Fig 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 and then infected with EV71 (MOI = 5) after 60 h. The knockdown efficiency of LAMTOR3 was determined by Western blotting. Viral RNA level was quantified by qPCR 5 h after infection. 3D expression was analyzed by Western blot 12 h after infection. 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. (J and K) U251 cells were transfected with LAMTOR3 siRNAs and then infected with EV71-VR784 (MOI = 5; J) or EV71-695F (MOI = 5; K) after 60 h. Viral RNA was quantified by qPCR 5 h after infection. VP1 expression was analyzed by Western blot 12 h after infection. 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. (L–N) RagB+/+ and RagB−/− HeLa cells were infected with EV71 (MOI = 5). Viral RNA was quantified by qPCR 5 h after infection. The knockout efficiency of RagB in HeLa cells and VP1 expression were detected by Western blot 12 h after infection (L). The quantitation analysis of Western blot was performed using ImageJ. Viral titers were determined by TCID50 assay at 12, 24, and 36 h after infection (M). Cell death induced by EV71 was detected by measuring ATP levels 18 h after infection (N). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. Source data are available for this figure: SourceData F2.

Fig 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 mock infected or infected with EV71 (MOI = 5) for 6 h. Colocalization of PI4KB and 3D was detected by staining cells with anti-PI4KB and anti-3D antibodies and visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm. Inset panels are magnified 4×. The Pearson’s correlation coefficient of PI4KB and 3D 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. (F) 293T cells were transfected with plasmids expressing HA-3×Flag-PI4KB, GFP-3D, and Myc-RagB, as indicated. Cell lysates were collected after 36 h and subjected to IP using anti-FLAG agarose beads. Interactions between PI4KB and 3D were detected by Western blot. The quantitation analysis of Western blot was performed using ImageJ. (G) HeLa cells were mock infected or infected with EV71 (MOI = 5) for 6 h. Colocalization of LAMP1, RagB, and 3D was assessed by staining cells with anti-LAMP1/RagB/3D antibodies followed by visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm. Inset panels are magnified 4×. (H) HeLa cells were mock infected or infected with EV71 (MOI = 5) for 6 h. Colocalization of PI4KB and LAMP1 was assessed by staining cells with anti-PI4KB and anti-LAMP1 antibodies and visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm. The Pearson's correlation coefficient of PI4KB 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. (I and J) Wild-type HeLa cells were infected with EV71 (MOI = 5). RagB−/− (I) and LAMTOR3−/− (J) HeLa cells were infected with EV71 (MOI = 20). Co-localization of 3D with LAMP1 was assessed by staining cells with anti-LAMP1 and anti-3D antibodies and visualizing by confocal microscopy. Nuclei were stained with DAPI. Scale bars, 10 μm. Inset panels are magnified 4×. 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. Source data are available for this figure: SourceData F3.

Fig 4: 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 and viral RNA was quantified by qPCR (E). All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. (F–H) RagA, RagC, and RagD 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 RagA (F), RagC (G), and RagD (H) was determined by qPCR 60 h after transfection. Viral RNA (F–H) was quantified by qPCR 5 h after infection. VP1 expression (F–H) was analyzed by Western blot 12 h after infection. All values are means ± SEM. Data were analyzed using two-tailed Student’s t test. Source data are available for this figure: SourceData FS2.

Fig 5: 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.