Genome-scale CRISPR screen identifies TMEM198 driving double membrane vesicle formation in swine alphacoronavirus and murine betacoronavirus infected cells

Figures

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  Fig 1. Genome-wide CRISPR screen identifies host factors associated with the Alphacoronavirus TGEV. (A) The identification of TGEV host factors utilized the porcine genome-scale CRISPR/Cas9 knockout (PigGeCKO) library. Transformed PK15-Cas9 cells were subjected to either mock treatment or TGEV challenge at a multiplicity of infection (MOI) of 0.01. Surviving cells from each round of virus challenge were isolated, followed by PCR amplification and sequencing of sgRNA. The figure was generated using resources from the OpenClipart website. (B) We assessed the optimal titer for TGEV-induced cell death in PK15 cells at an MOI of 0.01. TGEV-induced cell death was evaluated following infection at MOI values of 0.0001, 0.001, 0.01, and 0.1, revealing cytopathic effects for the various MOIs approximately 48h post-infection. (C to E) Scatter plots depict sgRNA-targeted sequence frequencies and the extent of enrichment in transformed PK15-Cas9 cells (mock-treated versus TGEV infected) across three rounds of TGEV screening. (C) First round. (D) Second round. (E) Third round. Counts_TGEV1st, Counts_TGEV2nd, and Counts_TGEV3rd represent the average reads from paired-end sequencing for each round.…

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  Fig 2. TMEM198 is a key factor required for TGEV replication. (A). The indicated host factors were overexpressed, followed by infection of the cells with TGEV at a MOI of 0.01. The absolute mRNA levels of the TGEV N gene were subsequently measured. (B) PK15 cells that overexpressed TMEM198 were infected with TGEV (MOI = 0.01), and the expression of TGEV N protein (green fluorescence) and TMEM198 (red fluorescence) was assessed via indirect immunofluorescence. (C) Following the knockdown of TMEM198 in PK15 cells, the cells were infected with TGEV (MOI = 0.01). The copy number of TGEV RNA in the infected cells was determined using RT-qPCR, normalized to actin. The Ctrl group was set to 1. (D) The detection of TGEV N protein (red fluorescence) and TMEM198 (green fluorescence) via indirect immunofluorescence in PK15 cells stably expressing TMEM198. The absolute mRNA levels of the TGEV N gene and the relative mRNA levels of the TMEM198 gene were detected by qPCR. (E) The TMEM198 knockout cell line was established, and the knockout clones were confirmed through nucleic acid sequencing. (F) Both TMEM198 knockout (KO-TMEM198) and Ctrl cells were infected with TGEV (MOI = 0.01). The expression…

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  Fig 3. TMEM198 enhances the transcription and replication of TGEV. (A) KO-TMEM198 and Ctrl cells were infected with TGEV at a MOI of 5 at 4°C for 1h to assess TGEV adsorption. Viral RNA was extracted to determine the attachment of virions to the cell surface. (B) Following attachment of TGEV, temperature is shifted to 37 °C and internalization of viral particles is allowed to take place. The internalization of TGEV in both KO-TMEM198 and Ctrl cells was evaluated using absolute quantitative real-time PCR. (C) The release stage of TGEV was assessed in KO-TMEM198 and Ctrl cells infected with TGEV. The extracellular and intracellular RNA copies of TGEV were measured by RT-qPCR at 36h post-infection, and the ratio of extracellular to intracellular viral RNA copies was calculated. (D) KO-TMEM198 and Ctrl cells were infected with TGEV at an MOI of 0.01. The early replication of TGEV was quantified by assessing the copy number of the TGEV N gene at indicated time point using RT-qPCR. (E) The positive (+vRNA) or negative (−vRNA) viral RNA was quantified at the indicated time point using RT-qPCR. (F) Left panel, confocal microscopy to evaluate early-stage TGEV replication by detecting dsRNA in…

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  Fig 4. TMEM198 is involved in the formation of double membrane vesicle. (A) TMEM198 protein (green fluorescence) and the endoplasmic reticulum marker protein Sec61 (red fluorescence) were co-expressed in PK15 cells, and their co-localization was analyzed using confocal microscopy. (B) Co-localization of double-stranded RNA (dsRNA, green fluorescence), TMEM198 protein (purple fluorescence), and Sec61 was observed by confocal microscopy assay after infection with TGEV for 12h. (C) Co-localization analysis was performed to assess the interaction of TMEM198 with TGEV nsp3c and nsp4. (D and E) (Co-IP) analysis was performed to assess the interaction between TMEM198 and the viral proteins nsp3c and nsp4. (F) Transmission electron microscopy (TEM) analysis revealed the presence of DMVs in both Ctrl and KO-TMEM198 cells post-TGEV infection. Quantification of the percentage of cells containing DMVs relative to the total number of cells counted for each condition is provided. ‘N’ represents the number of cells counted for that condition. Scale bars are indicated as 1 μm, 2 μm, or 500 nm. (G) TEM analysis demonstrated the presence of DMVs in Ctrl and KO-TMEM198 cells following the overexpression…

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  Fig 5. The first N-terminal 35 amino acids of TMEM198 are critical for DMVs formation and viral replication. (A) Schematic diagram of the TMEM198 truncations. GFP-tagged constructs were generated. Constructs were N-terminally tagged with GFP and named GFP-TMEM198-1. Constructs were C-terminally tagged with GFP and named GFP-TMEM198-2. The orange segments represent the transmembrane regions of TMEM198. HEK-293T cells were transfected with plasmids encoding indicated truncations and Flag-nsp4. Cell extracts were prepared 24h after transfection, followed by immunoblotting and IP-western analysis with indicated antibodies. (B) Schematic diagram of the TMEM198-1 truncation library. HA-tagged constructs were generated. Construct with deletion of the first N-terminal 35 amino acids is named HA-TMEM198△OR1. Construct with deletion of 36-61 amino acids residues is named HA-TMEM198△TM1. Construct with deletion of 62 to 89 amino acids residues is named HA-TMEM198△TM2. Construct with deletion of 90 to 142 amino acids residues is named HA-TMEM198△TM34. Construct with deletion of 116 to 142 amino acids residues is named HA-TMEM198△TM4. Construct with deletion of 143 to 174 amino acids residues is…

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  Fig 6. TMEM198 serves as a critical host factor essential for the replication of swine alphacoronavirus and murine betacoronavirus. (A) The alignment of the TMEM198 amino acid sequences from various species, including Sus scrofa, Homo sapiens, Mus musculus, Canis lupus familiaris, Felis catus, Nyctereutes procyonoides, and Rhinolophus sinicus, was conducted using multiple sequence alignment software (MEGA). The NCBI protein database accession numbers for TMEM198 are as follows: XM 021076309.1 for Sus scrofa, XP 001005209.3 for Homo sapiens, XP 036165696.1 for Mus musculus, XM 038650239.1 for Canis lupus familiaris, XM 003991183.6 for Felis catus, XM 055345425.1 for Nyctereutes procyonoides, and XM 019741814.1 for Rhinolophus sinicus. (B) TMEM198-expressing Vero cells were infected with PEDV and SADS at a MOI of 0.1 for 36h. The relative mRNA levels of the N gene for both PEDV and SADS were quantified using qPCR. (C) Knockdown of TMEM198 in Vero cells attenuates PEDV replication. Left panel, siRNA-mediated knockdown of TMEM198 in Vero cells and confirmed by RT-qPCR. Right panel, Vero cells were then infected with PEDV and viral replication was analyzed by viral mRNA levels determined by…

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  Fig 7. TMEM198 is required for murine betacoronavirus infection in vivo. (A) At 5 days post-infection, virus titers in liver tissue were evaluated using TCID50 assays (n = 6). (B) Left panel, line graphs showing the body weight of TMEM198−/− and WT mice during the 5 days post-inoculation with MHV (n = 8 mice per genotype). Right panel, the weight loss of wild type and KO-TMEM198 mice at day 5 post-inoculation of MHV was analyzed. (C) Survival curves for TMEM198−/− and WT mice infected with MHV (n = 8 mice per genotype). (D) Gross postmortem examination of liver tissue in TMEM198−/− and WT mice infection with MHV at day 5 post-inoculation of MHV. (E) Histopathological analysis of the degree of liver damage in TMEM198−/− and WT mice infection with MHV at day 5 post-inoculation of MHV. The histological examination was performed on liver tissue samples stained with hematoxylin and eosin (H&E). (F) Viral copies in liver tissue were evaluated using qPCR to detect the MHV N gene in the TMEM198−/− and WT mice at day 5 post-inoculation of MHV.