FIG 7.

The ACE2 receptor is required for SARS-CoV-2 S protein-induced translation control in lung cells. (a) RNAi-mediated depletion of ACE2 protein from A549 cells abrogates VAIT element-mediated translational control. In vitro translation reactions were performed as described for Fig. 2 with luciferase reporter cRNAs harboring S or ORF1a VAIT elements as indicated below lanes. Cell extracts added to in vitro translation reactions were prepared from A549 cells that were transfected with a mock (nontargeting) siRNA pool or a specific ACE2 siRNA pool and then transduced with SARS-CoV-2 spike protein pseudotyped lentivirus as indicated above lanes. (b) Reduced expression of ACE2 protein in A549 cells transfected with ACE2-specific siRNA (but not in A549 cells transfected with a nontargeting “mock” siRNA pool) was confirmed by Western blotting with anti-ACE2 antibody. Actin was used as a specificity/loading control. (c) RNA-EMSA performed with biotin-labeled S and OFR1a VAIT element RNA probes and extracts prepared from A549 cells treated as in (a) (transfected with ACE2-targeting or nontargeting siRNA pools and transduced with S pseudotyped lentivirus).