SPIKE MUTATION T403R ALLOWS BAT CORONAVIRUS RaTG13 to USE HUMAN ACE2

ABSTRACT

Background: The bat coronavirus RaTG13 shares 96% sequence identity to SARS-CoV-2, the causative agent of the COVID-19 pandemic. However, the RaTG13 Spike (S) protein interacts only weakly with the human SCoV-2 receptor Angiotensin-converting Enzyme 2 (ACE2) and does not mediate efficient infection of human cells. Here, we examined which alterations are required to allow the RaTG13 S protein to use human ACE2 for efficient entry into human cells. Methods: Sequence alignments showed that SARS-CoV-2 almost invariantly encodes a positively charged amino acid at position 403 of its S protein, while RaTG13 has a neutral Threonine (T). REAX based computational modeling suggested that S R403 contributes to binding of human ACE2. Wild-type and T403R mutant RaTG13 S proteins were investigated for their ability to bind ACE2 and to mediate infection of pseudotyped VSV particles in human lung-and intestine-derived cell lines as well as hPSC-derived gut organoids. Replication-competent recombinant SCoV2 S R403T was produced and replication monitored. In addition, we mutated human ACE2 to map the interacting residue of S R403. Finally, sera of vaccinated individuals were analyzed for their neutralizing potential against various WT CoV and RaTG13 S as well as mutant S containing pseudoparticles. Results: Our results show that a single amino acid change of T403R allows the RaTG13 S to utilize human ACE2 for viral entry. Spike T403R enhanced infection of VSV-based RatG13 S pseudotypes in human lung and colon cells as well as gut-derived organoids. Vice versa R403T mutation reduced infectivity of SCoV2 S pseudotypes and recombinant SCoV2 replication. The enhancing effect of T403R in RaTG13 S depends on E37 in ACE2. RaTG13 T403R S-mediated infection was blocked by the fusion inhibitor EK-1 but not by the SCoV-2 antibody Casirivimab. SARS-CoV-2 and the T403R RaTG13 S were equally susceptible to neutralization by sera from individuals vaccinated against COVID-19. Conclusion: A positively charged amino acid at position 403 in the S protein of bat coronaviruses is critical for efficient utilization of human ACE2. Our results help to better assess the zoonotic potential of bat sarbecoviruses and suggest that COVID-19 vaccination will also protect against closely bat relatives of SARS-CoV-2 that may emerge in the future.