YIPF5 is an essential host factor for porcine epidemic diarrhea virus double-membrane vesicle formation

Figures

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  Fig 1. Genome-scale CRISPR/Cas9 knockout screens identify host factors required for PEDV infection. (A) Flowchart for identification of PEDV essential host factors using genome-scale CRISPR/Cas9 KO library screens in IPEC-J2 cell lines. The IPEC-J2 KO library was subjected to three rounds of PEDV infection with escalating MOIs (0.01, 0.1, and 1). Surviving cells from the third round of virus challenge were selected, PCR amplified, and subjected to deep sequencing of sgRNAs. (B) MAGeCK analyses of the third round of PEDV screens in the IPEC-J2 KO library. (C) Venn diagram illustrating the overlap of differential genes identified in three screens. MOI, multiplicity of infection.

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  Fig 2. YIPF5 is an essential host factor required for PEDV infection. (A, D) Quantification of virus infectivity (TCID50) in cultural supernatant of WT and YIPF5 KO IPEC-J2 cell lines infected with PEDV CV777 (A) or LJX (D) at 48 h (MOI = 1). (B, E) RT-qPCR assay for determining the relative mRNA expression of N protein in WT and YIPF5 KO IPEC-J2 cell lines infected with PEDV CV777 (B) or LJX (E) at 48 h (MOI = 1). (C, F) Western blot assays for detecting the PEDV N protein in WT and YIPF5 KO IPEC-J2 cell lines infected with PEDV CV777 (C) or LJX (F) at 48 h (MOI = 1). Tubulin served as the internal control protein. (G) TCID50 in cultural supernatant of vector and overexpression of YIPF5-Flag IPEC-J2 cell lines infected with PEDV CV777 at 48 h (MOI = 1). (H) RT-qPCR assay for determining the relative mRNA expression of N protein in vector and overexpression of YIPF5-Flag IPEC-J2 cell lines infected with PEDV CV777 at 48 h (MOI = 1). (I) TCID50 in cultural supernatant of vector, YIPF5 KO, and YIPF5 knockout rescued (KO rescue) IPEC-J2 cell lines infected with PEDV CV777 at 48 h (MOI = 1). (J) RT-qPCR assay for determining the relative mRNA expression of PEDV CV777 N protein in vector,…

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  Fig 3. Knocking out YIPF5 does not affect PEDV adsorption and internalization. (A) Assessment of PEDV adsorption activity in WT and YIPF5 KO IPEC-J2 cell lines by RT-qPCR assay. WT and YIPF5 KO IPEC-J2 cell lines were incubated with PEDV CV777 at 4°C for 2 h (MOI = 5). (B) Immunofluorescence assays for detection of PEDV N protein in WT and YIPF5 KO IPEC-J2 cell lines incubated with PEDV CV777 at 4°C for 2 h (MOI = 5). Scale bars, 10 µm. (C) Normalized mean fluorescence intensity was measured using ImageJ. (D) Evaluation of the PEDV internalization stage in WT and YIPF5 KO IPEC-J2 cell lines by RT-qPCR assay. WT and YIPF5 KO IPEC-J2 cell lines were incubated with PEDV CV777 at 4°C for 2 h (MOI = 10) and transferred to 37°C for 1 h. (E) Immunofluorescence assays for detection of PEDV N protein in WT and YIPF5 KO IPEC-J2 cell lines incubated with PEDV CV777 at 4°C for 2 h (MOI = 10) and transferred to 37°C for 1 h. Scale bars, 10 µm. (F) Normalized mean fluorescence intensity was measured using ImageJ. WT, wild type; KO, knockout; MOI, multiplicity of infection; DAPI, 4’,6-diamidino-2-phenylindole; N, nucleocapsid; ns, not significant. P values were determined by two-sided Student’s…

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  Fig 4. The replication stage of PEDV was impaired in YIPF5 KO IPEC-J2 cell lines. (A) Assessment of PEDV replication stage in WT and YIPF5 KO IPEC-J2 cell lines by RT-qPCR assay. WT and YIPF5 KO IPEC-J2 cell lines were incubated with PEDV CV777 at 37°C for 24 h (MOI = 1). (B) Immunofluorescence assays for detection of PEDV dsRNA in WT and YIPF5 KO IPEC-J2 cell lines infected with PEDV CV777 at 37°C for 24 h (MOI = 1). Scale bars, 10 µm. (C) Normalized mean fluorescence intensity was measured using ImageJ. (D) Viral growth curves within 12 h in WT and YIPF5 KO IPEC-J2 cell lines. WT, wild type; KO, knockout; MOI, multiplicity of infection; DAPI, 4’,6-diamidino-2-phenylindole; dsRNA, double-stranded RNA. ***P < 0.001, ****P < 0.0001. P values were determined by two-sided Student’s t-test. Data are representative of at least three independent experiments.

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  Fig 5. YIPF5 contributes to the formation of PEDV DMVs. (A) Analyses of the subcellular location of YIPF5 and dsRNA in IPEC-J2 cell lines uninfected (above) or infected with PEDV after 48 h (MOI = 1) (bottom). The YIPF5 (indicated in green) was co-localized with dsRNA (indicated in red). Scale bar, 10 µm. (B) Measure of the fluorescence intensity of YIPF5 and dsRNA at the same location in panel A (PEDV-Merge, indicated by the black line). (C) Representative transmission electron microscope images depicting PEDV-induced DMVs (indicated by asterisks) in WT, YIPF5 KO, and YIPF5 KO rescue IPEC-J2 cell lines infected with PEDV after 48 h (MOI = 1). The black arrows indicate viral particles. The red arrow indicates stacked whorl. Scale bar, 1 µm (left) or 500 nm (right). (D) Quantification of the number of DMVs in WT, YIPF5 KO, and YIPF5 KO rescue IPEC-J2 cell lines infected with PEDV after 48 h (MOI = 1). N ≥ 16 cells for each cell line were calculated. (E) Representative immunoelectron microscope images depicting DMVs in IPEC-J2 cell lines infected with PEDV after 48 h (MOI = 1) and location of YIPF5. Scale bar, 200 nm. WT, wild type; KO, knockout; MOI, multiplicity of infection; DAPI,…

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  Fig 6. YIPF5 interacts with PEDV non-structural proteins 3, 4, and 6. HEK293T cells were co-transfected with YIPF5-Flag and PEDV HA-nsp3, HA-nsp4, or HA-nsp6 after 24 h for confocal assays (A) and co-IP assays (B). (A) Confocal fluorescence microscopy analyses of the co-localization of YIPF5-Flag (indicated in red) and HA-nsp3, HA-nsp4, or HA-nsp6 (indicated in green) in HEK293T cells. Measure of the fluorescence intensity of YIPF5 and nsp3, nsp4, or nsp6 at the same location (indicated by the black lines). Scale bar, 10 µm. (B) Protein interactions were analyzed by immunoprecipitation with anti-Flag beads and immunoblotting with anti-Flag and anti-HA. The co-transfection of YIPF5-Flag and PEDV HA-N served as the control group. (C) HEK293T cells were co-transfected with GFP-YIPF5, HA-nsp3, and Myc-nsp4 after 24 h for confocal assays. Confocal fluorescence microscopy analyses of the co-localization of GFP-YIPF5 (indicated in green), HA-nsp3 (indicated in red), and Myc-nsp4 (indicated in cyan) in HEK293T cells. Measure of the fluorescence intensity of YIPF5, nsp3, and nsp4 at the same location (indicated by the black lines). Scale bar, 10 µm. (D) HEK293T cells were co-transfected with…

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  Fig 7. Knocking out YIPF5 affects the interaction between nsp3 and nsp4, thereby impacting the formation of DMVs driven by nsp3 and nsp4. (A) Representative transmission electron microscope images depicting nsp3/nsp4-induced DMVs (indicated by asterisks) in WT, YIPF5 KO, and YIPF5 KO rescue IPEC-J2 cell lines. The red arrow indicates the zippered structure. Scale bar, 1 µm (left) or 500 nm (right). (B) Quantification of the number of DMVs driven by nsp3 and nsp4 in WT, YIPF5 KO, and YIPF5 KO rescue IPEC-J2 cell lines. N ≥ 14 cells for each cell line were calculated. (C) WT, YIPF5 KO, and YIPF5 KO rescue IPEC-J2 cell lines were co-transfected with HA-nsp3 and Myc-nsp4 after 24 h for confocal assays. Confocal fluorescence microscopy analyses of the co-localization of HA-nsp3 (indicated in green) and Myc-nsp4 (indicated in red) in WT, YIPF5 KO, and YIPF5 KO rescue IPEC-J2 cell lines. Scale bar, 10 µm. (D) The number of nsp3/nsp4 puncta was quantified. N ≥ 16 cells for each cell line were calculated. (E) WT, YIPF5 KO, and YIPF5 KO rescue IPEC-J2 cell lines were co-transfected with HA-nsp3 and Myc-nsp4 after 24 h for co-IP assays. Protein interactions were analyzed by immunoprecipitation…