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.