FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3131417871> ?p ?o ?g. }

• W3131417871 endingPage "100435" @default.
• W3131417871 startingPage "100435" @default.
• W3131417871 abstract "The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic represents a global threat, and the interaction between the virus and angiotensin-converting enzyme 2 (ACE2), the primary entry receptor for SARS-CoV-2, is a key determinant of the range of hosts that can be infected by the virus. However, the mechanisms underpinning ACE2-mediated viral entry across species remains unclear. Using infection assay, we evaluated SARS-CoV-2 entry mediated by ACE2 of 11 different animal species. We discovered that ACE2 of Rhinolophus sinicus (Chinese rufous horseshoe bat), Felis catus (domestic cat), Canis lupus familiaris (dog), Sus scrofa (wild pig), Capra hircus (goat), and Manis javanica (Malayan pangolin) facilitated SARS-CoV-2 entry into nonsusceptible cells. Moreover, ACE2 of the pangolin also mediated SARS-CoV-2 entry, adding credence to the hypothesis that SARS-CoV-2 may have originated from pangolins. However, the ACE2 proteins of Rhinolophus ferrumequinum (greater horseshoe bat), Gallus gallus (red junglefowl), Notechis scutatus (mainland tiger snake), or Mus musculus (house mouse) did not facilitate SARS-CoV-2 entry. In addition, a natural isoform of the ACE2 protein of Macaca mulatta (rhesus monkey) with the Y217N mutation was resistant to SARS-CoV-2 infection, highlighting the possible impact of this ACE2 mutation on SARS-CoV-2 studies in rhesus monkeys. We further demonstrated that the Y217 residue of ACE2 is a critical determinant for the ability of ACE2 to mediate SARS-CoV-2 entry. Overall, these results clarify that SARS-CoV-2 can use the ACE2 receptors of multiple animal species and show that tracking the natural reservoirs and intermediate hosts of SARS-CoV-2 is complex. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic represents a global threat, and the interaction between the virus and angiotensin-converting enzyme 2 (ACE2), the primary entry receptor for SARS-CoV-2, is a key determinant of the range of hosts that can be infected by the virus. However, the mechanisms underpinning ACE2-mediated viral entry across species remains unclear. Using infection assay, we evaluated SARS-CoV-2 entry mediated by ACE2 of 11 different animal species. We discovered that ACE2 of Rhinolophus sinicus (Chinese rufous horseshoe bat), Felis catus (domestic cat), Canis lupus familiaris (dog), Sus scrofa (wild pig), Capra hircus (goat), and Manis javanica (Malayan pangolin) facilitated SARS-CoV-2 entry into nonsusceptible cells. Moreover, ACE2 of the pangolin also mediated SARS-CoV-2 entry, adding credence to the hypothesis that SARS-CoV-2 may have originated from pangolins. However, the ACE2 proteins of Rhinolophus ferrumequinum (greater horseshoe bat), Gallus gallus (red junglefowl), Notechis scutatus (mainland tiger snake), or Mus musculus (house mouse) did not facilitate SARS-CoV-2 entry. In addition, a natural isoform of the ACE2 protein of Macaca mulatta (rhesus monkey) with the Y217N mutation was resistant to SARS-CoV-2 infection, highlighting the possible impact of this ACE2 mutation on SARS-CoV-2 studies in rhesus monkeys. We further demonstrated that the Y217 residue of ACE2 is a critical determinant for the ability of ACE2 to mediate SARS-CoV-2 entry. Overall, these results clarify that SARS-CoV-2 can use the ACE2 receptors of multiple animal species and show that tracking the natural reservoirs and intermediate hosts of SARS-CoV-2 is complex. Reply to Hayashi and Konishi: Noneffect of SARS-CoV-2 spike glycoprotein Y217N mutation on affinity between virus and ACE2Journal of Biological ChemistryVol. 296PreviewWe thank Hayashi and Konishi for their comments and interest in our article (1). We agree that by structural modeling analysis, angiotensin-converting enzyme 2 (ACE2 [Y217N]) mutant shows no changes in binding with receptor-binding domain (RBD) when compared with WT ACE2, which is depicted in Figure 4 in our article. We think the lower binding affinity of human ACE2 N217 with RBD was due to the following reasons. First, cell surface abundance of ACE2 Y217N was less compared with WT ACE2 (Fig. 5 in our article). Full-Text PDF Open AccessNoneffect of SARS-CoV-2 spike glycoprotein Y217N mutation on affinity between the virus and ACE2Journal of Biological ChemistryVol. 296PreviewThe interaction between the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and angiotensin-converting enzyme 2 (ACE2), the primary entry receptor for SARS-CoV-2, is a key determinant of the range of hosts that can be infected by the virus. Zhang et al. (1) reportedly constructed human ACE2 (hACE2) with the Y217N mutation and found that this mutation completely blocked SARS-CoV-2 entry. Zhang et al. (1) performed an receptor binding domain (RBD) binding assay and found that WT hACE2 potently bound the RBD; however, hACE2 Y217N almost lost the ability to bind the RBD. Full-Text PDF Open Access In December 2019, a novel pneumonia, termed coronavirus disease 2019 (COVID-19) by the World Health Organization (WHO), emerged from Wuhan, China, and the causative agent was soon identified as a novel coronavirus, which was named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by the International Committee on Taxonomy of Viruses (ICTV) (1Zhou P. Yang X.L. Wang X.G. Hu B. Zhang L. Zhang W. Si H.R. Zhu Y. Li B. Huang C.L. Chen H.D. Chen J. Luo Y. Guo H. Jiang R.D. et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature. 2020; 579: 270-273Crossref PubMed Scopus (13521) Google Scholar, 2Wu F. Zhao S. Yu B. Chen Y.M. Wang W. Song Z.G. Hu Y. Tao Z.W. Tian J.H. Pei Y.Y. Yuan M.L. Zhang Y.L. Dai F.H. Liu Y. Wang Q.M. et al.A new coronavirus associated with human respiratory disease in China.Nature. 2020; 579: 265-269Crossref PubMed Scopus (7027) Google Scholar). The SARS-CoV-2 outbreak has been speculatively associated with a seafood market where sales also include various wild animals (3Lu R. Zhao X. Li J. Niu P. Yang B. Wu H. Wang W. Song H. Huang B. Zhu N. Bi Y. Ma X. Zhan F. Wang L. Hu T. et al.Genomic characterisation and epidemiology of 2019 novel coronavirus: Implications for virus origins and receptor binding.Lancet. 2020; 395: 565-574Abstract Full Text Full Text PDF PubMed Scopus (7709) Google Scholar). Bats are recognized as a potential natural reservoir for SARS-CoV-2 (1Zhou P. Yang X.L. Wang X.G. Hu B. Zhang L. Zhang W. Si H.R. Zhu Y. Li B. Huang C.L. Chen H.D. Chen J. Luo Y. Guo H. Jiang R.D. et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature. 2020; 579: 270-273Crossref PubMed Scopus (13521) Google Scholar, 3Lu R. Zhao X. Li J. Niu P. Yang B. Wu H. Wang W. Song H. Huang B. Zhu N. Bi Y. Ma X. Zhan F. Wang L. Hu T. et al.Genomic characterisation and epidemiology of 2019 novel coronavirus: Implications for virus origins and receptor binding.Lancet. 2020; 395: 565-574Abstract Full Text Full Text PDF PubMed Scopus (7709) Google Scholar). However, recent studies have indicated that pangolins are also considered possible natural reservoirs of this coronavirus (4Lam T.T. Shum M.H. Zhu H.C. Tong Y.G. Ni X.B. Liao Y.S. Wei W. Cheung W.Y. Li W.J. Li L.F. Leung G.M. Holmes E.C. Hu Y.L. Guan Y. Identifying SARS-CoV-2 related coronaviruses in Malayan pangolins.Nature. 2020; 583: 282-285Crossref PubMed Scopus (1078) Google Scholar, 5Zhang T. Wu Q. Zhang Z. Probable pangolin origin of SARS-CoV-2 associated with the COVID-19 outbreak.Curr. Biol. 2020; 30: 1346-1351.e2Abstract Full Text Full Text PDF PubMed Scopus (902) Google Scholar). Discovering the potential intermediate animal hosts of SARS-CoV-2 and evaluating their possible cross-species transmissibility are scientifically very important. Unfortunately, we know little about this. A recent study revealed that ferrets and cats are sensitive to SARS-CoV-2 infection; however, these animals showed no clinical symptoms (6Shi J. Wen Z. Zhong G. Yang H. Wang C. Huang B. Liu R. He X. Shuai L. Sun Z. Zhao Y. Liu P. Liang L. Cui P. Wang J. et al.Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-coronavirus 2.Science. 2020; 368: 1016-1020Crossref PubMed Scopus (1182) Google Scholar). Whether other animals exist as a candidate SARS-CoV-2 infection model should be further explored. The interaction between receptors and viruses is a key determinant of the host range. SARS-CoV-2 resembles severe acute respiratory syndrome coronavirus (SARS-CoV), and both use angiotensin-converting enzyme 2 (ACE2) as the primary cell entry receptor (1Zhou P. Yang X.L. Wang X.G. Hu B. Zhang L. Zhang W. Si H.R. Zhu Y. Li B. Huang C.L. Chen H.D. Chen J. Luo Y. Guo H. Jiang R.D. et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature. 2020; 579: 270-273Crossref PubMed Scopus (13521) Google Scholar, 7Li W. Moore M.J. Vasilieva N. Sui J. Wong S.K. Berne M.A. Somasundaran M. Sullivan J.L. Luzuriaga K. Greenough T.C. Choe H. Farzan M. Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus.Nature. 2003; 426: 450-454Crossref PubMed Scopus (4339) Google Scholar, 8Hoffmann M. Kleine-Weber H. Schroeder S. Kruger N. Herrler T. Erichsen S. Schiergens T.S. Herrler G. Wu N.H. Nitsche A. Muller M.A. Drosten C. Pohlmann S. SARS-CoV-2 cell entry Depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease Inhibitor.Cell. 2020; 181: 271-280.e8Abstract Full Text Full Text PDF PubMed Scopus (12453) Google Scholar, 9Wan Y. Shang J. Graham R. Baric R.S. Li F. Receptor recognition by the novel coronavirus from Wuhan: An analysis based on Decade-Long structural studies of SARS coronavirus.J. Virol. 2020; 94: e00127-20Crossref PubMed Scopus (3013) Google Scholar). When we retrace the origin of a virus, the cell susceptibility to viruses conferred by receptors of speculated animals is preferentially investigated (10Li W. Wong S.K. Li F. Kuhn J.H. Huang I.C. Choe H. Farzan M. Animal origins of the severe acute respiratory syndrome coronavirus: Insight from ACE2-S-protein interactions.J. Virol. 2006; 80: 4211-4219Crossref PubMed Scopus (226) Google Scholar, 11Ren W. Qu X. Li W. Han Z. Yu M. Zhou P. Zhang S.Y. Wang L.F. Deng H. Shi Z. Difference in receptor usage between severe acute respiratory syndrome (SARS) coronavirus and SARS-like coronavirus of bat origin.J. Virol. 2008; 82: 1899-1907Crossref PubMed Scopus (116) Google Scholar). Before clarifying that Rhinolophus sinicus (Chinese rufous horseshow bat) is the natural reservoir of SARS-CoV, scientists first evaluated the susceptibility provided by ACE2 from different bat species to SARS-CoV. They found that the ACE2 of R. sinicus was responsible for the susceptibility to SARS-CoV and subsequently confirmed that R. sinicus was the natural reservoir of SARS-CoV (10Li W. Wong S.K. Li F. Kuhn J.H. Huang I.C. Choe H. Farzan M. Animal origins of the severe acute respiratory syndrome coronavirus: Insight from ACE2-S-protein interactions.J. Virol. 2006; 80: 4211-4219Crossref PubMed Scopus (226) Google Scholar, 12Ge X.Y. Li J.L. Yang X.L. Chmura A.A. Zhu G. Epstein J.H. Mazet J.K. Hu B. Zhang W. Peng C. Zhang Y.J. Luo C.M. Tan B. Wang N. Zhu Y. et al.Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor.Nature. 2013; 503: 535-538Crossref PubMed Scopus (1204) Google Scholar, 13Hou Y. Peng C. Yu M. Li Y. Han Z. Li F. Wang L.F. Shi Z. Angiotensin-converting enzyme 2 (ACE2) proteins of different bat species confer variable susceptibility to SARS-CoV entry.Arch. Virol. 2010; 155: 1563-1569Crossref PubMed Scopus (61) Google Scholar). The Middle East respiratory syndrome coronavirus (MERS-CoV) was also recognized as having a bat origin because MERS-CoV and two MERS-CoV-related viruses from bats could utilize human or bat dipeptidyl peptidase 4 (DPP4) for cell entry (14Raj V.S. Mou H. Smits S.L. Dekkers D.H. Muller M.A. Dijkman R. Muth D. Demmers J.A. Zaki A. Fouchier R.A. Thiel V. Drosten C. Rottier P.J. Osterhaus A.D. Bosch B.J. et al.Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC.Nature. 2013; 495: 251-254Crossref PubMed Scopus (1508) Google Scholar, 15Lau S.K.P. Fan R.Y.Y. Luk H.K.H. Zhu L. Fung J. Li K.S.M. Wong E.Y.M. Ahmed S.S. Chan J.F.W. Kok R.K.H. Chan K.H. Wernery U. Yuen K.Y. Woo P.C.Y. Replication of MERS and SARS coronaviruses in bat cells offers insights to their ancestral origins.Emerging microbes & infections. 2018; 7: 209Crossref PubMed Scopus (27) Google Scholar, 16Yang Y. Du L. Liu C. Wang L. Ma C. Tang J. Baric R.S. Jiang S. Li F. Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirus.Proc. Natl. Acad. Sci. U. S. A. 2014; 111: 12516-12521Crossref PubMed Scopus (211) Google Scholar). Therefore, in this study, we systemically evaluated the ability of SARS-CoV-2 to infect nonsusceptible HEK293T cells utilizing ACE2 proteins from nine different animal species and humans to determine its possible origin and to further explore its cross-species transmission. Our findings provide evidence that SARS-CoV-2 is able to engage the ACE2 receptor of different species, which poses a very large challenge to determine the natural reservoir of SARS-CoV-2 for the control and prevention of coronaviruses in the future. Furthermore, we found a natural isoform of ACE2 in Macaca mulatta (rhesus monkey; RhACE2) with the Y217N mutation, which was resistant to SARS-CoV-2 infection, and this amino acid residue is a novel critical determinant of the ability of ACE2 to mediate SARS-CoV-2 entry. To investigate which animal ACE2 proteins could facilitate SARS-CoV-2 entry, we synthesized full-length cDNA fragments of ACE2 from 11 animal species as well as from humans. These species were R. sinicus (Chinese rufous horseshoe bat), Rhinolophus ferrumequinum (greater horseshoe bat), Felis catus (domestic cat), Capra hircus (goat), Canis lupus familiaris (dog), Sus scrofa (wild pig), Manis javanica (Malayan pangolin), Gallus gallus (red junglefowl), Notechis scutatus (mainland tiger snake), Mus musculus (house mouse), M. mulatta (rhesus monkey), and Homo sapiens (human). Synthesized cDNA fragments were then subcloned into the pCAGGS-HA vector for expression in eukaryotic cells. The origins and GenBank accession numbers of these ACE2 molecules are listed in the Table 1. We first compared the nucleotide sequence of the ACE2 coding region of these 11 animals with that of humans. The sequence similarities of these ACE2 cDNAs are exhibited in the Table 1. Among these sequences, the sequence of RhACE2 was the most similar to that of human ACE2 (hACE2), and in contrast, the sequence of mainland tiger snake ACE2 was the least similar (Fig. 1A). It has been reported that two virus-binding hotspots, K31 and K353 in hACE2, are critical for SARS-CoV infection (17Li F. Structural analysis of major species barriers between humans and palm civets for severe acute respiratory syndrome coronavirus infections.J. Virol. 2008; 82: 6984-6991Crossref PubMed Scopus (137) Google Scholar, 18Wu K. Peng G. Wilken M. Geraghty R.J. Li F. Mechanisms of host receptor adaptation by severe acute respiratory syndrome coronavirus.J. Biol. Chem. 2012; 287: 8904-8911Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). In this study, we found that K31 was not conserved in ACE2 of all 11 animal species observed in this study. However, K353 was conserved in all ten animal species except mouse (Table 1).Table 1Nucleotide sequence similarity of the ACE-2 proteins of various animals to human ACE-2ACE-2 originLength of coding sequence (bp)Similarity to human ACE-2 (%)Position 31Position 353GenBank accession numberHomo sapiens (Human)2418100KKAB046569.1Rhinolophus ferrumequinum (Greater horseshoe bat)241886.2DKAB297479.1Rhinolophus sinicus (Chinese horseshoe bat)241885.5EKGQ262791.1Macaca mulatta (Rhesus monkey)aNOTE: The sequence for Macaca mulatta (Rhesus monkey) provided here contains the natural mutation Y217N.241896.6KKNM_001135696.1Sus scrofa (Pig)241884.5KKNM_001123070.1Canis lupus familiaris (Dog)241587KKNM_001165260.1Capra hircus (Goat)241585.5KKKF921008.1Felis catus (Cat)241886.8KKAY957464.1Gallus gallus (Chicken)242768.1EKMK560199.1Manis javanica (Malayan pangolin)241886.5KKXM_017,650,257.1Notechis scutatus (Mainland tiger snake)248766.5QKXM_026674969Mus musculus (Mouse)241885.2NHNM_001130513.1a NOTE: The sequence for Macaca mulatta (Rhesus monkey) provided here contains the natural mutation Y217N. Open table in a new tab Next, we tested whether ACE2 of the 11 animal species was able to facilitate SARS-CoV-2 entry to nonsusceptible HEK 293T cell lines. Different ACE2 proteins were expressed in HEK 293T cells (Fig. 1, B and C). Plasmids expressing hACE2 and mouse ACE2 were applied as the positive and negative controls of the entry assay, respectively. The infection ratio varied according to the ACE2 protein expressed (Fig. 1C). As expected, hACE2 supported SARS-CoV-2 entry, whereas mouse ACE2 did not (Fig. 1C). One previous study indicated that the SARS-CoV outbreak 17 years ago originated from Rhinolophus affinis (intermediate horseshoe bat) (12Ge X.Y. Li J.L. Yang X.L. Chmura A.A. Zhu G. Epstein J.H. Mazet J.K. Hu B. Zhang W. Peng C. Zhang Y.J. Luo C.M. Tan B. Wang N. Zhu Y. et al.Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor.Nature. 2013; 503: 535-538Crossref PubMed Scopus (1204) Google Scholar). A recent study further demonstrated that ACE2 of R. sinicus (Chinese rufous horseshoe bat) allowed both SARS-CoV-2 and SARS-CoV entry (1Zhou P. Yang X.L. Wang X.G. Hu B. Zhang L. Zhang W. Si H.R. Zhu Y. Li B. Huang C.L. Chen H.D. Chen J. Luo Y. Guo H. Jiang R.D. et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature. 2020; 579: 270-273Crossref PubMed Scopus (13521) Google Scholar). In this study, we were not able to synthesize the ACE2 cDNA of R. affinis due to the absence of its sequence. Therefore, we synthesized the ACE2 cDNA of R. sinicus and R. ferrumequinum (greater horseshoe bat) to test whether ACE2 of other bat species was responsible for the susceptibility to SARS-CoV-2. Interestingly, we found that the ACE2 of R. ferrumequinum did not support SARS-CoV-2 entry but R. sinicus did (Fig. 1C), suggesting that not all species of bat are sensitive to SARS-CoV-2 infection. A recent study indicated that SARS-CoV-2 did not replicate and shed in dogs, pigs, chickens, and ducks but replicated fairly well in ferrets and replicated effectively in cats (6Shi J. Wen Z. Zhong G. Yang H. Wang C. Huang B. Liu R. He X. Shuai L. Sun Z. Zhao Y. Liu P. Liang L. Cui P. Wang J. et al.Susceptibility of ferrets, cats, dogs, and other domesticated animals to SARS-coronavirus 2.Science. 2020; 368: 1016-1020Crossref PubMed Scopus (1182) Google Scholar). Our study demonstrated that cat ACE2 supported viral entry of SARS-CoV-2 (Fig. 1C). Although pigs, dogs, and chickens are not sensitive to SARS-CoV-2 infection, we know little about the molecular mechanisms and role of receptor avidity in resistance. Our data demonstrated that the ACE2 protein of dogs and pigs supported SARS-CoV-2 entry similar to that of cats. There is a debate regarding if SARS-CoV-2 originated from bats or pangolins (1Zhou P. Yang X.L. Wang X.G. Hu B. Zhang L. Zhang W. Si H.R. Zhu Y. Li B. Huang C.L. Chen H.D. Chen J. Luo Y. Guo H. Jiang R.D. et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature. 2020; 579: 270-273Crossref PubMed Scopus (13521) Google Scholar, 4Lam T.T. Shum M.H. Zhu H.C. Tong Y.G. Ni X.B. Liao Y.S. Wei W. Cheung W.Y. Li W.J. Li L.F. Leung G.M. Holmes E.C. Hu Y.L. Guan Y. Identifying SARS-CoV-2 related coronaviruses in Malayan pangolins.Nature. 2020; 583: 282-285Crossref PubMed Scopus (1078) Google Scholar, 5Zhang T. Wu Q. Zhang Z. Probable pangolin origin of SARS-CoV-2 associated with the COVID-19 outbreak.Curr. Biol. 2020; 30: 1346-1351.e2Abstract Full Text Full Text PDF PubMed Scopus (902) Google Scholar). It has been demonstrated that bat ACE2 mediates SARS-CoV-2 entry (1Zhou P. Yang X.L. Wang X.G. Hu B. Zhang L. Zhang W. Si H.R. Zhu Y. Li B. Huang C.L. Chen H.D. Chen J. Luo Y. Guo H. Jiang R.D. et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature. 2020; 579: 270-273Crossref PubMed Scopus (13521) Google Scholar). Therefore, we expressed ACE2 in M. javanica (Malayan pangolins) and tested its role in conferring susceptibility to SARS-CoV-2. We demonstrated that pangolin ACE2 could mediate the entry of SARS-CoV-2 (Fig. 1C). Finally, we demonstrated that ACE2 of N. scutatus (mainland tiger snake) could not support SARS-CoV-2 entry as previously predicted (19Ji W. Wang W. Zhao X. Zai J. Li X. Cross-species transmission of the newly identified coronavirus 2019-nCoV.J. Med. Virol. 2020; 92: 433-440Crossref PubMed Scopus (632) Google Scholar). Snakes may not be the natural reservoir of SARS-CoV-2. Old world monkeys (M. mulatta (rhesus monkey) and Macaca fascicularis (crab-eating macaque)) were used as experimental animal models for SARS-CoV-2 infection (20Lu S. Zhao Y. Yu W. Yang Y. Gao J. Wang J. Kuang D. Yang M. Yang J. Ma C. Xu J. Qian X. Li H. Zhao S. Li J. et al.Comparison of SARS-CoV-2 infections among 3 species of non-human primates.bioRxiv. 2020; Google Scholar). Surprisingly, we found that RhACE2 in our study did not support SARS-CoV-2 entry, as expected (Fig. 1C). By investigating the monkey ACE2 sequence, we found that the ACE2 isoform of rhesus monkey cloned in our study contained two natural variations (R192G and Y217N) (Fig. 2A). When we reverted the Y217N mutation to N217Y (wild-type sequence of ACE2) in RhACE2, RhACE2 with N217Y had the ability to support SARS-CoV-2 infection (Fig. 2B). We noticed that Y217 was conserved in the other species investigated in this study (data not shown), which suggests that residue Y217 is a key residue for SARS-CoV-2 entry. To further confirm this hypothesis, we constructed hACE2 with the Y217N mutation and found that this mutation completely blocked SARS-CoV-2 entry, as expected (Fig. 2B). Potential asparagine (N)-linked glycosylation may influence virus and receptor binding; however, when we analyzed the sequence, we found that ACE2 N217 was not a potential N-linked glycosylation site (N-X-T/S motif). We further constructed an RhACE2 with the N217Q mutation, and this mutation did not facilitate SARS-CoV-2 entry (Fig. 2B). This further demonstrated that the ability of N217 blocking viral entry is not due to N-linked glycosylation of this residue. A previous study indicated that the natural mutation of Y217N in RhACE2 dramatically alters RhACE2 expression and reduces the efficiency of SARS-CoV entry (21Chen Y. Liu L. Wei Q. Zhu H. Jiang H. Tu X. Qin C. Chen Z. Rhesus angiotensin converting enzyme 2 supports entry of severe acute respiratory syndrome coronavirus in Chinese macaques.Virology. 2008; 381: 89-97Crossref PubMed Scopus (22) Google Scholar). In the current study, we illustrated that both hACE2 and RhACE2 Y217N expressed as well as the corresponding wild-type ACE2 proteins (Fig. 2C). Next, we used the RBD as a probe to examine receptor-binding ability by using IFA. We performed an RBD binding assay and found that wild-type hACE2 potently bound the RBD, as expected (Fig. 3A); however, hACE2 Y217N, RhACE2 Y217N, and RhACE2 Y217Q almost lost the ability to bind the RBD (Fig. 3A). Receptor-binding ability was further confirmed and quantified by western blotting (Fig. 3B). Since the conformation of a recombinant RBD may not completely reflect its native conformation within an entire envelope glycoprotein, we also investigated ACE2 binding with more relevant systems of the full-length S (spike) protein, and we obtained a similar result as for the RBD (Fig. 3, C and D). The above data demonstrated that the Y217 residue of ACE2 significantly reduced the binding ability of the RBD and spike protein. To test whether position 217 of ACE2 influenced its RBD binding activities is due to N217 disrupting the interaction between ACE2 and RBD, we used homology-based structural modeling to analyze the effects of residue substitutions at position 217. Structural models of Y217N were generated based on the crystal structure of the SARS-CoV-2 RBD/ACE2 complex (22Lan J. Ge J. Yu J. Shan S. Zhou H. Fan S. Zhang Q. Shi X. Wang Q. Zhang L. Wang X. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor.Nature. 2020; 581: 215-220Crossref PubMed Scopus (3629) Google Scholar, 23Shang J. Ye G. Shi K. Wan Y. Luo C. Aihara H. Geng Q. Auerbach A. Li F. Structural basis of receptor recognition by SARS-CoV-2.Nature. 2020; 581: 221-224Crossref PubMed Scopus (2355) Google Scholar). We found that position 217 was not located at the interaction domain with RBD; furthermore, the predicted model of residue substitutions at position 217 seemed to have no influence on the interaction between ACE2 and RBD (Fig. 4). Overall, structural modeling analysis demonstrated that the substitutions at position 217 in ACE2 did not influence its corresponding receptor activities. To enter into cells, viruses need to first interact with the corresponding receptor at the cell surface; therefore, we tested the possibility that ACE2 Y217N fails to mediate SARS-CoV-2 entry due to a change in the cell surface localization of ACE2 induced by the Y217N mutation. To test this hypothesis, HEK 293T cells were first transfected with the indicated plasmids. Forty-eight hours later, the transfected cells were detached from the plate and then stained with a FITC-labeled anti-ACE2 antibody. We found that both wild-type hACE2 and RhACE2 Y217N were strongly positive for FITC, whereas the other mutants were weakly positive (Fig. 5A). To confirm that this difference was not due to transfection efficacy, the transfected cells were permeabilized with Triton X-100 and then stained with ACE2. The results indicated that all cells transfected with different ACE2 molecules were labeled with FITC at a high level (Fig. 5B).This demonstrated that RhACE2 Y217N failed to mediate SARS-CoV-2 entry, mainly due to its cell surface localization. The spike (S) protein features of coronaviruses and lysosomal proteases of hosts determine the tropism of coronaviruses (24Zheng Y. Shang J. Yang Y. Liu C. Wan Y. Geng Q. Wang M. Baric R. Li F. Lysosomal proteases are a determinant of coronavirus tropism.J. Virol. 2018; 92: e01504-18Crossref PubMed Scopus (42) Google Scholar). To date, the bat coronavirus RaTG13 in R. affinis (intermediate horseshoe bat) from Yunnan Province exhibits the highest sequence similarity to SARS-CoV-2 (1Zhou P. Yang X.L. Wang X.G. Hu B. Zhang L. Zhang W. Si H.R. Zhu Y. Li B. Huang C.L. Chen H.D. Chen J. Luo Y. Guo H. Jiang R.D. et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature. 2020; 579: 270-273Crossref PubMed Scopus (13521) Google Scholar). In this study, we found that ACE2 of R. ferrumequinum (greater horseshoe bat) failed to mediate SARS-CoV-2 entry, whereas ACE2 of R. sinicus (Chinese rufous horseshoe bat) facilitated SARS-CoV-2 entry to nonsusceptible cells. In fact, in contrast to genetically homologous hACE2, bat ACE2 proteins have great genetic diversity (13Hou Y. Peng C. Yu M. Li Y. Han Z. Li F. Wang L.F. Shi Z. Angiotensin-converting enzyme 2 (ACE2) proteins of different bat species confer variable susceptibility to SARS-CoV entry.Arch. Virol. 2010; 155: 1563-1569Crossref PubMed Scopus (61) Google Scholar). A number of ACE2 molecules isolated from different bat species failed to mediate SARS-CoV entry (13Hou Y. Peng C. Yu M. Li Y. Han Z. Li F. Wang L.F. Shi Z. Angiotensin-converting enzyme 2 (ACE2) proteins of different bat species confer variable susceptibility to SARS-CoV entry.Arch. Virol. 2010; 155: 1563-1569Crossref PubMed Scopus (61) Google Scholar). A study has reported that R. sinicus serves as a natural reservoir of SARS-CoV, and an isolated bat-origin SARS-CoV-like virus is able to use ACE2 proteins from humans and civets for cell entry (12Ge X.Y. Li J.L. Yang X.L. Chmura A.A. Zhu G. Epstein J.H. Mazet J.K. Hu B. Zhang W. Peng C. Zhang Y.J. Luo C.M. Tan B. Wang N. Zhu Y. et al.Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor.Nature. 2013; 503: 535-538Crossref PubMed Scopus (1204) Google Scholar). These results suggest that analysis of the receptor-conferred susceptibility to virus entry is important before investigating the origin of SARS-CoV-2. Recently, pangolins have also been considered a possible natural reservoir of SARS-CoV-2 (4Lam T.T. Shum M.H. Zhu H.C. Tong Y.G. Ni X.B. Liao Y.S. Wei W. Cheung W.Y. Li W.J. Li L.F. Leung G.M. Holmes E.C. Hu Y.L. Guan Y. Identifying SARS-CoV-2 related coronaviruses in Malayan pangolins.Nature. 2020; 583: 282-285Crossref PubMed Scopus (1078) Google Scholar, 5Zhang T. Wu Q. Zhang Z. Probable pangolin origin of SARS-CoV-2 associated with the COVID-19 outbreak.Curr. Biol. 2020; 30: 1346-1351.e2Abstract Full Text Full Text PDF PubMed Scopus (902) Google Scholar). Coronaviruses with high sequence homology were identified in M. javanica (Malayan pangolins), suggesting that Malayan pangolin is a possible source for the emergence of SARS-CoV-2 (4Lam T.T. Shum M.H. Zhu H.C. Tong Y.G. Ni X.B. Liao Y.S. Wei W. Cheung W.Y. Li W.J. Li L.F. Leung G.M. Holmes E.C. Hu Y.L. Guan Y. Identifying SARS-CoV-2 related coronaviruses in Malayan pangolins.Nature. 2020; 583: 282-285Crossref PubMed Scopus (1078) Google Scholar). We demonstrated that ACE2 of Malayan pangolin supported SARS-CoV-2 entry in nonsusceptible cells. SARS-CoV and MERS-CoV engage receptors of both human a" @default.
• W3131417871 created "2021-03-01" @default.
• W3131417871 creator A5000952627 @default.
• W3131417871 creator A5022855601 @default.
• W3131417871 creator A5023693252 @default.
• W3131417871 creator A5024167231 @default.
• W3131417871 creator A5026937923 @default.
• W3131417871 creator A5048966258 @default.
• W3131417871 creator A5062469117 @default.
• W3131417871 creator A5064463286 @default.
• W3131417871 creator A5073065036 @default.
• W3131417871 creator A5075557785 @default.
• W3131417871 creator A5076567651 @default.
• W3131417871 creator A5078848376 @default.
• W3131417871 date "2021-01-01" @default.
• W3131417871 modified "2023-10-18" @default.
• W3131417871 title "Evaluating angiotensin-converting enzyme 2-mediated SARS-CoV-2 entry across species" @default.
• W3131417871 cites W1966238900 @default.
• W3131417871 cites W1993577573 @default.
• W3131417871 cites W2065042116 @default.
• W3131417871 cites W2082231154 @default.
• W3131417871 cites W2090139119 @default.
• W3131417871 cites W2101166094 @default.
• W3131417871 cites W2103498752 @default.
• W3131417871 cites W2119111857 @default.
• W3131417871 cites W2121757696 @default.
• W3131417871 cites W2146242600 @default.
• W3131417871 cites W2783739334 @default.
• W3131417871 cites W2893432007 @default.
• W3131417871 cites W2949841045 @default.
• W3131417871 cites W3000771439 @default.
• W3131417871 cites W3003217347 @default.
• W3131417871 cites W3004280078 @default.
• W3131417871 cites W3004318991 @default.
• W3131417871 cites W3004348779 @default.
• W3131417871 cites W3006743526 @default.
• W3131417871 cites W3009912996 @default.
• W3131417871 cites W3010441732 @default.
• W3131417871 cites W3011680753 @default.
• W3131417871 cites W3013152614 @default.
• W3131417871 cites W3013800395 @default.
• W3131417871 cites W3014067025 @default.
• W3131417871 cites W3014233146 @default.
• W3131417871 cites W3015190630 @default.
• W3131417871 cites W3025798952 @default.
• W3131417871 cites W3043468445 @default.
• W3131417871 doi "https://doi.org/10.1016/j.jbc.2021.100435" @default.
• W3131417871 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7892319" @default.
• W3131417871 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/33610551" @default.
• W3131417871 hasPublicationYear "2021" @default.
• W3131417871 type Work @default.
• W3131417871 sameAs 3131417871 @default.
• W3131417871 citedByCount "28" @default.
• W3131417871 countsByYear W31314178712021 @default.
• W3131417871 countsByYear W31314178712022 @default.
• W3131417871 countsByYear W31314178712023 @default.
• W3131417871 crossrefType "journal-article" @default.
• W3131417871 hasAuthorship W3131417871A5000952627 @default.
• W3131417871 hasAuthorship W3131417871A5022855601 @default.
• W3131417871 hasAuthorship W3131417871A5023693252 @default.
• W3131417871 hasAuthorship W3131417871A5024167231 @default.
• W3131417871 hasAuthorship W3131417871A5026937923 @default.
• W3131417871 hasAuthorship W3131417871A5048966258 @default.
• W3131417871 hasAuthorship W3131417871A5062469117 @default.
• W3131417871 hasAuthorship W3131417871A5064463286 @default.
• W3131417871 hasAuthorship W3131417871A5073065036 @default.
• W3131417871 hasAuthorship W3131417871A5075557785 @default.
• W3131417871 hasAuthorship W3131417871A5076567651 @default.
• W3131417871 hasAuthorship W3131417871A5078848376 @default.
• W3131417871 hasBestOaLocation W31314178711 @default.
• W3131417871 hasConcept C116675565 @default.
• W3131417871 hasConcept C126322002 @default.
• W3131417871 hasConcept C134018914 @default.
• W3131417871 hasConcept C159047783 @default.
• W3131417871 hasConcept C170493617 @default.
• W3131417871 hasConcept C181199279 @default.
• W3131417871 hasConcept C185592680 @default.
• W3131417871 hasConcept C27016395 @default.
• W3131417871 hasConcept C2775946041 @default.
• W3131417871 hasConcept C2776980637 @default.
• W3131417871 hasConcept C2779134260 @default.
• W3131417871 hasConcept C2908929049 @default.
• W3131417871 hasConcept C2910482119 @default.
• W3131417871 hasConcept C3006700255 @default.
• W3131417871 hasConcept C3007834351 @default.
• W3131417871 hasConcept C3008058167 @default.
• W3131417871 hasConcept C524204448 @default.
• W3131417871 hasConcept C55493867 @default.
• W3131417871 hasConcept C71924100 @default.
• W3131417871 hasConcept C84393581 @default.
• W3131417871 hasConcept C86803240 @default.
• W3131417871 hasConceptScore W3131417871C116675565 @default.
• W3131417871 hasConceptScore W3131417871C126322002 @default.
• W3131417871 hasConceptScore W3131417871C134018914 @default.
• W3131417871 hasConceptScore W3131417871C159047783 @default.
• W3131417871 hasConceptScore W3131417871C170493617 @default.
• W3131417871 hasConceptScore W3131417871C181199279 @default.
• W3131417871 hasConceptScore W3131417871C185592680 @default.

• next