Journal: PLoS Biology
Article Title: The SARS-CoV-2 Spike protein has a broad tropism for mammalian ACE2 proteins
Figure Lengend Snippet: The SARS-CoV-2 binding site on ACE2 is highly variable. (A) A phylogenetic tree of ACE2 proteins assembled using the neighbor-joining method [ 51 ] conducted in MEGA7 (Temple University, USA) [ 52 ] with ambiguous positions removed. The tree is drawn to scale, and support was provided with 500 bootstraps. (B) Structure of human ACE2 ectodomain (green) in complex with the RBD of SARS-CoV-2 [ 10 ]. (C) Conservation of mammalian ACE2 amino acid residues, estimated from site-specific evolutionary rates [ 50 ], mapped onto the surface of the ACE2 ectodomain [ 10 ], and coloured from blue (divergent) to purple (conserved) and presented in 2 orientations. Inset depicts the SARS-CoV-2 binding region of ACE2 (outlined), with residues that contact the SARS-CoV-2 RBD highlighted [ 6 ]. (D) WebLogo (University of California, Berkeley, USA) [ 53 ] plots summarising the amino acid divergence within the mammalian and bird ACE2 sequences characterised in this study. The single letter amino acid (aa) code is used with the vertical height of the amino acid representing its prevalence at each position in the polypeptide (aa 18–46, 78–91, 324–358, and 392–394 are indicated). The aa sites bound by SARS-CoV and SARS-CoV-2 Spike [ 11 ] are indicated by red arrows. *SARS-CoV-specific interactions. (E) ACE2 sequences were cloned into the pDISPLAY expression construct in frame with an N-terminal signal peptide (the murine Ig κ-chain leader sequence) and HA-tag. (F) Expression of individual mammal or bird ACE2 proteins was confirmed at a whole cell level by western blot. (G) Flow cytometry was performed to examine surface expression of each ACE2 protein on non-permeabilised cells. For gated cells, the percentage positivity and MFI are plotted. The data underlying this figure may be found in S1 Data and S1 Raw Images . aa, amino acid; ACE2, angiotensin-converting enzyme 2; MFI, mean fluorescence intensity; RBD, receptor binding domain; SARS-CoV, SARS Coronavirus; SARS-CoV-2, SARS Coronavirus 2.
Article Snippet: For the initial optimisation of pseudoparticle activity, 3 conditions were tested: (1) ACE2 expression only: HEK293T target cells transfected with 500 ng of a human ACE2 expression plasmid (Addgene, USA) and were seeded at 2 × 104 in 100 μL DMEM-10% in a white-bottomed 96-well plate (Corning, USA) 1 day prior to infection; (2) Spike activation with TMPRSS2: HEK293T target cells were transfected with 25 ng TMPRSS2 alongside human ACE2 as above; (3) Spike activation by trypsin treatment: viral pseudoparticles were treated with 2.5 mg/mL trypsin for 1 h at 37°C before addition to target cells overexpressing human ACE2.
Techniques: Binding Assay, Clone Assay, Expressing, Construct, Sequencing, Western Blot, Flow Cytometry, Fluorescence