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

Biochemical and structural analyses suggest that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) efficiently infects humans, and the presence of four residues (PRRA) at the S1/S2 site within the spike (S) protein may lead to unexpected tissue or host tropism. Here, we report that SARS-CoV-2 efficiently utilized angiotensin-converting enzyme 2 (ACE2) of 9 species to infect 293T cells. Similarly, pseudoviruses bearing S protein derived from either bat RaTG13 or pangolin GX, two closely related animal coronaviruses, utilized ACE2 of a diverse range of animal species to gain entry. The removal of PRRA from SARS-CoV-2 S protein displayed distinct effects on pseudoviral entry into different cell types. Unexpectedly, the 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 S1 and 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 usages 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.