Galectin-9 is a restrictor of infection with multiple enteroviruses

Figures

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  Fig 1. Gal-9 exerted an inhibitory effect on EV-A71, EV-D68, and coxsackievirus B2 (CV-B2), but not on poliovirus 3 (PV3) in cells. (A) LGALS9-KO and control A549 cells were stimulated with 50 U/mL IFN-β. After 24 h stimulation, cells were infected with 0.1 MOI EV-A71 for 12 h. Cell lysates were collected and subjected to WB with antibodies against VP0/VP2, Gal-9, and β-actin. (B) LGALS9-KO and control A549 cells were stimulated with 50 U/mL IFN-β. After 24 h stimulation, cells were infected with 0.5 MOI EV-D68 for 12 h. Cell lysates were subjected to WB with antibodies against VP1, Gal-9, and β-actin. (C) LGALS9-KO and control A549 cells were infected with EV-A71, CV-B2, PV3, and EV-D68 for 24 h. Supernatants were collected, and viral titers were detected by using the TCID50 assay. (D) LGALS9-KO and control A549 cells were infected with EV-A71, CV-B2, PV3, and EV-D68. At 24 h post-infection, the cells were immunostained with dsRNA (green) and DAPI. Cell images were scanned using a Leica TCS SP8. Eighteen fields were imaged to calculate the percentages in each well. Scale bar, 5 μm. (E) LGALS9-KO and control THP-1 cells, which were differentiated into macrophages using PMA, were…

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  Fig 2. Gal-9 exhibits an inhibitory effect on viral replication at 4 h post-EV-A71 infection. (A) Schematic of EV-A71 infection in LGALS9-KO and control A549 cells. EV-A71 was allowed to bind to the cells on ice for 1 h. After washes, infection was allowed to proceed by incubating the cells at 37°C and collecting at indicated times. (B and C) LGALS9-KO and control A549 cells were infected with 0.5 MOI EV-A71 and performed as indicated in panel A. WB (B) and qRT-PCR (C) were performed. (D) Schematic of EV-A71 infection in LGALS9-KO A549 cells with Gal-9 transfection. LGALS9-KO A549 cells transfected with isoform Δ5 of Gal-9-Flag plasmids. After 24 h transfection, 0.5 MOI EV-A71 was allowed to bind to the cells on ice for 1 h. After washes, infection was allowed to proceed by incubating the cells at 37°C and collecting them at indicated times. (E and F) LGALS9-KO A549 cells were transfected with Gal-9 plasmids and infected with 0.5 MOI EV-A71 and performed as indicated in panel E. WB (E) and qRT-PCR (F) were performed. All experiments were done at least twice, and one representative is shown. Error bars indicate SD of technical triplicates. P-values of the two-tailed Student’s t-test are…

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  Fig 3. Gal-9 restricted EV-A71 replication. (A) Schematic of EV-A71 RNA transinfection in LGALS9-KO and control A549 cells. The full-genome RNA of EV-A71 was transcribed in vitro and purified viral RNA was transfected into LGALS9-KO and control A549 cells. After transfection, the cells were incubated at 37°C and collected at the indicated times. (B) Viral RNAs were detected by using qRT-PCR. The experiment was done at least twice, and one representative is shown. Error bars indicate SD of technical triplicates. P-values of the two-tailed Student’s t-test are indicated. (C) 293T cells were transfected with a vector or isoform Δ5 of Gal-9-Flag expression plasmid. At 24 h post-transfection, the cells were infected with 1 MOI EV-A71 for 12 h and then immunostained with the indicated antibodies. Scale bar, 5 μm. Colocalization between Gal-9 and 3A of the Manders’ colocalization coefficient above auto-threshold was indicated (at least 10 cells from two independent experiments).

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  Fig 4. Gal-9 interacted with VP2 of EV-D68, EV-A71, but not PV3. (A) Protein–protein interactions of Gal-9 with host factors (gray) or viral protein (green) after EV-D68 infection are shown. (B) 293T cells were transfected with a vector, isoform Δ5 of Gal-9-Flag and EV-D68 VP2-HA. Cell lysates were IP with anti-Flag antibody. Immunoprecipitates and aliquots of cell lysates were subjected to WB with antibodies against Flag, HA, and β-actin. (C) 293T cells were transfected with a vector, isoform Δ5 of Gal-9-HA and Flag-EV-A71 VP2, Flag-CV-B2 VP2, or Flag-PV3 VP2. Cell lysates were immunoprecipitated (IP) with anti-HA antibody. Immunoprecipitates and aliquots of cell lysates were subjected to WB with antibodies against Flag, HA, and β-actin. (D) 293T cells were transfected with isoform Δ5 of Gal-9-HA and/or Flag-EV-A71 VP2 expression plasmid. At 24 h post-transfection, the cells were immunostained with the indicated antibodies. Scale bar, 5 μm. Colocalization between Gal-9 and VP2 using the Manders’ colocalization coefficient above auto-threshold was indicated (at least 10 cells from two independent experiments).

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  Fig 5. Gal-9 interacted with VP2 to restrict EVs replication. (A) Schematic of the isoform Δ5 of Gal-9, which contains an NCRD domain, a CCRD domain, and a linker region. (B) 293T cells were transfected with a vector, Flag-EV-A71 VP2, and Gal-9-HA, NCRD (1–148), or CCRD (195–323) without the linker region. Cell lysates were IP with anti-HA antibody. Immunoprecipitates and aliquots of cell lysates were subjected to WB with antibodies against Flag, HA, and β-actin. (C) 293T cells were transfected with a vector, Flag-EV-A71 VP2, and Gal-9-HA, NCRD, or CCRD with the linker region. Cell lysates were IP with anti-HA antibody. Immunoprecipitates and aliquots of cell lysates were subjected to WB with antibodies against Flag, HA, and β-actin. (D) 293T cells were transfected with a vector, Flag-EV-A71 VP2, and Gal-9-HA or truncation mutants. Cell lysates were IP with anti-HA antibody. Immunoprecipitates and aliquots of cell lysates were subjected to WB with antibodies against Flag, HA, and β-actin. (E) RD cells were transfected with a vector, Gal-9-HA, or Δ179–194 truncation mutant for 24 h. Cells were then infected with 0.5 MOI of EV-A71. At 24 h after infection, cell lysates were analyzed by WB…

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  Fig 6. Gal-9 is important for EV-A71 infection in mice. Three-day-old suckling WT and LGALS9−/− mice were challenged with 3.5 × 106 pfu EV-A71 (strain SHAPHC 695F) or Dulbecco’s modified Eagle’s medium by intraperitoneal route. The mRNA levels of LGALS9 in mock or EV-A71-infected mice were detected using qRT-PCR in the small intestine (A), hind-limb skeletal muscle (B), and lung (C) at 5 days post-infection. The survival rates (D), clinical scores (E), and body weights (F) of the infected mice were monitored. * Compared to the mock-infected group, LGALS9−/− mice with EV-A71 infection exhibited delayed weight gain at 8 and 9 days post-infection. P = 0.0126 and P = 0.0157. The viral load of EV-A71 present in the small intestine (G), spinal cord (H), hind-limb skeletal muscle (I), and lung (J) at 5 days post-infection of the indicated mice was detected using qRT-PCR. Concentrations of TNF-α in the small intestine (K), serum (L), and lung (M) of EV-A71- or mock-infected 3-day-old suckling WT and LGALS9−/− mice were determined by ELISA. The samples were collected at 5 days post-infection. The red horizontal dotted line indicates the limit of detection for this assay (7 pg/mL).