Sphingosine prevents binding of SARS–CoV-2 spike to its cellular receptor ACE2

Abstract

Sphingosine has been shown to prevent and eliminate bacterial infections of the respiratory tract, but it is unknown whether sphingosine can be also employed to prevent viral infections. To test this hypothesis, we analyzed whether sphingosine regulates the infection of cultured and freshly isolated ex vivo human epithelial cells with pseudoviral particles expressing SARS–CoV-2 spike (pp-VSV–SARS–CoV-2 spike) that served as a bona fide system mimicking SARS–CoV-2 infection. We demonstrate that exogenously applied sphingosine suspended in 0.9% NaCl prevents cellular infection with pp-SARS–CoV-2 spike. Pretreatment of cultured Vero epithelial cells or freshly isolated human nasal epithelial cells with low concentrations of sphingosine prevented adhesion of and infection with pp-VSV–SARS–CoV-2 spike. Mechanistically, we demonstrate that sphingosine binds to ACE2, the cellular receptor of SARS–CoV-2, and prevents the interaction of the receptor-binding domain of the viral spike protein with ACE2. These data indicate that sphingosine prevents at least some viral infections by interfering with the interaction of the virus with its receptor. Our data also suggest that further preclinical and finally clinical examination of sphingosine is warranted for potential use as a prophylactic or early treatment for coronavirus disease-19. Sphingosine has been shown to prevent and eliminate bacterial infections of the respiratory tract, but it is unknown whether sphingosine can be also employed to prevent viral infections. To test this hypothesis, we analyzed whether sphingosine regulates the infection of cultured and freshly isolated ex vivo human epithelial cells with pseudoviral particles expressing SARS–CoV-2 spike (pp-VSV–SARS–CoV-2 spike) that served as a bona fide system mimicking SARS–CoV-2 infection. We demonstrate that exogenously applied sphingosine suspended in 0.9% NaCl prevents cellular infection with pp-SARS–CoV-2 spike. Pretreatment of cultured Vero epithelial cells or freshly isolated human nasal epithelial cells with low concentrations of sphingosine prevented adhesion of and infection with pp-VSV–SARS–CoV-2 spike. Mechanistically, we demonstrate that sphingosine binds to ACE2, the cellular receptor of SARS–CoV-2, and prevents the interaction of the receptor-binding domain of the viral spike protein with ACE2. These data indicate that sphingosine prevents at least some viral infections by interfering with the interaction of the virus with its receptor. Our data also suggest that further preclinical and finally clinical examination of sphingosine is warranted for potential use as a prophylactic or early treatment for coronavirus disease-19. Infections with a novel member of the Coronaviridae family, named severe acute respiratory syndrome coronavirus-2 (SARS–CoV-2), are a serious global health problem and are responsible for the coronavirus disease 2019 (COVID-19) pandemic. Many details of the infection with SARS–CoV-2 remain to be defined, but it is now clear that a substantial number of patients develop severe symptoms and pneumonia upon infection, eventually requiring intensive care and ventilator treatment with a high mortality rate (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 (32015507): 270-27310.1038/s41586-020-2012-7Crossref PubMed Scopus (12486) Google Scholar). COVID-19 seems to be a high risk for certain groups of individuals, for instance the elderly, those with high blood pressure, and those who are overweight, but even healthy and young individuals develop severe disease (2Yang X. Yu Y. Xu J. Shu H. Xia J. Liu H. Wu Y. Zhang L. Yu Z. Fang M. Yu T. Wang Y. Pan S. Zou X. Yuan S. et al.Clinical course and outcomes of critically ill patients with SARS–CoV-2 pneumonia in Wuhan, China: a single-centered, retrospective, observational study.Lancet Respir. Med. 2020; 8 (32105632): 475-48110.1016/S2213-2600(20)30079-5Abstract Full Text Full Text PDF PubMed Scopus (6085) Google Scholar). It is therefore of outstanding interest to develop a prophylactic treatment to prevent SARS–CoV-2 infections or to prevent at least a severe course of the disease after diagnosis. SARS–CoV-2 infects cells by the initial interaction of the surface unit S1 of the viral spike glycoprotein with its cellular receptor angiotensin-converting enzyme 2 (ACE2) (3Hoffmann M. Kleine-Weber H. Schroeder S. Krüger N. Herrler T. Erichsen S. Schiergens T.S. Herrler G. Wu N.H. Nitsche A. Müller M.A. Drosten C. Pöhlmann S. SARS–CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.Cell. 2020; 181 (32142651): 271-28010.1016/j.cell.2020.02.052Abstract Full Text Full Text PDF PubMed Scopus (11332) Google Scholar, 4Wrapp D. Wang N. Corbett K.S. Goldsmith J.A. Hsieh C.L. Abiona O. Graham B.S. McLellan J.S. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.Science. 2020; 367 (32075877): 1260-126310.1126/science.abb2507Crossref PubMed Scopus (74) Google Scholar, 5Lan 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 (32225176): 215-22010.1038/s41586-020-2180-5Crossref PubMed Scopus (3256) Google Scholar, 6Wang Q. Zhang Y. Wu L. Niu S. Song C. Zhang Z. Lu G. Qiao C. Hu Y. Yuen K.Y. Wang Q. Zhou H. Yan J. Qi J. Structural and functional basis of SARS–CoV-2 entry by using human ACE2.Cell. 2020; 181 (32275855): 894-90410.1016/j.cell.2020.03.045Abstract Full Text Full Text PDF PubMed Scopus (1713) Google Scholar). The interaction of the two proteins allows viral entry and cellular infection. Studies employing antibodies and mutations of the S1 protein proved that abrogating the interaction between the S1 unit of the viral spike protein and ACE2 prevents infection with SARS–CoV-2 (3Hoffmann M. Kleine-Weber H. Schroeder S. Krüger N. Herrler T. Erichsen S. Schiergens T.S. Herrler G. Wu N.H. Nitsche A. Müller M.A. Drosten C. Pöhlmann S. SARS–CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.Cell. 2020; 181 (32142651): 271-28010.1016/j.cell.2020.02.052Abstract Full Text Full Text PDF PubMed Scopus (11332) Google Scholar, 7Walls A.C. Park Y.J. Tortorici M.A. Wall A. McGuire A.T. Veesler D. Structure, function, and antigenicity of the SARS–CoV-2 Spike glycoprotein.Cell. 2020; 181 (32155444): 281-29210.1016/j.cell.2020.02.058Abstract Full Text Full Text PDF PubMed Scopus (5041) Google Scholar). Previous studies have used replication-deficient vesicular stomatitis virus (VSV) pseudoviral particles (pp-VSV) presenting coronavirus spike protein on their surface, abbreviated pp-VSV–SARS–CoV-2 spike (3Hoffmann M. Kleine-Weber H. Schroeder S. Krüger N. Herrler T. Erichsen S. Schiergens T.S. Herrler G. Wu N.H. Nitsche A. Müller M.A. Drosten C. Pöhlmann S. SARS–CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.Cell. 2020; 181 (32142651): 271-28010.1016/j.cell.2020.02.052Abstract Full Text Full Text PDF PubMed Scopus (11332) Google Scholar). The studies showed that these particles accurately reflect key aspects of the entry of coronavirus into host cells (3Hoffmann M. Kleine-Weber H. Schroeder S. Krüger N. Herrler T. Erichsen S. Schiergens T.S. Herrler G. Wu N.H. Nitsche A. Müller M.A. Drosten C. Pöhlmann S. SARS–CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.Cell. 2020; 181 (32142651): 271-28010.1016/j.cell.2020.02.052Abstract Full Text Full Text PDF PubMed Scopus (11332) Google Scholar); in particular they bind to ACE2 for infectious entry, which was inhibited by anti-ACE2 antibodies. Thus, this system allows a safe and easy method to study principle biomedical questions of SARS–CoV-2 infections. We used VSV pseudoviral particles carrying the spike protein of SARS-2 on their surface and recombinant proteins of spike and ACE2 to test whether sphingosine has an impact on the infection of human epithelial cells with SARS–CoV-2. Sphingolipids localize into cellular membranes. They determine biophysical membrane properties and are involved in diverse cellular processes, including proliferation, cellular differentiation, apoptosis, signal transduction, and membrane trafficking (8Hannun Y.A. Obeid L.M. Sphingolipids and their metabolism in physiology and disease.Nat. Rev. Mol. Cell Biol. 2018; 19 (29165427): 175-19110.1038/nrm.2017.107Crossref PubMed Scopus (838) Google Scholar, 9Kolesnick R.N. Goñi F.M. Alonso A. Compartmentalization of ceramide signaling: physical foundations and biological effects.J. Cell. Physiol. 2000; 184 (10.1002/1097-4652(200009)184:3<285::AID-JCP2>3.0.CO;2-3 10911359): 285-300Crossref PubMed Scopus (378) Google Scholar, 10Gulbins E. Li P.L. Physiological and pathophysiological aspects of ceramide.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2006; 290 (16352856): R11-R2610.1152/ajpregu.00416.2005Crossref PubMed Scopus (185) Google Scholar). We and others have shown that ceramide often promotes bacterial and viral infections, for instance by mediating bacterial or viral adhesion and internalization (11Grassmé H. Jendrossek V. Riehle A. von Kürthy G. Berger J. Schwarz H. Weller M. Kolesnick R. Gulbins E. Host defense against Pseudomonas aeruginosa requires ceramide-rich membrane rafts.Nat. Med. 2003; 9 (12563314): 322-33010.1038/nm823Crossref PubMed Scopus (462) Google Scholar, 12Teichgräber V. Ulrich M. Endlich N. Riethmüller J. Wilker B. De Oliveira-Mundin C.C. van Heeckeren A.M. Barr M.L. von Kürthy G. Schmid K.W. Weller M. Tümmler B. Lang F. Grassmé H. Döring G. et al.Ceramide accumulation mediates inflammation, cell death and infection susceptibility in cystic fibrosis.Nat. Med. 2008; 14 (18376404): 382-39110.1038/nm1748Crossref PubMed Scopus (455) Google Scholar, 13Simonis A. Hebling S. Gulbins E. Schneider-Schaulies S. Schubert-Unkmeir A. Differential activation of acid sphingomyelinase and ceramide release determines invasiveness of Neisseria meningitidis into brain endothelial cells.PLoS Pathog. 2014; 10 (24945304): e100416010.1371/journal.ppat.1004160Crossref PubMed Scopus (56) Google Scholar, 14Peng H. Li C. Kadow S. Henry B.D. Steinmann J. Becker K.A. Riehle A. Beckmann N. Wilker B. Li P.L. Pritts T. Edwards M.J. Zhang Y. Gulbins E. Grassmé H. Acid sphingomyelinase inhibition protects mice from lung edema and lethal Staphylococcus aureus sepsis.J. Mol. Med. (Berl.). 2015; 93 (25616357): 675-68910.1007/s00109-014-1246-yCrossref PubMed Scopus (61) Google Scholar, 15Grassmé H. Henry B. Ziobro R. Becker K.A. Riethmüller J. Gardner A. Seitz A.P. Steinmann J. Lang S. Ward C. Schuchman E.H. Caldwell C.C. Kamler M. Edwards M.J. Brodlie M. et al.β1-Integrin accumulates in cystic epithelial and bacterial Host Full Text Full Text PDF PubMed Scopus Google Scholar, H. Riehle A. Wilker B. Gulbins E. human epithelial cells membrane Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, S. requires acid sphingomyelinase and membrane for PubMed Scopus Google Scholar). sphingosine which is from ceramide by the of or N. W. C. Y.A. in metabolism and in health and Biol. Regul. PubMed Scopus Google bacterial and is an of the defense against bacterial in the respiratory H. Henry B. Ziobro R. Becker K.A. Riethmüller J. Gardner A. Seitz A.P. Steinmann J. Lang S. Ward C. Schuchman E.H. Caldwell C.C. Kamler M. Edwards M.J. Brodlie M. et al.β1-Integrin accumulates in cystic epithelial and bacterial Host Full Text Full Text PDF PubMed Scopus Google Scholar, R. H.R. of Full Text PDF PubMed Scopus Google Scholar, C.L. K.S. K.A. are by Staphylococcus aureus and Physiol. PubMed Scopus Google Scholar, Y. S. Grassmé H. Becker K.A. L. Steinmann J. T. Lang S. B. Schuchman E.H. B. Edwards M.J. Gulbins E. prevent lung infection by Pseudomonas Mol. Med. 2014; PubMed Scopus Google Scholar, S. Seitz A. P. C. S. Lang S. Edwards E. Grassmé H. Gulbins E. Becker K.A. of sphingosine susceptibility to Staphylococcus aureus infections in cystic Physiol. PubMed Scopus Google Scholar, P. H. M. T. H. M. T. A. acid protects epithelial cells from in 2018; PubMed Scopus Google Scholar, A.P. F. J. M. Wilker B. Caldwell C.C. Kamler M. Becker K.A. S. B. Edwards M.J. Gulbins E. of prevents Mol. Med. (Berl.). PubMed Scopus Google Scholar). We also showed an of which in and to J. P. H. H. B. F. J. M. D. A. V. Becker K.A. A. Edwards M.J. et of virus in and protects from severe disease.Nat. 2020; PubMed Scopus Google Scholar). it is unknown whether sphingosine be also as an The studies whether exogenously applied sphingosine prevents pp-SARS–CoV-2 spike infection of epithelial We that treatment of Vero epithelial or freshly isolated human nasal epithelial cells with sphingosine prevents infection with spike. Sphingosine binds to ACE2, the cellular receptor for SARS–CoV-2, and the interaction of the viral spike protein with ACE2. Sphingosine on the epithelial of was on infection of cells with spike and the interaction of ACE2 with the viral spike Thus, sphingosine be a novel for and infections with SARS–CoV-2 in the respiratory To whether sphingosine prevents the infection of epithelial cells with SARS–CoV-2, we Vero epithelial cells with sphingosine for and the cells with pp-VSV–SARS–CoV-2 spike. The that sphingosine prevented the infection with pp-VSV–SARS–CoV-2 spike concentrations of sphingosine the infection of Vero epithelial cells with spike by we sphingosine and of inhibited the infection of Vero cells with pp-VSV–SARS–CoV-2 spike Sphingosine at concentrations of was on cultured Vero epithelial cells and we whether sphingosine also prevented infection of freshly isolated human nasal epithelial cells with spike. The studies that or 2 sphingosine also prevented infection of freshly isolated human nasal epithelial cells and Sphingosine on nasal epithelial cells and Sphingosine is a that is at or The on or epithelial cell is K.A. Zhu L. M.J. host in cystic PubMed Scopus Google and sphingosine be on these The domain of the spike protein that bind to in the of the ACE2 but also between the receptor-binding domain of the viral spike protein and ACE2 are for viral 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 (32225176): 215-22010.1038/s41586-020-2180-5Crossref PubMed Scopus (3256) Google Scholar). Thus, we that which is an that is bind to ACE2 and prevent the interaction of the spike protein with ACE2, infection. We this by of sphingosine to with recombinant ACE2 protein or from Vero cells and by whether ACE2 binds to The the and showed of cellular or recombinant ACE2 to sphingosine we a of the recombinant receptor-binding domain of spike to sphingosine The of sphingosine prevented of ACE2 to the of a interaction between ACE2 and sphingosine of recombinant ACE2 protein with sphingosine a of sphingosine to ACE2 We also the of ACE2 to to sphingosine or to The that bound recombinant ACE2 we whether sphingosine with the of viral spike protein with ACE2. To this recombinant was with 2 sphingosine or and and the recombinant receptor-binding domain of spike S1 was of ACE2 with sphingosine of the recombinant of spike S1 to ACE2 that as sphingosine and of recombinant receptor-binding domain of spike S1 to recombinant ACE2 We a recombinant protein of the receptor-binding domain of the viral spike protein on and recombinant in the or of 2 The that sphingosine the interaction of the receptor-binding domain of spike protein with ACE2 of sphingosine or the interaction of recombinant spike protein with ACE2 we whether sphingosine also with the of spike to recombinant ACE2. The studies that or 2 sphingosine of spike to recombinant ACE2, in of Vero cells with the spike particles we adhesion studies of spike on Vero cells in the or of We Vero cells with spike for in the or of sphingosine or with S1 and of the virus to the cells by The that sphingosine prevented of spike to the cell surface, of the an on the interaction of the virus with the Our demonstrate that sphingosine prevents infection of cultured epithelial cells also freshly isolated human nasal epithelial cells with spike. Sphingosine inhibited infection of epithelial cells at concentrations as low as and an inhibition of infection was with 2 Sphingosine or on human nasal epithelial cells or cultured Vero cells at these a of and cellular we demonstrate that the of recombinant ACE2 to viral spike protein and the adhesion of virus to cells is by with sphingosine at low Sphingosine with ACE2, we of sphingosine to the receptor-binding domain of the viral spike it is unknown sphingosine prevents of spike to ACE2. It be that the sphingosine binds to and in the of ACE2 that with the receptor-binding domain of the viral spike protein 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 (32225176): 215-22010.1038/s41586-020-2180-5Crossref PubMed Scopus (3256) Google but studies are to the details of the interaction of sphingosine with ACE2. data on cellular and recombinant proteins suggest that sphingosine on ACE2 to prevent of data that sphingosine with the ACE2 domain that with the receptor-binding domain of spike. It is also that sphingosine with of ACE2 of the interaction between ACE2 and the spike The interaction of sphingosine with ACE2 in a of the domain ACE2, of viral spike Sphingosine has been shown to bacterial at concentrations between and 10 H. Henry B. Ziobro R. Becker K.A. Riethmüller J. Gardner A. Seitz A.P. Steinmann J. Lang S. Ward C. Schuchman E.H. Caldwell C.C. Kamler M. Edwards M.J. Brodlie M. et al.β1-Integrin accumulates in cystic epithelial and bacterial Host Full Text Full Text PDF PubMed Scopus Google Scholar, R. H.R. of Full Text PDF PubMed Scopus Google Scholar, C.L. K.S. K.A. are by Staphylococcus aureus and Physiol. PubMed Scopus Google Scholar, Y. S. Grassmé H. Becker K.A. L. Steinmann J. T. Lang S. B. Schuchman E.H. B. Edwards M.J. Gulbins E. prevent lung infection by Pseudomonas Mol. Med. 2014; PubMed Scopus Google Scholar, S. Seitz A. P. C. S. Lang S. Edwards E. Grassmé H. Gulbins E. Becker K.A. of sphingosine susceptibility to Staphylococcus aureus infections in cystic Physiol. PubMed Scopus Google Scholar, P. H. M. T. H. M. T. A. acid protects epithelial cells from in 2018; PubMed Scopus Google Scholar, A.P. F. J. M. Wilker B. Caldwell C.C. Kamler M. Becker K.A. S. B. Edwards M.J. Gulbins E. of prevents Mol. Med. (Berl.). PubMed Scopus Google Scholar). Mechanistically, it was that the sphingosine with as in bacterial these to and and a of the bacterial membrane R. Becker K.A. Edwards M.J. Gulbins E. Sphingosine by to Biol. 2020; Full Text Full Text PDF PubMed Scopus Google Scholar). using recombinant we demonstrate that sphingosine with cellular as ACE2 and prevents interaction of the viral spike protein with its receptor an of sphingosine on ACE2 membrane in vivo it be that sphingosine also with in the cell which the and of ACE2, and this to the inhibition of spike to ACE2. We have shown that sphingosine also has an J. P. H. H. B. F. J. M. D. A. V. Becker K.A. A. Edwards M.J. et of virus in and protects from severe disease.Nat. 2020; PubMed Scopus Google Scholar). These studies that sphingosine binds to virus and the virus the cell to the virus upon of and sphingosine a in the cellular defense against SARS–CoV-2 to be but it is that sphingosine also binds to SARS–CoV-2 and the virus to in to the interaction of the virus with its receptor. Thus, sphingosine have a in the defense against SARS–CoV-2 on ACE2 and also SARS–CoV-2. Previous studies sphingosine to the of mice and of sphingosine that even high concentrations of sphingosine cell death or in epithelial cells Y. S. Grassmé H. Becker K.A. L. Steinmann J. T. Lang S. B. Schuchman E.H. B. Edwards M.J. Gulbins E. prevent lung infection by Pseudomonas Mol. Med. 2014; PubMed Scopus Google Scholar, S. Seitz A. P. C. S. Lang S. Edwards E. Grassmé H. Gulbins E. Becker K.A. of sphingosine susceptibility to Staphylococcus aureus infections in cystic Physiol. PubMed Scopus Google Scholar, A.P. F. J. M. Wilker B. Caldwell C.C. Kamler M. Becker K.A. S. B. Edwards M.J. Gulbins E. of prevents Mol. Med. (Berl.). PubMed Scopus Google Scholar, H. F. S. M. C. C. M. Wilker B. D. A. B. R. G. Edwards M.J. M. et al.Clinical of sphingosine as of sphingosine in has Physiol. PubMed Scopus Google Scholar). We also a of sphingosine or nasal which be by of sphingosine into sphingosine and of even high concentrations of sphingosine applied to the epithelial cells upon of with sphingosine in of epithelial cells or of the of the epithelial cell A.P. F. J. M. Wilker B. Caldwell C.C. Kamler M. Becker K.A. S. B. Edwards M.J. Gulbins E. of prevents Mol. Med. (Berl.). PubMed Scopus Google Scholar). These studies suggest that of sphingosine or of sphingosine into the be Our data demonstrate that of sphingosine prevents infection with spike. These particles a bona fide system to study early that cellular infection with SARS–CoV-2. Thus, data suggest that a nasal of sphingosine or of sphingosine be a novel to prevent or infection with SARS–CoV-2. viral particles on a replication-deficient vesicular stomatitis virus as H. Wang L. Graham B.S. Müller M.A. Drosten C. Pöhlmann S. M. in the spike protein of respiratory syndrome coronavirus in to 93 Google Scholar). The particles for and of was by of and M. G. vesicular stomatitis virus for the and of PubMed Scopus Google Scholar). cells for using the method to or The cells with for at and The was and the cells with and cultured in at and for of the was with from to finally and cellular was by 10 The pp-VSV–SARS–CoV-2 used for the Sphingosine was suspended in 0.9% NaCl and in a we a Sphingosine was at and for 10 to The of 0.9% NaCl as used for the was to The also as in 0.9% NaCl and to nasal epithelial cells from healthy by nasal with a The cells suspended in and in The cells with or sphingosine or for and in with pp-VSV–SARS–CoV-2 spike and the of sphingosine as used in the The cells for in and cultured for in with to of the by the was analyzed on a by the of epithelial cells in at least epithelial cells in The of the the number The studies in with the of Vero cells epithelial cultured in with 10 2 from and The cells to on in a for to the The cells with and with or sphingosine or for in further we the cells with sphingosine or for to the infection. The was and the cells with pp-VSV–SARS–CoV-2 spike in the of the of sphingosine or the as used the was after the and the cells cultured for an in as to of The was the cells in in with for 10 in and analyzed with a with a and We cells in in cells a and of nasal epithelial or Vero epithelial cells as with sphingosine for in or and in with sphingosine for or The cells and was for in The cells in for with and analyzed by The for with at with V. nasal epithelial cells or Vero epithelial cells with sphingosine or as for The cells with in in for 10 in with in for at and The was the of the using recombinant and with recombinant or for 10 at the The analyzed by To determine of sphingosine to ACE2, 2 of by to protein in of with 10 and at for The in in and sphingosine was at a of 2 To this sphingosine was suspended at in 0.9% for 10 in a and into the The ACE2 with sphingosine for and in protein that with The in and sphingosine was by a The cells in of and of The was and in a in The was by of sphingosine in and 10 of The for at with The was by of of of and of and 2 The between The and the was in of and on with as The analyzed with a Sphingosine with a of The to sphingosine ceramide sphingosine or with 2 of in of at for The in in for at and at for The on and blocked in The for in NaCl with for with anti-ACE2 antibodies for 10 in with for with antibodies and using an from Vero cells in 10 10 10 and 10 with a of for on and at for at and the of in and with of sphingosine to or for at The analyzed by for ACE2 as To of the between sphingosine and ACE2, we also 2 suspended sphingosine to some To test whether sphingosine of viral spike to human ACE2, was on of protein for in of at The in 2 of sphingosine was or the The at for The in and of was The for at by for 2 at and the used to Vero cells as a functional virus was from the as a of to To test whether sphingosine of viral spike to human ACE2, we 2 of on of protein in of at for in the or of 2 sphingosine or protein The in in and with 2 of recombinant receptor-binding domain of spike for at The in in for at and at for at The on and analyzed for of spike by using antibodies we used a S1 to further the by antibodies as an to that sphingosine of viral spike to human ACE2, we 2 of which is a on in of and 2 of recombinant in the or of 2 sphingosine or The for in in and analyzed by for ACE2 as using anti-ACE2 antibodies. Vero cells for with 2 sphingosine or or and pp-VSV–SARS–CoV-2 spike was the to the as and the cells for The and the cells from the with cell with with S1 antibodies for at in and with for at The in and analyzed by using a The data are as the of from we used by for and the for The for the after was at the of for the was on of to the of the in data are in the or upon We of and for replication-deficient vesicular stomatitis with Becker with J. Edwards severe acute respiratory syndrome coronavirus pseudoviral vesicular stomatitis virus angiotensin-converting enzyme receptor-binding domain of

Topics & Concepts