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. 2008 Sep;82(17):8721-32.
doi: 10.1128/JVI.00818-08. Epub 2008 Jun 25.

Pathways of cross-species transmission of synthetically reconstructed zoonotic severe acute respiratory syndrome coronavirus

Timothy Sheahan  1 , Barry Rockx, Eric Donaldson, Davide Corti, Ralph Baric
Affiliations

Pathways of cross-species transmission of synthetically reconstructed zoonotic severe acute respiratory syndrome coronavirus

Timothy Sheahan et al. J Virol. 2008 Sep.

Abstract

Zoonotic severe acute respiratory syndrome coronavirus (SARS-CoV) likely evolved to infect humans by a series of transmission events between humans and animals in markets in China. Virus sequence data suggest that the palm civet served as an amplification host in which civet and human interaction fostered the evolution of the epidemic SARS Urbani strain. The prototypic civet strain of SARS-CoV, SZ16, was isolated from a palm civet but has not been successfully cultured in vitro. To propagate a chimeric recombinant SARS-CoV bearing an SZ16 spike (S) glycoprotein (icSZ16-S), we constructed cell lines expressing the civet ortholog (DBT-cACE2) of the SARS-CoV receptor (hACE2). Zoonotic SARS-CoV was completely dependent on ACE2 for entry. Urbani grew with similar kinetics in both the DBT-cACE2 and the DBT-hACE2 cells, while icSZ16-S only grew in DBT-cACE2 cells. The SZ16-S mutant viruses adapted to human airway epithelial cells and displayed enhanced affinity for hACE2 but exhibited severe growth defects in the DBT-cACE2 cells, suggesting that the evolutionary pathway that promoted efficient hACE2 interactions simultaneously abolished efficient cACE2 interactions. Structural modeling predicted two distinct biochemical interaction networks by which zoonotic receptor binding domain architecture can productively engage hACE2, but only the Urbani mutational repertoire promoted efficient usage of both hACE2 and cACE2 binding interfaces. Since dual species tropism was preserved in Urbani, it is likely that the virus evolved a high affinity for cACE2/hACE2 receptors through adaptation via repeated passages between human and civet hosts. Furthermore, zoonotic SARS-CoV was variably neutralized by antibodies that were effective against the epidemic strain, highlighting their utility for evaluating passive immunization efficacy.

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Figures

FIG. 1.
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.
FIG. 2.
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 μl 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.
FIG. 3.
FIG. 3.
Assessment of virus growth in DBT, DBT-cACE2, DBT-hACE2, and Vero cells, the spike amino acid variation in our recombinant virus panel, and the ACE2 contact residues with the Urbani spike. DBT-cACE2 (A), DBT-hACE2 (B), Vero E6 (C), or DBT (data not shown) cells were infected with icCUHK-W1, icGD03-S, icGZ02, icSZ16-S K479N p6, icSZ16-S K479N D22, icSARS, or icSZ16-S at an MOI of 0.01 for 1 h at 37°C. Cell medium (25 μl) was removed at 0, 6, 12, 24, and 36 hpi, and samples were stored at −80°C until titers were determine by plaque assay. Growth curves were performed in duplicate on two separate occasions, and the data shown are representative of one experiment. (D) The location of spike amino acid differences among the recombinant virus panels. (E) Urbani, cACE2, primate-ACE2, and hACE2 contact residues, adapted from the crystal structure published by Li et al. (17).
FIG. 4.
FIG. 4.
icGD03-S and icSZ16-S are dependent on ACE2 for entry. DBT-cACE2 cells were seeded at 5 × 105 cells/well in six-well dishes. The following day, cell medium was removed, and cells were incubated with 10, 5, 2.5, 1.25, or 0.625 μg/ml polyclonal anti-ACE2 or anti-ACE or DBPS for 1 h at 37°C. After the 1-h pretreatment with antibody, 100 PFU/50 μl of icSARS, icSZ16-S, or icGD03-S was added to the monolayer and incubated for 1 h at 37°C. After the infection, the inoculum was removed and the monolayer was rinsed with DPBS and then overlaid with 0.9% agarose in complete growth medium. At 48 hpi, plates were stained with neutral red, and plaques were counted. The average percentage of blockade was calculated by dividing the average number of plaques per Ab dilution by the average number of plaques in the DPBS no-Ab controls. Blockade experiments were performed in duplicate on two separate occasions.
FIG. 5.
FIG. 5.
hu-MAb S230.15 and S3.1 neutralization profiles differ between the epidemic, the in vitro-evolved, and the zoonotic strains of SARS-CoV. Neutralizing titers were determined by PRNT assay. Twenty-four hours before cells were infected, six-well plates were seeded with 5 × 105 DBT-cACE2 cells/well. hu-MAb S230.15 and S3.1 and an isotype control antibody directed against cholera toxin, D2.2, were serially diluted twofold and incubated with 100 PFU of either icSARS, icSZ16-S, or icGD03-S for 1 h at 37°C. Virus and antibodies were then added to six-well plates of DBT-cACE2 cells in duplicate and incubated at 37°C for 1 h, after which the cells were overlaid with 3 ml of 0.8% agarose in medium. After 48 h, plates were stained with neutral red, and plaques were counted. The percentage of neutralization was calculated as 1 − (number of plaques with antibody/number of plaques without antibody) × 100%.
FIG. 6.
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.
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