Dipeptidyl Peptidase 4 Is a Functional Receptor for the Emerging Human Coronavirus-EMC
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LETTER doi:10.1038/nature12005 Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC V. Stalin Raj1*, Huihui Mou2*, Saskia L. Smits1,3, Dick H. W. Dekkers4, Marcel A. Mu¨ller5, Ronald Dijkman6, Doreen Muth5, Jeroen A. A. Demmers4, Ali Zaki7, Ron A. M. Fouchier1, Volker Thiel6,8, Christian Drosten5, Peter J. M. Rottier2, Albert D. M. E. Osterhaus1, Berend Jan Bosch2 & Bart L. Haagmans1 Most human coronaviruses cause mild upper respiratory tract dis- antigen-related cell adhesion molecules (CEACAM)10 to enter cells, ease but may be associated with more severe pulmonary disease in whereas for several Alpha- and Betacoronaviruses, two peptidases immunocompromised individuals1. However, SARS coronavirus have been identified as receptors (aminopeptidase N (APN, CD13) caused severe lower respiratory disease with nearly 10% mortality for hCoV-229E and several animal coronaviruses11,12, and angiotensin and evidence of systemic spread2. Recently, another coronavirus (human coronavirus-Erasmus Medical Center (hCoV-EMC)) was a identified in patients with severe and sometimes lethal lower res- )] Vero –1 piratory tract infection3,4. Viral genome analysis revealed close 9 ml 8 5 50 relatedness to coronaviruses found in bats . Here we identify 7 dipeptidyl peptidase 4 (DPP4; also known as CD26) as a functional 6 5 receptor for hCoV-EMC. DPP4 specifically co-purified with the 4 receptor-binding S1 domain of the hCoV-EMC spike protein from 3 Relative cell number log[GE (TCID 0 20 40 lysates of susceptible Huh-7 cells. Antibodies directed against 0 101 102 103 104 DPP4 inhibited hCoV-EMC infection of primary human bronchial b )] COS-7 –1 epithelial cells and Huh-7 cells. Expression of human and bat 9 (Pipistrellus pipistrellus) DPP4 in non-susceptible COS-7 cells ml 8 50 enabled infection by hCoV-EMC. The use of the evolutionarily 7 6 conserved DPP4 protein from different species as a functional 5 receptor provides clues about the host range potential of hCoV- 4 3 Relative cell number EMC. In addition, it will contribute critically to our understanding log[GE (TCID 0 101 102 103 104 0 20 40 of the pathogenesis and epidemiology of this emerging human coronavirus, and may facilitate the development of intervention c Huh-7 )] strategies. –1 9 ml 8 Coronaviruses infect a wide range of mammals and birds. Their 50 7 tropism is primarily determined by the ability of the spike (S) entry 6 protein to bind to a cell surface receptor. Coronaviruses have zoonotic 5 potential due to the adaptability of their S protein to receptors of other 4 Relative cell number 3 6 1 2 3 4 species, most notably demonstrated by SARS-CoV , the causative 0 10 10 10 10 log[GE (TCID 0 20 40 7 agent of the SARS epidemic, which probably originated from bats . d Recently, a new coronavirus, named hCoV-EMC, has been identified Bat )] in 13 patients to date with seven fatalities that suffered from severe res- –1 9 3,4,8 ml 8 piratory illness, in some cases accompanied with renal dysfunction . 50 7 Genetically the virus is similar to bat coronaviruses HKU4 and 6 HKU5 and—based on phylogenetic analysis using a small fragment 5 4 of the virus—to a bat coronavirus found in Pipistrellus pipistrellus Relative cell number 3 1 2 3 4 in the Netherlands5. In recent years molecular surveillance studies 0 10 10 10 10 log[GE (TCID 02040 revealed the existence of at least 60 novel bat coronaviruses, including Fluorescence intensity Time after infection (h) some closely related to SARS-CoV9. As the human cases do not seem to originate from transmission from one source, the epidemiology of Figure 1 | Binding of hCoV-EMC S1 to cells is correlated with infection of hCoV-EMC may be explained by a concealed circulation in the human hCoV-EMC. a–d, Shown in the left panels are the FACS analyses of hCoV- population or by the repeated introduction from an intermediate ani- EMC S1–Fc binding (red line) to Vero (a), COS-7 (b), Huh-7 (c) and bat cells mal host. For a better understanding of the biology of this novel coro- (d). A feline CoV S1–Fc protein (blue line) and mock-incubated cells (grey shading) were used as controls. In the middle panels, hCoV-EMC-infected cells navirus, timely identification of the receptor could reveal important are visualized using an antiserum that recognizes the non-structural protein clues to its zoonotic transmission potential and pathogenesis and to NSP4. In the right panels, hCoV-EMC RNA levels in supernatants of the the design of possible intervention strategies. Two types of corona- infected cells at 0, 20 and 40 h after infection were quantified using a TaqMan virus protein receptors have been identified: the Betacoronavirus assay and expressed as genome equivalents (GE; half-maximal tissue-culture mouse hepatitis virus uses immunoglobulin-related carcinoembryonic infectious dose (TCID50) per ml). Error bars indicate s.e.m. 1Department of Viroscience, Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands. 2Virology Division, Department of Infectious Diseases & Immunology, Faculty of Veterinary Medicine, Utrecht University, 3508 TD Utrecht, the Netherlands. 3Viroclinics Biosciences BV, 3029 AK Rotterdam, The Netherlands. 4Proteomics Department, Erasmus Medical Center, 3000 CA Rotterdam, The Netherlands. 5Institute of Virology, University of Bonn Medical Centre, 53105 Bonn, Germany. 6Institute of Immunobiology, Kantonal Hospital St Gallen, 9007 St Gallen, Switzerland. 7Virology Laboratory, Dr Soliman Fakeeh Hospital, Jeddah, Saudi Arabia. 8Vetsuisse Faculty, University of Zu¨rich, 8057 Zu¨ rich, Switzerland. *These authors contributed equally to this work. 14 MARCH 2013 | VOL 495 | NATURE | 251 ©2013 Macmillan Publishers Limited. All rights reserved RESEARCH LETTER a b green monkey kidney (Vero) and human liver (Huh-7) cells by immu- hCoV-EMC S1–Fc – – + + + – – – nofluorescence and fluorescence-activated cell sorting (FACS) analysis, SARS-CoV S1–Fc – – – – – + + + whereas kidney cells of the P. pipistrellus bat showed intermediate sDPP4 + – – + – – + – staining (Fig. 1). No S1 binding was detectable to COS-7 African green sACE2 – + – – + – – + SARS-CoV S1–Fc S1–Fc hCoV-EMC monkey kidney cells (Fig. 1b). Furthermore, no specific binding to any S1–Fc of these cells was observed with a feline coronavirus S1 domain, whereas kDa kDa dimer feline cells (Felis catus whole fetus, FCWF) showed strong reactivity 170 - 170 - 130 - 130 - sDPP4 (Supplementary Table 1). Binding of hCoV-EMC S1 was shown to correlate with susceptibility to hCoV-EMC infection and with viral 100 - 100 - sACE2 70 - 70 - genome detection in the culture medium of infected cells (Fig. 1). The hCoV-EMC S1 domain was demonstrated also to bind to cells from 55 - 55 - other species but its overall reactivity was more restricted compared to 40 - 40 - that observed for SARS-CoV S1 (Supplementary Table 1). 35 - 35 - To identify the cell surface protein(s) binding to S1 we affinity- 25 - 25 - isolated proteins from Vero and Huh-7 cells using the S1–Fc chimae- 15 - 15 - ric proteins. The hCoV-EMC S1–Fc protein—but not SARS-CoV , Figure 2 | hCoV-EMC S1 binding to DPP4. a, Huh-7 cell lysates were S1–Fc—extracted a protein of 110 kDa when analysed under non- incubated with hCoV-EMC and SARS-CoV S1–Fc proteins and affinity- reducing conditions from Huh-7 cell lysates (Fig. 2a). Mass spectro- isolated proteins were subjected to protein electrophoresis under non-reducing metric analysis identified this protein as dipeptidyl peptidase 4 (DPP4 conditions. The arrowhead indicates the position of the ,110-kDa DPP4 or DPP IV, also called CD26; Supplementary Fig. 2). Similar results protein specifically isolated using the hCoV-EMC S1–Fc protein. b, hCoV- were obtained using Vero cells (data not shown). We subsequently EMC and SARS-CoV S1–Fc proteins were mock-incubated or incubated with produced soluble (that is, non-membrane-anchored) forms of DPP4 soluble DPP4 (sDPP4) or soluble ACE2 (sACE2) followed by protein A and ACE2 and found that the hCoV-EMC S1–Fc protein bound the sepharose affinity isolation and subjected to protein electrophoresis under non- former but not the latter, whereas the opposite was seen with the SARS reducing conditions. S1–Fc protein (Fig. 2b). Soluble DPP4, but not soluble ACE2, inhibited infection of Vero cells by hCoV-EMC (Supplementary Fig. 3). More- converting enzyme 2 (ACE2) for SARS-CoV13). In addition, sialic acid over, transient expression of human DPP4 in the non-susceptible may act as a receptor for some coronaviruses14. COS-7 cells rendered these cells susceptible to binding the hCoV- Our initial experiments indicated that hCoV-EMC does not use EMC S1–Fc protein to their surface (Supplementary Fig. 4). These ACE2 as an entry receptor15. Therefore we first examined whether data indicate a direct and specific binding of hCoV-EMC S1 to human the amino-terminal receptor-binding spike domain S1 binds to cells DPP4. and investigated its correlation with cell susceptibility. We expressed The DPP4 protein displays high amino acid sequence conservation the S1 domain fused to the Fc region of human IgG, yielding a recom- across different species, including the sequence that we obtained from binant disulphide-bonded dimer of approximately 280 kDa (Sup- P. pipistrellus bat cells (Supplementary Fig. 5), particularly towards the plementary Fig. 1). Highly specific binding was observed to African carboxy-terminal end (Supplementary Fig. 6). Next, we tested surface a S1 binding DPP4 staining b Figure 3 | DPP4 is present on hCoV-EMC-susceptible cell lines hDPP4 hDPP4 COS-7 Huh-7 and human bronchiolar epithelial cells. a, COS-7 cells transfected with plasmids encoding human DPP4 number number (hDPP4), bat DPP4 (bDPP4) or a Relative cell Relative cell control plasmid (pcDNA) were 1 2 3 4 1 2 3 4 010 10 10 10 0 10 10 10 10 tested for S1 binding and staining with a polyclonal antiserum against bDPP4 bDPP4 Vero Bat DPP4.