Inhibition of lysosome-tethered Ragulator-Rag-3D complex restricts the replication of Enterovirus 71 and Coxsackie A16

Figures

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  Figure S1.. Loss of Ragulator components blocks EV71-induced apoptosis and pyroptosis. (A) Schematic diagram of the genome-wide CRISPR screening process and Western blot analysis of Cas9 expression in HeLa cells. (B and C) Sequencing analysis of knockout efficiency of LAMTOR3 and LAMTOR1. (D–F) LAMTOR2 and LAMTOR4 were silenced in HeLa cells using siRNAs and then the indicated HeLa cells were infected with EV71 (MOI = 5) 60 h after transfection. The knockdown efficiency of LAMTOR2 (D) and LAMTOR4 (E) was determined by qPCR 60 h after transfection. Cell death induced by EV71 was detected by measuring ATP levels (D and E) and PI staining at 18 h after infection (F). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. Source data are available for this figure: SourceData FS1.

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  Figure 1.. Loss of Ragulator components blocks EV71-induced apoptosis and pyroptosis. (A) A genome-wide CRISPR screening identified a total of 13 genes in which more than three sgRNAs were expressed at least fivefold higher in EV71-infected cells compared to mock-infected cells (left panel). In addition to LAMTOR3, other hits were identified that were either involved in enterovirus replication or acted as enterovirus receptors (right panel). The light red color represents genes that serve as receptors for enteroviruses. The blue color represents genes associated with enterovirus replication. The deep red color represents LAMTOR3. (B) LAMTOR3+/+ or LAMTOR3−/− HeLa cells were infected (MOI = 5) with EV71 for 18 h and cell death was determined by measuring ATP levels (upper panel). The KO efficiency of LAMTOR3 in HeLa cells was assessed by Western blot analysis (lower panel). All values are means ± SEM. Data were analyzed using a two-tailed Student’s t test. (C) LAMTOR3+/+ or LAMTOR3−/− HeLa cells stably transfected with an empty vector (LAMTOR3−/−-1+Vector) or a vector expressing LAMTOR3 (LAMTOR3−/−-1+LAMTOR3) were infected with EV71 (MOI = 5) for 18 h and cell death was determined by…

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  Figure 2.. The Ragulator-Rag complex is required for EV71 replication. (A and B) Wild-type, LAMTOR1−/− (A), and LAMTOR3−/− (B) HeLa cells were infected with EV71 (MOI = 5) and the viral titers were determined by TCID50 assay at 12, 24, and 36 h after infection. (C–F) Wild-type, LAMTOR1−/−, and LAMTOR3−/− HeLa cells were incubated with EV71 (MOI > 100) at 4°C for 1 h. For the virus binding assay, cells were washed with 1×PBS three times, then total cellular RNA was extracted and viral RNA was quantified by qPCR (C and D). For the virus entry assay, these cells were then transferred to a 37°C incubator for 20 min after 1 h of incubation at 4°C and were rinsed three times with 1×PBS-HCl. Total RNA was extracted, and viral RNA was quantified by qPCR (E and F). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (G and H) Wild-type, LAMTOR1−/− (G), and LAMTOR3−/− (H) HeLa cells were infected with EV71 (MOI = 5). Viral RNA was quantified by qPCR 5 h after infection. VP1 expression was analyzed by Western blot 12 h after infection. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (I) SK-N-SH cells were transfected with LAMTOR3 siRNAs…

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  Figure S2.. The Ragulator-Rag complex is required for EV71 replication. (A) LAMTOR3+/+ or LAMTOR3−/− RD cells were infected with EV71 (MOI = 2). The viral RNA was quantified by qPCR 5 h after infection and the VP1 protein was analyzed by Western blot 8 h after EV71 infection. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (B) WT and LAMTOR3-KO cells were infected with EV71 for 24 h. The virus titers in the culture supernatant and cells were measured by TCID50 assay. The release rate represents the proportion of virus in the supernatant relative to the total virus in WT cells and LAMTOR3-KO cells. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (C) Sequencing analysis of knockout efficiency of RagB. (D and E) RagB+/+ and RagB−/− HeLa cells were incubated with EV71 (MOI > 100) at 4°C for 1 h. For the virus binding assay, cells were washed with 1×PBS three times, then total cellular RNA was extracted and viral RNA was quantified by qPCR (D). For the virus entry assay, these cells were transferred to a 37°C incubator for 20 min after 1 h of incubation at 4°C and then rinsed three times with 1×PBS-HCl. Total RNA was extracted…

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  Figure 3.. The Ragulator-Rag complex recruits EV71 3D protein and PI4KB. (A) HeLa cells were infected with EV71 (MOI = 5) for 6 h and then the colocalization of LAMTOR3, RagB, and 3D was examined by staining cells with anti-LAMTOR3/RagB/3D antibodies followed by confocal microscopy analysis. Nuclei were stained with DAPI. Scale bars, 10 μm. Inset panels are magnified 4×. (B) GST pull-down assay of in vitro translated Flag-tagged 3D and GST-tagged RagB/LAMTOR3. The quantitation analysis of Western blot was performed using ImageJ. (C) The plasmid encoding HA-3×FLAG-RagB and the plasmid encoding GFP-tagged 3D were cotransfected into 293T cells. Cell lysates were collected after 36 h for IP with anti-FLAG agarose and the interaction between 3D and RagB was detected by Western blotting. The quantitation analysis of Western blot was performed using ImageJ. (D) RD cells were harvested 6 h after EV71 infection and analyzed using the in situ PLA. Representative images of in situ PLA show the endogenous interaction between RagB and 3D (red). Nuclei were stained with DAPI. Scale bars, 10 and 40 μm. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (E) HeLa cells were…

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  Figure S3.. The Ragulator-Rag complex recruits EV71 3D protein and PI4KB. (A) HeLa cells were mock infected or infected with EV71 (MOI = 5) and the changes in cellular morphology were observed under a microscope at 6 h after infection. Scale bars, 200 μm. (B) SK-N-SH cells were mock infected or infected with EV71 (MOI = 5) for 12 h. Colocalization of RagB, LAMTOR3, and 3D was detected by staining cells with anti-RagB/LAMTOR3/3D antibodies and visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm. (C–G) GST pull-down assay of in vitro translated Flag-tagged 3D and GST-tagged RagA, RagC, LAMTOR1, LAMTOR2, and LAMTOR4. The quantitation analysis of Western blot was performed using ImageJ. (H) Schematic diagram of EV71 3D protein binding to Ragulator-Rag complex. (I) SK-N-SH cells were mock-infected or infected with EV71 (MOI = 5) for 12 h. Colocalization of RagB, LAMP1, and 3D was detected by staining cells with anti-LAMP1/RagB/3D antibodies and visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm. Source data are available for this figure: SourceData FS3.

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  Figure 4.. Disrupting lysosomal tethering of the Ragulator-Rag-3D complex inhibits EV71 replication. (A) Co-localization of LAMTOR1 with LAMP1 was assessed by confocal microscopy in indicated HeLa cells (LAMTOR1−/−-1+LAMTOR1-FLAG and LAMTOR1−/−-1+ LAMTOR1-3A-FLAG). FLAG (green), LAMP1 (red), and DAPI-stained nuclei are shown. Scale bars, 10 μm (left panel). The Pearson’s correlation coefficient of FLAG and LAMP1 were analyzed by Image pro plus 6.0 (right panel). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (B) Western blot analysis of the ectopic expression of LAMTOR1 and LAMTOR1-3A in LAMTOR1−/− cells. (C) LAMTOR1−/−-1+LAMTOR1-FLAG and LAMTOR1−/−-1+ LAMTOR1-3A-FLAG HeLa cells were infected with EV71 (MOI = 5 and MOI = 40, respectively) for 6 h. Co-localization of 3D with LAMP1 was assessed by staining cells with anti-LAMP1 and anti-3D antibodies followed by visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm (left panel). The Pearson’s correlation coefficient of 3D and LAMP1 was analyzed by Image pro plus 6.0 (right panel). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (D and E) The…

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  Figure 5.. The lysosomal tethered Ragulator-Rag complex mediates CVA16 replication. (A–C) Wild-type, LAMTOR1−/−-1 (A), LAMTOR3−/−-1 (B), and RagB−/−-1 (C) HeLa cells were infected with CVA16 (MOI = 5). Viral RNA was quantified by qPCR 3 h after infection (upper panel). Expression of viral 3D was detected by Western blot analysis 12 h after infection (lower panel). The quantitation analysis of Western blot was performed using ImageJ. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (D–F) Wild-type, LAMTOR1−/−-1 (D), LAMTOR3−/−-1 (E), and RagB−/−-1 (F) HeLa cells were infected with CVA16 (MOI = 5). Cell viability was determined by measuring ATP levels 24 h after infection. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (G–I) Wild-type, LAMTOR1−/−-1 (G), LAMTOR3−/−-1 (H), and RagB−/−-1(I) HeLa cells were infected with CVA16 (MOI = 5). Cleavage of GSDME and caspase3 were analyzed by Western blot 12 h after infection. (J) LAMTOR1−/−-1+LAMTOR1-FLAG and LAMTOR1−/−-1+3A-FLAG HeLa cells were infected with CVA16 (MOI = 5 and MOI = 20, respectively). Colocalization of 3D with LAMP1 was detected by staining cells with anti-LAMP1 and…

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  Figure S4.. The lysosomal tethered Ragulator-Rag complex mediates CVA16 replication. (A and B) LAMTOR3+/+ and LAMTOR3−/−-1, LAMTOR3−/−-2 HeLa cells were infected with HSV (MOI = 2; A) or VSV (MOI = 0.5; B) and viral titers were determined by TCID50 assay 24 h after infection. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test.

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  Figure 6.. ZHSI-1 is a novel EV71 inhibitor that interacts with 3D and blocks the replication of EV71 and CVA16. (A) ZHSI-1 inhibits EV71-induced cell death. The schematic shows the chemical structure of ZHSI-1. (B) RD cells were pretreated with the indicated concentrations of ZHSI-1 and then infected with EV71 (MOI = 2). Cell viability was determined by measuring ATP levels 24 h after infection. (C and D) RD cells were pretreated with ZHSI-1 for 1 h and then infected with EV71 (MOI = 2) or CVA16 (MOI = 2). Viral RNA was quantified by qPCR 5 h after infection (C) and viral titers were determined by TCID50 assay 24 h after infection (D). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (E) HeLa cells were infected with EV71 (MOI = 5) and then treated with ZHSI-1 at 5 μM 2 h after infection. Co-localization of LAMP1 and 3D was determined by staining cells with anti-LAMP1 and anti-3D antibodies and visualizing by confocal microscopy 7 h after infection. Nuclei were stained with DAPI. Scale bars, 10 μm. The Pearson’s correlation coefficient of 3D and LAMP1 was analyzed by Image pro plus 6.0 (right panel). All values are means ± SEM. Data were analyzed using…

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  Figure S5.. ZHSI-1 is a novel EV71 inhibitor that interacts with 3D and blocks replication of EV71 and CVA16. (A) The synthesis route for ZHSI-1. (B) RD cells were pretreated with ZHSI-1 for 1 h and then infected with EV71 (MOI = 2) for 8 h. EV71 RNA was quantified by semi-quantitative PCR. (C) RD cells were pretreated with ZHSI-1 for 1 h and then infected with EV71 (MOI = 5) or CVA16 (MOI = 5). Western blot analysis was performed to detect the expression of 3D or VP1 12 h after infection. (D) HeLa cells were treated with ZHSI-1 at 5 μM for 6 h. Cells were stained with anti-LAMP1 antibody and visualized by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 8 μm. (E) The synthesis route for Biotin-ZHSI-1. (F) RD cells were pretreated with Biotin-ZHSI-1 or Biotin-NC for 1 h and then infected with EV71 (MOI = 2) for 8 h. The expression of VP1 was detected by Western blot analysis. Source data are available for this figure: SourceData FS5.

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  Figure 7.. ZHSI-1 inhibits EV71 replication and viral pathogenesis in vivo. (A) Schematic diagram of the mouse model of EV71-695F infection. (B–E) 3-d-old C57BL/6J mice were injected intraperitoneally with EV71-695F or PBS and administered intraperitoneally with indicated concentrations of ZHSI-1-HCl 2 h after infection. Symptoms of paralysis were scored on day 6 (B). Clinical disease was scored as follows: 0, healthy; 1, ruffled hair and hunchbacked appearance; 2, limb weakness; 3, paralysis in one limb; 4, paralysis in two limbs; and 5, death. To minimize animal suffering, mice were euthanized if they were paralyzed in two limbs. Postmortem tissue was collected on day 6 and processed and viral RNA was quantified by qPCR (C–E). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (F) Schematic diagram for the mouse model of EV71 (VR-784) infection. (G–I) 11-d-old AG129 mice were injected intraperitoneally with EV71 (VR-784) or PBS and administered intraperitoneally with ZHSI-1-HCl 2 h after infection. Postmortem tissue was collected on day 4 and processed and viral RNA was quantified by qPCR. All values are means ± SEM. Data were analyzed using two-tailed…