Validation of pseudovirus preparation and ACE2 expression
In this study, we investigated the ACE2 utilization of three SARSr-CoVs, including SARS-CoV-BJ01, SARS-CoV-2 and Pangolin CoV. Pangolin CoV was tested since pangolins were suspected to be a potential intermediate host of SARS-CoV-2 (Zhang, Wu et al. 2020). Notably, the RBM of Pangolin CoV spike is almost identical with SARS-CoV-2 spike except for the 498th aa residue, but they are different from SARS-CoV spike on multiple aa sites (Figure 1A ).
In order to validate the successful preparation of the three pseudovirus, the HEK293T cells for virus package were lysed after virus harvest the cells were lysed and subjected to Western blot detection of HA-tagged spike proteins. As shown in Figure 1B , samples from all three package cells showed typical bands (about 180 kDa) of SARSr-CoV spike proteins, indicating the success of pseudovirus preparation.
Then, we continued to validate the ectopic expression of 20 ACE2s from different animals in HeLa cells, an ACE2-negative human cell line. We transfected the HeLa cells with plasmids harboring the coding gene of ACE2s from different animals. At 48 h post transfection, he cells were lysed and subjected to Western blot detection of 6*His-tagged ACE2 protein. As shown in Figure 1C , bands of all ACE2s (about 150 kDa) could be observed, indicating the successful expression of all ACE2s in HeLa cells.