Analysis of angiotensin-converting enzyme 2 (ACE2) from different species sheds some light on cross-species receptor usage of a novel coronavirus 2019-nCoV
Authors: Rui Li, Songlin Qiao, Gaiping Zhang
Journal: Journal of Infection (2020)

A novel coronavirus from Wuhan in central China, named 2019-nCoV, has recently caused an epidemic of pneumonia in humans and posed a huge threat to global public health.1Wu F. Zhao S. Yu B. Chen Y.-.M. Wang W. Song Z.-.G. et al.A new coronavirus associated with human respiratory disease in China.Nature. 2020; (PubMed PMID:32015508)https://doi.org/10.1038/s41586-020-2008-3Crossref Scopus (7481) Google Scholar,2Li Q. Guan X. Wu P. Wang X. Zhou L. Tong Y. et al.Early transmission dynamics in Wuhan, China, of novel coronavirus-infected pneumonia.N Engl J Med. 2020; (PubMed PMID:31995857)https://doi.org/10.1056/NEJMoa2001316Crossref Scopus (10590) Google Scholar To the date 06/02/2020, 2019-nCoV has led to more than 31,000 confirmed cases and 637 deaths in China according to National Health Commission of the People's Republic of China (http://en.nhc.gov.cn/index.html). Cases have also been documented in a growing number of other international locations, including the United States (https://www.cdc.gov/coronavirus/2019-ncov/index.html). As a consequence, it is urgent to develop effective measures to control this novel coronavirus on the basis of its pathogenesis. Host receptor recognition is a determinant for virus infection. During the time of this letter preparation, three works have just been published to explore the receptor usage of 2019-nCoV. A work by Zheng-Li Shi et al. has shown that angiotensin-converting enzyme 2 (ACE2), the receptor for severe acute respiratory syndrome coronavirus (SARS-CoV),3Li W. Moore M.J. Vasilieva N. Sui J. Wong S.K. Berne M.A. et al.Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus.Nature. 2003; 426 (PubMed PMID:14647384): 450-454Crossref PubMed Scopus (4499) Google Scholar from human, Rhinolophus sinicus bat, civet, swine but not mouse mediate 2019-nCoV infection in vitro, while the detailed mechanisms are not yet determined.4Zhou P. Yang X.-.L. Wang X.-.G. Hu B. Zhang L. Zhang W. et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature. 2020; (PubMed PMID:32015507)https://doi.org/10.1038/s41586-020-2012-7Crossref Scopus (14262) Google Scholar The other two works have reported or predicted human ACE2 usage of 2019-nCoV in a similar way to SARS-CoV mainly based on the coronavirus spike (S) glycoproteins.5Letko M, Munster V. Functional assessment of cell entry and receptor usage for lineage b β-coronaviruses, including 2019-nCoV. bioRxiv. 2020:2020.01.22.915660.Google Scholar,6Wan Y. Shang J. Graham R. Baric R.S. Li F. Receptor recognition by novel coronavirus from Wuhan: an analysis based on decade-long structural studies of SARS.J Virol. 2020; (JVI.00127-20. PubMed PMID:31996437. eng)Crossref Scopus (3133) Google Scholar Considering the fact that the S proteins mutate and gain capability to recognize host receptors among species,7Hulswit R.J.G. de Haan C.A.M. Bosch B.J. Coronavirus spike protein and tropism changes.Adv Virus Res. 2016; 96 (PubMed PMID:27712627. Epub 2016/09/13. eng): 29-57Crossref PubMed Scopus (305) Google Scholar,8Li F. Structure, function, and evolution of coronavirus spike proteins.Annu Rev Virol. 2016; 3 (PubMed PMID:27578435. Epub 2016/08/25. eng): 237-261Crossref PubMed Scopus (1788) Google Scholar there is still a lack of analyses on receptor usage of 2019-nCoV from the receptor perspective, which does not evolve as quickly as viruses. Here, we firstly performed amino acid sequence alignment of ACE2 from different species, including human, five non-human primates (gibbon, green monkey, macaque, orangutan and chimpanzee), two companion animals (cat and dog), six domestic animals (bovine, sheep, goat, swine, horse and chicken), three wild animals (ferret, civet and Chinese horseshoe bat) and two rodents (mouse and rat). The alignment by Clustal W 2.1 shows that they share a high sequence similarity except chicken (data not shown). The result suggests that 2019-nCoV of probable bat origin may not interact with chicken ACE2 and subsequently infect them, which were not considered in the following analyses. In ACE2, the regions at position 30–41, 82–84 and 353–357 are demonstrated to be involved in the interaction with SARS-CoV S protein, where the residues at positions 31, 35, 38, 82 and 353 are critical.9Li F. Li W. Farzan M. Harrison S.C. Structure of sars coronavirus spike receptor-binding domain complexed with receptor.Science. 2005; 309 (PubMed PMID:16166518. eng): 1864-1868Crossref PubMed Scopus (1495) Google Scholar Therefore, we took a close comparison in these regions and residues. As shown in Fig. 1, human and non-human primates share the identity sequences in the regions and residues, implying that ACE2 from non-human primates may recognize 2019-nCoV and mediate its infection. As a result, non-human primates may be susceptible to 2019-nCoV and serve as animal models for antiviral research or intermediate hosts for cross-species transmission. In Fig. 1, the residues of most companion, domestic and wild animals are conserved, especially for the critical ones stated above, while certain ones are variable. For example, Lys31, Glu35, Asp/Glu38 and Lys353 are conserved, which probably form salt bridges. Interestingly, the changes at positions 31, 38 and 82 are observed. These changes suggest steric hindrance and electrostatic interference for host-virus interaction. Taking civet ACE2 as an example, the change of Lys31 to Thr31 is likely to form a hydrogen bond instead of a salt bridge. In addition, the polar side chain of Thr82 may influence the hydrophobic interaction of the original Met82. All these changes may result in a lower binding affinity. However, an additional region covering residues 90–93 has been shown to be involved in civet ACE2 binding to SARS-CoV and enhance their interaction.10Li F. Structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections.J Virol. 2008; 82 (PubMed PMID:18448527. Epub 2008/04/30. eng): 6984-6991Crossref PubMed Scopus (140) Google Scholar Consequently, we can't preclude the existence of other regions to compensate for the residue changes. With most residues in human ACE2, the ones from these compaion, domestic and wild animals may be favorable for 2019-nCoV recognition, which is in consistent with the recent work by Zheng-Li Shi et al. In case cross-species transmission, close contact with sick or asymptomatic companion, domestic and wild animals should be cautious, such as for workers in livestock farming and travellers in the wild. In contrast, certain significant changes occur in the mouse and/or rat ACE2 compared to the human one (Fig. 1). The Asn31 and Ser82 in mouse ACE2 may not form favorable interactions with 2019-nCoV due to their electrostatic or hydrophilic characteristics. Importantly, the change into His353 in both mouse and rat ACE2 does not form a strong salt bridge as Lys353 does. Since the structural information for mouse and rat ACE2 is unavailable, we carried out homology modeling using human ACE2 (PDB code 2AJF) as template on online (https://swissmodel.expasy.org) for further analyses. In Fig. 2A, the change into Ser82 in mouse ACE2 may interfere with the hydrophobic interaction of the original Met82. Additionally, the changes into Asn31 and His353 definitely affect the salt bridge formation and electrostatic potential. The change into His353 in rat ACE2 is similar in the effect on receptor-virus interaction (Fig. 2B). These analyses partially explain why mouse ACE2 does not mediate 2019-nCoV infection reported by Zheng-Li Shi et al. and assume that rodents are not likely to be the susceptible host. In conclusion, we conducted sequence and structural analyses of angiotensin-converting enzyme 2 (ACE2) from different species, which sheds some light on cross-species receptor usage of 2019-nCoV. All these analyses raise an alert on a potential interspecies transmission of 2019-nCoV and propose further surveillance in other animal populations. Structural studies on human and other species ACE2 in complex with 2019-nCoV spike protein will contribute to understanding cross-species receptor usage of this novel coronavirus. The authors declare no conflict of interest. This work was supported by Henan Emergency Project for Prevention and Control of 2019 Novel Coronavirus, the Earmarked Fund for Modern Agro-industry Technology Research System of China (CARS-35) and the Special Fund for Henan Agriculture Research System (S2012-06). The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.