1
The novel coronavirus 2019 (2019-nCoV) uses the SARS-coronavirus receptor 1
ACE2 and the cellular protease TMPRSS2 for entry into target cells 2
3
Markus Hoffmann,
1*
†
Hannah Kleine-Weber
1,2
†
, Nadine Krüger,
3,4
Marcel Müller,
5,6,7
4
Christian Drosten,
5,6
Stefan Pöhlmann
1,2*
5
6
1
Infection Biology Unit, German Primate Center – Leibniz Institute for Primate Research, 7
Göttingen, Germany 8
2
Faculty of Biology and Psychology, University Göttingen, Göttingen, Germany 9
3
Institute of Virology, University of Veterinary Medicine Hannover, Hannover, Germany. 10
4
Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine 11
Hannover, Hannover, Germany. 12
4
Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-13
Universität zu Berlin, and Berlin Institute of Health, Institute of Virology, Berlin, Germany. 14
5
German Centre for Infection Research, associated partner Charité, Berlin, Germany. 15
6
Martsinovsky Institute of Medical Parasitology, Tropical and Vector Borne Diseases, Sechenov 16
University, Moscow, Russia. 17
18
Corresponding authors. E-mail: mhoffmann@dpz.eu (M.H.), spoehlmann@dpz.eu (S.P.) 19
† These authors contributed equally 20
21
22
23
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted January 31, 2020. ; https://doi.org/10.1101/2020.01.31.929042doi: bioRxiv preprint
2
Abstract: The emergence of a novel, highly pathogenic coronavirus, 2019-nCoV, in China, 24
and its rapid national and international spread pose a global health emergency. 25
Coronaviruses use their spike proteins to select and enter target cells and insights into 26
nCoV-2019 spike (S)-driven entry might facilitate assessment of pandemic potential and 27
reveal therapeutic targets. Here, we demonstrate that 2019-nCoV-S uses the SARS-28
coronavirus receptor, ACE2, for entry and the cellular protease TMPRSS2 for 2019-nCoV-29
S priming. A TMPRSS2 inhibitor blocked entry and might constitute a treatment option. 30
Finally, we show that the serum form a convalescent SARS patient neutralized 2019-nCoV-31
S-driven entry. Our results reveal important commonalities between 2019-nCoV and 32
SARS-coronavirus infection, which might translate into similar transmissibility and disease 33
pathogenesis. Moreover, they identify a target for antiviral intervention. 34
35
One sentence summary: The novel 2019 coronavirus and the SARS-coronavirus share central 36
biological properties which can guide risk assessment and intervention. 37
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preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted January 31, 2020. ; https://doi.org/10.1101/2020.01.31.929042doi: bioRxiv preprint
3
Several members of the family Coronaviridae constantly circulate in the human population and 47
usually cause mild respiratory disease (1). In contrast, the severe acute respiratory syndrome-48
associated coronavirus (SARS-CoV) and the Middle East respiratory syndrome-associated 49
coronavirus (MERS-CoV) are transmitted from animals to humans and cause severe respiratory 50
diseases in afflicted human patients, SARS and MERS, respectively (2). SARS emerged in 2002 51
in Guangdong province, China, and its subsequent global spread was associated with 8096 cases 52
and 774 deaths (3, 4). The virus uses Chinese horseshoe bats as natural reservoir (5, 6) and is 53
transmitted via intermediate hosts to humans. Thus, SARS-CoV was identified in Civet cats and 54
raccoon dogs, which are sold as food sources in Chinese wet markets (7). No specific antivirals 55
or approved vaccines are available to combat SARS and the SARS pandemic in 2002/2003 was 56
stopped by conventional control measures, including travel restrictions and patient isolation. 57
In December 2019 a new infectious respiratory disease emerged in Wuhan, Hubei 58
province, China (8-10). Initial infections occurred at Huanan seafood market, potentially due to 59
animal contact. Subsequently, human-to-human transmission occurred (11) 60
and the disease rapidly spread within China. A novel coronavirus, 2019-nCoV, which is closely 61
related to SARS-CoV, was detected in patients and is believed to be the etiologic agent of the 62
new lung disease (10). On January 28, 2020, at total of 4593 laboratory confirmed infections 63
were reported, including 976 severe cases and 106 deaths (12). Infections were also detected in 64
14 countries outside China and were associated with international travel. At present, it is 65
unknown whether the sequence similarities between 2019-nCoV and SARS-CoV translate into 66
similar biological properties, including pandemic potential (13). 67
The spike (S) protein of coronaviruses facilitates viral entry into target cells. Entry 68
depends on S protein binding to a cellular receptor and on S protein priming by a cellular 69
protease. SARS-S engages angiotensin-converting enzyme 2 (ACE2) as entry receptor (14) and 70
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted January 31, 2020. ; https://doi.org/10.1101/2020.01.31.929042doi: bioRxiv preprint
4
employs the cellular serine protease TMPRSS2 for S protein priming (15-17). The SARS-71
S/ACE2 interface has been elucidated and the efficiency of ACE2 usage was found to be a key 72
determinant of SARS-CoV transmissibility (6, 18). SARS-S und 2019-nCoV-S share ~76% 73
amino acid identity. However, it is unknown whether 2019-nCoV-S like SARS-S employs ACE2 74
and TMPRSS2 for host cell entry. 75
Replication-defective vesicular stomatitis virus (VSV) particles bearing coronavirus S 76
proteins faithfully reflect key aspects of coronavirus host cell entry (19). We employed VSV 77
pseudotypes bearing 2019-nCoV-S to study cell entry of 2019-nCoV. Both 2019-nCoV-S and 78
SARS-S were comparably expressed (Fig. 1A) and incorporated into VSV particles (Fig. 1B), 79
allowing a meaningful side-by-side comparison. We first focused on 2019-nCoV cell tropism. 80
Transduction of cell lines of animal and human origin revealed that all cell lines were readily 81
susceptible to entry driven by the pantropic VSV glycoprotein (VSV-G) (Fig. 1C), as expected. 82
(Fig. 1C). Notably, 2019-nCoV-S facilitated entry into an identical spectrum of cell lines as 83
SARS-S (Fig. 1C), suggesting similarities in receptor choice. 84
Sequence analysis revealed that 2019-nCoV clusters with SARS-CoV-related viruses 85
from bats (SARSr-CoV), of which some but not all can use ACE2 for host cell entry (Fig. 2A and 86
fig. S1). Analysis of the receptor binding motif (RBM), a portion of the receptor binding domain 87
(RBD) that makes contact with ACE2, revealed that most amino acid residues essential for ACE2 88
binding were conserved in 2019-nCoV-S but not in the S proteins of SARSr-CoV previously 89
found not to use ACE2 for entry (Fig. 2B). In agreement with these findings, directed expression 90
of human and bat ACE2 but not human DPP4, the entry receptor used by MERS-CoV (20), or 91
human APN, the entry receptor used by HCoV-229E (21), allowed 2019-nCoV-S- and SARS-S-92
driven entry into otherwise non-susceptible BHK-21 cells (Fig. 2C), indicating that 2019-nCoV-S 93
like SARS-S uses ACE2 for cellular entry. 94
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted January 31, 2020. ; https://doi.org/10.1101/2020.01.31.929042doi: bioRxiv preprint
5
We next investigated protease dependence of 2019-nCoV entry. SARS-CoV can use the 95
endosomal cysteine proteases cathepsin B and L (CatB/L) for S protein priming in TMPRSS2
-
96
cell lines (22). However, TMPRSS2 is expressed in viral target cells in the lung (23) and entry 97
into TMPRSS2
+
cell lines is promoted by TMPRSS2 (15-17) and is partially CatB/L 98
independent, although blockade of both proteases is required for efficient entry inhibition (24). 99
Moreover, TMPRSS2 but not CatB/L activity is essential for spread of SARS-CoV and other 100
coronaviruses in the infected host (25, 26). For initial insights into 2019-nCoV-S protease choice, 101
we employed ammonium chloride, which elevates endosomal pH and thereby blocks CatB/L 102
activity. Ammonium chloride treatment blocked VSV-G-driven entry into both cell lines studied 103
while entry driven by the Nipah virus F and G proteins was not affected (Fig. 3A), in keeping 104
with expectations. Moreover, ammonium chloride treatment strongly inhibited 2019-nCoV-S- 105
and SARS-S-driven entry into TMPRSS2
-
293T cells while inhibition of entry into TMPRSS2
+
106
Caco-2 cells was less efficient, which would be compatible with 2019-nCoV-S priming by 107
TMPRSS2 in Caco-2 cells. Indeed, the serine protease inhibitor camostat mesylate, which is 108
active against TMPRSS2 (24), efficiently blocked 2019-nCoV-S-driven entry into Caco-2 109
(TMPRSS2
+
) but not 293T (TMPRSS2
-
) cells while the CatB/L inhibitor E64d had the opposite 110
effect (Fig. 3B). Moreover, directed expression of TMPRSS2 rescued 2019-nCoV-S-driven entry 111
from inhibition by E64d (Fig. 3C), demonstrating that 2019-nCoV-S uses TMPRSS2 for priming. 112
Convalescent SARS patients exhibit a neutralizing antibody response directed against the 113
viral S protein (27). We investigated whether such antibodies block 2019-nCoV-S-driven entry. 114
Serum from a convalescent SARS patient inhibited SARS-S- but not VSV-G-driven entry in a 115
concentration dependent manner (Fig. 4). In addition, the serum reduced 2019-nCoV-S-driven 116
entry, although with somewhat lower efficiency as compared to SARS-S (Fig. 4). Thus, antibody 117
preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for thisthis version posted January 31, 2020. ; https://doi.org/10.1101/2020.01.31.929042doi: bioRxiv preprint
