VMP1 and TMEM41B are required for β-coronavirus infection. (A) Immunostaining results show that GFP expression from an MHV-A59/GFP reporter virus and virus-specific dsRNA is detected in control (Ctrl) HeLa cells, but not in VMP1 KO or TMEM41B KO cells 9 h after MHV-A59 infection. Efficient expression of the MHV-A59 receptor Flag-mCEACAM1 is verified by immunostaining with Flag antibody. Bars: 10 μm. (B) 16 h after MHV-A59/GFP infection at the indicated multiplicity of infection (MOI), high levels of nsp9 precursors, mature nsp9, and GFP are detected in control HeLa cells, while no expression of nsp9 or GFP is detected in VMP1 KO or TMEM41B KO cells. The expression levels of mCEACAM1 are comparable in control and KO cells. (C) Control, VMP1 KO, and TMEM41B KO cells were infected with MHV-A59 for 3 h. Viral RNA levels were determined by quantitative PCR using virus-specific primers. Values were normalized to GAPDH mRNA levels (n = 3). (D and E) TEM analysis shows that numerous DMVs are observed in control cells 6 h after MHV-A59 infection. No DMVs are formed in VMP1 KO or TMEM41B KO cells (D). Virus (red arrow) is occasionally detected in endolysosomes of VMP1 KO cells. White arrows indicate DMVs. N, nucleus. Quantification of DMV numbers per cell is shown as mean ± SD (Ctrl, n = 33; VMP1 KO, n = 42; TMEM41B KO, n = 25; E). ****, P < 0.0001. Bars: 500 nm; inserts, 200 nm. Source data are available for this figure: SourceData F1.