a Inhibition of entry of SARS-CoV-2 S pseudovirion on 293/hACE2 by lysosomotropic agents (20 mM NH4Cl and 100 nM bafilomycin A). b Inhibition of entry of SARS-CoV, MERS-CoV, and MHV S pseudovirions by a PIKfyve inhibitor apilimod. HeLa/mCEACAM, 293/hACE2, HeLa/hDPP4 cells were pretreated with different concentrations of apilimod and transduced with MHV S, SARS-CoV S, MERS-CoV S pseudovirions, respectively. The luciferase activity was measured 40 h post transduction. VSV-G pseudovirions were used as a control. Experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM. c Inhibition of MHV A59 infection by apilimod. The 17Cl.1 cells were pretreated with 3, 10, 30, 100, 300 nM apilimod for 30 min and infected by MHV A59 at MOI = 0.01. Viral infection and cell viability were determined by using qPCR and MTT assay, respectively. Experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM. d, e Inhibition of entry of SARS-CoV-2 S protein pseudovirions by apilimod, YM201636, and tetrandrine. HEK 293/hACE2 cells were pretreated with either apilimod (d), YM201636 (e), or tetrandrine (f), then inoculated with SARS-CoV-2 S pseudovirons in the presence of drug. The luciferase activity were measured 40 h post transduction. YM201636, PIKfyve inhibitor; tetrandrine, TPC2 inhibitor. The experiments were done in triplicates and repeated at least three times. One representative is shown with error bars indicating SEM of technical triplicates. Source data are provided as a Source Data file.