Functional and genetic analysis of viral receptor ACE2 orthologs reveals a broad potential host range of SARS-CoV-2

The pandemic of COVID-19, caused by SARS-CoV-2, is a major global health threat. Epidemiological studies suggest that bats (Rhinolophus affinis) are the natural zoonotic reservoir for SARS-CoV-2. However, the host range of SARS-CoV-2 and intermediate hosts that facilitate its transmission to humans remain unknown. The interaction of coronavirus with its host receptor is a key genetic determinant of host range and cross-species transmission. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) as the receptor to enter host cells in a species-dependent manner. In this study, we characterized the ability of ACE2 from diverse species to support viral entry. By analyzing the conservation of five residues in two virus-binding hotspots of ACE2 (hotspot 31Lys and hotspot 353Lys), we predicted 80 ACE2 proteins from mammals that could potentially mediate SARS-CoV-2 entry. We chose 48 ACE2 orthologs among them for functional analysis, and showed that 44 of these orthologs—including domestic animals, pets, livestock, and animals commonly found in zoos and aquaria—could bind the SARS-CoV-2 spike protein and support viral entry. In contrast, New World monkey ACE2 orthologs could not bind the SARS-CoV-2 spike protein and support viral entry. We further identified the genetic determinant of New World monkey ACE2 that restricts viral entry using genetic and functional analyses. These findings highlight a potentially broad host tropism of SARS-CoV-2 and suggest that SARS-CoV-2 might be distributed much more widely than previously recognized, underscoring the necessity to monitor susceptible hosts to prevent future outbreaks.

Coronaviruses are a group of positive-stranded, enveloped RNA viruses that circulate broadly among humans, other mammals, and birds, causing respiratory, enteric, or hepatic diseases (1). In the last two decades, coronaviruses have caused three major outbreaks: severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and the recent coronavirus disease 2019 (COVID-19) (2, 3). As of December 7, 2020, COVID-19 has already caused 50 million infections, leading to 1 million deaths globally. The pathogen responsible is a novel coronavirus-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (4, 5). Phylogenetic and epidemiological analyses suggest that SARS-CoV, MERS-CoV, and SARS-CoV-2 likely originated from bats, with SARS-CoV spreading from bats to palm civets to humans, and MERS-CoV spreading from bats to camel to humans (6). However, the intermediate host of SARS-CoV-2, fueling spillover to humans, remains unknown.The SARS-CoV-2 genome encodes a spike (S) protein, the receptor-binding domain (RBD) of which binds the cellular receptor angiotensin-converting enzyme 2 (ACE2) to mediate viral entry (5, 7). Following binding of ACE2, the S protein is subsequently cleaved by the host transmembrane serine protease 2 (TMPRSS2) to release the spike fusion peptide, promoting virus entry into target cells (7). It has been demonstrated that the interaction of a virus with species-specific receptors is a primary determinant of host tropism and therefore constitutes a major interspecies barrier at the level of viral entry (8). For example, murine ACE2 does not efficiently bind the SARS-CoV or SARS-CoV-2 S protein, hindering viral entry into murine cells; consequently, a human ACE2 transgenic mouse was developed as an in vivo model to study the infection and pathogenesis of these two viruses (9, 10).ACE2 is expressed in a diverse range of species throughout the subphylum Vertebrata. Several recent studies demonstrated that ferrets, cats, dogs, and some nonhuman primates are susceptible to SARS-CoV-2 (11–15). However, the exact host tropism of SARS-CoV-2 remains unknown and it is urgent to identify the putative zoonotic reservoirs to prevent future outbreaks. Numerous studies have predicted ACE2 orthologs/SARS-CoV-2 S binding affinity or energies but lack of support by virus infection experimentation (16–21). In this study, we experimentally assessed ACE2 orthologs from a broad range of species for their ability to support SARS-CoV-2 entry. Our data demonstrate that an evolutionarily diverse set of ACE2 species variants can mediate SARS-CoV-2 entry, suggesting that SARS-CoV-2 has a broad host range at the level of virus entry that may contribute to cross-species transmission and viral evolution.