siPTDSS1 reverses the DMV defects in VMP1-depleted cells. (A and B) The enlarged LD phenotype in siVMP1 cells is suppressed by siPTDSS1, but not siPTDSS2. siPTDSS2 alone causes bigger LDs, compared with control cells (A). Quantitative data are shown as mean ± SD (NC, n = 54; siPTDSS1, n = 55; siPTDSS2, n = 53; siVMP1, n = 52; siVMP1&siPTDSS1, n = 50; siVMP1&siPTDSS1, n = 52; B). ****, P < 0.0001; N.S., not significant. Bars: 5 μm. (C) Compared with NC-treated VMP1 KO cells, concentric membrane structures (red arrows) are reduced and the formation of DMVs (white arrow) is increased after siPTDSS1. N, nucleus. Bars: 200 nm. (D–F) In FPP assays, nsp4–mCherry immediately disappears upon pK addition in NC-treated VMP1 KO cells (D), while nsp4-mCherry shows partial resistance to pK treatment after knocking down of PTDSS1 (E). Quantitative data are shown as mean ± SEM (NC, n = 18; siPTDSS1, n = 12; F). Bars: 5 μm. (G) The proposed model of the roles of VMP1 and TMEM41B in DMV biogenesis. Once β-coronaviruses enter the cell, the polyprotein containing nsp3 and nsp4 is translated, and then cleaved into individual proteins by viral genome-encoded proteases. The host ER protein TMEM41B facilitates the binding of nsp3 and nsp4 to each other through their luminal domains. This leads to the separation of nsp3 and nsp4 and their concentration on the opposite sides of the ER by unknown mechanisms. nsp3/4 interaction and separation drive the zippering and bending of the ER. The paired ER finally closes into vesicles, possibly regulated by VMP1-modulated PS distribution.