Cleavage and activation of the severe acute respiratory syndrome coronavirus spike protein by human airway trypsin-like protease.

Abstract

The highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) poses a constant threat to human health. The viral spike protein (SARS-S) mediates host cell entry and is a potential target for antiviral intervention. Activation of SARS-S by host cell proteases is essential for SARS-CoV infectivity but remains incompletely understood. Here, we analyzed the role of the type II transmembrane serine proteases (TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2), in SARS-S activation. We found that HAT activates SARS-S in the context of surrogate systems and authentic SARS-CoV infection and is coexpressed with the viral receptor angiotensin-converting enzyme 2 (ACE2) in bronchial epithelial cells and pneumocytes. HAT cleaved SARS-S at R667, as determined by mutagenesis and mass spectrometry, and activated SARS-S for cell-cell fusion in cis and trans, while the related pulmonary protease TMPRSS2 cleaved SARS-S at multiple sites and activated SARS-S only in trans. However, TMPRSS2 but not HAT expression rendered SARS-S-driven virus-cell fusion independent of cathepsin activity, indicating that HAT and TMPRSS2 activate SARS-S differentially. Collectively, our results show that HAT cleaves and activates SARS-S and might support viral spread in patients.

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Published Ahead of Print 12 October 2011.
2011, 85(24):13363. DOI: 10.1128/JVI.05300-11.
J. Virol.
Pöhlmann
Elizabeth J. Soilleux, Olaf Jahn, Imke Steffen and Stefan
Niemeyer, Yuxian He, Graham Simmons, Christian Drosten,
Hayley Lavender, Kerstin Gnirss, Inga Nehlmeier, Daniela
Stephanie Bertram, Ilona Glowacka, Marcel A. Müller,
Trypsin-Like Protease
Spike Protein by Human Airway
Acute Respiratory Syndrome Coronavirus
Cleavage and Activation of the Severe
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JOURNAL OF VIROLOGY, Dec. 2011, p. 13363–13372
0022-538X/11/$12.00doi:10.1128/JVI.05300-11
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 24
Cleavage and Activation of the Severe Acute Respiratory Syndrome
Coronavirus Spike Protein by Human Airway
Trypsin-Like Protease?
Stephanie Bertram,1,2† Ilona Glowacka,1† Marcel A. Mu ¨ller,3† Hayley Lavender,4Kerstin Gnirss,1
Inga Nehlmeier,2Daniela Niemeyer,3Yuxian He,5Graham Simmons,6Christian Drosten,3
Elizabeth J. Soilleux,7,8Olaf Jahn,9Imke Steffen,1,6and Stefan Po ¨hlmann1,2*
Institute of Virology, Hannover Medical School, Hannover, Germany1; German Primate Center, Go ¨ttingen, Germany2; Institute of
Virology, University of Bonn Medical Centre, Bonn, Germany3; Oxfabs, Nuffield Department of Clinical Laboratory Sciences,
John Radcliffe Hospital, University of Oxford, Oxford, United Kingdom4; Institute of Pathogen Biology, Chinese Academy of
Medical Sciences, and Peking Union Medical College, Beijing, China5; Blood Systems Research Institute and
Department of Laboratory Medicine, University of California, San Francisco, California6; Department of
Cellular Pathology, John Radcliffe Hospital, Oxford, United Kingdom7; Nuffield Department of
Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford,
United Kingdom8; and Proteomics Group, Max Planck Institute of
Experimental Medicine, Go ¨ttingen, Germany9
Received 6 June 2011/Accepted 21 September 2011
The highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) poses a constant threat
to human health. The viral spike protein (SARS-S) mediates host cell entry and is a potential target for
antiviral intervention. Activation of SARS-S by host cell proteases is essential for SARS-CoV infectivity but
remains incompletely understood. Here, we analyzed the role of the type II transmembrane serine proteases
(TTSPs) human airway trypsin-like protease (HAT) and transmembrane protease, serine 2 (TMPRSS2), in
SARS-S activation. We found that HAT activates SARS-S in the context of surrogate systems and authentic
SARS-CoV infection and is coexpressed with the viral receptor angiotensin-converting enzyme 2 (ACE2) in
bronchial epithelial cells and pneumocytes. HAT cleaved SARS-S at R667, as determined by mutagenesis and
mass spectrometry, and activated SARS-S for cell-cell fusion in cis and trans, while the related pulmonary
protease TMPRSS2 cleaved SARS-S at multiple sites and activated SARS-S only in trans. However, TMPRSS2
but not HAT expression rendered SARS-S-driven virus-cell fusion independent of cathepsin activity, indicating
that HAT and TMPRSS2 activate SARS-S differentially. Collectively, our results show that HAT cleaves and
activates SARS-S and might support viral spread in patients.
The severe acute respiratory syndrome coronavirus (SARS-
CoV) is the causative agent of a novel lung disease, SARS,
which was first observed in Guangdong Province, southern
China, in 2002 (32, 33). Subsequently, SARS spread to more
than 30 countries with 8,096 cases. About 10% of the afflicted
individuals died from the disease, with elderly patients being
disproportionally affected (32, 33). The emergence of the
SARS-CoV was traced back to palm civets and other animals
sold in Chinese wet markets (15). It is believed that these
animals serve as intermediate hosts, while Chinese horseshoe
bats, which harbor SARS-CoV-related viruses, constitute a
natural reservoir (27, 29). The circulation of SARS-CoV in an
animal reservoir poses the continuous threat of reintroduction
of the virus into the human population. Therefore, the devel-
opment of preventive and therapeutic measures is required,
and host cell factors essential for spread of SARS-CoV and
potentially other respiratory viruses are attractive targets.
The SARS-CoV spike protein (SARS-S), jointly with the
viral M and E proteins, is incorporated into the viral mem-
brane, and SARS-S mediates infectious viral entry into target
cells (20). For this, SARS-S needs to bind to a receptor on the
host cell surface and to fuse the viral membrane with a host cell
membrane, thereby allowing delivery of SARS-CoV proteins
and genomic information into the host cell cytoplasm, the
location of SARS-CoV replication (20, 43). Angiotensin-con-
verting enzyme 2 (ACE2) has been identified to be the SARS-
CoV receptor (28) and was found to be expressed on type II
pneumocytes and enterocytes, major viral target cells (16, 31,
46, 47). ACE2 expression protects against experimentally in-
duced lung disease, and viral interference with receptor ex-
pression might contribute to SARS pathogenesis (22, 25).
Receptor engagement and membrane fusion are accom-
plished by two separate subunits in SARS-S, the N-terminal
surface unit S1 (receptor binding) and the C-terminal trans-
membrane unit S2 (membrane fusion). In order to transit into
an active state, viral glycoproteins with an architecture similar
to that of SARS-S, termed class I fusion proteins, frequently
depend on cleavage by host cell proteases (20). An initial study
indeed suggested that efficient SARS-CoV spread might de-
pend on SARS-S activation by furin (3), but these findings
were not substantiated by subsequent work. A seminal study by
Simmons and colleagues revealed that cathepsins, pH-depen-
* Corresponding author. Mailing address: Infection Biology Unit,
German Primate Center, Kellnerweg 4, 37077 Go ¨ttingen, Germany.
Phone: 49 551 3851 150. Fax: 49 551 3851 184. E-mail: s.poehlmann
@dpz.eu.
† S.B., I.G., and M.A.M. contributed equally to this work.
?Published ahead of print on 12 October 2011.
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dent, endo-/lysosomal cysteine proteases, activate SARS-S
(40). Thus, binding of SARS-CoV to ACE2 is thought to trig-
ger receptor-mediated endocytosis and transport of virions
into host cell endosomes (48), where SARS-S is activated for
membrane fusion upon cleavage by cathepsin L (40). Conse-
quently, it was proposed that cathepsin inhibitors could be
developed for therapy of SARS-CoV infection (40), and such
efforts are under way (36).
Type II transmembrane serine proteases (TTSPs) play an
important role in development and homeostasis, and dysregu-
lated TTSP expression is a hallmark of different cancers (10).
Human influenza viruses parasitize transmembrane protease,
serine 2 (TMPRSS2), and human airway trypsin-like protease
(HAT), members of the TTSP family, to facilitate their acti-
vation (5, 7, 9). TMPSS2 has also recently been shown to cleave
and activate SARS-S for cell-cell and virus-cell fusion, thereby
allowing cathepsin-independent host cell entry (14, 30, 38).
Whether HAT plays a role in SARS-S activation is at present
unknown.
Here, we report that HAT cleaves SARS-S at position R667
and activates SARS-S for cell-cell fusion, while TMPRSS2 has
multiple cleavage sites in SARS-S and activates SARS-S for
cell-cell and virus-cell fusion. HAT was found to be coex-
pressed with ACE2 in human lung epithelium, indicating that
this protease could promote viral spread in patients.
MATERIALS AND METHODS
Plasmids. Expression plasmids pCAGGS-SARS-S, encoding the spike protein
of the Frankfurt strain of SARS-CoV, and pcDNA3-hACE2, encoding human
ACE2 (hACE2), have been described previously (18, 19). The plasmids encoding
human TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS6, and hepsin have also
been described previously (9, 24, 35, 37). HAT was PCR amplified from cDNA
from human bronchus, employing primers p5_EcoRI_HAT (GCGAATTCACC
ATGTATAGGCCAGCACGTGTAACTTCG) and p3_NheI_HAT (GCGCTA
GCGCCTAGATCCCAGTTTGTTGCCTAATCC). The PCR product was in-
serted into pCAGGS via EcoRI and NheI and controlled by sequencing.
Cell culture. 293T cells were propagated in Dulbecco’s modified Eagle’s me-
dium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin,
and streptomycin and were grown in a humidified atmosphere containing 5%
CO2. 293T cells stably expressing ACE2, 293T-hACE2, were generated by trans-
fection of plasmid pcDNA3.1zeo-hACE2 (18) into 293T cells, followed by se-
lection of resistant cells with zeocin (Invitrogen) at 50 ?g/ml, as previously
reported (13). Homogeneous surface expression of ACE2 on stably transfected
cells was confirmed by flow cytometry.
Cleavage of cellular SARS-S by HAT. For detection of cleavage of SARS-S by
HAT and other TTSPs, 293T cells were cotransfected with SARS-S expression
plasmid jointly with a TTSP expression plasmid or empty plasmid. At 6 to 8 h
posttransfection, the medium was changed, and at 48 h posttransfection, the cells
were washed once with 1? phosphate-buffered saline (PBS) and lysed in 2?
sodium dodecyl sulfate (SDS) loading buffer. For immunoblotting, the lysates
were separated by SDS-gel electrophoresis and transferred onto nitrocellulose
membranes. SARS-S protein was detected by staining with a rabbit serum spe-
cific for the S1 subunit, generated by immunization with a peptide comprising
SARS-S amino acids 19 to 48 (17). For loading control, the stripped membranes
were incubated with an anti-?-actin antibody (Sigma).
VLPs. For production of virus-like particles (VLPs), 293T cells were cotrans-
fected with the HIV-1 Gag (p55)-encoding plasmid p96ZM651gag-opt (12),
SARS-S expression plasmid, TTSP expression plasmid, or empty vector. The
cellular supernatants were harvested at 48 h posttransfection and concentrated
by ultrafiltration employing VivaSpin centrifugal concentrators (Sartorius). Sub-
sequently, VLPs were purified from concentrated supernatants by ultracentrif-
ugation through a 20% sucrose cushion for 2 h at 25,000 rpm and 4°C. Finally,
supernatants and pellets of the ultracentrifuge reactions were treated with PBS
or trypsin, followed by addition of soybean trypsin inhibitor (Sigma) and analysis
by Western blotting, employing a commercially available antibody directed
against the S2 subunit of SARS-S (Imgenex). In addition, the presence of HIV
Gag in pellets and supernatants was detected using anti-p24 hybridoma super-
natant 183-H12-5C.
HAT-dependent activation of SARS-S for cell-cell fusion. The cell-cell fusion
assay was carried out essentially as described previously (14, 21, 39). For analysis
of SARS-S-driven cell-cell fusion in the absence of TTSPs, 293T effector cells
seeded in 6-well plates at 1.2 ? 105/well were CaPO4transfected with either
SARS-S expression plasmid or empty plasmid (negative control) in combination
with plasmid pGAL4-VP16, which encodes the herpes simplex virus VP16 trans
activator fused to the DNA binding domain of the Saccharomyces cerevisiae
transcription factor GAL4 (21). In parallel, 293T target cells were seeded in
48-well plates at 0.8 ? 105/well and transfected with either hACE2-encoding
plasmid or empty vector (negative control) together with plasmid pGal5-luc,
which encodes the luciferase reporter gene under the control of a promoter
containing five GAL4 binding sites. For analysis of SARS-S activation by HAT
and other TTSPs in cis, the fusion assay was carried out as described above, but
effector cells were additionally transfected with a TTSP expression plasmid. In
order to determine SARS-S activation by HAT and other TTSPs in trans, the
fusion assay was carried out as described above, but target cells were additionally
transfected with a TTSP expression plasmid. At 24 h posttransfection, the effec-
tor cells were detached by pipetting, resuspended in fresh medium, and added to
the target cells. At 6 h postcocultivation, medium supplemented with trypsin
(final concentration, 100 ng/ml; Sigma) or PBS was added to the samples. At 16 h
after trypsin treatment, medium was completely removed and fresh culture
medium without trypsin was added. Finally, at 24 h after medium change, the
cell-cell fusion was quantified by determination of luciferase activities in cell
lysates using a commercially available kit (Promega).
Activation of SARS-S-bearing lentiviral pseudotypes by HAT. Lentiviral pseu-
dotypes bearing SARS-S were essentially generated as described previously (14,
21, 39, 41). In brief, 293T cells were transiently cotransfected with pNL4-3
E-R-Luc (11), SARS-S expression plasmid, and either TTSP expression plasmid
or empty plasmid. At 16 h posttransfection, the culture medium was replaced by
fresh medium, and at 48 h posttransfection, culture supernatants were harvested.
The supernatants were passed through 0.45-?m-pore-size filters, aliquoted, and
stored at ?80°C. In order to analyze cis activation of virus-cell fusion, pseu-
dotypes generated in TTSP-expressing cells were used for infection experiments.
Specifically, equal volumes of pseudotypes normalized for comparable infectivity
for 293T-hACE2 cells were employed for infection of 293T-hACE2 cells pre-
treated for 30 min at 37°C with the indicated concentrations of ammonium
chloride or the cathepsin inhibitor MDL28170 (Calbiochem). At 16 h postinfec-
tion, the infection medium was replaced by fresh culture medium without inhib-
itor, and at 72 h postinfection, luciferase activities in cell lysates were determined
employing a commercially available kit (Promega). For the assessment of trans
activation of virus-cell fusion, the assay was carried out as described for the cis
setting, except that pseudotypes produced in the absence of TTSP expression
were used for infection of 293T-hACE2 target cells transfected to express TTSPs.
HAT-dependent syncytium formation. 293T-hACE2 cells (2 ? 105cells per
ml) were seeded on cover slides in 24 wells and were transfected with 1 ?g HAT,
TMPRSS2, or TMPRSS4 expression plasmids or transfected with empty control
plasmid (pCAGGS) using X-tremeGENE reagent (Roche), according to the
manual instructions. After 24 h, cells were infected with SARS-CoV (Frank-
furt-1; multiplicity of infection, 0.1) for 30 min at 4°C, and slides were fixed with
paraformaldehyde (8%) after another 24 h. Cells were permeabilized with 0.1%
Triton X-100, and SARS-CoV antigen was detected by immunostaining by applying
a SARS patient antiserum diluted 1:100. In parallel, proteases were detected with
mouse anti-HAT (R&D Systems), mouse anti-TMPRSS2 (Santa Cruz), or mouse
anti-Myc (for Myc-tagged TMPRSS4; Life Technologies), all diluted 1:100, and
nuclei were stained with 4?,6-diamidino-2-phenylindole (DAPI). Secondary detec-
tion was performed with Cy3-conjugated goat anti-human (1:200) and Cy2-labeled
goat anti-mouse (1:200) antibodies (Dianova). All pictures were taken with the help
of an Axio Imager.M1 fluorescence microscope (Zeiss).
Digest of recombinant SARS-S by recombinant HAT. Eight micrograms of
recombinant SARS-S (rSARS-S [Beijing 02]), produced in 293T cells and ob-
tained from a commercial source (Geneimmune), was incubated with 200 ng
HAT (R&D Systems) in assay buffer, 50 mM Tris, 0.05% (wt/vol) Brij 35, pH 9.5,
for 2 h at 37°C in a total volume of 25 ?l. Subsequently, the reactions were
stopped by addition of SDS-loading buffer and the reaction products were ana-
lyzed by 12.5% SDS-PAGE and Western blotting. Alternatively, the reaction
products were separated on NuPAGE gradient gels and analyzed by mass spec-
trometry (MS), as described below.
Mass spectrometric analysis of SARS-S cleavage products. SARS-S cleavage
products were separated on precast NuPAGE bis-Tris 4 to 12% gradient gels
using a morpholinepropanesulfonic acid buffer system according to the manu-
facturer (Invitrogen). After colloidal Coomassie staining, gel plugs were excised
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and subjected to tryptic in-gel digest using our automated platform for the
identification of gel-separated proteins by matrix-assisted laser desorption ion-
ization–time of flight (MALDI-TOF) MS and Mascot database search (23). The
remainders of the bands were subjected to manual in-gel digest with sequencing-
grade endoproteinase Asp-N (Roche) under standard conditions (including prior
reduction and carboxamidomethylation of Cys residues). Extracted peptides
were dried, redissolved in 0.5% trifluoroacetic acid–0.1% octyl-glucopyranoside,
and prepared for MALDI MS by thin-layer affinity preparation on an alpha-
cyano-4-hydroxycinnamic acid matrix (23). Peptide survey and fragment ion mass
spectra were acquired on an Ultraflex MALDI-TOF/TOF mass spectrometer
(Bruker Daltonics).
Immunostaining of tissue sections. Formalin-fixed paraffin-embedded lung
tissue was obtained with full ethical approval from the National Research and
Ethics Service (Oxfordshire Research and Ethics Committee A, reference 04/
Q1604/21) and immunostained for HAT and ACE2. Antigen retrieval was per-
formed by pressure cooking in different antigen retrieval solutions. Slides were
mounted in Aquatex mounting medium (Merck, United Kingdom). ACE2 im-
munostaining (affinity-purified goat polyclonal serum; R&D Systems, Abingdon,
United Kingdom) was performed, and ACE2 was detected using a mouse anti-
goat Ig (GTI-75) (16) and a Novolink max polymer detection system (Leica
Microsystems, Newcastle, United Kingdom), according to the manufacturer’s
instructions, after antigen retrieval in citrate, pH 6.0. HAT immunostaining
FIG. 1. HAT cleaves SARS-S. (A) Cleavage of SARS-S by HAT in cotransfected 293T cells. Expression plasmids coding for SARS-S and the
proteases indicated or empty vector (pcDNA) were transiently cotransfected into 293T cells, which were then treated with trypsin or PBS.
Subsequently, S-protein cleavage was detected by Western blot analysis of cell lysates using a serum specific for the S1 subunit of SARS-S.
Detection of ?-actin served as a loading control. SFl, full-length SARS-S; S1, S1 subunit of SARS-S; asterisks, SARS-S cleavage fragments
generated by TMPRSS2. (B) HAT cleaves SARS-S at arginine 667. Plasmids encoding wild-type SARS-S or the SARS-S R667A mutation were
transfected into 293T cells jointly with TMPRSS2, HAT expression plasmids, or empty vector (pcDNA). Subsequently, the cells were treated with
PBS or trypsin, and SARS-S cleavage was analyzed by Western blot analysis of cell lysates, using an S1-specific antiserum. Expression of ?-actin
in cell lysates was assessed as a loading control. (C) Cleavage of recombinant SARS-S (rSARS-S) by recombinant HAT. Recombinant SARS-S
was incubated with the indicated concentrations of recombinant HAT, and cleavage products were analyzed by Western blot analysis employing
a SARS-S1-specific serum. Recombinant HAT was also detected. (D) Separation of SARS-S cleavage products for mass spectrometric analysis.
SARS-S cleavage products were separated by gel electrophoresis and visualized by colloidal Coomassie staining. Major differential bands appearing
upon HAT treatment (see apparent molecular mass annotations on the right) were subjected to in-gel digest with trypsin or Asp-N, followed by
mass spectrometric analysis. Thereby, we identified the 130-kDa band to be S2 with its N terminus starting at S668 and the 95-kDa band to be S1
with its C terminus ending at R667. Untreated SARS-S (170-kDa band in the left lane) was processed in parallel as a control.
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(mouse monoclonal antibody 337029; R&D Systems, Abingdon, United King-
dom) was performed using the Novolink max polymer detection system, after
antigen retrieval in Dako Target retrieval solution, pH 6.0 (Dako, Cambridge,
United Kingdom). Alveolar macrophages in lung tissue were used as an internal
positive control for ACE2 immunostaining (45), while bronchus tissue was used
as a positive control for HAT immunostaining (44). As negative controls for
immunostaining, normal goat polyclonal serum was substituted for the anti-
ACE2 primary antibody and an irrelevant mouse monoclonal (anti-ALK1 anti-
body, clone ALK1 [34]) was substituted for the anti-HAT primary antibody.
Stained sections were photographed with a Nikon DS-FI1 camera with a Nikon
DS-L2 control unit (Nikon United Kingdom Limited, Kingston-upon-Thames,
United Kingdom) and an Olympus BX40 microscope (Olympus United Kingdom
Limited, Watford, United Kingdom).
RESULTS
HAT cleaves SARS-S. We first investigated whether HAT
cleaves SARS-S. For this, the protease and the S protein were
coexpressed in 293T cells and the cell lysates were analyzed by
Western blotting for cleavage products. As a positive control
for SARS-S cleavage, lysates from cells transfected with
SARS-S alone were treated with trypsin, which cleaves
SARS-S at the boundary between the S1 and S2 subunits (2,
39). In the absence of TTSP coexpression and trypsin treat-
ment of cell lysates, only full-length SARS-S (180 kDa) was
detected (Fig. 1A). Trypsin digestion generated the S1 subunit
(120 kDa). Coexpression of TMPRSS2 resulted in SARS-S
cleavage at multiple sites, as expected (14), while coexpression
of HAT produced a cleavage pattern indistinguishable from
the one observed upon trypsin treatment (Fig. 1A). Thus,
SARS-S is a substrate for HAT and trypsin and HAT cleave
SARS-S at similar or identical sites.
HAT cleaves SARS-S at arginine 667. Trypsin and HAT
generated indistinguishable SARS-S cleavage fragments, and
the cleavage site of trypsin has previously been mapped to
arginine 667 (2, 39). Therefore, we investigated whether this
amino acid is also a target for SARS-S proteolysis by HAT.
While coexpression of wild-type (wt) SARS-S and HAT in
293T cells or trypsin treatment of wt SARS-S-expressing cells
resulted in comparable SARS-S cleavage, no cleavage of the
SARS-S mutant with the R667A mutation by trypsin or HAT
was observed (Fig. 1B). In contrast, mutation R667A had little
impact on SARS-S processing by TMPRSS2 (Fig. 1B). These
results indicate that R667 is essential for SARS-S proteolysis
by trypsin and HAT but not TMPRSS2.
We next sought to provide direct proof that HAT indeed
cleaves SARS-S at R667. To this end, we digested soluble
FIG. 2. Mass spectrometric analysis of Asp-N-digested SARS-S cleavage products. (A and B) Zoomed-in peptide survey spectra of the Asp-N
digests. The mass signals at m/z 1,644.00 (A) and m/z 2,043.29 (B) represent the N-terminal Asp-N fragment of S2 (668-STSQKSIVAYTMSLGA-
683, M ? H?calculated ? 1,643.83 Da) and the C-terminal Asp-N fragment of S1 (649-DIPIGAGICASYHTVSLLR-667, M ? H?calculated ?
2,043.069 Da with carboxamidomethylated Cys), respectively, as they were exclusively detected in the corresponding fractions. Note that the mass
signal at m/z 1,659.99 in panel A represents the Met-oxidized variant of 668-STSQKSIVAYTMSLGA-683 (?16 mass units). (C and D) Mass
spectrometric sequencing of the terminal Asp-N fragments. (C) The fragment ion mass spectrum of m/z 1,644 clearly confirmed the identity of the
peptide to be 668-STSQKSIVAYTMSLGA-683 through a conclusive N-terminal b-ion series. The predominant occurrence of b-type ions is in
agreement with the charge localization at Lys in position 5, and only this ion series is annotated for the sake of clarity (P, precursor). (D) The
fragment ion mass spectrum of m/z 2,043 clearly confirmed the identity of the peptide to be 649-DIPIGAGICASYHTVSLLR-667 through a
conclusive C-terminal y-ion series. The predominant occurrence of y-type ions is in agreement with the charge localization at the C-terminal Arg,
and only this ion series is annotated for the sake of clarity. Mascot database searches of both fragment ion mass spectra against the Swiss-Prot
database (with enzyme set to Arg-C plus Asp-N to mimic the cleavage scenario) identified spike glycoprotein (Swiss-Prot database accession
number P59594) with significant MS/MS ion scores. a.u., arbitrary units.
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SARS-S with recombinant HAT and analyzed the cleavage
products by gel electrophoresis and mass spectrometry. Digest
of SARS-S with HAT generated the S1 subunit in a concen-
tration-dependent manner (Fig. 1C), indicating that the re-
combinant proteins used adequately model SARS-S processing
by HAT in cells. Next, we repeated the digest and visualized
the cleavage fragments by gel electrophoresis, followed by col-
loidal Coomassie staining. As expected, two prominent bands
(?130 kDa, ?95 kDa), potentially corresponding to the S1 and
S2 subunits, were detected upon SARS-S incubation with HAT
and were absent in reactions carried out in the absence of HAT
(Fig. 1D). These bands, together with the bands most likely
representing intact SARS-S (?170 kDa) and recombinant
HAT (?25 kDa), were subjected to tryptic in-gel digest and
mass spectrometric protein identification. By database search
against the Swiss-Prot database, all four bands analyzed were
identified to be spike glycoprotein (Swiss-Prot database acces-
sion number P59594). However, closer inspection of the se-
quence coverage (i.e., the distribution of the detected tryptic
peptides) revealed that the 170-kDa band represents full-
length SARS-S, while the 130-kDa and 95-kDa bands corre-
spond to S2 and S1, respectively. The slower migration of the
S2 fragment relative to the S1 fragment, which was confirmed
by Western blot analysis (data not shown), might be due to its
more extensive glycosylation and/or the particular gel electro-
phoresis conditions used. In the 25-kDa band, a C-terminal
fragment of SARS-S (matching amino acids 800 to 1000) and
HAT (identified to be transmembrane protease serine 11D,
Swiss-Prot database accession number O60235) were detected.
If HAT indeed cleaves SARS-S C terminally of R667, a digest
of the S2 subunit with endoproteinase Asp-N (which cleaves N
terminally of Asp) should yield the peptide 668-STSQKSIVA
YTMSLGA-683 (M ? H?calculated ? 1,642.8 Da) as a cleav-
age product from the N terminus of S2. By in-gel digest with
Asp-N and mass spectrometry, the respective mass signal was
exclusively detected from the 130-kDa S2 band but not from
the bands corresponding to S1 and intact SARS-S, and its
identity was further confirmed by mass spectrometric sequenc-
ing (Fig. 2A and C). Consistently, we detected and sequenced
the C-terminal Asp-N fragment of S1 (649-DIPIGAGICASY
HTVSLLR-667, M ? H?calculated ? 2,042.1 Da with car-
boxamidomethylated Cys) only from the 95-kDa S1 band (Fig.
2B and D). Thus, our mass spectrometric data unambiguously
demonstrated that HAT generates a SARS-S cleavage frag-
ment compatible only with cleavage at R667, confirming the
results of our mutagenic analysis.
SARS-S cleaved by HAT is incorporated into virus-like par-
ticles. Coexpression of SARS-S and TMPRSS2 produces sev-
eral S-protein fragments, the largest of which is shed into
culture supernatants (14). Therefore, we next examined
whether the SARS-S cleavage products generated by HAT
remained particle associated or were shed into culture super-
natants. For this, HIV Gag protein-derived VLPs carrying the
SARS-S protein were produced in the absence or presence of
HAT and TMPRSS2. Subsequently, particles were pelleted
through a 20% sucrose cushion, and pellets as well as super-
natants were treated with trypsin or PBS, followed by Western
blot analysis for the presence of S protein (Fig. 3). Particles
produced in the presence of TMPRSS2 were found to harbor
SARS-S fragments of different sizes (pellet), including the
full-length spike protein of 180 kDa. Upon coexpression of
HAT, full-length SARS-S and its S2 subunit (90 kDa) were
detected in the pellet, similarly to the cleavage products re-
sulting from trypsin treatment of particles produced in the
absence of TMPRSS2 or HAT (Fig. 3). The supernatants of
particle preparations produced in TMPRSS2-expressing cells
contained the 150-kDa subunit of SARS-S as well as smaller
cleavage products (Fig. 3), indicating that these fragments
were released into culture supernatants. In contrast, SARS-S
was largely absent from supernatants produced in control or
HAT-expressing cells. Thus, SARS-S cleaved by HAT retains
its particle association, while a substantial proportion of the
SARS-S fragments generated by TMPRSS2 is shed into cul-
ture supernatants.
HAT activates SARS-S in cis and trans for cell-cell fusion.
We next investigated if cleavage of SARS-S by HAT activates
the S protein for cell-cell fusion. Our previous studies demon-
strated that in the 293T cell system, both receptor expression
and proteolytic activation limit the rate of SARS-S-driven cell-
cell fusion (14, 39). Thus, S-protein-mediated fusion with 293T
cells, which express small amounts of endogenous ACE2 (28),
is detectable but inefficient and can be rescued by ACE2 over-
expression on target cells (14, 39). Under these conditions,
fusion is driven by an as yet unidentified leupeptin-sensitive
protease endogenously expressed by 293T cells (39). Alterna-
tively, fusion can be rescued by expression of TMPRSS2 on
FIG. 3. Expression of HAT does not induce SARS-S shedding.
VLPs were produced in 293T cells by coexpression of HIV p55-Gag
and SARS-S in the absence and presence of coexpressed TMPRSS2,
HAT, or cotransfected empty vector. Supernatants were collected and
subjected to ultrafiltration followed by ultracentrifugation through a
20% sucrose cushion. Afterwards, pellets and supernatants of the
ultracentrifuge reactions were treated with trypsin or PBS and ana-
lyzed for the presence of S protein, using a serum specific for the S2
subunit of SARS-S. In parallel, the presence of HIV p55-Gag was
determined. UC pellet, VLP preparation subjected to ultrafiltration
followed by ultracentrifugation and analysis of the pellets; UC-Sup,
VLP preparation subjected to ultrafiltration followed by ultracentrif-
ugation and analysis of the supernatants of ultracentrifuge reactions;
SFl, full-length SARS-S; S2, S2 subunit of SARS-S; asterisks, SARS-S
cleavage fragments generated by TMPRSS2.
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target cells or by trypsin treatment (14, 39), which both ensure
efficient SARS-S activation.
Employing this cell-cell fusion assay, we first tested if coexpres-
sion of HAT in S-protein-expressing effector cells enhances cell-
cell fusion activity (cis activation). For this, effector cells were
transfected with SARS-S expression plasmid and a TTSP expres-
sion plasmid or empty plasmid. Effector cells transfected with
empty plasmid alone served as negative controls. Target cells
were transfected with an ACE2 expression plasmid or empty
plasmid. Subsequently, effector and target cells were mixed and
treated with trypsin or PBS, and the rate of efficiency of cell-cell
fusion was determined at 48 h after cell mixing. The presence of
SARS-S in effector cells was critical for cell-cell fusion, as ex-
pected (Fig. 4A, pcDNA). In the absence of exogenous ACE2
expression on target cells and trypsin treatment or expression of
TTSPs, no notable S-protein-driven cell-cell fusion was observed
(pcDNA/SARS-S, white bar). Trypsin treatment overcame low-
level receptor expression on target cells and led to a substantial
increase in the fusogenic activity of SARS-S (pcDNA/SARS-S,
gray bar), as expected (39) (Fig. 4A). Similarly, ACE2 overex-
pression augmented cell-cell fusion, which was further enhanced
by trypsin treatment (pcDNA/SARS-S, lined bar and black bar,
respectively), in agreement with our recent findings (14, 39). Ex-
pression of TMPRSS2, TMPRSS3, TMPRSS4, TMPRSS6, and
hepsin, TTSPs previously examined for their ability to activate
SARS-S (14) and influenza virus hemagglutinin (4), in SARS-S-
transfected effector cells had no marked influence on the fuso-
genic activity of SARS-S (Fig. 4A). In contrast, HAT efficiently
activated SARS-S for fusion with target cells expressing endoge-
nous ACE2, indicating that SARS-S cleavage in cis by HAT but
not TMPRSS2 activates SARS-S for cell-cell fusion.
To address whether HAT can also activate SARS-S in trans,
the cell-cell fusion assay was carried out as described above,
but TTSPs were expressed in target instead of effector cells.
Expression of TMPRSS3, TMPRSS6, and hepsin in target cells
had no effect on SARS-S-driven cell-cell fusion, while expres-
sion of TMPRSS2 or TMPRSS4 markedly increased the fuso-
genic activity of SARS-S (Fig. 4B), in agreement with our
published results (14). The same observation was made for
expression of HAT, which induced efficient SARS-S-driven
cell-cell fusion with target cells expressing endogenous, low
levels of ACE2 (Fig. 4B). Thus, HAT activates the SARS spike
protein both in cis and in trans, while activation of SARS-S by
TMPRSS2 occurs only in trans.
FIG. 4. HAT activates SARS-S for cell-cell fusion in cis and in trans. (A) cis activation of SARS-S by HAT. 293T effector cells were
cotransfected with pGAL4-VP16 expression plasmid, SARS-S plasmid, and plasmids encoding the indicated proteases or empty plasmid (pcDNA)
and mixed with target cells cotransfected with ACE2 expression plasmid or empty plasmid and a plasmid encoding luciferase under the control
of a promoter with five GAL4 binding sites. The cell mixtures were then treated with PBS or trypsin, and the luciferase activities in cell lysates
were quantified at 48 h after cell mixing. The results of a representative experiment performed in triplicate are shown; error bars indicate standard
deviations (SDs). Similar results were observed in an independent experiment. (B) trans activation of SARS-S by HAT. The cell-cell fusion assay
was performed as described in the legend for panel A, but proteases were expressed in target cells. The results of a representative experiment
performed in triplicate are shown and were confirmed in two separate experiments. Error bars indicate SDs.
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Cleavage activation of SARS-S by HAT does not bypass the
need for cathepsin activity. The pH-dependent protease cathep-
sin L can activate SARS-S for virus-cell fusion (40). Accordingly,
SARS-S-driven virus-cell fusion can be inhibited by the lysoso-
motropic agent ammonium chloride (NH4Cl) and the cathepsin
inhibitor MDL28170 (40, 42). Since HAT activated SARS-S for
cell-cell fusion in cis, we next investigated whether viruses pro-
duced in HAT-expressing cells and thus bearing activated
SARS-S were no longer dependent on cathepsin activity for in-
fection of target cells. Infectious entry of pseudotypes bearing
SARS-S was efficiently inhibited by both NH4Cl and MDL28170
in a concentration-dependent manner independent of the expres-
sion of HAT or TMPRSS2 in virus-producing cells (Fig. 5A).
Thus, cis cleavage of SARS-S by TMPRSS2 or HAT does not
render virions independent of cathepsin activity for infectious
host cell entry. In contrast, expression of TMPRSS2 on target
cells (trans cleavage) allowed efficient infection of NH4Cl- or
MDL28170-treated cells by SARS-S-bearing pseudotypes (Fig.
5B), in accordance with our previous results (14). Notably, this
observation did not hold true for infection of HAT-expressing
target cells, which was efficiently blocked by both agents (Fig. 5B).
These results suggest that HAT activates cell- but not virus-asso-
ciated SARS-S in trans and emphasize that TMPRSS2 and HAT
activate SARS-S differentially.
ACE2 is coexpressed with HAT in human lung tissue and
activates the SARS coronavirus for cell-cell fusion. Finally, we
explored if activation of SARS-S by HAT could modulate viral
spread in infected patients. Immunostaining for HAT demon-
strated strong expression by extra- and intrapulmonary bron-
chial epithelial cells and alveolar macrophages (Fig. 6A and
data not shown). Weaker positive immunostaining suggested
expression at a lower level by type I and type II pneumocytes
(Fig. 6A). Analysis of a serial section of lung demonstrated
coexpression of ACE2 with HAT by intrapulmonary bronchial
epithelial cells, alveolar macrophages, and type II pneumo-
cytes, although no expression of ACE2 by type I pneumocytes
was detected (Fig. 6A). Thus, HAT is expressed in or near
potential SARS-CoV target cells and might modulate viral
spread in infected humans. Indeed, expression of HAT and
TMPRSS2 promoted formation of syncytia in ACE2-transfected
and SARS-CoV-infected 293T cells; syncytia were absent in un-
infected cells and much less frequent in infected cells transfected
with empty plasmid (Fig. 6B). Similarly, syncytium formation was
inefficient in TMPRSS4-transfected cells (Fig. 6B), in agreement
withourpreviousfindingthatTMPRSS4doesnotactivateSARS-
CoV (14). In summary, HAT is expressed in viral target cells in
human lung and can promote fusion of SARS-CoV-infected cells
with neighboring permissive cells.
FIG. 5. Activation of SARS-S by HAT does not bypass the requirement for cathepsin activity for virus-cell fusion. (A) cis cleavage of SARS-S
by HAT does not rescue SARS-S-driven infectious entry from blockade by cathepsin inhibitors. Lentiviral pseudotypes bearing SARS-S were
generated in 293T cells coexpressing the proteases indicated or cotransfected with empty plasmid (pcDNA). The pseudotypes were used to infect
293T cells engineered to express high levels of ACE2 and preincubated with the indicated concentrations of the cathepsin B/L inhibitor MDL28170
or the lysosomotropic agent NH4Cl. Luciferase activities in cell lysates were determined at 72 h postinfection. The results of a representative
experiment performed in triplicate are shown; error bars indicate SDs. Comparable results were obtained in two independent experiments.
(B) trans cleavage of SARS-S by HAT does not rescue SARS-S-driven infectious entry from blockade by cathepsin inhibitors. The experiment was
carried out as described in the legend for panel A, but proteases were expressed in viral target cells. The results of a representative experiment
performed in triplicate are shown and were confirmed in two separate experiments. Error bars indicate SDs.
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DISCUSSION
TMPRSS2 activates influenza virus (4, 7, 8), human metap-
neumovirus (37), and SARS-CoV (14, 30, 38) in cell culture
and is coexpressed with ACE2 in type II pneumocytes, major
SARS-CoV target cells (14, 30). It is thus likely that this pro-
tease modulates SARS-CoV spread in infected humans. The
related protease HAT, which can regulate the structure and
function of the urokinase receptor and potentially other recep-
tors (1), also activates influenza viruses (7), but its potential
role in other viral infections has not been analyzed. Here, we
show that HAT, like TMPRSS2, cleaves and activates SARS-S,
but the mode and outcome of SARS-S activation by these
proteases are notably different.
First, HAT and TMPRSS2 cleave different motifs in
SARS-S. Mutagenic analysis and mass spectrometry revealed
that HAT, like trypsin, cleaves SARS-S at arginine 667. In
contrast, TMPRSS2 cleaves SARS-S at multiple, at present
unidentified sites, and it is unknown which of the cleavage
FIG. 6. HAT and ACE2 are coexpressed in human lung, and HAT activates the SARS coronavirus for cell-cell fusion. (A, left) Section of lung
immunostained for HAT, using the peroxidase technique (brown), demonstrating strong expression by intrapulmonary bronchial epithelial cells
(B) and alveolar macrophages (M). Weaker positive immunostaining suggested expression at a lower level by type 1 pneumocytes (P1; thin flat
cells) and type 2 pneumocytes (P2; plumper cells). (A, right) Serial section of lung immunostained for ACE2, using the peroxidase technique
(brown), demonstrating expression by intrapulmonary bronchial epithelial cells (B), alveolar macrophages (M), and type 2 pneumocytes (P2),
although no expression of ACE2 by type 1 pneumocytes is seen. Bar, 50 ?m (the bar pertains to both panels). (B) ACE2-expressing 293T cells were
transfected with HAT, TMPRSS2, or TMPRSS4 expression plasmids or transfected with empty plasmid (control) and infected with SARS-CoV
(Frankfurt-1; multiplicity of infection, 0.1). At 24 h postinfection, the cells were fixed with paraformaldehyde (8%) and permeabilized, and
SARS-CoV antigen was detected by immunostaining using a human antiserum and a goat anti-human Cy3-labeled secondary antibody (red). In
parallel, proteases were detected with mouse anti-HAT, mouse anti-TMPRSS2, or mouse anti-Myc (for Myc-tagged TMPRSS4) and a Cy2-
conjugated goat anti-mouse antibody (green). Nuclei were stained with DAPI (blue). Arrows, examples of syncytium formation that were partially
magnified (white squares). Bars, 25 ?m. Similar results were obtained in an independent experiment.
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events is important for SARS-S activation. A recent study
suggests that activation of SARS-S involves two cleavage
events, one at arginine 667 and one at arginine 797 (2), and
one can speculate that the latter amino acid residue defines
the cleavage site recognized by TMPRSS2. Alternatively,
TMPRSS2 might recognize the same motif as cathepsin L,
which has been shown to cleave at R678 (6). Differential cleav-
age of SARS-S by HAT and TMPRSS2 most likely reflects
differential substrate specificity, and only the substrate speci-
ficity of HAT has been determined (49). Alternatively, SARS-S
sequences distant from the cleavage sites might modulate rec-
ognition by TTSPs, a possibility that remains to be investi-
gated.
Second, HAT activates cellular SARS-S in cis and in trans,
while TMPRSS2 is active only in trans. The inability of
TMPRSS2 to activate SARS-S in cis could be explained by
aberrant S-protein cleavage or lack of cleavage under these
conditions. Our previous analysis of cleavage fragments argues
against but does not exclude these possibilities (14). A more
likely explanation could be that activation of SARS-S by
TMPRSS2 is a spatially and temporally restricted process
which can proceed only once SARS-S makes contact with
ACE2 and TMPRSS2 on target cells. The recent finding that
ACE2 binds to and is cleaved by TMPRSS2 lends support to
such a scenario (38).
Third, TMPRSS2 but not HAT expression allows SARS-S-
driven virus fusion with target cells treated with a cathepsin
inhibitor. This observation is particularly striking, since trypsin
treatment of cell-bound, SARS-S-bearing virions bypasses the
need for cathepsin activity for infectious entry (2, 39, 40). A
possible explanation for this apparent discrepancy is that tryp-
sin sequentially cleaves SARS-S at arginines 667 and 797, while
cleavage by HAT might be limited to arginine 667 and might
be sufficient to activate SARS-S for cell-cell but not virus-cell
fusion. Alternatively, we have previously provided evidence for
a requirement for conformational rearrangements (induced
upon ACE2 binding) within SARS-S for proteolysis and infec-
tious cell entry (40). These conformational changes may not
proceed to completion in the relatively short time that the virus
has available to make contact with HAT during virus-cell fu-
sion, precluding proteolytic activation of SARS-S. In contrast,
substantial amounts of S protein, receptor, and protease are
stably available at the cell surface for prolonged time periods
during cell-cell fusion, potentially allowing conformational
changes and subsequent proteolysis to occur.
Regardless of the differences in mode and outcome of
SARS-S activation by HAT and TMPRSS2, both proteases
were able to activate SARS-S for cell-cell fusion in the context
of authentic SARS-CoV. The resulting syncytia were readily
detectable in the infected cultures and, as determined by im-
munostaining, were positive for both SARS-CoV and protease,
as expected. Numeric analysis revealed that syncytium forma-
tion was about 15-fold more frequent in TMPRSS2- and HAT-
transfected cells than control cells. Given that HAT was de-
tectedin ACE2-positivebronchial
agreement with a report describing HAT expression in the
bronchus (44), it is conceivable that HAT contributes to the
previously documented syncytium formation in SARS-CoV-
infected patients (26). Similarly, the documented findings that
TMPRSS2 is expressed on SARS-CoV target cells and acti-
epithelialcells, in
vates the virus in cell culture suggest that this protease might
promote viral spread in humans (14, 30). In sum, HAT and
TMPRSS2 activate SARS-S by different mechanisms and
might support viral spread in infected humans.
ACKNOWLEDGMENTS
We thank T. F. Schulz, S. Treue, and T. Liepold for support.
Hayley Lavender was funded by the NIHR Biomedical Research
Centre Programme. S.P. and C.D. were funded by BMBF (SARS-
Verbund, 01KI1005C). Y.H. was supported by China 973 program
(2010CB530100). G.S. was supported by grant R01AI074986 from the
NIAID. C.D. was funded by DFG Africa (DR 772/3-1) and EMPERIE
(project code 223498).
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