Fig 4. The internalization and early-stage replication of TGEV were impaired on TMEM41B KO cells.

(A) Evaluation of the adsorption activity of TGEV on TMEM41B KO and WT cells by absolute quantitative real-time PCR assay. WT and TMEM41B KO cells were infected with TGEV (MOI = 5) at 4°C for 1 h and assessed for TGEV adsorption. (B) Confocal microscopy assay for detection of TGEV N protein (Red) on WT and TMEM41B KO cells infected with TGEV (MOI = 5) at 4°C for 1 h. Scale bars = 5 μm. (C) Evaluation of the TGEV endocytosis stage in TMEM41B KO cells. WT and KO cells were infected with TGEV (MOI = 50) at 4°C for 1 h and transferred to 37°C for 30 min. Left: Confocal microscopy assay for detection of TGEV N protein (Red) expression in TMEM41B KO and WT cells. Right: Normalized mean fluorescence intensity was standardized by the ratio of each fluorescence intensity was divided by the maximum fluorescence intensity, n≧3. Scale bars = 10 μm. (D) RT-qPCR validation of mRNA expression of endocytic pathway genes enriched by RNA-seq. (E) Evaluation of the effects of TMEM41B KO cells on virus particle assembly by transmission electron microscope. Compared with WT cells, no virus-like particles (red arrows) wrapped in vesicles of varying sizes were found in TMEM41B KO cells. Scale bar, 2 μm or 500 nm as indicated. (F) Assessment of TGEV release stage of replication in TMEM41B KO and WT cells infected with TGEV (MOI = 5). Intracellular and extracellular viral titers were evaluated by virus TCID₅₀ assays at 24 hpi. (G and H) Confocal microscopy to evaluate early-stage TGEV replication by detecting (G) dsRNA formation and (H) TGEV N protein expression in WT and TMEM41B KO cells infected with TGEV (MOI = 5) at 3 hpi. WT, wild-type; KO, knock out; hpi, hours post-infection; MOI, multiplicity of infection; Mock, uninfected cells; TGEV, Transmissible gastroenteritis virus infected cells; N, Nucleus, dsRNA, double-stranded RNA; DAPI, 4’,6-diamidino-2-phenylindole. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, no significant. P values were determined by two-sided Student’s t-test. Data are representative of at least three independent experiments.