Figure 1. Cholesterol dependent inhibition of SARS-CoV-2 pseudovirus (SARS2-PV) infection.

(A) Cartoon diagram showing the experimental setup for loading cultured cells with cholesterol. i., Cholesterol (yellow shading) loaded into lipoprotein (e.g., low- and high-density lipoprotein (LDL and HDL respectively)) from blood serum. ii., Cholesterol free human apolipoprotein E (apoE, brown shading) a cholesterol transport protein is exposed to cholesterol from blood serum and iii, ApoE transports cholesterol into cells (grey shading) (see also Supplemental Figure S1B). (B–C) SARS-CoV-2 pseudovirus (SARS2-PV) entry assay in HEK293T cells (B) and Vero E6 cells (C). Cells were treated with a luciferase expressing retrovirus pseudotyped with the SARS-CoV-2 spike protein that recapitulates viral entry. Infectivity was monitored by a luciferase activity in cells treated with or without apoE. Viral infection in cells with high cholesterol (apoE + serum) was more than 3-fold higher compared to low cholesterol. Data are expressed as mean ± s.e.m., *P<0.05, **P<0.01, one-way ANOVA. (D) Depletion of cellular cholesterol with methyl-beta-cyclodextrin (MβCD) blocked almost all viral entry measured by pseudotyped luciferase assay. Data are expressed as mean ± s.e.m., **P<0.01, two-sided Student’s t-test. (E) ACE2 activity assay was performed to detect ACE2’s expression in wild type HEK293T cells. Cells incubated with ACE2 inhibitor showed significantly decreased ACE2 activity, suggesting ACE2 is expressed in the HEK293T cells. Data are expressed as mean ± s.e.m., ****P<0.0001, two-sided Student’s t-test. (F) ACE2’s enzymatic activity is regulated by membrane cholesterol. ApoE mediated cholesterol depletion from cell membrane inhibits ACE2’s activity. Data are expressed as mean ± s.e.m., *P<0.05, one-way ANOVA.