Fig. 3.

Activation of the Nsp5 reporter assay during hCoV infection. (a) Relative levels and (b) absolute copy numbers of SARS-CoV-2 RNA present in the supernatant of infected HEK293T reporter cells transfected with plasmid encoding human ACE2 or ACE2-Gal4 fusion protein 48 h after infection. MOI multiplicity of infection of input virus. Mean of one independent experiment measured by qRT PCR in technical triplicates, + SD. (c) Activation of the Nsp5 Gaussia luciferase reporter assay 18 h after SARS-CoV-2 infection with indicated MOI or by transfection of 500 ng of Nsp5 expression construct. Mean of 3 infections + SD. ***P < 0.001, unpaired Student’s t-test. (d) Western blot of reporter transfected and infected cell lysates showing cleavage of the ACE2-Gal4 reporter 3 days after SARS-CoV-2 infection (indicated by N staining). Protein concentrations were adjusted prior to loading to account for infection-induced cytotoxicity. Hsp70 served as a protein loading control. (e) Quantification of ACE2-Gal4 cleavage efficiency detected by western blot shown in panel (d).