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. 2021 Nov 4;22(11):e52948.
doi: 10.15252/embr.202152948. Epub 2021 Sep 1.

Inhibition of IRGM establishes a robust antiviral immune state to restrict pathogenic viruses

Parej Nath #  1   2 , Nishant Ranjan Chauhan #  1 , Kautilya Kumar Jena #  1 , Ankita Datey  3 , Nilima Dinesh Kumar  4 , Subhash Mehto  1 , Saikat De  3 , Tapas Kumar Nayak  3 , Swatismita Priyadarsini  1 , Kshitish Rout  1 , Ramyasingh Bal  1 , Krushna C Murmu  5 , Manjula Kalia  6 , Srinivas Patnaik  2 , Punit Prasad  5 , Fulvio Reggiori  4 , Soma Chattopadhyay  3 , Santosh Chauhan  1
Affiliations

Inhibition of IRGM establishes a robust antiviral immune state to restrict pathogenic viruses

Parej Nath et al. EMBO Rep. .

Abstract

The type I interferon (IFN) response is the major host arsenal against invading viruses. IRGM is a negative regulator of IFN responses under basal conditions. However, the role of human IRGM during viral infection has remained unclear. In this study, we show that IRGM expression is increased upon viral infection. IFN responses induced by viral PAMPs are negatively regulated by IRGM. Conversely, IRGM depletion results in a robust induction of key viral restriction factors including IFITMs, APOBECs, SAMHD1, tetherin, viperin, and HERC5/6. Additionally, antiviral processes such as MHC-I antigen presentation and stress granule signaling are enhanced in IRGM-deficient cells, indicating a robust cell-intrinsic antiviral immune state. Consistently, IRGM-depleted cells are resistant to the infection with seven viruses from five different families, including Togaviridae, Herpesviridae, Flaviviverdae, Rhabdoviridae, and Coronaviridae. Moreover, we show that Irgm1 knockout mice are highly resistant to chikungunya virus (CHIKV) infection. Altogether, our work highlights IRGM as a broad therapeutic target to promote defense against a large number of human viruses, including SARS-CoV-2, CHIKV, and Zika virus.

Keywords: CHIKV; IRGM; IRGM1; SARS-CoV-2; ZIKV.

PubMed Disclaimer

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1. Viruses and viral PAMP‐induced IRGM expression suppress IFN response
  1. A–C

    Western blot analysis of THP‐1 cells infected with (A) HSV‐1 at MOI 5 for 3 h, 6 h, 12 h, 18 h, and 24 h (B) CHIKV at MOI 5 for 3 h, 6 h, 12 h, 18 h, and 24 h (C) JEV at MOI 5 for 3 h, 6 h, 12 h, and 24 h.

  2. D–H

    Western blot analysis of THP‐1 cells untreated or treated with (D) 5’pppRNA (1 μg/ml) for 3 h, 6 h, 12 h, 18 h and 24 h (E) Poly I:C (1 μg/ml) for 3 h, 6 h, 9 h, 12 h and 24 h (F) Poly dA:dT (1 μg/ml) for 3 h, 6 h, 9 h,12 h and 24 h (G) cGAMP (1 μg/ml) for 6 h, 12 h, 18 h, and 24 h (H) dsDNA (1 μg/ml) for 3 h, 6 h, and 12 h and probed with antibodies as indicated.

  3. I–L

    Control and IRGM knockdown THP‐1 IFN reporter cells were treated with (I) Poly I:C (1 μg/ml) for 8 h, 12 h and 24 h or (J) Poly dA:dT (1 μg/ml) for 12 h and 24 h or (K) IFN‐β (500 ng/ml) for 8 h and 12 h or (L) cGAMP (1 μg/ml) for 12 h and 24 h and the supernatant was subjected to luciferase reporter assay using QUANTI‐Luc reagent (n = 3, mean ± SD, *P < 0.05, **P < 0.005, ***P < 0.0005, Student’s unpaired t‐test).

  4. M–P

    Control and IRGM knockdown THP‐1 cells were untreated and treated with Poly I:C (1 μg/ml) for 8 h and the total RNA was subjected to qRT–PCR with primers of (M) MX2 (N) ISG15 (O) OAS1 and (P) IFN‐β. (n = 3, mean ± SD, *P ≤ 0.05, **P ≤ 0.005, ***P < 0.0005, Student’s unpaired t‐test).

  5. Q–T

    Control and IRGM knockdown THP‐1 cells were untreated and treated with Poly dA:dT (1 μg/ml) for 8 h and the total RNA was subjected to qRT–PCR with primers of (Q) MX2 (R) ISG15 (S) OAS1 and (T) IFN‐β. (n = 3, mean ± SD, **P ≤ 0.005, ***P < 0.0005, Student’s unpaired t‐test).

Source data are available online for this figure.
Figure EV1
Figure EV1. Viruses and viral PAMP‐induced IRGM expression suppress the interferon response

  1. THP‐1 cells were untreated or treated with Poly I:C (1 μg/ml) for 4 h and were subjected to qRT–PCR with IRGM (n = 3, mean ± SD, **P ≤ 0.005 Student’s unpaired t‐test).

  2. THP‐1 cells untreated or treated with 5’ppp‐dsRNA (1 μg/ml) for 4 h and was subjected to qRT–PCR with IRGM (n = 3, mean ± SD, **P ≤ 0.005, Student’s unpaired t‐test).

  3. THP‐1 cells untreated or treated with cGAMP (1 μg/ml) for 4 h and was subjected to qRT–PCR with IRGM (n = 3, mean ± SD, *P < 0.05, Student’s unpaired t‐test).

  4. The graph depicts the knockdown efficiency upon transfection of control and IRGM siRNA in THP‐1 cells (n = 4, mean ± SD, ***P < 0.0005, Student’s unpaired t‐test).

Figure 2
Figure 2. Depletion of IRGM induces key viral restriction factors and antiviral mechanisms
  1. A

    Pictorial representation of stages (black font) of a typical life cycle of RNA viruses with host viral restriction factors (red font) induced in IRGM/Irgm1 depleted cells. Created using Biorender.com.

  2. B, C

    Heatmaps representing the expression pattern of viral restriction factors from RNA sequencing data in (B) control and IRGM knockdown shRNA stable HT‐29 cells (3 biological replicates) and (C) irgm1+/+ and irgm1 −/− mice BMDMs (2 biological replicates).

  3. D

    RNA was isolated from irgm1+/+ and irgm1 −/− BMDMs and subjected to qRT–PCR with indicated viral restriction factor genes (n = 3, mean ± SD, *P < 0.05, **P ≤ 0.005, ***P < 0.0005, Student’s unpaired t‐test).

  4. E

    Western blot analysis with lysates of control and IRGM +/− HT‐29 cells with indicated antibodies of viral restriction factors.

  5. F

    Western blot analysis with lysates of control or IRGM +/− HT‐29 cells or Flag IRGM complemented IRGM +/− HT‐29 cells with indicated antibodies of viral restriction factors.

  6. G

    RNA isolated from control or Flag IRGM transfected or Flag IRGM S47N transfected THP‐1 cells subjected to qRT–PCR with indicated genes. (n = 3, mean ± SD, **P < 0.005, ***P < 0.0005, Student’s unpaired t‐test).

  7. H–J

    RNA isolated from control and IRGM knockdown HT‐29 cells and subjected to qRT–PCR with indicated genes of (H) Immunoproteasome complex (I) TAP complex (J) human leukocyte antigen (HLA) system. (n = 3, mean ± SD, *P < 0.05, **P < 0.005, ***P < 0.0005, Student’s unpaired t‐test).

  8. K

    Antigen uptake assay shown by representative flow cytometry analysis of control and IRGM siRNA transfected THP‐1 cells treated with OVA conjugate AF488 (5 µg/ml, 30 min). The graph depicts the mean fluorescence intensity of control and IRGM knockdown THP‐1 cells treated with OVA conjugate AF488 (n = 3, mean ± SD, **P < 0.005, Student’s unpaired t‐test).

  9. L, M

    Antigen processing assay shown by representative confocal images of control and IRGM siRNA transfected THP‐1 cells treated with DQ‐OVA (green) (10 µg/ml, 30 min). Scale Bar, 10 µm. (M) Graph depicts percentage of control and IRGM knockdown THP‐1 cells with DQ‐OVA puncta’s (n = 3, mean ± SD, ***P < 0.0005, Student’s unpaired t‐test).

  10. N, O

    SIINFEKL based Antigen presentation assay shown by representative flow cytometry analysis of H‐2Kb‐SIINFEKL on the surface of Irgm1+/+ and irgm1 −/− mouse BMDMs treated with OVA (2 mg/ml, 3 h). (O) The graph depicts the mean fluorescence intensity of H‐2Kb‐ SIINFEKL on the surface of Irgm1+/+ and irgm1 −/− mouse BMDMs treated with OVA (2 mg/ml, 3 h) (n = 3, mean ± SE, **P < 0.005, Student’s unpaired t‐test).

  11. P

    Representative confocal images of H‐2Kb‐SIINFEKL (red) on the surface of Irgm1+/+ and irgm1 −/− mouse BMDMs treated with OVA (2 mg/ml, 3 h), Scale bar 5 µm or 8 µm (as indicated).

  12. Q

    Western blot analysis with lysates of control and IRGM siRNA knockdown HT‐29 cells with indicated antibodies of stress granules signaling pathway.

  13. R

    Representative Immunofluorescence confocal images of irgm1+/+ and irgm1 −/− mouse BMDMs immunostained with dsRNA (green) and TIA‐1 (red) antibody. Scale bar 3 µm.

Source data are available online for this figure.
Figure EV2
Figure EV2. Key antiviral mechanisms are upregulated upon IRGM depletion

  1. RNA isolated from control and IRGM knockdown HT‐29 cells and subjected to qRT–PCR with indicated viral restriction factor genes (n = 3, mean ± SD, **P < 0.005, ***P < 0.0005, Student’s unpaired t‐test).

  2. The graph depicts the knockdown efficiency of control and IRGM shRNA stable HT‐29 cells (n = 3, mean ± SD, ****P < 0.00005, Student’s unpaired t‐test).

  3. The graph depicts the IFN‐β levels in serum of Irgm1 wild‐type and KO mice (n = 3 mice each group, mean ± SD, ****P < 0.00005, Student’s unpaired t‐test).

  4. The qRT–PCR analysis of APOBEC3G, OAS1, ISG15, MX1 and IFITM3 with RNA isolated from control or Flag IRGM overexpressing HT‐29 cells (n = 3, Mean ± SD, *P < 0.05, **P < 0.005, ***P < 0.0005, Student's unpaired t‐test).

  5. The qRT–PCR analysis of OAS1, ISG15 and MX1 with RNA isolated from control or IRGM shRNA or Flag IRGM complemented IRGM shRNA HT‐29 cells. n = 3, Mean ± SD, *P < 0.05, **P < 0.005, ***P < 0.0005, Student's unpaired t‐test.

  6. Pictorial representation of sequential events of antigen uptake, processing, and presentation via Class I and Class II MHC Pathways. Created using Biorender.com.

  7. The graph depicts the knockdown efficiency upon transfection of control and si‐IRGM in THP‐1 cells (n = 3, mean ± SD, ***P < 0.0005, Student’s unpaired t‐test).

  8. Transferrin uptake assay shown by representative confocal images and flow cytometry analysis of control and si‐IRGM transfected THP‐1 cells treated with AF488 Transferrin (green) (10 μg/ml, 30 min). Graph depicts the mean fluorescence intensity of transferrin uptake in control and si‐IRGM transfected THP‐1 cells treated with AF488 transferrin. Scale, 5 μm (upper panel); Scale, 3 μm, (lower panel).

  9. Representative confocal images of H‐2Kb‐SIINFEKL (red) in Irgm1+/+ and Irgm1−/− BMDMs treated with OVA (2 mg/ml, 3 h). Scale, 25 μm.

Figure 3
Figure 3. IRGM‐depleted cells can restrict infection with CHIKV, HSV‐1, JEV, VSV, ZIKV, WNV, and SARS‐CoV‐2

  1. Total RNA was isolated from mock and CHIKV (MOI 1, 24 h) infected control and IRGM knockdown HT‐29 cells and subjected to qRT–PCR with CHIKV specific primers to quantitate total viral load (n = 3, mean ± SE, **P < 0.005, Student’s unpaired t‐test).

  2. The graph depicts quantification of CHIKV plaque assays (plaque‐forming units/ml) in Vero cells performed from the culture supernatant of CHIKV (MOI 1, 24 h) infected HT‐29 control and IRGM knockdown cell (n = 3, mean ± SE, ****P < 0.00005, Student’s unpaired t‐test).

  3. Representative images of the plaque assay in Vero cells performed from the culture supernatant of CHIKV (MOI 1, 24 h) infected HT‐29 control and IRGM knockdown cells.

  4. Total RNA was isolated from mock and HSV‐1 (MOI 1, 24 h) infected control and IRGM knockdown HT‐29 cells and subjected to qRT–PCR with HSV‐1 specific primers to quantitate total viral load (n = 3, mean ± SE, ***P < 0.0005, Student’s unpaired t‐test).

  5. The graph depicts quantification of HSV‐1 plaque assays (plaque‐forming unit/ml) in Vero cells performed from culture supernatant of HSV‐1 (MOI 1, 24 h) infected HT‐29 control and IRGM knockdown cell (n = 3, mean ± SE, ***P < 0.0005, Student’s unpaired t‐test).

  6. Representative images of plaque assay in Vero cells performed from the culture supernatant of HSV‐1 (MOI 1, 24 h) infected HT‐29 control and IRGM knockdown cells.

  7. Total RNA was isolated from mock and JEV (MOI 1, 48 h) infected control and IRGM knockdown HT‐29 cells subjected to qRT–PCR with JEV specific primers to quantitate total viral load (n = 3, mean ± SE, **P < 0.005, Student’s unpaired t‐test).

  8. The graph depicts quantification of JEV plaque assays (plaque‐forming unit/ml) in Vero cells performed from the culture supernatant of JEV (MOI 1, 24 h) infected HT‐29 control and IRGM knockdown cell (n = 3, mean ± SE, ***P < 0.0005, Student’s unpaired t‐test).

  9. Representative images of plaque assay in Vero cells performed from the culture supernatant of JEV (MOI 1, 24 h) infected HT‐29 control and IRGM knockdown cells.

  10. The graph depicts the percentage of WNV (MOI 0.3, 16 h) infected control and IRGM knockdown Huh7 cells stained with 4G2 antibody analyzed by flow cytometry (n = 3, mean ± SE, ***P < 0.0005, Student’s unpaired t‐test).

  11. The graph depicts quantification of WNV plaque assays (plaque‐forming units/ml) in Vero cells performed from culture supernatant of WNV (MOI 0.3, 16 h) infected Huh7 control and IRGM knockdown cell (n = 3, mean ± SE, ***P < 0.0005, Student’s unpaired t‐test).

  12. Representative images of plaque assay in Vero cells were performed from the culture supernatant of WNV (MOI 0.3, 16 h) infected HuH7 control and IRGM knockdown cells.

  13. The graph depicts the percentage of ZIKV (MOI 5, 48 h) infected control and IRGM knockdown HuH7 cells stained with 4G2 antibody analyzed by flow cytometry (n = 3, mean ± SD, *P < 0.05, Student’s unpaired t‐test).

  14. The graph depicts quantification of ZIKV plaque assays (plaque‐forming units/ml) in Vero cells performed from the culture supernatant of ZIKV (MOI 5, 48 h) infected Huh7 control and IRGM knockdown cells (n = 3, mean ± SD, *P < 0.05, Student’s unpaired t‐test).

  15. Representative images of plaque assay in Vero cells were performed from the culture supernatant of ZIKV (MOI 5, 48 h) infected Huh7 control and IRGM knockdown cells.

  16. Total RNA was isolated from mock and SARS‐CoV‐2 (MOI 1, 24 h) infected control and IRGM knockdown THP‐1 cells and subjected to qRT–PCR with envelope specific primers of SARS‐CoV‐2 to quantitate total viral load (n = 3, mean ± SE, **P < 0.005, Student’s unpaired t‐test).

  17. Total RNA was isolated from mock and SARS‐CoV‐2 (MOI 1, 24 h) infected control and IRGM knockdown THP‐1 cells and subjected to qRT–PCR with nucleocapsid specific primers of SARS‐CoV‐2 to quantitate total viral load (n = 3, mean ± SE, **P < 0.005, Student’s unpaired t‐test).

  18. Total RNA was isolated from the supernatant of mock and SARS‐CoV‐2 (MOI 1, 10 h and 20 h) infected control and IRGM knockdown THP‐1 cells and subjected to qRT–PCR with nucleocapsid specific primers of SARS‐CoV‐2 to quantitate total viral load (n = 3, mean ± SE, **P < 0.005, Student’s unpaired t‐test).

  19. Representative images of plaque assay in Vero E6 cells were performed from the culture supernatant of SARS‐CoV‐2 (MOI 1, 24 h) infected THP‐1 control and IRGM knockdown cells.

  20. The graph depicts quantification of SARS‐CoV‐2 plaque assays (plaque‐forming units/ml) in Vero E6 cells performed from the culture supernatant of SARS‐CoV‐2 (MOI 1, 24 h) infected THP‐1 control and IRGM knockdown cells (n = 3, mean ± SE, **P < 0.005, Student’s unpaired t‐test).

Figure EV3
Figure EV3. IRGM‐depleted cells are resistant to viral infection

  1. Total RNA was isolated from mock HT‐29 cells or CHIKV (MOI 1, 24 h) infected HT‐29 cells or Flag IRGM overexpressing HT‐29 cells and subjected to qRT–PCR with VSV specific primers to quantitate total viral load (n = 3, Mean ± SD, *P < 0.05, Student's unpaired t‐test).

  2. Total RNA was isolated from mock and VSV (MOI 2.5, 24 h) infected control and IRGM knockdown HeLa cells and subjected to qRT–PCR with VSV specific primers to quantitate total viral load (n = 3, mean ± SE, ***P < 0.0005, Student’s unpaired t‐test).

  3. Left panels, representative fluorescence microscopic images of control and IRGM knockdown HeLa (MOI 2.5) and THP‐1 (MOI 5) cells infected with VSV‐eGFP for 4 h. Right panel, the graph depicts quantitative analysis of percentage of cells that are VSV‐eGFP positive (n = 3, mean ± SD, **P < 0.005, ***P < 0.0005 Student’s unpaired t‐test).

  4. Left panels, representative fluorescence microscopic images of control or IRGM KD or Flag IRGM complemented IRGM KD THP‐1 cells infected with VSV‐eGFP (MOI 5.0) for 4 h. Scale, 400 μm. Right panel, the graph depicts VSV‐eGFP viral mRNA fold change. n = 3, Mean ± SD, *P < 0.05, Student's unpaired t‐test.

  5. Meta‐analysis using Metascape, comparing the transcriptome significantly induced in Irgm1−/− mice (> 1.5 folds; P < 0.05) and the genes induced significantly upon SARS‐CoV‐2 infection (seven different conditions from four different studies). The gene expression from different conditions and studies cluster together along with the query gene list (marked with red).

  6. Circos plot depicting the gene overlap analysis between the genes induced in Irgm1−/− mice (> 1.5 folds; P < 0.05) and the genes induced significantly upon SARS‐CoV‐2 infection (seven different conditions from four different studies). On the outside, each arc represents the identity of each gene list. On the inside, each arc represents a gene list, where each gene has a spot on the arc. Dark orange color represents the genes that appear in multiple lists and light orange color represents genes that are unique to that gene list. Purple lines link the same gene that is shared by multiple gene lists. Blue lines link the different genes where they fall into the same ontology term (the term has to be statistically significantly enriched and with a size no larger than 100). The greater the number of purple links and the longer the dark orange arcs imply greater overlap among the input gene lists. Blue links indicate the amount of functional overlap among the input gene list

  7. The heatmap depicts statistically enriched terms (GO or KEGG or Reactome) obtained when induced genes in Irgm1−/− mice (> 1.5 folds; P < 0.05) were compared with the genes induced upon SARS‐CoV‐2 infection (seven different conditions from four different studies). The heatmap cells are colored by their P‐values (P‐values are calculated based on the accumulative hypergeometric distribution, and q‐values are calculated using the Benjamini‐Hochberg procedure to account for multiple testings as described at https://metascape.org/COVID/), white cells indicate the lack of enrichment for that term in the corresponding gene list.

  8. Amplification plot of qRT–PCR analysis conducted with SARS‐CoV‐2 nucleocapsid primers and RNA isolated from the supernatant of THP‐1 cells infected with SARS‐CoV‐2 for 10 h and 20 h. The horizontal line (with values in red font) indicates threshold value of qPCR.

  9. Representative images of the plaque assay in Vero E6 cells performed from the culture supernatant of uninfected and SARS‐CoV‐2 (MOI 1, 24 h) infected THP‐1 cells.

  10. Western blot analysis with cell lysates of control and si‐IRGM transfected THP‐1 and HT‐29 cells and probed with the indicated antibodies.

  11. qRT–PCR analysis showing the percentage of viral infection in control or IRGM siRNA transfected THP‐1 cells untreated or treated with N‐acetyl‐l‐cysteine (NAC, 1 mM, 2 h) followed by VSV‐eGFP infection (MOI 5.0) for 4 h. Mean ± SD, n = 3 (biological replicates), *P < 0.05, Student's unpaired t‐test.

Source data are available online for this figure.
Figure 4
Figure 4. Irgm1 −/− mice are resistant to CHIKV infection
  1. A

    The stages of paralysis in C57BL/6 mice post CHIKV infection (MOI 1 × 107 PFU/mouse, 6 dpi) with scoring according to the severity of the disease.

  2. B

    The graph depicts percentage change in body weight of CHIKV‐infected irgm1+/+ and irgm1 −/− neonates for duration as indicated (n = 12, mean ± SD, *P < 0.05, **P < 0.005, ***P < 0.0005, Student’s unpaired t‐test).

  3. C

    The graph depicts total paralysis scores of CHIKV‐infected irgm1+/+ and irgm1 −/− neonates (n = 12) until 5 days post‐infection.

  4. D

    Kaplan–Meier survival graph depicts percentage survival of mock and CHIKV‐infected irgm1+/+ and irgm1 −/− neonates during the course of infection (n = 12, ****P < 0.0001, Log‐rank (Mantel‐Cox) test).

  5. E, F

    Total RNA isolated from muscles of mock and CHIKV‐infected (MOI 1 × 107 PFU/mouse, 6 and 11 dpi) irgm1+/+ and irgm1 −/− mice and was subjected to qRT–PCR to quantitate the viral load (n = 3, mean ± SD, ***P < 0.0005, Student’s unpaired t‐test).

  6. G

    Total RNA isolated from the brain of mock and CHIKV‐infected (MOI 1 × 107 PFU/mouse, 6 dpi) irgm1+/+ and irgm1 −/− mice and was subjected to qRT–PCR for quantitation of viral load (n = 3, mean ± SD, ***P < 0.0005, Student’s unpaired t‐test). The total RNA used for qRT–PCR with the brain is four times more than muscles.

  7. H, I

    Total RNA isolated from muscles and brains of mock and CHIKV‐infected (MOI 1 × 107 PFU/mouse) (6 dpi) irgm1+/+ and irgm1 −/− mice and was subjected to qRT–PCR with MX2 (n = 3, mean ± SD, *P < 0.05, ***P < 0.0005, Student’s unpaired t‐test). The total RNA used for qRT–PCR with the brain is four times more than muscles.

Figure 5
Figure 5. Type I IFN response is blunted in virus‐infected IRGM knockdown cells
  1. A–D

    Control and si‐IRGM transfected THP‐1 IFN reporter cells were kept uninfected (Mock) or infected with (A) JEV (MOI 5) or (B) CHIKV (MOI 5) or (C) HSV‐1 (MOI 2.5) or (D) VSV‐eGFP (MOI 1) and the supernatant collected 8 hpi were subjected to luciferase assay. The graphs depict fold change in interferon response. (n = 3, mean ± SD, *P < 0.05, **P < 0.005, ***P < 0.0005, Student’s unpaired t‐test).

  2. E–J

    Control and si‐IRGM transfected HT‐29 cells uninfected or infected with CHIKV and qRT–PCR analysis were performed with several ISG’s (E) SAMHD1 (F) HERC5 (G) ISG15 (H) viperin/RSAD2 (I) MX1 (J) APOBEC3G. (n = 3, mean ± SE, **P < 0.005, ***P < 0.0005, ****P < 0.00005, Student’s unpaired t‐test).

  3. K

    Western blot analysis with cell lysates of mock and CHIKV (MOI 5, 24 h) infected control and si‐IRGM transfected THP‐1 cells and probed with the indicated antibodies. S.E, short exposure; L.E, long exposure.

  4. L

    Western blot analysis with cell lysates of mock and CHIKV (MOI 5, 24 h) infected control and IRGM knockdown HT‐29 cells and probed with the indicated antibodies.

  5. M

    Western blot analysis with cell lysates of mock and JEV (MOI 5, 24 h) infected control and si‐IRGM transfected HT‐29 cells and probed with the indicated antibodies.

  6. N

    Western blot analysis with cell lysates of THP‐1 control or IRGM knockdown cells, untransfected or transfected with, heat‐killed whole CHIKV or CHIKV viral RNA and probed with the indicated antibodies.

  7. O

    qRT–PCR analysis to determine the knockdown efficiencies of PRR’s and adaptor proteins as indicated (n = 3, mean ± SE, ***P < 0.0005, Student’s unpaired t‐test).

  8. P

    qRT–PCR analysis with total RNA isolated from control and IRGM knockdown HT‐29 cells transfected with siRNA combinations as indicated that were infected with CHIKV (MOI 5, 24 h). (n = 3, mean ± SE, ***P < 0.0005, ****P < 0.00005, Student’s unpaired t‐test).

  9. Q

    Western blot analysis with cell lysates of mock and CHIKV (MOI 5, 24 h) infected control and IRGM knockdown HT‐29 cells transfected with indicated siRNA and probed with the indicated antibodies.

Source data are available online for this figure.
See this image and copyright information in PMC

References

    1. Bafica A, Feng CG, Santiago HC, Aliberti J, Cheever A, Thomas KE, Taylor GA, Vogel SN, Sher A (2007) The IFN‐inducible GTPase LRG47 (Irgm1) negatively regulates TLR4‐triggered proinflammatory cytokine production and prevents endotoxemia. J Immunol 179: 5514–5522 - PubMed
    1. Bastard P, Rosen LB, Zhang Q, Michailidis E, Hoffmann HH, Zhang Y, Dorgham K, Philippot Q, Rosain J, Beziat V et al (2020) Autoantibodies against type I IFNs in patients with life‐threatening COVID‐19. Science 370: eabd4585 - PMC - PubMed
    1. Blanco‐Melo D, Nilsson‐Payant BE, Liu W‐C, Uhl S, Hoagland D, Møller R, Jordan TX, Oishi K, Panis M, Sachs D et al (2020) Imbalanced host response to SARS‐CoV‐2 drives development of COVID‐19. Cell 181: 1036–1045 - PMC - PubMed
    1. Boso G, Kozak CA (2020) Retroviral restriction factors and their viral targets: restriction strategies and evolutionary adaptations. Microorganisms 8: 1965 - PMC - PubMed
    1. Boumaza A, Gay L, Mezouar S, Bestion E, Diallo AB, Michel M, Desnues B, Raoult D, La Scola B, Halfon P et al (2021) Monocytes and macrophages, targets of SARS‐CoV‐2: the clue for Covid‐19 immunoparalysis. J Infect Dis 224: 395–406 - PMC - PubMed

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