Article

SARS-CoV-2 Cell Entry Depends on ACE2 andTMPRSS2 and Is Blocked by a Clinically ProvenProtease Inhibitor
Graphical Abstract

Authors
Markus Hoffmann, Hannah Kleine-Weber,Simon Schroeder, ..., Marcel A. Müller,
Christian Drosten, Stefan Pöhlmann

Correspondence
mhoffmann@dpz.eu (M.H.),
spoehlmann@dpz.eu (S.P.)

In Brief
The emerging SARS-coronavirus 2(SARS-CoV-2) threatens public health.
Hoffmann and coworkers show that
SARS-CoV-2 infection depends on thehost cell factors ACE2 and TMPRSS2 andcan be blocked by a clinically provenprotease inhibitor. These findings might
help to establish options for prevention
and treatment.

Highlightsd

SARS-CoV-2 uses the SARS-CoV receptor ACE2 for hostcell entryd

The spike protein of SARS-CoV-2 is primed by TMPRSS2d

Antibodies against SARS-CoV spike may offer some
protection against SARS-CoV-2

Hoffmann et al., 2020, Cell 181, 271–280
April 16, 2020 ª 2020 Elsevier Inc.
https://doi.org/10.1016/j.cell.2020.02.052

Article
SARS-CoV-2 Cell Entry Depends on ACE2
and TMPRSS2 and Is Blocked by a Clinically
Proven Protease Inhibitor
Markus Hoffmann,1,13,* Hannah Kleine-Weber,1,2,13 Simon Schroeder,3,4 Nadine Krüger,5,6 Tanja Herrler,7Sandra Erichsen,8,9 Tobias S. Schiergens,10 Georg Herrler,5 Nai-Huei Wu,5 Andreas Nitsche,11 Marcel A. Müller,3,4,12
Christian Drosten,3,4 and Stefan Pöhlmann1,2,14,*
1Infection Biology Unit, German Primate Center – Leibniz Institute for Primate Research, Göttingen, Germany
2Faculty of Biology and Psychology, University Göttingen, Göttingen, Germany
3Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of
Health, Institute of Virology, Berlin, Germany
4German Centre for Infection Research, associated partner Charité, Berlin, Germany
5Institute of Virology, University of Veterinary Medicine Hannover, Hannover, Germany
6Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine Hannover, Hannover, Germany
7BG Unfallklinik Murnau, Murnau, Germany
8Institute for Biomechanics, BG Unfallklinik Murnau, Murnau, Germany
9Institute for Biomechanics, Paracelsus Medical University Salzburg, Salzburg, Austria
10Biobank of the Department of General, Visceral, and Transplant Surgery, Ludwig-Maximilians-University Munich, Munich, Germany
11Robert Koch Institute, ZBS 1 Highly Pathogenic Viruses, WHO Collaborating Centre for Emerging Infections and Biological Threats, Berlin,
Germany
12Martsinovsky Institute of Medical Parasitology, Tropical and Vector Borne Diseases, Sechenov University, Moscow, Russia
13These authors contributed equally
14Lead Contact*Correspondence: mhoffmann@dpz.eu (M.H.), spoehlmann@dpz.eu (S.P.)
https://doi.org/10.1016/j.cell.2020.02.052

SUMMARY

The recent emergence of the novel, pathogenic
SARS-coronavirus 2 (SARS-CoV-2) in China and its
rapid national and international spread pose a global
health emergency. Cell entry of coronaviruses depends on binding of the viral spike (S) proteins tocellular receptors and on S protein priming by hostcell proteases. Unravelling which cellular factors
are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutictargets. Here, we demonstrate that SARS-CoV-2
uses the SARS-CoV receptor ACE2 for entry andthe serine protease TMPRSS2 for S protein priming.
A TMPRSS2 inhibitor approved for clinical use
blocked entry and might constitute a treatment
option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV
infection and identify a potential target for antiviralintervention.
INTRODUCTION
Several members of the family Coronaviridae constantly circulate in the human population and usually cause mild respiratory

disease (Corman et al., 2019). In contrast, the severe acute respiratory syndrome coronavirus (SARS-CoV) and the Middle East
respiratory syndrome coronavirus (MERS-CoV) are transmitted
from animals to humans and cause severe respiratory diseases
in afflicted individuals, SARS and MERS, respectively (Fehret al., 2017). SARS emerged in 2002 in Guangdong province,
China, and its subsequent global spread was associated with
8,096 cases and 774 deaths (de Wit et al., 2016; WHO, 2004).
Chinese horseshoe bats serve as natural reservoir hosts for
SARS-CoV (Lau et al., 2005; Li et al., 2005a). Human transmission was facilitated by intermediate hosts like civet cats andraccoon dogs, which are frequently sold as food sources in Chinese wet markets (Guan et al., 2003). At present, no specific antivirals or approved vaccines are available to combat SARS, andthe SARS pandemic in 2002 and 2003 was finally stopped byconventional control measures, including travel restrictions andpatient isolation.
In December 2019, a new infectious respiratory disease
emerged in Wuhan, Hubei province, China (Huang et al., 2020;
Wang et al., 2020; Zhu et al., 2020). An initial cluster of infections
was linked to Huanan seafood market, potentially due to animal
contact. Subsequently, human-to-human transmission occurred(Chan et al., 2020) and the disease, now termed coronavirus disease 19 (COVID-19) rapidly spread within China. A novel coronavirus, SARS-coronavirus 2 (SARS-CoV-2), which is closely
related to SARS-CoV, was detected in patients and is believed
to be the etiologic agent of the new lung disease (Zhu et al.,2020). On February 12, 2020, a total of 44,730 laboratoryconfirmed infections were reported in China, including 8,204

Cell 181, 271–280, April 16, 2020 ª 2020 Elsevier Inc. 271

Figure 1. SARS-2-S and SARS-S Facilitate Entry into a Similar Panel of Mammalian Cell Lines(A) Schematic illustration of SARS-S including functional domains (RBD, receptor binding domain; RBM, receptor binding motif; TD, transmembrane domain) andproteolytic cleavage sites (S1/S2, S20 ). Amino acid sequences around the two protease recognition sites (red) are indicated for SARS-S and SARS-2-S (asterisks
indicate conserved residues). Arrow heads indicate the cleavage site.
(B) Analysis of SARS-2-S expression (upper panel) and pseudotype incorporation (lower panel) by western blot using an antibody directed against the C-terminal
hemagglutinin (HA) tag added to the viral S proteins analyzed. Shown are representative blots from three experiments. b-Actin (cell lysates) and VSV-M (particles)
served as loading controls (M, matrix protein). Black arrow heads indicate bands corresponding to uncleaved S proteins (S0) whereas gray arrow heads indicate
bands corresponding to the S2 subunit.
(C) Cell lines of human and animal origin were inoculated with pseudotyped VSV harboring VSV-G, SARS-S, or SARS-2-S. At 16 h postinoculation, pseudotype
entry was analyzed by determining luciferase activity in cell lysates. Signals obtained for particles bearing no envelope protein were used for normalization. The
average of three independent experiments is shown. Error bars indicate SEM. Unprocessed data from a single experiment are presented in Figure S1.

severe cases and 1,114 deaths (WHO, 2020). Infections werealso detected in 24 countries outside China and were associatedwith international travel. At present, it is unknown whether thesequence similarities between SARS-CoV-2 and SARS-CoVtranslate into similar biological properties, including pandemic
potential (Munster et al., 2020).
The spike (S) protein of coronaviruses facilitates viral entry into
target cells. Entry depends on binding of the surface unit, S1, of
the S protein to a cellular receptor, which facilitates viral attachment to the surface of target cells. In addition, entry requires S
protein priming by cellular proteases, which entails S protein
cleavage at the S1/S2 and the S2’ site and allows fusion of viraland cellular membranes, a process driven by the S2 subunit (Figure 1A). SARS-S engages angiotensin-converting enzyme 2(ACE2) as the entry receptor (Li et al., 2003) and employs the

272 Cell 181, 271–280, April 16, 2020

cellular serine protease TMPRSS2 for S protein priming (Glowacka et al., 2011; Matsuyama et al., 2010; Shulla et al., 2011).
The SARS-S/ACE2 interface has been elucidated at the atomic
level, and the efficiency of ACE2 usage was found to be a key
determinant of SARS-CoV transmissibility (Li et al., 2005a,
2005b). SARS-S und SARS-2-S share 76% amino acid identity. However, it is unknown whether SARS-2-S like SARS-S employs ACE2 and TMPRSS2 for host cell entry.
RESULTS
Evidence for Efficient Proteolytic Processingof SARS-2-S
The goal of our study was to obtain insights into how SARS-2-S
facilitates viral entry into target cells and how this process can beblocked. For this, we first asked whether SARS-2-S is robustly
expressed in a human cell line, 293T, commonly used for experimentation because of its high transfectability. Moreover, weanalyzed whether there is evidence for proteolytic processingof the S protein because certain coronavirus S proteins arecleaved by host cell proteases at the S1/S2 cleavage site in infected cells (Figure 1A). Immunoblot analysis of 293T cells
expressing SARS-2-S protein with a C-terminal antigenic tag revealed a band with a molecular weight expected for unprocessed S protein (S0) (Figure 1B). A band with a size expected
for the S2 subunit of the S protein was also observed in cells
and, more prominently, in vesicular stomatitis virus (VSV) particles bearing SARS-2-S (Figure 1B). In contrast, an S2 signal
was largely absent in cells and particles expressing SARS-S(Figure 1B), as previously documented (Glowacka et al., 2011;
Hofmann et al., 2004b). These results suggest efficient proteolytic processing of SARS-2-S in human cells, in keeping withthe presence of several arginine residues at the S1/S2 cleavage
site of SARS-2-S but not SARS-S (Figure 1A). In contrast, the S20
cleavage site of SARS-2-S was similar to that of SARS-S.
SARS-2-S and SARS-S Mediate Entry into a Similar
Spectrum of Cell Lines
Replication-defective VSV particles bearing coronavirus S proteins faithfully reflect key aspects of coronavirus host cell entry(Kleine-Weber et al., 2019). We employed VSV pseudotypes
bearing SARS-2-S to study cell entry of SARS-CoV-2. Both
SARS-2-S and SARS-S were robustly incorporated into VSV
particles (Figure 1B), allowing a meaningful side-by-side comparison; although, formally, comparable particle incorporationof the S1 subunit remains to be demonstrated. We first asked
which cell lines were susceptible to SARS-2-S-driven entry, using a panel of well-characterized cell lines of human and animal
origin, respectively. All cell lines were readily susceptible to entry
driven by the glycoprotein of the pantropic VSV (VSV-G) (Figure 1C; Figure S1), as expected. Most human cell lines and theanimal cell lines Vero and MDCKII were also susceptible to entry
driven by SARS-S (Figure 1C). Moreover, SARS-2-S facilitatedentry into an identical spectrum of cell lines as SARS-S (Figure 1C), suggesting similarities in choice of entry receptors.
SARS-CoV-2 Employs the SARS-CoV Receptor for Host
Cell Entry
In order to elucidate why SARS-S and SARS-2-S mediated entry
into the same cell lines, we next determined whether SARS-2-S
harbors amino acid residues required for interaction with the
SARS-S entry receptor ACE2. Sequence analysis revealed that
SARS-CoV-2 clusters with SARS-CoV-related viruses frombats (SARSr-CoV), of which some but not all can use ACE2 forhost cell entry (Figure 2A; Figure S2). Analysis of the receptor
binding motif (RBM), a portion of the receptor binding domain(RBD) that makes contact with ACE2 (Li et al., 2005a), revealed
that most amino acid residues essential for ACE2 binding by
SARS-S were conserved in SARS-2-S (Figure 2B). In contrast,
most of these residues were absent from S proteins of SARSrCoV previously found not to use ACE2 for entry (Figure 2B) (Geet al., 2013; Hoffmann et al., 2013; Menachery et al., 2020). In
agreement with these findings, directed expression of humanand bat (Rhinolophus alcyone) ACE2 but not human DPP4, theentry receptor used by MERS-CoV (Raj et al., 2013), or human
APN, the entry receptor used by HCoV-229E (Yeager et al.,1992), allowed SARS-2-S- and SARS-S-driven entry into otherwise non-susceptible BHK-21 cells (Figure 3A). Moreover, antiserum raised against human ACE2 blocked SARS-S- and
SARS-2-S- but not VSV-G- or MERS-S-driven entry (Figure 3B).
Finally, authentic SARS-CoV-2 infected BHK-21 cells transfected to express ACE2 cells but not parental BHK-21 cellswith high efficiency (Figure 3C), indicating that SARS-2-S, like
SARS-S, uses ACE2 for cellular entry.
The Cellular Serine Protease TMPRSS2 Primes SARS-2S for Entry, and a Serine Protease Inhibitor Blocks
SARS-CoV-2 Infection of Lung Cells
We next investigated protease dependence of SARS-CoV-2 entry. SARS-CoV can use the endosomal cysteine proteases
cathepsin B and L (CatB/L) (Simmons et al., 2005) and the serine
protease TMPRSS2 (Glowacka et al., 2011; Matsuyama et al.,2010; Shulla et al., 2011) for S protein priming in cell lines, andinhibition of both proteases is required for robust blockade ofviral entry (Kawase et al., 2012). However, only TMPRSS2 activity is essential for viral spread and pathogenesis in the infectedhost whereas CatB/L activity is dispensable (Iwata-Yoshikawa
et al., 2019; Shirato et al., 2016; Shirato et al., 2018; Zhou
et al., 2015).
In order to determine whether SARS-CoV-2 can use CatB/L forcell entry, we initially employed ammonium chloride, which elevates endosomal pH and thereby blocks CatB/L activity. 293T
cells (TMPRSS2, transfected to express ACE2 for robust S protein-driven entry) and Caco-2 cells (TMPRSS2+) were used astargets. Ammonium chloride blocked VSV-G-dependent entry
into both cell lines whereas entry driven by Nipah virus F and G
proteins was not affected (Figure S3A; data not shown), consistent with Nipah virus but not VSV being able to fuse directly withthe plasma membrane (Bossart et al., 2002). Ammonium chloride
treatment strongly inhibited SARS-2-S- and SARS-S-driven entry into TMPRSS2 293T cells (Figure S3 A), suggesting CatB/L
dependence. Inhibition of entry into TMPRSS2+ Caco-2 cells
was less efficient compared to 293T cells (Figure S3 A), which
would be compatible with SARS-2-S priming by TMPRSS2 in
Caco-2 cells. Indeed, the clinically proven serine protease inhibitor camostat mesylate, which is active against TMPRSS2 (Kawase et al., 2012), partially blocked SARS-2-S-driven entry into
Caco-2 (Figure S3 B) and Vero-TMPRSS2 cells (Figure 4A). Fullinhibition was attained when camostat mesylate and E-64d, an
inhibitor of CatB/L, were added (Figure 4A; Figure S3B), indicating that SARS-2-S can use both CatB/L as well as TMPRSS2
for priming in these cell lines. In contrast, camostat mesylate did
not interfere with SARS-2-S-driven entry into the TMPRSS2 celllines 293T (Figure S3B) and Vero (Figure 4A), which was efficiently blocked by E-64d and therefore is CatB/L dependent.
Moreover, directed expression of TMPRSS2 rescued SARS-2S-driven entry from inhibition by E-64d (Figure 4B), confirmingthat SARS-2-S can employ TMPRSS2 for S protein priming.
We next analyzed whether TMPRSS2 usage is required for
SARS-CoV-2 infection of lung cells. Indeed, camostat mesylate
significantly reduced MERS-S-, SARS-S-, and SARS-2-S- but

Cell 181, 271–280, April 16, 2020 273

Figure 2. SARS-2-S Harbors Amino Acid Residues Critical for ACE2 Binding(A) The S protein of SARS-CoV-2 clusters phylogenetically with S proteins of known bat-associated betacoronaviruses (see Figure S2 for more details).
(B) Alignment of the receptor binding motif of SARS-S with corresponding sequences of bat-associated betacoronavirus S proteins, which are able or unable touse ACE2 as cellular receptor, reveals that SARS-CoV-2 possesses crucial amino acid residues for ACE2 binding.

not VSV-G-driven entry into the lung cell line Calu-3 (Figure 4C)
and exerted no unwanted cytotoxic effects (Figure S3 C). Similarly, camostat mesylate treatment significantly reduced Calu-3

274 Cell 181, 271–280, April 16, 2020

infection with authentic SARS-CoV-2 (Figure 4D). Finally, camostat mesylate treatment inhibited SARS-S- and SARS-2-S- but
not VSV-G-driven entry into primary human lung cells (Figure 4E).

Collectively, SARS-CoV-2 can use TMPRSS2 for S protein priming and camostat mesylate, an inhibitor of TMPRSS2, blocks
SARS-CoV-2 infection of lung cells.
Evidence that Antibodies Raised against SARS-CoV Will
Cross-Neutralize SARS-CoV-2
Convalescent SARS patients exhibit a neutralizing antibody
response directed against the viral S protein (Liu et al., 2006).
We investigated whether such antibodies block SARS-2-Sdriven entry. Four sera obtained from three convalescent
SARS patients inhibited SARS-S- but not VSV-G-driven entry
in a concentration-dependent manner (Figure 5). In addition,these sera also reduced SARS-2-S-driven entry, although withlower efficiency compared to SARS-S (Figure 5). Similarly, rabbit
sera raised against the S1 subunit of SARS-S reduced both
SARS-S- and SARS-2-S-driven entry with high efficiency, andagain inhibition of SARS-S-driven entry was more efficient.
Thus, antibody responses raised against SARS-S during infection or vaccination might offer some level of protection against
SARS-CoV-2 infection.
DISCUSSION
The present study provides evidence that host cell entry of SARSCoV-2 depends on the SARS-CoV receptor ACE2 and can beblocked by a clinically proven inhibitor of the cellular serine protease TMPRSS2, which is employed by SARS-CoV-2 for S protein
priming. Moreover, it suggests that antibody responses raised
against SARS-CoV could at least partially protect against SARSCoV-2 infection. These results have important implications forour understanding of SARS-CoV-2 transmissibility and pathogenesis and reveal a target for therapeutic intervention.
The finding that SARS-2-S exploits ACE2 for entry, which was
also reported by Zhou and colleagues (Zhou et al., 2020) while
the present manuscript was in revision, suggests that the virus
might target a similar spectrum of cells as SARS-CoV. In thelung, SARS-CoV infects mainly pneumocytes and macrophages(Shieh et al., 2005). However, ACE2 expression is not limited to
the lung, and extrapulmonary spread of SARS-CoV in ACE2+ tissues was observed (Ding et al., 2004; Gu et al., 2005; Hamming
et al., 2004). The same can be expected for SARS-CoV-2,although affinity of SARS-S and SARS-2-S for ACE2 remains

Figure 3. SARS-2-S Utilizes ACE2 as Cellular Receptor(A) BHK-21 cells transiently expressing ACE2 of human or bat origin, human
APN, or human DPP4 were inoculated with pseudotyped VSV harboring VSVG, SARS-S, SARS-2-S, MERS-S, or 229E-S. At 16 h postinoculation, pseu-

dotype entry was analyzed (normalization against particles without viral envelope protein).
(B) Untreated Vero cells as well as Vero cells pre-incubated with 2 or 20 mg/mLof anti-ACE2 antibody or unrelated control antibody (anti-DC-SIGN, 20 mg/mL)
were inoculated with pseudotyped VSV harboring VSV-G, SARS-S, SARS-2S, or MERS-S. At 16 h postinoculation, pseudotype entry was analyzed(normalization against untreated cells).
(C) BHK-21 cells transfected with ACE2-encoding plasmid or control transfected with DsRed-encoding plasmid were infected with SARS-CoV-2 andwashed, and genome equivalents in culture supernatants were determined byquantitative RT-PCR.
The average of three independent experiments conducted with triplicate
samples is shown in (A–C). Error bars indicate SEM. Statistical significancewas tested by two-way ANOVA with Dunnett posttest. Cells transfected withempty vector served as reference in (A) whereas cells that were not treatedwith antibody served as reference in (B).

Cell 181, 271–280, April 16, 2020 275

(legend on next page)

276 Cell 181, 271–280, April 16, 2020

to be compared. It has been suggested that the modest ACE2
expression in the upper respiratory tract (Bertram et al., 2012;
Hamming et al., 2004) might limit SARS-CoV transmissibility.
In light of the potentially increased transmissibility of SARSCoV-2 relative to SARS-CoV, one may speculate that the new virus might exploit cellular attachment-promoting factors withhigher efficiency than SARS-CoV to ensure robust infection of
ACE2+ cells in the upper respiratory tract. This could comprisebinding to cellular glycans, a function ascribed to the S1 domainof certain coronaviruses (Li et al., 2017; Park et al., 2019). Finally,it should be noted that ACE2 expression protects from lung injury
and is downregulated by SARS-S (Haga et al., 2008; Imai et al.,2005; Kuba et al., 2005), which might promote SARS. It will thus
be interesting to determine whether SARS-CoV-2 also interfereswith ACE2 expression.
Priming of coronavirus S proteins by host cell proteases isessential for viral entry into cells and encompasses S protein
cleavage at the S1/S2 and the S20 sites. The S1/S2 cleavage
site of SARS-2-S harbors several arginine residues (multibasic),
which indicates high cleavability. Indeed, SARS-2-S was efficiently cleaved in cells, and cleaved S protein was incorporated
into VSV particles. Notably, the cleavage site sequence candetermine the zoonotic potential of coronaviruses (Menacheryet al., 2020; Yang et al., 2014, 2015), and a multibasic cleavage
site was not present in RaTG13, the coronavirus most closely
related to SARS-CoV-2. It will thus be interesting to determine
whether the presence of a multibasic cleavage site is required
for SARS-CoV-2 entry into human cells and how this cleavage
site was acquired.
The S proteins of SARS-CoV can use the endosomal cysteine
proteases CatB/L for S protein priming in TMPRSS2 cells (Simmons et al., 2005). However, S protein priming by TMPRSS2 but
not CatB/L is essential for viral entry into primary target cells andfor viral spread in the infected host (Iwata-Yoshikawa et al., 2019;
Kawase et al., 2012; Zhou et al., 2015). The present study indicates that SARS-CoV-2 spread also depends on TMPRSS2 activity, although we note that SARS-CoV-2 infection of Calu-3 cells
was inhibited but not abrogated by camostat mesylate, likely reflecting residual S protein priming by CatB/L. One can speculatethat furin-mediated precleavage at the S1/S2 site in infected cells
might promote subsequent TMPRSS2-dependent entry into
target cells, as reported for MERS-CoV (Kleine-Weber et al.,2018; Park et al., 2016). Collectively, our present findings andprevious work highlight TMPRSS2 as a host cell factor that is critical for spread of several clinically relevant viruses, including influenza A viruses and coronaviruses (Gierer et al., 2013; Glowacka
et al., 2011; Iwata-Yoshikawa et al., 2019; Kawase et al., 2012;
Matsuyama et al., 2010; Shulla et al., 2011; Zhou et al., 2015).
In contrast, TMPRSS2 is dispensable for development and homeostasis (Kim et al., 2006) and thus constitutes an attractive
drug target. In this context, it is noteworthy that the serine protease inhibitor camostat mesylate, which blocks TMPRSS2 activity(Kawase et al., 2012; Zhou et al., 2015), has been approved inJapan for human use, but for an unrelated indication. This compound or related ones with potentially increased antiviral activity(Yamamoto et al., 2016) could thus be considered for off-label
treatment of SARS-CoV-2-infected patients.
Convalescent SARS patients exhibit a neutralizing antibody
response that can be detected even 24 months after infection(Liu et al., 2006) and that is largely directed against the S protein.
Moreover, experimental SARS vaccines, including recombinant
S protein (He et al., 2006) and inactivated virus (Lin et al.,2007), induce neutralizing antibody responses. Although confirmation with infectious virus is pending, our results indicate thatneutralizing antibody responses raised against SARS-S could
offer some protection against SARS-CoV-2 infection, which
may have implications for outbreak control.
In sum, this study provided key insights into the first step of
SARS-CoV-2 infection, viral entry into cells, and defined potential targets for antiviral intervention.
STAR+METHODS
Detailed methods are provided in the online version of this paper
and include the following:d KEY RESOURCES TABLE
d LEAD CONTACT AND MATERIALS AVAILABILITY
d EXPERIMANTAL MODEL AND SUBJECT DETAILSB Cell cultures, primary cells, viral strains
d METHOD DETAILSB Plasmids
B Pseudotyping of VSV and transduction experiments
B Quantification of cell viability

Figure 4. SARS-2-S Employs TMPRSS2 for S Protein Priming in Human Lung Cells(A) Importance of activity of CatB/L or TMPRSS2 for host cell entry of SARS-CoV-2 was evaluated by adding inhibitors to target cells prior to transduction. E-64d
and camostat mesylate block the activity of CatB/L and TMPRSS2, respectively (additional data for 293T cells transiently expressing ACE2 and Caco-2 cells areshown in Figure S3).
(B) To analyze whether TMPRSS2 can rescue SARS-2-S-driven entry into cells that have low CatB/L activity, 293T cells transiently expressing ACE2 alone or in
combination with TMPRSS2 were incubated with CatB/L inhibitor E-64d or DMSO as control and inoculated with pseudotypes bearing the indicated viral surface
proteins.
(C) Calu-3 cells were pre-incubated with the indicated concentrations of camostat mesylate and subsequently inoculated with pseudoparticles harboring theindicated viral glycoproteins.
(D) Calu-3 cells were pre-incubated with camostat mesylate and infected with SARS-CoV-2. Subsequently, the cells were washed and genome equivalents inculture supernatants were determined by quantitative RT-PCR.
(E) In order to investigate whether serine protease activity is required for SARS-2-S-driven entry into human lung cells, primary human airway epithelial cells wereincubated with camostat mesylate prior to transduction.
The average of three independent experiments conducted with triplicate or quadruplicate samples is shown in (A–E). Error bars indicate SEM. Statistical significance was tested by two-way ANOVA with Dunnett posttest. Cells that did not receive inhibitor served as reference in (A), (C), (D), and (E) whereas cells
transfected with empty vector and not treated with inhibitor served as reference in (B).

Cell 181, 271–280, April 16, 2020 277

Figure 5. Sera from Convalescent SARS Patients Cross-Neutralize SARS-2-S-Driven Entry
Pseudotypes harboring the indicated viral surface proteins were incubated with different dilutions of sera from three convalescent SARS patients or sera fromrabbits immunized with the S1 subunit of SARS-S and subsequently inoculated onto Vero cells in order to evaluate cross-neutralization potential. The average ofthree independent experiments performed with triplicate samples is shown. Error bars indicate SEM. Statistical significance was tested by two-way ANOVA with
Dunnett posttest.

B Analysis

of SARS-2-S expression and particle
incorporation
B Infection with authentic SARS-CoV-2
B SeraB Phylogenetic analysis
d QUANTIFICATION AND STATISTICAL ANALYSIS
d DATA AND CODE AVAILABILITYACKNOWLEDGMENTS
We thank Heike Hofmann-Winkler for discussion, Andrea Maisner for Nipah F
and G expression plasmids, Anette Teichmann for technical assistance, andRoberto Cattaneo for plasmid pCG1. We acknowledge the support of the
non-profit foundation HTCR, which holds human tissue on trust, making it
broadly available for research on an ethical and legal basis. We gratefully
acknowledge the authors and the originating and submitting laboratories fortheir sequence and metadata shared through GISAID, on which this researchis based. This work was supported by BMBF (RAPID Consortium, 01KI1723D

278 Cell 181, 271–280, April 16, 2020

and 01KI1723A to C.D. and S.P.) and German Research Foundation (DFG)(WU 929/1-1 to N.-H.W.).
AUTHOR CONTRIBUTIONS
Conceptualization, M.H. and S.P.; Formal analysis, M.H., H.K.-W., M.A.M.,
and S.P.; Investigation, M.H., H.K.-W., S.S., N.K., T.H., N.-H.W., and
M.A.M.; Resources, T.H., S.E., T.S.S., G.H., A.N., M.A.M., and C.D.; Writing– Original Draft, M.H. and S.P.; Writing – Review & Editing, all authors; Funding
acquisition, S.P., N.-H.W., and C.D.
DECLARATION OF INTERESTS
The authors declare no competing interests.
Received: February 6, 2020
Revised: February 13, 2020
Accepted: February 25, 2020
Published: March 5, 2020

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STAR+METHODSKEY RESOURCES TABLEREAGENT or RESOURCESOURCEIDENTIFIER

Monoclonal anti-HA antibody produced in mouse

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Monoclonal anti-VSV-M (23H12) antibody

KeraFast

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Polyclonal anti-ACE2 antibody

R&D Systems

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Polyclonal anti-DC-SIGN antibody

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Patient serum, CSS-5

Laboratory of Andreas Nitsche

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Rabbit serum, anti-SARS-S1 rabbit I

Laboratory of Stefan Pöhlmann

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Rabbit serum, anti-SARS-S1 rabbit II

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Ammonium chloride

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Sigma-Aldrich

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SARS-2-S-HA (XbaI) R AAGGCCTCTAGATTACGCATAATCC
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WH-Ssyn 1380F GTCGGCGGCAACTACAATTAC

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WH-Ssyn 1992F CTGTCTGATCGGAGCCGAGCAC

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WH-Ssyn 2648F TGAGATGATCGCCCAGTACAC

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WH-Ssyn 3286F GCCATCTGCCACGACGGCAAAG

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ThermoFisher Scientific (GeneArt)

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Laboratory of Andrea Maisner

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Plasmid: pCAGGS-NiV-G

Laboratory of Andrea Maisner

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Plasmid: pCG1-hACE2(Hoffmann et al., 2013)

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Plasmid: pCG1-batACE2(Hoffmann et al., 2013)

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Plasmid: pCG1-hAPN(Hofmann et al., 2004a)

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Plasmid: pQCXIBL-hTMPRSS2(Kleine-Weber et al., 2018)

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Plasmid: pCG1

Laboratory of Roberto Cattaneo

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Plasmid: pCAGGS-DsRed

Laboratory of Stefan Pöhlmann

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Plasmid: pCAGGS-eGFP

Laboratory of Stefan Pöhlmann

N/A

Hidex Sense Microplate Reader Software

Hidex Deutschland Vertrieb GmbHhttps://www.hidex.de/

ChemoStar Imager Software (version v.0.3.23)

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GraphPad Software

https://www.graphpad.com/

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Microsoft Corporation

https://products.office.com/

Software and Algorithms

LEAD CONTACT AND MATERIALS AVAILABILITY
Requests for material can be directed to Markus Hoffmann (mhoffmann@dpz.eu) and the lead contact, Stefan Pöhlmann(spoehlmann@dpz.eu). All materials and reagents will be made available upon installment of a material transfer agreement (MTA).
EXPERIMANTAL MODEL AND SUBJECT DETAILS
Cell cultures, primary cells, viral strains
All cell lines were incubated at 37 C and 5% CO2 in a humidified atmosphere. 293T (human, kidney), BHK-21 (Syrian hamster, kidneycells), Huh-7 (human, liver), LLC-PK1 (pig, kidney), MRC-5 (human, lung), MyDauLu/47.1 (Daubenton’s bat [Myotis daubentonii],lung), NIH/3T3 (Mouse, embryo), RhiLu/1.1 (Halcyon horseshoe bat [Rhinolophus alcyone], lung) and Vero (African green monkey,
kidney) cells were incubated in Dulbecco’s’ modified Eagle medium (PAN-Biotech). Calu-3 (human, lung), Caco-2 (human, colon),
MDBK (cattle, kidney) and MDCKII (Dog, kidney) cells were incubated in Minimum Essential Medium (ThermoFisher Scientific).
A549 (human, lung), BEAS-2B (human, bronchus) and NCI-H1299 (human, lung) cells were incubated in DMEM/F-12 Medium
with Nutrient Mix (ThermoFisher Scientific). Vero cells stably expressing human TMPRSS2 were generated by retroviral transduction
and blasticidin-based selection. All media were supplemented with 10% fetal bovine serum (Biochrom), 100 U/mL of penicillin and
0.1 mg/mL of streptomycin (PAN-Biotech), 1x non-essential amino acid solution (10x stock, PAA) and 10 mM sodium pyruvate(ThermoFisher Scientific). For seeding and subcultivation, cells were first washed with phosphate buffered saline (PBS) and thenincubated in the presence of trypsin/EDTA solution (PAN-Biotech) until cells detached. Transfection was carried out by calciumphosphate precipitation. Lung tissue samples were obtained and experimental procedures were performed within the frameworkof the non-profit foundation HTCR, including the informed patient’s consent.
For preparation of human airway epithelial cells, bronchus tissue was derived from patients undergoing pulmonary resection andwas provided by the Biobank of the Department of General, Visceral, and Transplant Surgery, Ludwig-Maximilians- University Munich. Primary human airway epithelial cells were subsequently isolated as described (Wu et al., 2016). In brief, tissue with a length ofapproximately 10 mm and a diameter of 8mm was collected and incubated for 24 h at 4 C with DMEM (GIBCO) containing 1 mg/mL
protease type XIV and 10 mg/mL DNase I, 100 units/mL penicillin and 100 mg/mL streptomycin, 2.5 mg/mL amphotericin B, and
50 mg/mL gentamicin (GIBCO). The epithelial cells were then harvested from the mucosal surface using the scalpel and were resuspended in growth medium. After incubation at 37 C, 5% CO2 for 2 h to remove adherent fibroblast cells, non-adherent cells wereseeded on a collagen I coated flask and maintained at 37 C, 5% CO2. The growth medium was refreshed every 2 days and consistedof a 1:1 mixture of DMEM (GIBCO) and Airway Epithelial Cell basal medium (Promocell, Heidelberg, Germany) supplemented with
52 mg/mL bovine pituitary extract, 15 ng/mL retinoic acid, 5mg/mL insulin, 0.5 mg/mL hydrocortisone, 0.5 mg/mL epinephrine,
10 mg/mL transferrin, 1 ng/mL human epidermal growth factor (Corning), 1.5 ng/mL bovine serum albumin, 100 units/mL penicillinand 100 mg/mL streptomycin, with or without 5 mM Rho-associated protein kinase inhibitor (Y-27632), as previously described(Wu et al., 2016). If not stated otherwise all materials were purchased from Sigma-Aldrich.
For infection experiments with SARS-CoV-2, the SARS-CoV-2 isolate Munich 929 was propagated in VeroE6 cells (passage 1) after
primary isolation from patient material on Vero-TMPRSS2 cells (passage 0).
METHOD DETAILS
Plasmids
Expression plasmids for vesicular stomatitis virus (VSV, serotype Indiana) glycoprotein (VSV-G), Nipah virus (NiV) fusion (F)and attachment glycoprotein (G), SARS-S (derived from the Frankfurt-1 isolate) with or without a C-terminal HA epitope tag,
HCoV-229E-S, MERS-S, human and bat angiotensin converting enzyme 2 (ACE2), human aminopeptidase N (APN), human

Cell 181, 271–280.e1–e5, April 16, 2020 e3

dipeptidyl-peptidase 4 (DPP4) and human TMPRSS2 have been described elsewhere (Bertram et al., 2010; Brinkmann et al., 2017;
Gierer et al., 2013; Hoffmann et al., 2013; Hofmann et al., 2005; Kleine-Weber et al., 2019). For generation of the expression plasmids
for SARS-2-S with or without a C-terminal HA epitope tag we PCR-amplified the coding sequence of a synthetic, codon-optimized(for human cells) SARS-2-S DNA (GeneArt Gene Synthesis, ThermoFisher Scientific) based on the publicly available protein
sequence in the National Center for Biotechnology Information database (NCBI Reference Sequence: YP_009724390.1) and cloned
in into the pCG1 expression vector via BamHI and XbaI restriction sites.
Pseudotyping of VSV and transduction experiments
For pseudotyping, VSV pseudotypes were generated according to a published protocol (Berger Rentsch and Zimmer, 2011). In brief,
293T transfected to express the viral surface glycoprotein under study were inoculated with a replication-deficient VSV vector that
contains expression cassettes for eGFP (enhanced green fluorescent protein) and firefly luciferase instead of the VSV-G open reading
frame, VSV*DG-fLuc (kindly provided by Gert Zimmer, Institute of Virology and Immunology, Mittelhäusern/Switzerland). After an incubation period of 1 h at 37 C, the inoculum was removed and cells were washed with PBS before medium supplemented with antiVSV-G antibody (I1, mouse hybridoma supernatant from CRL-2700; ATCC) was added in order to neutralize residual input virus (noantibody was added to cells expressing VSV-G). Pseudotyped particles were harvested 16 h postinoculation, clarified from cellulardebris by centrifugation and used for experimentation.
For transduction, target cells were grown in 96-well plates until they reached 50%–75% confluency before they were inoculatedwith respective pseudotyped VSV. For experiments addressing receptor usage, cells were transfected with expression plasmids 24 h
before transduction. In order to block ACE2 on the cell surface, cells were pretreated with 2 or 20 mg/mL anti-ACE2 antibody (R&D
Systems, goat, AF933). As control, an unrelated anti-DC-SIGN antibody (Serotec, goat, 20 mg/mL) was used. For experiments
involving ammonium chloride (final concentration 50 mM) and protease inhibitors (E-64d, 25 mM; camostat mesylate, 1-500 mM),target cells were treated with the respective chemical 2 h before transduction. For neutralization experiments, pseudotypes werepre-incubated for 30 min at 37 C with different serum dilutions. Transduction efficiency was quantified 16 h posttransduction bymeasuring the activity of firefly luciferase in cell lysates using a commercial substrate (Beetle-Juice, PJK) and a Hidex Sense plate
luminometer (Hidex).
Quantification of cell viability
Cell viability following treatment of Calu-3 cells with camostat mesylate was analyzed using the CellTiter-Glo Luminescent Cell
Viability Assay (Promega). In brief, Calu-3 cells grown to 50% confluency in 96-well plates were incubated for 24 h in the absenceor presence of different concentrations (1-500 mM) of camostat mesylate. Next, the culture medium was aspirated and 100 ml of fresh
culture medium was added before an identical volume of the assay substrate was added. Wells containing only culture medium
served as a control to determine the assay background. After 2 min of incubation on a rocking platform and additional 10 min without
movement, samples were transferred into white opaque-walled 96-well plates and luminescent signal were recorded using a Hidex
Sense plate luminometer (Hidex).
Analysis of SARS-2-S expression and particle incorporation
To analyze S protein expression in cells, 293T cells were transfected with expression vectors for HA-tagged SARS-2-S or SARS-S or
empty expression vector (negative control). The culture medium was replaced at 16 h posttransfection and the cells were incubatedfor an additional 24 h. Then, the culture medium was removed and cells were washed once with PBS before 2x SDS-sample buffer(0.03 M Tris-HCl, 10% glycerol, 2% SDS, 0.2% bromophenol blue, 1 mM EDTA) was added and cells were incubated for 10 min atroom temperature. Next, the samples were heated for 15 min at 96 C and subjected to SDS-PAGE and immunoblotting.
For analysis of S protein incorporation into pseudotyped particles, 1 mL of the respective VSV pseudotypes were loaded onto a
20% (w/v) sucrose cushion (volume 50 ml) and subjected to high-speed centrifugation (25.000 g for 120 min at 4 C). Thereafter, 1 mLof supernatant was removed and the residual volume was mixed with 50 ml of 2x SDS-sample buffer, heated for 15 min at 96 C andsubjected to SDS-PAGE and immunoblotting. After protein transfer, nitrocellulose membranes were blocked in 5% skim milk solution(5% skim milk dissolved in PBS containing 0.05% Tween-20, PBS-T) for 1 h at room temperature and then incubated over night at
4 C with the primary antibody (diluted in in skim milk solution)). Following three washing intervals of 10 min in PBS-T the membranes
were incubated for 1 h at room temperature with the secondary antibody (diluted in in skim milk solution), before the membranes were
washed and imaged using an in in house-prepared enhanced chemiluminescent solution (0.1 M Tris-HCl [pH 8.6], 250 mg/mL luminol,
1 mg/mL para-hydroxycoumaric acid, 0.3% H2O2) and the ChemoCam imaging system along with the ChemoStar Professional software (Intas Science Imaging Instruments GmbH). The following primary antibodies were used: Mouse anti-HA tag (Sigma-Aldrich,
H3663, 1:2,500), mouse anti-b-actin (Sigma-Aldrich, A5441, 1:2,000), mouse anti-VSV matrix protein (Kerafast, EB0011, 1:2,500).
As secondary antibody we used a peroxidase-coupled goat anti-mouse antibody (Dianova, 115-035-003, 1:10000).
Infection with authentic SARS-CoV-2
BHK-21 cells (1.6 x105 cells/mL) were transfected with ACE2 and DsRed as a negative control. After 24 h, cells were washed with
PBS and infected with 8x107 genome equivalents (GE) per 24-well of SARS-CoV-2 isolate Munich 929 for 1 h at 37 C. Calu-3 cells(5 x105 cells/mL) were mock treated or treated with 100 mM camostat mesylate (Sigma Aldrich) 2 h prior to infection with SARS-CoV-2
e4 Cell 181, 271–280.e1–e5, April 16, 2020

isolate Munich 929 at a multiplicity of infection (MOI) of 0.001 for 1 h at 37 C. After infection, cells were washed three times with PBS
before 500 ml of DMEM medium was added. At 16 or 24 h post infection, 50 ml culture supernatant was subjected to viral RNA extraction using a viral RNA kit (Macherey-Nagel) according to the manufacturer’s instructions. GE per ml were detected by real time RTPCR using a previously reported protocol (Corman et al., 2020).
Sera
The convalescent human anti-SARS-CoV sera (CSS-2 to CSS-5) stemmed from the serum collection of the national consiliary laboratory for coronavirus diagnostics at Charité, Berlin, Germany or the Robert Koch Institute, Berlin, Germany. All sera were previously
tested positive using a recombinant S-based immunofluorescence test (Buchholz et al., 2013). CSS-2 was taken from a SARS patient
3.5 years post onset of disease. CSS-3 and CSS-4 originated from a second SARS patient 24 and 36 days post onset of disease.
CSS-5 was collected from a third SARS patient 10 days post onset of disease. Rabbit sera were obtained by immunizing rabbitswith purified SARS-S1 protein fused to the Fc portion of human immunoglobulin.
Phylogenetic analysis
Phylogenetic analysis (neighbor-joining tree, bootstrap method with 5,000 iterations, Poisson substitution model, uniform rates
among sites, complete deletion of gaps/missing data) was performed using the MEGA7.0.26 software. Reference sequenceswere obtained from the National Center for Biotechnology Information and GISAID (Global Initiative on Sharing All Influenza Data)
databases. Reference numbers are indicated in the figures.
QUANTIFICATION AND STATISTICAL ANALYSIS
One-way or two-way analysis of variance (ANOVA) with Dunnett posttest was used to test for statistical significance. Only p values of
0.05 or lower were considered statistically significant (p > 0.05 [ns, not significant], p % 0.05 [*], p % 0.01 [**], p % 0.001 [***]). For all
statistical analyses, the GraphPad Prism 7 software package was used (GraphPad Software).
DATA AND CODE AVAILABILITY
The study did not generate unique datasets or code.

Cell 181, 271–280.e1–e5, April 16, 2020 e5

Supplemental Figures

Figure S1. Representative Experiment Included in the Average, Related to Figure 1C
The indicated cells lines were inoculated with pseudoparticles harboring the indicated viral glycoprotein or harboring no glycoprotein (no protein) andluciferase activities in cell lysates were determined at 16 h posttransduction. The experiment was performed with quadruplicate samples, the average ± SD
is shown.

Figure S2. Extended Version of the Phylogenetic Tree, Related to Figure 2B

Figure S3. Protease Requirement for SARS-2-S-Driven Entry and Absence of Unwanted Cytotoxicity of Camostat Mesylate, Related to
Figure 4(A and B) Importance of endosomal low pH (A) and activity of CatB/L or TMPRSS2 (B) for host cell entry of SARS-CoV-2 was evaluated by adding inhibitors totarget cells prior to transduction. Ammonium chloride (A) blocks endosomal acidification while E-64d and camostat mesylate (B) block the activity of CatB/L and
TMPRSS2, respectively. Entry into cells not treated with inhibitor was set as 100%.
(C) Absence of cytotoxic effects of camostat mesylate. Calu-3 cells were treated with camostat mesylate identically as for infection experiments and cell viability
was measured using a commercially available assay (CellTiter-Glo, Promega).