Journal: Nature Communications
Article Title: Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV
Figure Lengend Snippet: Endocytosis of SARS-CoV-2 S pseudovirions on 293/hACE2 cells. a Inhibition of entry of SARS-CoV-2 S pseudovirion on 293/hACE2 by lysosomotropic agents (20 mM NH 4 Cl 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.
Article Snippet: MHV S proteins were detected using polyclonal goat anti-MHV S antibody AO4 (1:2000); SARS-CoV S proteins were blotted with either polyclonal anti-SARS S1 antibodies T62 (1:2000) (Sinobiological Inc, Beijing, China) or mouse monoclonal against SARS S1 antibody MM02 (1:1000) (Sinobiological Inc, Beijing, China), MERS-CoV and SARS-CoV-2 S proteins were detected using mouse monoclonal anti-MERS S (1:1000) (Sinobiological Inc, Beijing, China) and anti-FLAG M2 antibody (1:1000) (Sigma, St. Louis, MO, USA), respectively.
Techniques: Inhibition, Transduction, Luciferase, Activity Assay, Infection, Real-time Polymerase Chain Reaction, MTT Assay