A genome-wide CRISPR/Cas9 knockout screen identifies TMEM239 as an important host factor in facilitating African swine fever virus entry into early endosomes
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.