MOIs) for 6 hours before the abundance of miR-301a was measured by qRT-PCR analysis and normalized to that of SNORD68 snRNA. *P < 0.05, **P < 0.01 compared to uninfected cells. (B and C) Primary neuronal cells were isolated from postnatal day 0 (P0) to P2 BALB/c mouse pups, cultured for 7 days, and exposed to JEV for the indicated times (B) or were infected with JEV at the indicated MOIs for 6 hours (C). In both cases, miR-301a abundance was quantified by qRT-PCR analysis, and the results are expressed as the fold change compared to that in uninfected cells. *P < 0.05, **P < 0.01. (D) ISH of miR-301a (purple chromogen) in neuronal cells (brown chromogen) from mouse brain. Brain samples from P10 mice were collected from MI mice or from mice 2 days postinfection (d.p.i.) with JEV, and sections were hybridized with the miRCURY LNA miR-301a probe or the LNA U6 snRNA probe, which was followed by IHC analysis of neurons with DAB (3,3’-diaminobenzidine). Scale bars, 20 μm; magnification, ×40. The ubiquitously expressed U6 snRNA was used as a positive control for miRNA ISH. Data are representative of four mice per group. (E) SH-SY5Y cells were left uninfected (MI) or were exposed to JEV for the indicated times, and the abundance of miR-301a was evaluated by qRT-PCR analysis. *P < 0.05, **P < 0.01 compared to uninfected cells. (F) SH-SY5Y cells were left uninfected (MI) or were infected with JEV at the indicated MOIs for 6 hours. Relative miR-301a abundance was then determined by qRT-PCR analysis. *P < 0.05, **P < 0.01 compared to uninfected cells. h.p.i., hours postinfection. All data in bar graphs are means ± SD of three biological replicates. P values are calculated by analysis of variance (ANOVA) followed by Bonferroni’s post hoc test.

  • Fig. 2 miR-301a inhibits the antiviral IFN response and enhances viral replication.

    (A) HT22 cells transfected with an miR-301a inhibitor (anti–miR-301a) or a negative control (anti–miR-Con) were left uninfected (MI) or were infected with JEV for the indicated times. Relative miR-301a abundance was then determined by qRT-PCR analysis. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the inhibitor control. (B and C) HT22 cells were transfected with the miR-301a inhibitor or the nonspecific inhibitor control. Twenty-four hours later, the cells were infected with JEV for the indicated times before the cells were subjected to qRT-PCR analysis of the relative abundances of IFN-α (B) and IFN-β (C) mRNAs. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the inhibitor control. (D) Cell culture medium from the cells shown in (B) and (C) was analyzed by ELISA to determine the amount of secreted IFN-β protein. **P < 0.01, ***P < 0.001 compared to the inhibitor control. (E) HT22 cells were transfected with the miR-301a inhibitor or the nonspecific inhibitor control. Twenty-four hours later, the cells were left uninfected (MI) or were infected with JEV for the indicated times before the cells were subjected to qRT-PCR analysis of the relative abundances of IFIT1, ISG15, and OAS1 mRNAs. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the inhibitor control. (F) HT22 cells were transfected with the miR-301a inhibitor or the nonspecific inhibitor control. Twenty-four hours later, the cells were infected with JEV for the indicated times before the cells were subjected to qRT-PCR analysis of the relative abundances of viral RNA. *P < 0.05, ***P < 0.001 compared to the inhibitor control. (G) HT22 cells were transfected with the miR-301a inhibitor or the nonspecific inhibitor control. Twenty-four hours later, the cells were infected with JEV for the indicated times before the cell culture medium was collected for plaque assays, which were performed as described in Materials and Methods. ***P < 0.001 compared to the inhibitor control. All data are means ± SD of three biological replicates. P values were calculated by two-way ANOVA followed by the Holm-Sidak method.

  • Fig. 3 SOCS5 and IRF1 are targets of miR-301a.

    (A and B) Predicted miR-301a binding sites in the 3′UTRs of SOCS5 mRNA (A) and IRF1 mRNA (B). Perfect matches in the seed regions are indicated in orange. (C and D) Top: Diagrams of constructs containing the 3′UTR of SOCS5 (C) or IRF1 (D) downstream of a luciferase reporter. The WT 3′UTR (WT UTR) contains an intrinsic miR-301a binding site, whereas the mutant 3′UTR (Mut UTR) contains mutations that eliminated the seed match with miR-301a. Mutations (magenta) in the 3′UTRs of both SOCS5 and IRF1 were generated for reporter gene assays. Bottom: Dual luciferase assays of HT22 cells cotransfected with firefly luciferase constructs containing the WT or mutant 3′UTRs of SOCS5 (C) or IRF1 (D) and either the miR-301a mimic (Mimic) or the control mimic (Mimic Con) were performed. Firefly luciferase activity was normalized to Renilla luciferase activity. Data are shown as the relative luciferase activity of cells transfected with the miR-301a mimic compared to that of cells transfected with the control mimic. Data are means ± SD of nine experiments from three independent transfections. **P < 0.01, ***P < 0.001, by Student’s t test. ns, not significant. (E) HT22 cells were subjected to mock transfection [mock-transfected (MT)] or were transfected with either the miR-301a miRNA mimic or the negative control mimic. Top: Twenty-four hours later, the cells were subjected to Western blotting analysis of the abundances of SOCS5 and IRF1 proteins. Bottom: Densitometric analysis of the Western blots from three experiments was performed to determine the fold changes in SOCS5 and IRF1 protein abundance (normalized to that of β-actin) relative to the MT cells. The relative abundance of miR-301a as determined by qRT-PCR analysis of each set of cells is shown above the blots to confirm effective transfection. Data are means ± SD of three independent experiments. ***P < 0.001, by one-way ANOVA followed by Bonferroni’s post hoc test. (F) HT22 cells were left untransfected (MI) or were transfected with either the miR-301a mimic or the negative control mimic. Twenty-four hours later, the cells were subjected to qRT-PCR analysis of the relative abundances of SOCS5 and IRF1 mRNAs. Data are means ± SD of three independent experiments. Statistical analysis of the data by one-way ANOVA with Bonferroni’s multiple comparisons showed that there were no statistically significant differences.

  • Fig. 4 JEV-induced miR-301a suppresses SOCS5 and IRF1 protein production.

    (A and B) HT22 cells were cotransfected with either the miR-301a inhibitor (anti–miR-301a) or the inhibitor control (anti–miR-Con) together with a firefly luciferase reporter plasmid encoding the WT or mutant 3′UTRs of SOCS5 (A) or IRF1 (B). Twenty-four hours later, the cells were infected with JEV for 6 hours before luciferase activities were measured with a dual luciferase assay kit and normalized to that of Renilla luciferase. Data are expressed as the relative luciferase activity of the anti–miR-301a–transfected cells compared to that of the anti–miR-Con–transfected cells. Data are means ± SD of nine experiments from three independent transfections. **P < 0.01, by Student’s t test. (C and D) HT22 cells were transfected with either the miR-301a inhibitor or the inhibitor control and then were either infected with JEV at an MOI of 5 for the indicated times (C) or infected for 6 hours with JEV at the indicated MOIs (D). Top: The cells were analyzed by Western blotting to determine the relative abundances of SOCS5 and IRF1 proteins. β-Actin was used as a loading control. Western blots are representative of three independent experiments. The relative abundance of miR-301a in each set of cells was determined by qRT-PCR analysis and is shown below the blots to confirm effective transfection. Bottom: Densitometric analysis of the Western blots was performed to determine the fold changes in the indicated protein abundances (normalized to that of β-actin) in infected cells compared to those in uninfected (MI) cells. Data are means ± SD of three individual experiments. *P < 0.05, **P < 0.01, ***P < 0.001, by one-way ANOVA followed by Bonferroni’s post hoc test.

  • Fig. 5 miR-301a inhibits the IFN response by repressing SOCS5 and IRF1 protein production.

    (A to E) HT22 cells were left untransfected (MT) or were transfected with anti–miR-301a together with Con-esiRNA or esiRNAs specific for SOCS5 or IRF1 alone or in combination before being infected with JEV at an MOI of 5. Six hours after infection, the relative abundances of IFN-β (A), IFIT1 (B), ISG15 (C), OAS1 (D) mRNAs, and viral RNA (E) were quantified by qRT-PCR analysis. (A) Cells were also subjected to Western blotting (WB) analysis of SOCS5 and IRF1 abundances to confirm effective transfection of the cells. Bar graphs show densitometric analysis of the fold changes in the abundance of the indicated proteins (normalized to that of β-actin) compared to that in the MT, JEV-infected cells. ##P < 0.01, ###P < 0.001 compared to MT cells; *P < 0.05, **P < 0.01, ***P < 0.001 compared to cells transfected with anti–miR-301a and Con-esiRNA. (F) HT22 cells transfected as described in (A) to (E) were infected with JEV for 48 hours before the culture medium was collected for plaque assays. Data are means ± SD of three independent experiments. #P < 0.05 compared to MT cells; *P < 0.05 compared to cells transfected with anti–miR-301a and Con-esiRNA. (G to K) HT22 cells were left untransfected (MT) or were transfected with the indicated combinations of plasmids and anti–miR-301a. Twenty-four hours later, the cells were infected with JEV at an MOI of 5. At 6 hours after infection, the relative abundances of IFN-β (G), IFIT1 (H), ISG15 (I), and OAS1 (J) mRNAs, and of viral RNA (K) were measured by qRT-PCR analysis. (G) Cells were also subjected to Western blotting analysis of SOCS5 and IRF1 abundances to confirm effective transfection of the cells. Bar graphs show densitometric analysis of the fold changes in the abundance of the indicated proteins (normalized to that of β-actin) compared to that in the MT, JEV-infected cells. #P < 0.05, ##P < 0.01, ###P < 0.001 compared to untransfected cells (MT); *P < 0.05, **P < 0.01, ***P < 0.001 compared to cells transfected with anti–miR-301a and Con-vector. (L) HT22 cells that were transfected as described in (G) to (K) were infected with JEV for 48 hours before the culture medium was collected for plaque assays. Data are means ± SD of three independent experiments. ##P < 0.01 compared to untransfected cells (MT); *P < 0.05, **P < 0.01, ***P < 0.001 compared to cells transfected with anti–miR-301a and Con-vector. P values were calculated by one-way ANOVA followed by Bonferroni’s post hoc test.

  • Fig. 6 Loss of SOCS5 facilitates the suppression of IRF1 production through EGFR activation.

    (A) HT22 cells were left uninfected (MI) or were infected with JEV at an MOI of 5 for 24 hours. Top: Cells were analyzed by Western blotting with antibodies against the indicated proteins. Bottom: Densitometric analysis was performed to show the fold change in SOCS5 protein abundance (normalized to that of β-actin) in infected cells compared to that in uninfected cells (MI). *P < 0.05, **P < 0.01, ***P < 0.001 compared to uninfected cells. (B and C) HT22 cells were left untransfected or were transfected with either SOCS5-specific or Con-esiRNA (B) or with either the SOCS5 expression plasmid or the control plasmid (C). Top: Twenty-four hours later, the cells were infected with JEV for 6 hours before the relative abundance of IRF1 mRNA was quantified by qRT-PCR analysis. ***P < 0.001 compared to untransfected cells. Bottom: Transfection efficiency was determined by Western blotting analysis of SOCS5 abundance. Histograms show the densitometric analysis of the fold change in SOCS5 protein abundance (normalized to that of β-actin) in transfected cells compared to that in untransfected cells. **P < 0.01 compared to untransfected cells. (D) HT22 cells were left untransfected or were transfected with the indicated esiRNAs or expression plasmids before being infected with JEV for 6 hours. Top: Samples were then analyzed by Western blotting with antibodies against the indicated proteins. Equivalent gel loading was confirmed by analysis with an antibody against β-actin. Bottom: Densitometric quantification of the abundance of p-EGFR normalized to that of β-actin. **P < 0.01, ***P < 0.001. (E and F) HT22 cells were treated with 10 μM AG 1478 or vehicle before being left uninfected of infected with JEV for 6 hours. The relative abundance of IRF1 mRNA and viral RNA were analyzed by qRT-PCR (E). **P < 0.01, ***P < 0.001 compared to untreated control cells; ##P < 0.01 compared to JEV-infected cells. In addition, the amount of IFN-β secreted by the indicated cells was measured by ELISA (F). **P < 0.01 comparison to untreated control cells; ##P < 0.01 compared to JEV-infected cells. (G) HT22 cells treated with or without 10 μM AG 1478 were infected with JEV for the indicated times before the culture medium was collected for plaque assays. All data are means ± SD of three biological replicates. P values were obtained by one-way ANOVA followed by Bonferroni’s multiple comparisons.

  • Fig. 7 JEV induces miR-301a expression through the activation of NF-κB.

    (A) HT22 cells were left uninfected (MI) or were infected with JEV at an MOI of 5 for the indicated times. Cells were then analyzed by Western blotting with antibodies specific for the indicated proteins. β-Actin served as a loading control. Western blots are representative of three independent experiments. (B) Densitometric analysis of the indicated bands in the Western blots shown in (A). Fold changes in the abundance of the indicated proteins (normalized to that of β-actin) are shown relative to those in uninfected cells. Data are means ± SD of three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to uninfected cells. Data were analyzed by one-way ANOVA followed by Bonferroni’s post hoc test. (C) Schematic representation of the predicted RelA binding site in the miR-301a promoter region within the host gene Ska2. The WT sequence GGTTCTTCCC in the miR-301a promoter was replaced by GGTTCTTAAA to generate the mutant construct. (D) Top: HT22 cells were transfected with either the WT or mutant miR-301a promoter construct alone or together with plasmid encoding Flag-tagged RelA (RelA cFlag pcDNA3). Twenty-four hours later, promoter activity was analyzed by dual luciferase assay. Firefly luciferase activity was normalized to Renilla luciferase activity, and the fold change in luciferase activity compared to that in cells transfected with the empty plasmid pGL3 was determined. Data are means ± SD of nine experiments from three independent transfections. **P < 0.01 compared to plasmid control, by one-way ANOVA with Bonferroni’s corrections. Bottom: Transfection efficiency was determined by Western blotting analysis of Flag-p65 abundance. (E) HT22 cells were transfected with the WT or mutant miR-301a promoter reporter. Six to 8 hours later, the cells were left uninfected or were infected with JEV at an MOI of 5 for the indicated times before luciferase assays were performed. The relative luciferase activity of the mutant reporter compared to that of the WT reporter after normalization to Renilla luciferase was determined. Data are means ± SD nine experiments from three independent transfections. ***P < 0.001 by two-way ANOVA followed by the Holm-Sidak method. (F) HT22 cells pretreated with or without 50 μM PDTC were left uninfected or were infected with JEV for 6 hours. Nuclear and cytoplasmic fractions of the cells were then isolated and analyzed by Western blotting with antibodies specific for the indicated proteins. PCNA and β-actin served as loading controls for the nuclear and cytoplasmic extracts, respectively. Values below the blots show miR-301a abundance in the corresponding samples as determined by qRT-PCR analysis. (G) Densitometric analysis of the indicated bands in the Western blots shown in (F). The fold changes in the abundance of the indicated proteins (normalized to that of the appropriate loading control) compared to those in uninfected control cells were determined. Data are means ± SD of three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 compared to JEV-infected, untreated cells, as analyzed by one-way ANOVA followed by Bonferroni’s post hoc test. (H to J) HT22 cells were left uninfected or were infected with JEV at an MOI of 5 for 6 hours before being subjected to chromatin immunoprecipitation (ChIP) assay with an anti–p-p65 antibody to measure recruitment to the miR-301a promoter. The nuclear extract before treatment with antibody served as input. (I) PCR analysis of the DNA isolated by ChIP compared to input DNA with primers flanking the RelA binding site in the predicted miR-301a promoter region. (J) The relative abundance of the miR-301a promoter fragment in the indicated samples, normalized to that in the input, was determined by qRT-PCR analysis. ***P < 0.001 compared to the uninfected cells, as determined by Student’s t test. Data are means ± SD of three independent experiments, each with triplicate measurements.

  • Fig. 8 Inhibition of miR-301a in vivo restores the IFN response and improves survival in JEV-infected mice.

    (A) Stem-loop sequence of pre–miR-301a. Bottom: Sequences of the miR-301a Vivo-Morpholino (miR-301a-VM), which targets mature miR-301a (blue), and a scrambled Vivo-Morpholino that was designed as a negative control (VM-NC). (B) BALB/c mice were intracranially treated with miR-301a-VM or VM-NC (18 mg/kg) at the indicated times after they were infected with JEV (3 × 105 PFU) and then were observed daily to determine their survival rate. The asterisk represents comparison between the JEV/VM-NC group versus the JEV/miR-301a-VM 12 h.p.i. and JEV/miR-301a-VM 24 h.p.i. groups. P values were determined by one-way ANOVA followed by Bonferroni’s post hoc test. (C) Brain samples from the mice shown in (B) were collected on day 3 (72 h.p.i.) of infection, and their abundance of miR-301a was quantified by qRT-PCR analysis. Data are represented as the fold change in miR-301a abundance (normalized to that of SNORD68) in the infected samples relative to that in the uninfected samples. Data are means ± SD of four mice from each group. **P < 0.01, ***P < 0.001 compared to the JEV + VM-NC group, as analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons. (D) Viral loads in the brain tissues of the mice shown in (B) were evaluated by plaque assay in porcine kidney cells. Plaque assays were performed twice, and the data are means ± SD of three mice from each group. *P < 0.05 when compared to the JEV + VM-NC group, as determined by one-way ANOVA with Bonferroni’s correction. (E to G) Mice were left uninfected or were infected with JEV in the presence of VM-NC or were infected with JEV and treated with miR-301a-VM at the indicated times. Three days after infection, brains were collected from the mice and analyzed by qRT-PCR to determine the abundances of viral RNA (E), IFN-β mRNA (F), and IFIT1, ISG15, and OAS1 mRNAs (G). Data are means ± SD of four mice from each group. *P < 0.05, **P < 0.01, ***P < 0.001 when compared to the VM-NC group. Data were analyzed by one-way ANOVA followed by Bonferroni’s post hoc test. (H) Brain samples from the mice described in (E) to (G) were analyzed by Western blotting with antibodies specific for SOCS5 and IRF1. β-Actin served as loading control. Blots are representative of four mice from each group. (I and J) Densitometric analysis of the blots shown in (H). The histograms show the fold changes in SOCS5 (I) and IRF1 (J) protein abundance (normalized to that of β-actin) in infected samples compared to those in uninfected samples. Data are means ± SD of four mice from each group. ##P < 0.01, ###P < 0.001, **P <0.01, ***P < 0.001, as calculated by one-way ANOVA followed by Bonferroni’s multiple comparisons.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/466/eaaf5185/DC1

    Fig. S1. Expression profile of miR-301 family members during JEV infection.

    Fig. S2. miR-301a inhibits the antiviral type I IFN response and increases viral replication.

    Fig. S3. SOCS5 and IRF1 are targets of miR-301a.

    Fig. S4. Suppression of SOCS5 and IRF1 protein production is reversed by inhibition of miR-301a.

    Fig. S5. Loss of SOCS5 protein early during JEV infection.

    Fig. S6. JEV induces miR-301a expression through the activation of NF-κB.

    Fig. S7. In vivo dose standardization and treatment with miR-301a-VM in JEV-infected mice.

    Fig. S8. Aberrant regulation of miR-301a expression during JEV infection in mouse liver and kidney.

    Fig. S9. The regulation of miR-301a expression is not ubiquitous in the peripheral organs of JEV-infected mice.

    Fig. S10. The role of miR-301a early in JEV infection.

    Fig. S11. Examples of Western blots used for densitometric analysis.

    Fig. S12. Further examples of Western blots used for densitometric analysis.

    Table S1. Potential targets of mouse miR-301a.

    Table S2. Prediction of transcription factor binding sites within the miR-301a promoter by PROMO software.

    Table S3. Primer sequences.

  • Supplementary Materials for:

    The host microRNA miR-301a blocks the IRF1-mediated neuronal innate immune response to Japanese encephalitis virus infection

    Bibhabasu Hazra, Kanhaiya Lal Kumawat, Anirban Basu*

    *Corresponding author. Email: anirban{at}nbrc.ac.in

    This PDF file includes:

    • Fig. S1. Expression profile of miR-301 family members during JEV infection.
    • Fig. S2. miR-301a inhibits the antiviral type I IFN response and increases viral replication.
    • Fig. S3. SOCS5 and IRF1 are targets of miR-301a.
    • Fig. S4. Suppression of SOCS5 and IRF1 protein production is reversed by inhibition of miR-301a.
    • Fig. S5. Loss of SOCS5 protein early during JEV infection.
    • Fig. S6. JEV induces miR-301a expression through the activation of NF-κB.
    • Fig. S7. In vivo dose standardization and treatment with miR-301a-VM in JEV-infected mice.
    • Fig. S8. Aberrant regulation of miR-301a expression during JEV infection in mouse liver and kidney.
    • Fig. S9. The regulation of miR-301a expression is not ubiquitous in the peripheral organs of JEV-infected mice.
    • Fig. S10. The role of miR-301a early in JEV infection.
    • Fig. S11. Examples of Western blots used for densitometric analysis.
    • Fig. S12. Further examples of Western blots used for densitometric analysis.
    • Table S1. Potential targets of mouse miR-301a.
    • Table S2. Prediction of transcription factor binding sites within the miR-301a promoter by PROMO software.
    • Table S3. Primer sequences.

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    Citation: B. Hazra, K. L. Kumawat, A. Basu, The host microRNA miR-301a blocks the IRF1-mediated neuronal innate immune response to Japanese encephalitis virus infection. Sci. Signal. 10, eaaf5185 (2017).

    © 2017 American Association for the Advancement of Science

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