Fig. 3.

ACE2 primes the proteolytic cleavage of virion spike. (A) A schematic diagram illustrating the SARS-CoV-2 PP infection system. HEK293T cells without or with ACE2 expression were incubated with PPs at 37 °C or 4 °C for different time (hours post infection, hpi) as indicated; supernatants or cell lysates were collected for immunoblotting detection of postfusion proteins. (B) Immunoblots showing the full-length S, S2, S2′, ACE2, and tubulin detected from supernatants (Top) and cell lysates (Bottom) from HEK293T-ACE2 cells infected with PP for 0, 2, 4, 6, or 8 h as indicated; negative control was performed by incubating PPs alone at 37 °C for 8 h without cells (lane 1). Blots are representative of three individual repeats. (C) Immunoblots showing S, S2, S2′, ACE2 and tubulin, detected from HEK293T cells expressing human or mouse ACE2-V5-6his infected with PPs for 6 h. ACE2 variants were detected using anti-his antibody. Blots are representative of four individual repeats. (D) Fluorescent images showing eGFP expression as a result of SARS-CoV-2 PP infection in HEK293T cells expressing human or mouse ACE2-V5-6his (Top); representative dot plot from the FACS showing eGFP-positive cells expressing human ACE2 and PP infection rate from three individual experiments was summarized as the mean ± SD (Bottom). (E) Immunoblots showing the full-length S, S2, S2′, ACE2, SPC, and tubulin detected from cell lysates of hACE2 KI murine lung epithelial cells infected with or without SARS-CoV-2 PPs for 12 h. Asterisk denotes nonspecific bands, blots are representative results obtained from two independent experiments.