Broad host tropism of ACE2-using MERS-related coronaviruses and determinants restricting viral recognition

Abstract

Phylogenetically distant coronaviruses have evolved to employ ACE2 as their common receptors, including NL63 and many Severe acute respiratory syndrome (SARS) coronavirus-related viruses. Recently, we found two Middle East respiratory syndrome coronaviruses (MERS-CoV)-related bat coronaviruses, NeoCoV and PDF-2180, also use Angiotensin-converting enzyme 2(ACE2) but not MERS-CoV receptor dipeptidyl peptidase 4 (DPP4) for entry. Receptor binding domain (RBD)-binding and pseudovirus entry assays based on a wide range of bat ACE2 orthologs revealed that the two viruses strongly prefer ACE2 from Yangochiropteran bats as compared with Yinpterochiropteran bats, which is not observed in NL63 and SARS-CoV-2. Genetic and structural analyses of the virus-receptor interactions of 50 bat ACE2 orthologs pointed to four crucial host range determinants in two viral binding loops on ACE2. Subsequent functional verifications via mutagenesis on representative ACE2 orthologs confirmed the importance of these determinants on human and bat cells. Remarkably, NeoCoV-T510F, a mutation previously shown to acquire human ACE2 recognition, displayed an expanded potential host range covering most tested bat ACE2, probably due to its reinforced interaction with an evolutionary conserved hydrophobic pocket. Our results elucidated the molecular mechanisms for the species-specific ACE2 usage of MERS-related viruses, offering basic information for assessing the zoonotic risk of these ACE2 utilizing merbecoviruses.

Multiple independent acquisitions of ACE2 usage in MERS-related coronaviruses

“…To determine whether MOW-15-22 and PnNL2018B utilize ACE2 as their receptors, we conducted a series of cell-based experiments to evaluate the functionality of various ACE2 orthologue in facilitating viral RBD binding and pseudovirus entry. As the complete genome sequences or coding sequences of ACE2 and DPP4 orthologues from P. nathusii are currently unavailable, we utilized a well-characterized receptor library consisting of 103 ACE2 and 7 DPP4 orthologues from 52 bats and 53 non-bat mammals to comprehensively assess RBD binding and pseudovirus entry efficiency 33 . These assays were carried out in 293T cells transiently transfected with plasmids expressing the receptors, all of which were previously confirmed to be properly expressed.. Our findings indicate that MOW-15-22 pseudovirus entry occurred in cells expressing bat ACE2 from P. par, P. dav, L. bor, and several other bat species, while the the PnNL2018B pseudovirus entry was supported by bat ACE2 from L. bor, N. hum, P. pip, and several other bat species ( Fig.…”

“…To determine whether MOW-15-22 and PnNL2018B utilize ACE2 as their receptors, we conducted a series of cell-based experiments to evaluate the functionality of various ACE2 orthologue in facilitating viral RBD binding and pseudovirus entry. As the complete genome sequences or coding sequences of ACE2 and DPP4 orthologues from P. nathusii are currently unavailable, we utilized a well-characterized receptor library consisting of 103 ACE2 and 7 DPP4 orthologues from 52 bats and 53 non-bat mammals to comprehensively assess RBD binding and pseudovirus entry efficiency 33 .…”

“…While these two viruses exhibit limited efficiency in using human ACE2 (hACE2), a single mutation (e.g., T510F) in the receptor binding motif (RBM) enhances their ability to enter human cells. Moreover, NeoCoV and PDF-2180 display a broad ACE2 recognition spectrum across various mammals, underscoring a relatively high zoonotic risk 32,33 .…”

“…[19][20][21][22][23] We recently revealed that NeoCoV and PDF-2180 also use the ACE2 receptor with a broad tropism across mammals but low efficiency in using human ACE2 (hACE2). 9,24 Cryo-electron microscopy (cryo-EM) analysis of the NeoCoV and the PDF-2180 RBDs in complex with Pipistrellus pipistrellus (P.pip) ACE2 unveiled a distinct binding mode from that of SARS-CoV-1/SARS-CoV-2 and of NL63 involving extensive interactions with ACE2 N linked-glycans. The footprint of their RBDs shared few residues with that of SARS-CoV-1/2 or NL63, highlighting a convergent evolutionary history of ACE2 acquisition in these viruses.…”

Sarbecovirus RBD indels and specific residues dictating ACE2 multi-species adaptiveness

“…ACE2 stable expression cell lines were established as previously reported 30,59 . Briefly, lentivirus carrying the ACE2 genes was generated by co-transfecting pLVX-IRES-puro-ACE2 orthologues, pMD2G (plasmid no.…”