Disrupted glycosylphosphatidylinositol anchoring induces ER stress and restricts enterovirus infection

Figures

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  Fig 1. GPI-related genes are required for enterovirus infection. (A) CRISPR genetic screening for Echo7 host factors in RD cells. Each dot represents a specific gene and size corresponds to the number of sgRNAs that were enriched for each gene. Red color dots represent known enterovirus host factors. Green color dots represent GPI-related genes. (B-E) Control, PIGS-KO (B, D), or PIGK-KO (C, E) cells were infected with Echo7 virus at MOI = 1 and VP3 expression (B, C) or virus titer (D, E) was determined at 20 h.p.i. The underlined sgRNA sequences were selected for downstream knockout experiments. ***, p < 0.001, compared to control cells. (F) Diagram of GPI anchor synthesis, transfer and maturation. Enzymes catalyzing each step were listed above. (G-J) Control, PGAP1-KD (G, I), or PGAP3-KD (H, J) cells were infected with Echo7 virus at MOI = 1 and viral infection (G, H) or cell viability (I, J) was determined at 20 h.p.i. ***, p < 0.001, compared to control cells (H) or uninfected cells (I, J). (K-N) Control, PIGS-KO, or PIGK-KO cells were infected with EV71 (K, L) or CVB5 virus (M, N) at MOI = 1 and vial protein expression (K, M) or virus titer (L, N) was determined at 20 h.p.i. ***, p…

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  Fig 2. PIGS or PIGK supports Echo7 infection not just through CD55. (A) Control, PIGS- or PIGK-KO cells were immunoblotted with anti-CD55 antibody. (B) Control, CD55-KO or CD55-KO cells supplemented with sgRNA-resistant CD55 were infected with EV71, Echo7, or CVB5 and immunoblotted with indicated antibodies. (C) Diagram of GPI anchored CD55 (CD55/GPI) or chimeric CD55 with transmembrane domain (CD55/TM). (D) Surface expression of CD55 from control (orange line) or CD55-KO cells supplemented with CD55/GPI (red line) or CD55/TM (cyan line) was determined with FACS. Cells stained with isotype control antibody were used as control (grey line). (E-F) Control, CD55-KO or CD55-KO cells supplemented with CD55/GPI or CD55/TM were infected with Echo7 for 20 hrs, and CD55 and VP3 expression (E), or cell viability were determined (F). ***, p < 0.001, compared to uninfected cells. (G-H) PIGS-KO, PIGK-KO cells, or CD55/TM supplemented cells were infected with Echo7, CD55 and VP3 expression (G) or cell viability (H) was determined at 20 h.p.i. ***, p < 0.001, compared to uninfected cells. (I) Cells as described in (G) were tested for virus attachment and internalization. viral RNA was quantified by…

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  Fig 3. PIGS or PIGK is required for enterovirus translation. (A) Upper panel, diagram of full-length EV71 with NanoLuc reporter. Lower panel, control, PIGS-KO or PIGK-KO cells infected with NanoLuc-EV71 were monitored for luciferase activity at indicated time points. Inset, enlargement of the first 6 hrs. (B-C) Upper panels: Diagrams of EV71-SGR or EV71-SGR (GND). Lower panels: control, PIGS-KO or PIGK-KO cells were transfected with EV71-SGR (B) or EV71-SGR (GND) (C) and luciferase was monitored. (D) RD cells stably expressing PIGS-HA (upper panels) or PIGK-HA (lower panels) infected with EV71 (MOI = 1) for 8 hrs before they were immunostained with anti-HA (Green) and dsRNA (Red). Nuclei were counterstained with DAPI. Bar, 10μm. (E) Uninfected (upper) or EV71-infected (lower) RD cell homogenates (H) were centrifuged to prepare a “water soluble” supernatant (WS) or “detergent soluble” (DS) fraction. Detergent resistant membranes were then fractionated on a density gradient. Fractions (numbered in order from light to heavy) were analyzed by immunoblotting for the indicated proteins. The numbers below represent the protein mass distribution across the fractions, expressed as a percentage…

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  Fig 4. PIGS or PIGK is required for enterovirus RO biogenesis. (A) Diagram of pTM1 (2A-3D). The P2 and P3 regions of EV71 genome together with EV71 3’UTR were put downstream of T7 promoter and EMCV IRES, followed by HDV ribozyme and T7 terminator. (B) RD cells, PIGS-KO or PIGK-KO cells stably transduced with T7 polymerase were transfected with pTM1 (2A-3D) and immunoblotted with anti-3AB or anti-3D antibody.β-actin was used as loading control. (C) RD cells infected with EV71 were immunostained with anti-3AB antibody with nuclei counterstaining (left panel). RD/T7 cells were transfected with pTM1 (2A-3D) and immunostained with anti-3AB antibody (right panel). Bar, 10μm. (D) Representative electron microscopy image of uninfected RD cell. (E) Representative electron microscopy images of EV71 induced membrane alterations. Area with extensive membrane alterations was circled with red dashed lines. Enlargement of the boxed area was shown on right. Arrowheads indicate electron-dense membrane vesicles. (F) Representative image of EV71 infected RD cells with membrane vesicles. Empty arrowheads indicate electron-translucent DMVs. (G) Representative images of RD/T7 cells transfected pTM1 (2A-3D).…

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  Fig 5. PIGS or PIGK maintains ER integrity. (A) RD cells were infected with EV71 at MOI = 1 for 6 hrs and they were immunostained with anti-CALR, GM130 or TGN46 together with anti-dsRNA. Nuclei were counterstained with DAPI. Bar, 10μm. (B) Quantification of pearson’s correlation coefficient of CALR, HSP60, GM130 or TGN46 with dsRNA. Each data point corresponds to the coefficient derived from a single image. (C) Control, PIGS- or PIGK-KO cells were immunostained with CALR with nuclei counterstaining. Bar, 10μm. (D) Control, PIGS- or PIGK-KO cells were transfected with Sec61β-GFP and nuclei were counterstained with DAPI. 0.5μM TG treatment for 6 hrs was used to induce ER whorl formation. Bar, 10μm.

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  Fig 6. Enterovirus RNA is degraded via RIDD pathway. (A-C) Control, PIGS-, PIGK-KO cells were immunoblotted with indicated antibodies (A), transfected with IRE1-GFP and evaluated with confocal microscopy (B) or tested for spliced XBP1 expression with q-RT-PCR (C). Control cells treated with 0.15μM TG for 6 hrs were used as positive control for ER stress. ns, not significant; ***, p < 0.001, compared to control. (D) Upper panel, diagram of IRE1-splicing dependent NanoLuc expression system (XBP1-ΔDBD-NL). Lower panel, indicated cells were transfected with XBP1-ΔDBD-NL and luciferase was monitored and normalized to control cells. ns, not significant; *, p < 0.05; ***, p < 0.001, compared to control. (E) Diagram of IRE1α activation pathways. IRE1α-mediated XBP1 alternative splicing and RIDD pathways were shown. (F) SCRAR, Col6A1 expression in control, PIGS- or PIGK-KO cells were determined with q-RT-PCR. **, p < 0.01; ***, p < 0.001, compared to control group. (G) GFP and SPARC-GFP expression in control, PIGS- or PIGK-KO cells determined with immunoblotting. (H) GFP, or SPARC-GFP expression with or without 20μM 4μ8C treatment was determined with q-RT-PCR in control, PIGS- or PIGK-KO cells.…

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  Fig 7. Knockout of PIGC or PIGH does not activate RIDD. (A) Control, PIGC KO (upper panels), or PIGH KO (lower panels) cells were infected with Echo7 virus at MOI = 1 for 20 hrs and viral VP3 expression was detected with immunoblotting. sgRNAs underlined were chosen for downstream experiments. (B) Control, PIGC KO, or PIGH KO cells transfected with IRE1-GFP and evaluated with confocal microscopy. TG was used as a positive control. Bar, 10μm. (C-D) Control, PIGC KO, or PIGH KO cells were immunoblotted with indicated antibodies (C) or tested for spliced XBP1 expression with q-RT-PCR (D). ns, not significant; ***, p < 0.001, compared to values in control group. (E) Indicated cells were transfected with XBP1-ΔDBD-NL and luciferase was measured. Cell viability was measured with Cell Titer Glo. ns, not significant; ***, p < 0.001, compared to values in control group. (F-G) SCARA (F) or Col6A1 (G) expression in indicated cells were determined with q-RT-PCR. ns, not significant; ***, p < 0.001, compared to values in control group. (H) Indicated cells were transfected with GFP or SPARC-GFP and the expression was determined with immunoblotting.

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  Fig 8. Enterovirus infection activates PERK pathway during late stage of infection. (A) RD cells infected with EV71, Echo7 or CVB5 (MOI = 1) were immunoblotted with indicated antibodies. (B) RD cells transfected with IRE1-GFP were infected with EV71, Echo7 or CVB5 (MOI = 1) and immunostained with dsRNA (red). Nuclei were counterstained with DAPI. Bar, 10μm. (C) RD cells infected with EV71, Echo7 or CVB5 were tested for spliced XBP1 expression with q-RT-PCR. ns, not significant, compared to uninfected cells. (D) RD cells transfected with XBP1-ΔDBD-NL were infected with EV71, Echo7 or CVB5 and luciferase was determined. Cell viability was measured with Cell Titer Glo. *, p < 0.05; **, p < 0.01, compared to uninfected cells. (E-F) RD cells infected with EV71, Echo7 or CVB5 were tested for SCRAR (E) or Col6A1 (F) expression with q-RT-PCR. ns, not significant; *, p < 0.05, compared to uninfected cells. (G) RD cells transfected GFP or SPARC-GFP were infected with EV71, Echo7 or CVB5 and immunoblotted with indicated antibodies. (H) RD cells infected with EV71, Echo7 or CVB5 were immunoblotted with indicated antibodies. (I-J) RD cells infected with EV71, Echo7 or CVB5 were tested for CHOP (I)…

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  Fig 9. The UTRs are responsible for enterovirus sensitivity to ER stress. (A) Control, PIGS- or PIGK-KO Huh 7 cells were immunoblotted with indicated antibodies. (B) Control, PIGS- or PIGK-KO Huh 7 cells were transfected with HCV-SGR and luciferase activity was monitored at indicated time points. ns, not significant, compared to control cells. (C) Control, PIGS- or PIGK-KO Huh 7 cells were immunoblotted with IRE1α antibodies. (D) Control, PIGS- or PIGK-KO Huh 7 cells were transfected with GFP or SPARC-GFP and relative mRNA was determined with q-RT-PCR. ns, not significant; ***, p < 0.001, compared to values in control group. (E) Diagrams of luciferase expressing RNA with different combination of 5’- or 3’-UTR. (F) Expression of different luciferase RNAs in control, PIGS- or PIGK KO cells. Values were normalized to control groups. ns, not significant; *, p < 0.05; ***, p < 0.001, compared to values in control group.