The PRRA Insert at the S1/S2 Site Modulates Cellular Tropism of SARS-CoV-2 and ACE2 Usage by the Closely Related Bat RaTG13

Abstract

Biochemical and structural analyses suggest that SARS-CoV-2 is well-adapted to infecting humans and the presence of four residues (PRRA) at the S1/S2 site within the spike (S) protein, which may lead to unexpected tissue or host tropism. Here we report that SARS-CoV-2 efficiently utilized ACE2 of 9 species to infect 293T cells. Similarly, pseudoviruses bearing S protein derived from either the bat RaTG13 or pangolin GX, two closely related animal coronaviruses, utilized ACE2 of a diverse range of animal species to gain entry. Removal of PRRA from SARS-CoV-2 S protein displayed distinct effects on pseudoviral entry into different cell types. Unexpectedly, insertion of PRRA into the RaTG13 S protein selectively abrogated the usage of horseshoe bat and pangolin ACE2 but enhanced the usage of mouse ACE2 by the relevant pseudovirus to enter cells. Together, our findings identified a previously unrecognized effect of the PRRA insert on SARS-CoV-2 and RaTG13 S proteins. Importance The four-residue insert (PRRA) at the boundary between the S1and S2 subunits of SARS-CoV-2 has been widely recognized since day 1 for its role in SARS-CoV-2 S protein processing and activation. As this PRRA insert is unique to SARS-CoV-2 among group b betacoronaviruses, it is thought to affect the tissue and species tropism of SARS-CoV-2. We compared the usage of 10 ACE2 orthologs and found that the presence of PRRA not only affects the cellular tropism of SARS-CoV-2 but also modulates the usage of ACE2 orthologs by the closely related bat RaTG13 S protein. The binding of pseudovirions carrying RaTG13 S with a PRRA insert to mouse ACE2 was nearly 2-fold higher than that of pseudovirions carrying RaTG13 S.

Characterization of entry pathways, species-specific ACE2 residues determining entry, and antibody neutralization evasion of Omicron BA.1, BA.1.1, BA.2, and BA.3 variants

“…293T cells transiently transfected with ACE2 receptors of each species were infected with pseudoviruses bearing spike proteins of D614G and the Omicron variants. We found that D614G pseudoviruses infected cells expressing ACE2 receptors of all the species well above the background except mouse, consistent with prior studies 3,33,42 (Figure 5A). Conversely, ACE2 receptors of all species, except horseshoe bat, supported infection by Omicron pseudoviruses (Figure 5B-E).…”

“…The HIV gag/pol packaging (pCMV∆R8.2) and firefly luciferase encoding transfer vector (pHR'CMV-Luc) plasmids 62,63 were obtained from the Vaccine Research Center (National Institutes of Health, Bethesda, MD, USA). ACE2 genes of various species (African green monkey (AGM), Chinese rufous horseshoe bat (Rhinolophus sinicus), ferret, mouse, Chinese hamster, Syrian golden hamster, white-tailed deer, swine, bovine, and pangolin) with a C-terminal V5 tag were synthesized by GenScript as described previously 42 .…”

Phage-Displayed Mimotopes of SARS-CoV-2 Spike Protein Targeted to Authentic and Alternative Cellular Receptors

“…Our current understanding of the molecular mechanisms of virus adaptation to human host cells [30] and immuno-resistant human populations is not sufficient to rationally design efficient countermeasures against viral pandemics, such as the SARS-CoV-2 virus. To empower increased insight into the evolutionary mechanisms of the SARS-CoV-2 spike protein, we suggest using recombinant filamentous bacteriophage-based probes [31], displaying on their surface an array of ~4000 foreign peptides, which produces a unique molecular landscape across the viral surface that can mimic the structure of viral receptor-binding sites [32][33][34][35][36][37], as illustrated in Figures 2 and 3.…”

Structure–Function Analysis of Resistance to Bamlanivimab by SARS-CoV-2 Variants Kappa, Delta, and Lambda

“…SARS-CoV-2 pseudovirus production and neutralization assay human codon-optimized cDNA encoding SARS-CoV-2 S glycoprotein of the Wuhan-Hu-1 isolate (NC_045512) or the Kappa variant was synthesized by GenScript and cloned into eukaryotic cell expression vector pcDNA 3.1 between the BamHI and XhoI sites. 38 Pseudovirions were produced by co-transfection of Lenti-X 293T cells with psPAX2, pTRIP-luc, and SARS-CoV-2 S expressing plasmid using Lipofectamine 3000. The supernatants were harvested at 48 and 72 h post-transfection and filtered through 0.45 μm membranes.…”