Fig 1.
GeCKO screening for host factors associated with ASFV replication.
(A) Construction of the WSL-Cas9 cell line. The primer pairs F1/R1 and F2/R2 were used to confirm the correct insertion of the Cas9 expression cassette into the Rosa26 locus of the cellular genome. (B) PCR identification of the WSL-Cas9 cell line. Lanes #1 and #2, amplified by the F1/R1 and F2/R2 primers, respectively, were indicative of the presence of WSL-Cas9 cells, with sizes of 1561 bp and 1909 bp, respectively. Lanes #3 and #4 are the amplification results of WSL cells identified by F1/R1 and F2/R2 primer PCR, and the amplification results are negative. M: DL5,000 DNA Marker. (C) Western blot analysis of Cas9 expression in WSL-Cas9 cells. (D) Validation of nuclease cleavage ability in WSL-Cas9 cells by using the T7 endonuclease 1 (T7E1) cleavage assay. A sgRNA targeting GGTA1 with a well-documented high gene editing efficiency was selected to evaluate the cleavage activity of the Cas9 protein expressed in the WSL-Cas9 cells used in the T7 endonuclease 1 (T7E1) cleavage assay. ***p < 0.001. (E) Schematic of the Genome-wide CRISPR/Cas9 knockout screen for host factors associated with ASFV infection. (F) Coverage of CRISPR knockout library plasmids and library cells compared with synthetic sgRNAs lists. (G) Homogeneity of library plasmids and library cells was assessed by using Lorenz curve analysis. (H) Results of the MAGeCK algorithm for candidate gene ranking. Results of the MAGeCK algorithm that assesses the statistical significance of candidate gene rankings through the robust ranking aggregation (RRA).
Fig 2.
TMEM239 plays an important role in ASFV replication.
(A and B) RT-qPCR analysis of TMEM239 and Rab14 transcription in TMEM239-/- cells and Rab14-/- cells. (C) Fluorescence micrographs illustrating ad-7GD replication. WSL cells, WSL cells with a non-target control sgRNA (WSL-NC-sgRNA cells), TMEM239-/- cells, and Rab14-/- cells were infected with ad-7GD (MOI = 1), and fluorescence images were collected at 48 hpi. (D) Quantification of viral genome copies (p72) in cell culture supernatants. WSL cells, WSL-NC-sgRNA cells, TMEM239-/- cells, and Rab14-/- cells were infected with ad-7GD (MOI = 1), and cell culture supernatants were collected at 48 hpi to detect viral genome copies. ***p < 0.001. (E and F) Multistep growth curves of ad-7GD in WSL cells and TMEM239-/- cells. Cells were infected with ad-7GD (MOI = 1), and samples were collected daily until the cells were destroyed by the virus. Viral replication was assessed on the basis of quantified viral genome copies (p72) and viral titers. (G) Efficiency of TMEM239 knockdown using various siRNAs. Three siRNAs specifically targeting TMEM239 were designed, synthesized, and subsequently transfected into PAMs. The mRNA level of TMEM239 was measured by RT-qPCR at 36 hpt to evaluate the knockdown efficiency. (H and I) Knockdown of TMEM239 using the three siRNAs significantly affects viral replication in PAMs. PAMs were transfected with negative control siRNA (NC-siRNA) and siRNAs specifically targeting TMEM239 for 36 h; subsequently the PAMs were infected with WT-HLJ18 (MOI = 0.1) for 48 h. *p < 0.05, ***p < 0.001.
Fig 3.
TMEM239 is not required for ASFV adsorption and internalization.
(A and B) TMEM239 is not required for ASFV adsorption. WSL cells and TMEM239-/- cells were incubated with ad-HRB1 strain (MOI = 5) for 2 h at 4°C, followed by washing with ice-cold PBS to eliminate unbound virus particles. The quantification of virus particles adsorbed on the cell surface was performed using viral genome copy quantification (p72) and Western blot analysis (p54). ns: not significant. (C and D) TMEM239 is not required for ASFV internalization. WSL cells and TMEM239-/- cells were initially incubated with an ad-HRB1 isolate (MOI = 5) for 2 h at 4°C, followed by extensive washing with ice-cold PBS. Subsequently, the cells were shifted to 37°C for 2 h to facilitate the internalization of viral particles, and a trypsin treatment was applied to eliminate the remaining surface-bound virus particles. The control group (Con) underwent trypsin treatment after the 4°C incubation to determine the efficiency of removing the ASFV particles adsorbed on the cell surface. The quantification of virus particles internalized into cells was performed using viral genome copy quantification (p72) and Western blot analysis (p54). ns: not significant.
Fig 4.
TMEM239 is important for the early step of ASFV replication.
(A) Knockout of TMEM239 affects the formation of viral factories. WSL cells and TMEM239-/- cells were infected with ad-HRB1 isolate (MOI = 1) for 18 h, followed by fixation with methanol. Subsequent immunofluorescence analysis targeted p72 (red), whereas nuclear and viral DNA was visualized with DAPI staining (blue). The arrows indicate virus factories. (B and C) Knockout of TMEM239 affects the transcription of the viral early gene CP204L (p30) and late gene B646L (p72). WSL cells and TMEM239-/- cells were incubated with ad-HRB1 isolate (MOI = 5) for 2 h at 4°C, followed by extensive washing with ice-cold PBS. Then, the cells were shifted to 37°C and collected at multiple timepoints (0 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12 h). Total RNA was extracted to detect the transcription of CP204L and B646L. *p < 0.05, ***p < 0.001. (D) Knockout of TMEM239 affects the translation of the viral early gene CP204L (p30) and the late gene B646L (p72). WSL cells and TMEM239-/- cells were incubated with ad-HRB1 isolate (MOI = 5) for 2 h at 4°C, followed by extensive washing with ice-cold PBS. Then, the cells were shifted to 37°C and collected at multiple timepoints (0 h, 1 h, 2 h, 4 h, 6 h, 8 h, 10 h, and 12h). Total protein was extracted to detect the expression of viral genes p30 and p72. (E and F) Quantitative analysis of the expression of the viral proteins (p30 and p72) at different timepoints in WSL cells and TMEM239-/- cells. ***p < 0.001.
Fig 5.
Proteomic analysis of host proteins interacting with TMEM239.
(A) Detection of exogenous TMEM239 expression in WSL cells and visualization of proteins that interact with TMEM239 by using silver staining. pTMEM239-Flag (6μg) and empty pCAGGS-Flag (6μg) were transfected into WSL cells, respectively. At 24 hpt, ad-HRB1 was inoculated at MOI = 5 into one of the groups of cells transfected with TMEM239 plasmid. Anti-Flag (M2) agarose beads were used to pull down TMEM239 and its binding proteins 24 h after transfection. (B) Gene ontology (GO) analysis based on the interaction between TMEM239 and its binding proteins in the uninfected virus group. The GO distributions of all proteins were divided into three types (biological processes, composition category, and function category). (C) KEGG pathway enrichment analysis based on the interaction between TMEM239 and its binding proteins in the uninfected virus group. Cellular processes, Environmental information processing, and organismal systems are listed.
Fig 6.
TMEM239 interacts with Rab5A and the virus protein pE248R.
(A) TMEM239 and Rab5A co-immunoprecipitation (Co-IP) assay. HEK-293T cells were transfected with pTMEM239-Flag and pRab5A-Myc either together or separately. At 36 hpt, cell lysates were collected for co-immunoprecipitation with beads conjugated with Myc antibody. Tubulin was used as an internal loading control. (B) Colocalization analysis of TMEM239 and Rab5A. Colocalization of TMEM239 and Rab5A was assessed in HEK-293T cells co-transfected with pTMEM239-Flag and pRab5A-Myc by use of confocal microscopy. Immunofluorescence analysis was performed against Flag (red) and Myc (Green). Nuclear DNA was visualized by DAPI staining (blue). (C and D) Dynasore inhibits ASFV internalization into WSL cells. WSL cells pre-treated for 15 minutes with the inhibitor for clathrin-dependent endocytosis dynasore were incubated with ad-HRB1 isolate (MOI = 5) for 2 h at 4°C, followed by extensive washing with ice-cold PBS. Subsequently, the cells were shifted to 37°C for 30 min to facilitate the internalization of viral particles, and a trypsin treatment was applied to eliminate the remaining surface-bound virus particles. Western blot analysis (p54) and quantification of viral genome copies (p72) were used to quantify virus particles internalized into cells. ***p < 0.001. (E) TMEM239 and viral proteins co-immunoprecipitation (Co-IP) assay. HEK-293T cells were transfected with pTMEM239-Myc and HA-tagged viral protein expression plasmids either together or separately. At 36 hpt, cell lysates were collected for co-immunoprecipitation with beads conjugated with Myc antibody. Tubulin was used as an internal loading control.
Fig 7.
Knockout of TMEM239 affects colocalization of the viral p72 protein with Rab5A.
(A and B) Knockout of TMEM239 affects the colocalization of the viral p72 protein with Rab5A. WSL cells (A) and TMEM239-/- cells (B) were incubated with ad-HRB1 isolates (MOI = 20) for 2 h at 4°C and then washed with ice-cold PBS to remove unbound virus particles. Then, ice-cold medium was added, and the cells were incubated at 37°C for 30, 45, and 60 minutes before being fixed in methanol. Immunofluorescence analysis was performed against p72 (red) and Rab5A (Green). Nuclear DNA was visualized by DAPI staining (blue). (C) Quantification of p72-Rab5A Co-localization. p72-Rab5A co-localization was quantified by using Pearson’s correlation coefficient method. Cells co-stained with p72 and Rab5A antibodies were randomly selected for co-localization analysis by using ImageJ software. ***p < 0.001.
Fig 8.
ASFVs exhibit lower replication in TMEM239-/- PBMCs.
(A) SCNT schematic for TMEM239 knockout piglets. sgRNAs and Cas9 protein (RNP) were co-electroporated into PEF cells, generating cells that completely lacked the TMEM239 coding region. Then, SCNT technology was used to generate TMEM239 knockout piglets. F3/R3, F4/R4, and F3/R4 are primer pairs used to identify the genotype of the SCNT-generated piglets. (B) Genotype identification of SCNT-producing Piglets. DNA extracted from the ear margin of the piglets was subjected to PCR identification. F1/R1 and F2/R2 primers were used to ascertain the genotypes of the piglets. PCR amplification products of wild-type piglets were 615 bp (left panel) and 930 bp (right panels) bands, respectively, whereas the PCR amplification results for the TMEM239 knockout piglets were negative. The red arrows indicate the wild-type bands from piglet #355–4. WT, Wild-type piglet, NC, Negative control. (C) Quantification of ad-7GD Strain Viral Genome Copies (p72). WT PBMCs and TMEM239-/- PBMCs were individually infected with the ad-7GD strain at an MOI of 1. Supernatants were collected at 72 hpi, and viral genome copies (p72) were quantified. (D and E) Quantification of WT-HLJ18 strain viral genome copies (p72) and HAD50. WT PBMCs and TMEM239-/- PBMCs were infected with the WT-HLJ18 strain at an MOI of 0.1. At 72 hpi, supernatants were collected, and viral genome copies, as well as the HAD50 values, were quantified. WT-1, WT-2, and WT-3 indicate the numbers of wild-type piglets selected for the current experiment. Piglet numbers 355-(1–4) and 393-(1–5) correspond to piglets born to surrogate sows numbered #355 and #393.