Journal: Journal of Virology

Article Title: Pathways of Cross-Species Transmission of Synthetically Reconstructed Zoonotic Severe Acute Respiratory Syndrome Coronavirus

doi: 10.1128/jvi.00818-08

Figure Lengend Snippet: FIG. 2. Resurrection of icSZ16-S with DBT-cACE2 cells. RT-PCR to detect subgenomic leader-containing transcripts was performed to detect viral replication. DBT, DBT-GFP-His, DBT-cACE2, DBT-hACE2, or Vero E6 cells were infected with 100 l of viral supernatant from the initial icSZ16-S transfection, or the icSARS supernatants (B), or they were mock infected (A). At 24 hpi, total RNA was isolated, and cDNA was generated and then used as template for PCR. Evidence of SARS-CoV replication and subgenomic transcription, ACE2 gene expression, and control GAPDH gene expression was detected by the production of amplicons (SARS-CoV, 3a [1,796 bp], E [947 bp], M [666 bp]; control, 235 bp GAPDH; ACE2, 258 bp) visualized by electrophoresis using a 1.8% agarose TAE gel.

Article Snippet: Membranes were blocked with 5% milk and probed with either polyclonal goat anti-human ACE2 (R&D Systems, Minneapolis, MN) or mouse anti-penta-His antibody (Qiagen, Valencia, CA).

Techniques: Reverse Transcription Polymerase Chain Reaction, Infection, Transfection, Isolation, Generated, Gene Expression, Control, Electrophoresis

Journal: Journal of Virology

Article Title: Pathways of Cross-Species Transmission of Synthetically Reconstructed Zoonotic Severe Acute Respiratory Syndrome Coronavirus

doi: 10.1128/jvi.00818-08

Figure Lengend Snippet: FIG. 1. Characterization of cACE2- and hACE2-expressing DBT cells by flow cytometry and Western blotting. ACE2 expression levels in Vero E6 cells or DBT cells stably expressing cACE2, hACE2, or GFP were assessed by flow cytometry and Western blotting. DBT cells stably transfected with plasmids carrying cACE2-His, hACE2, or GFP-His were passaged four times, after which cells were stained for ACE2 expression (primary antibody, polyclonal anti-hACE2; secondary antibody, anti-goat-FITC) and sorted for mid-to-high ACE2 expression (FITC). GFP control cells were also sorted for mid-to-high expression. (A and C) To assess ACE2 expression in expanded postsorted cell stocks and in Vero E6 cells, cells were stained as described above and analyzed for FITC/GFP expression by flow cytometry. (B) To assess ACE2 expression in Vero E6 cells and in postsorted cell stocks by Western blotting, similar numbers of Vero E6, DBT, DBT-hACE2, DBT-cACE2-His, and DBT-GFP-His cells were lysed and separated on a NuPage 12% bis-Tris SDS-polyacrylamide gel. After membrane transfer, blots were probed with either polyclonal goat anti-human ACE2 or mouse anti-penta-His antibody. After membranes were washed, they were probed with either rabbit anti-goat HRP or anti-mouse IgG-HRP antibody. Membranes were rinsed and treated with ECL Plus reagent and exposed to radiographic film.

Article Snippet: Membranes were blocked with 5% milk and probed with either polyclonal goat anti-human ACE2 (R&D Systems, Minneapolis, MN) or mouse anti-penta-His antibody (Qiagen, Valencia, CA).

Techniques: Expressing, Cytometry, Western Blot, Stable Transfection, Transfection, Staining, Control, Membrane

Journal: Journal of Virology

Article Title: Pathways of Cross-Species Transmission of Synthetically Reconstructed Zoonotic Severe Acute Respiratory Syndrome Coronavirus

doi: 10.1128/jvi.00818-08

Figure Lengend Snippet: FIG. 6. Molecular modeling demonstrating structural mechanisms of ACE2 tropism. Based on the reported crystal coordinates of SARS Urbani RBD interacting with the hACE2 receptor, we generated models of Urbani, SZ16, SZ16-K479N, and SZ16 K479N D22 RBD interactions with either cACE2 or hACE2, using RosettaDesign and Modeler software. Ribbon structures and “space filling” schematics of each RBD and ACE2 combination are shown. Dotted spheres around the RBD and ACE2 residues indicate they are within 4 Å and thus are predicted to interact. Red spheres around the RBD and ACE2 residues indicate a steric clash. (A) Urbani RBD and hACE2 architecture. (B) Additional methyl groups of the cACE2 E30 and Y34 mutations add a surface protrusion to the contact interface. The Urbani RBD can accommodate the increased surface protrusion of cACE2, thereby retaining an efficient binding interface. (C) Similar to the Urbani RBD, the SZ16 RBD can accommodate the increased surface protrusion of cACE2 for efficient binding. (D) The N479 mutation in SZ16 K479N remodels the SZ16 binding interface to promote binding to hACE2. (E) The remodeling of the SZ16 K479N binding interface by the N479 mutation creates a clash between S residues (V404 and Y440) and cACE2 residues (E30 and Y34), blocking S and ACE2 binding. The SZ16 K479N RBD cannot accommodate the extended surface protrusion of the cACE2 RBD. (F) In addition to the N479 mutation, the F442 and F472 mutations further remodel the SZ16 K479N D22 RBD, further enhancing the binding efficiency to hACE2. (G) Similar to the SZ16 K479N RBD interaction with cACE2, the interaction of the SZ16 K479N D22 RBD cannot accommodate the protrusion of cACE2, abrogating binding.

Article Snippet: Membranes were blocked with 5% milk and probed with either polyclonal goat anti-human ACE2 (R&D Systems, Minneapolis, MN) or mouse anti-penta-His antibody (Qiagen, Valencia, CA).

Techniques: Generated, Software, Binding Assay, Mutagenesis, Blocking Assay