INTRODUCTION
Influenza A virus (IAV) is a global health problem that causes significant morbidity, mortality, and economic losses annually. In addition, the recurrent transmission of avian IAV to other host species provides the basis for the emergence of new viruses posing unpredictable public health threats and is likely to engender future influenza pandemics. IAVs belong to the family
Orthomyxoviridae and contain a segmented, negative-sense, single-stranded RNA genome. IAVs circulate in a wide range of animal species with wild aquatic birds being the natural reservoir [reviewed in (
1)].
IAV infection is initiated by the viral surface glycoprotein hemagglutinin (HA) through binding to cellular receptors and mediating the fusion of the viral lipid envelope with the endosomal membrane following endocytosis of the virus (reviewed in reference (
2)). Like most viral fusion proteins, HA is synthesized as a fusion-incompetent precursor and must be cleaved by a host cell protease to acquire its fusion capacity. Proteolytic cleavage is essential for virus infectivity and spread and the proteases responsible represent potential drug targets [reviewed in reference (
3)]. The HA of most IAVs including human and other mammalian IAVs as well as low pathogenic avian IAVs is cleaved at a single arginine or rarely a lysine residue designated as “monobasic cleavage site” by trypsin-like proteases (
4,
5). By contrast, HA of highly pathogenic avian influenza viruses of subtypes H5 and H7 that cause fowl plague (bird flu) possesses multiple basic amino acids at the cleavage site. HA with such a “multibasic cleavage site” is cleaved by ubiquitously expressed furin and related proprotein convertases, supporting the systemic spread of infection with often fatal outcomes (
6,
7).
We first identified the trypsin-like transmembrane serine protease 2 (TMPRSS2) as a protease present in the human airways that cleaves the HA of human IAV with a monobasic cleavage site (
8). Subsequently, TMPRSS2 was shown to activate the fusion proteins of a number of other respiratory viruses such as human metapneumovirus, human parainfluenza viruses as well as coronaviruses (CoVs) including severe acute respiratory syndrome (SARS) CoV, Middle East respiratory syndrome (MERS) CoV, and more recently SARS-CoV-2 (
9–11). Remarkably, TMPRSS2-deficient mice are protected from pathogenesis upon infection with various IAVs as well as SARS-CoV, MERS-CoV, and SARS-CoV-2 due to inhibition of the activation of progeny virus and consequently inhibition of virus spread along the respiratory tract (
11–14). Moreover, we demonstrated that TMPRSS2 is the major activating protease of IAV with monobasic HA cleavage site in human airways and of influenza B virus in human lungs (
15,
16). The physiological role of TMPRSS2, however, is still unclear.
In 2003, angiotensin-converting enzyme 2 (ACE2) was shown to be the functional entry receptor of SARS-CoV (
17). Later, ACE2 was identified as an entry receptor for human coronavirus (HCoV) NL63 and more recently for SARS-CoV-2 (
10,
18). ACE2 is a zinc metalloproteinase and carboxypeptidase that plays a central role in the renin-angiotensin system (RAS) that maintains blood pressure and fluid balance. ACE2 converts the vasoconstrictor angiotensin II into the vasodilatory heptapeptide Ang 1–7, thereby counterbalancing the action of ACE (
19,
20). In addition, ACE2 hydrolyzes a number of other bioactive peptides including neurotensin, ghrelin, and apelin (
19–22). Interestingly, ACE2 has also been shown to perform non-catalytic physiological functions, such as regulating the transport of neutral amino acids in the intestine by acting as a chaperone for the trafficking of the neutral amino acid transporter B
0AT1 (
23,
24). ACE2 is widely expressed in many different tissues with high expression in the intestinal tract, testis, kidney, heart, thyroid, and adipose tissue (
25–27). It is also found in the liver and brain, albeit at typically lower levels overall (
27–31). In the respiratory tract, ACE2 is highly expressed in the upper airways, while expression in the lung is low and mainly confined to type II pneumocytes (
25,
32). ACE2 is a type I transmembrane protein comprised of a short cytoplasmic domain, a transmembrane domain, and a large ectodomain consisting of an N-terminal protease domain followed by a collectrin domain (
Fig. 1) (
22,
26,
33). It is shed from the plasma membrane as a catalytically active form after cleavage by ADAM17 (also known as TACE) within the collectrin domain (
34).
Interestingly, previous studies demonstrated that ACE2 and TMPRSS2 interact with each other and that TMPRSS2 cleaves ACE2 within the collectrin domain (
Fig. 1) (
35,
36). Furthermore, ACE2 cleavage by TMPRSS2 did not enhance ACE2 shedding, rather it interfered with the cleavage and shedding of ACE2 by ADAM17 (
36).
We were intrigued that ACE2 and TMPRSS2 interact and wondered whether this interaction produced an enhancement of TMPRSS2-mediated events. In the present study, we further characterized ACE2-TMPRSS2 interaction by enzyme kinetic measurements and co-immunoprecipitation analyses. Furthermore, we developed peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) to knockdown ACE2 expression in Calu-3 human airway cells and to investigate IAV replication and HA cleavage in the absence and presence of ACE2 expression. We demonstrate that ACE2 acts as a co-factor or stabilizer of TMPRSS2 activity in a non-catalytic manner and is involved in TMPRSS2-mediated IAV HA cleavage in Calu-3 human airway cells. Our data also suggest that ACE2 is involved in TMPRSS2-mediated activation of certain CoVs, exemplified here for MERS-CoV.
DISCUSSION
During the SARS-CoV-2 pandemic, TMPRSS2 and ACE2 received considerable attention as viral entry factors. Interestingly, previous studies showed that ACE2 and TMPRSS2 interact with each other (
35,
36). Here, we demonstrate that ACE2 increases the enzymatic activity of TMPRSS2 in a protease activity assay and is involved in TMPRSS2-mediated IAV HA cleavage in Calu-3 human airway cells. Transient-expressed ACE2 was also able to enhance TMPRSS2-mediated IAV activation in Caco-2 human colon carcinoma cells. Furthermore, we show that knockdown of ACE2 inhibits MERS-CoV S cleavage and reduces virus titers in Calu-3 cells, indicating that ACE2 is involved in the proteolytic activation of different respiratory viruses by TMPRSS2.
Knockdown of ACE2 expression inhibited cleavage of HA with monobasic cleavage site and strongly reduced virus titers. Cleavage of HA by TMPRSS2 takes place intracellularly prior to IAV release from the infected cell, most likely in the trans-Golgi network (TGN) or during transport to the plasma membrane (
47–49). We found that the ACE2-∆Glyco7 mutant, which is not transported to the cell surface but accumulates in the ER, is cleaved by TMPRSS2 and co-precipitated with TMPRSS2, suggesting that these proteins already interact with each other within the cell. Thus, the knockdown of ACE2 in Calu-3 cells likely results in decreased TMPRSS2 activity in the TGN, reducing canonical HA cleavage (
Fig. 7A and B). By contrast, proteolytic activation and multicycle replication of the H7N7 IAV were not substantially reduced by the knockdown of ACE2, suggesting that furin and related proprotein convertases do not interact with ACE2 in a manner similar to that of TMPRSS2. Furthermore, inhibition of MERS-CoV S activation and virus replication by ACE2 knockdown in Calu-3 cells suggest that ACE2-mediated regulation of TMPRSS2 enzymatic activity occurs, at least to some extent, on the cell surface (
Fig. 7C).
Although the molecular mechanism underlying ACE2-mediated enhancement of TMPRSS2 activity has yet to be established, this study helps to elucidate the biochemical basis. In the enzyme kinetic measurements at 37°C, a strong decrease in TMPRSS2 activity was observed if ACE2 was absent. Presumably, TMPRSS2 was less stable under these experimental conditions. At room temperature, the marked decrease in TMPRSS2 activity was not observed. Interestingly, the activity of TMPRSS2 was increased to the same extent at both room temperature and 37°C if ACE2 was present. Thus, our data indicate that ACE2 may somehow stabilize the TMPRSS2 protein overall or its enzymatic activity in some respect. In addition, our data show that the presence of ACE2, but not its carboxypeptidase activity, is relevant for supporting enhanced TMPRSS2 activity, further indicating that ACE2 functions as a chaperone required for stabilization, trafficking, or membrane internalization of TMPRSS2. Although the best-known function of ACE2 is to counterbalance ACE by decreasing Ang II levels, it performs a number of other functions independent of the renin-angiotensin system. ACE2 is a chimeric protein: its catalytic domain is homologous with ACE and its membrane-proximal domain is homologous with collectrin (
50). Similar to its renal paralogue collectrin, ACE2 acts as a molecular chaperone of the neutral amino acid transporter B
0AT1 (also designated as SLC6A19) in the small intestine and is responsible for the trafficking of B
0AT1 to the surface of intestinal epithelial cells (
23,
24,
51). Thereby, ACE2 and B
0AT1 regulate the uptake of neutral amino acids in the intestine. Hence, ACE2 may act as a trafficking chaperone for TMPRSS2 in airway cells. However, this hypothesis warrants further investigation. Interestingly, cleavage of ACE2 by TMPRSS2 has been shown to interfere with ACE2 cleavage by ADAM17 and subsequent ACE2 shedding (
34,
36). Based on our results here, one could speculate that the TMPRSS2-ACE2 interaction prevents ACE2 shedding to promote TMPRSS2 activity. However, the exact nature of how this interaction affects the subcellular localization of both proteins remains to be investigated.
Furthermore, it remains to be determined whether cleavage of ACE2 by TMPRSS2 is required for ACE2-mediated enhancement of TMPRSS2 activity. TMPRSS2 was shown to cleave ACE2 at amino acids 697–716 within the collectrin domain (
36). Here, we found that ACE2 lacking the collectrin domain is still cleaved by TMPRSS2, whereas ACE2 lacking both the collectrin and transmembrane domain remains un-cleaved. Thus, ACE2 cleavage by TMPRSS2 appears to have flexibility. Our co-IP analysis revealed that the (i) enzymatic activity of TMPRSS2 and (ii) the peptidase domain of ACE2 are crucial for ACE2-TMPRSS2 interaction, whereas the collectrin domain and the transmembrane domain of ACE2 were found to be dispensable. Notably, we observed that cleavage of ACE2 by TMPRSS2 and interaction of ACE2 with TMPRSS2 were not necessarily correlated with each other. ACE2-∆PD mutant was cleaved by TMPRSS2 but not co-precipitated, while ACE2∆CD∆TMD mutant was co-precipitated, but not cleaved by TMPRSS2.
In the protease activity assay, we observed a slight reduction in ACE2 activity in the presence of TMPRSS2. It is possible that the interaction of TMPRSS2 with the peptidase domain of ACE2 sterically influences ACE2 activity, and we hope to investigate this in future studies. Yan et al. solved the cryo-electron microscopy structure of the ACE2-B
0AT1 complex and demonstrated that it is assembled as a dimer of heterodimers (
52). Dimerization is entirely mediated by ACE2, which forms homodimers mainly
via amino acids 616 to 726 of its collectrin domain (
52,
53). Thus, cleavage of ACE2 by TMPRSS2 at amino acids 697–716 could interfere with ACE2 dimerization (
36,
52).
In the present study, we focused primarily on the influence of ACE2 on TMPRSS2-mediated activation of IAV. However, we were able to show that ACE2 is involved in S cleavage and multicycle replication of MERS-CoV S in Calu-3 cells. Thus, it will be interesting to determine whether ACE2 is involved in TMPRSS2-mediated activation of other respiratory viruses such as human metapneumovirus, human parainfluenza viruses, and further CoVs including SARS-CoV-2 (
9–11).
Protection of TMPRSS2-deficient mice from pathogenesis after infection with IAV of various HA subtypes as well as with CoVs including MERS-CoV and SARS-CoV-2 impressively demonstrated that TMPRSS2 represents a promising drug target for the treatment of respiratory virus infections (reviewed in references (
11,
12). A number of broad-spectrum serine protease inhibitors, such as aprotinin and camostat as well as potent peptide or peptide-mimetic inhibitors of TMPRSS2, were shown to prevent IAV activation and multicycle replication in cell cultures during the last decade (reviewed in references (
54,
55)). Finally, the critical role of TMPRSS2 in SARS-CoV-2 activation has further accelerated the search for TMPRSS2 inhibitors, and several promising candidates for further drug development to treat both influenza and COVID-19 have been described (
9,
56,
57). The physiological role of TMPRSS2 in humans is not yet clear. TMPRSS2-deficient mice do not exhibit a phenotype, suggesting a non-essential function or functional redundancy (
58). Hence, inhibition of TMPRSS2 during acute infection is likely to be well-tolerated. Although the molecular basis of ACE2-TMPRSS2 interaction and its effect on TMPRSS2 activity remains unclear, our results may help to identify a physiological role for TMPRSS2.
Our data immediately raise the question of whether ACE2 is also a promising target for inhibition of HA cleavage; the answer is probably no. In the lung, Ang II is known to promote the development of pulmonary hypertension and pulmonary fibrosis, and ACE2 was shown to play a critical role in the development of acute respiratory distress syndrome (ARDS) (
59–61). ACE2-knockout mice show more severe ARDS symptoms when compared to wild-type mice, whereas ACE2 overexpression or administration of recombinant ACE2 was shown to be protective in rodent models (
59,
60). Downregulation of ACE2 by the viral spike protein (possibly
via internalization of ACE2 upon virus attachment) and accumulation of Ang II is also associated with ARDS and acute lung failure in SARS-CoV and SARS-CoV-2 infections (
62,
63). Conversely, administration of recombinant ACE2, which provides both a decoy to neutralize the virus and a supplement to ACE2 carboxypeptidase activity, was found to ameliorate virus-induced lung injury in mice. Meanwhile, recombinant soluble human ACE2 is under development for the treatment of moderate to severe COVID-19 infections as well as acute lung injury, ARDS and pulmonary arterial hypertension (
64–66).
To summarize, we were able to show that ACE2 increases or stabilizes the enzymatic activity of TMPRSS2 and is involved in TMPRSS2-mediated IAV HA cleavage in human airway cells. Thus, our data identify ACE2 as a new host cell factor of IAV replication. Our data also suggest that ACE2 is involved in proteolytic activation of other respiratory viruses by TMPRSS2, exemplified here for MERS-Co V. In addition, our results may also stimulate further studies addressing the physiological function of the “virus-activating protease” TMPRSS2.
MATERIAL AND METHODS
Cells and viruses
Propagation of all cells was carried out at 37°C and 5% CO2. Calu-3 human airway epithelial cells (ATCC number HTB55) were cultured in DMEM/F-12 Ham (1:1) (Gibco) supplemented with 10% fetal calf serum (FCS), penicillin, streptomycin, and glutamine, with fresh culture medium replenished every 2 to 3 days. HEK293 human embryonic kidney cells, Caco-2 human colorectal adenocarcinoma cells, and Madin-Darby canine kidney II (MDCK(II)) cells were maintained in DMEM supplemented with 10% FCS, antibiotics, and glutamine.
IAVs used here were recombinant Aichi/H3N2/WSN (HA and NA of A/Aichi/2/68 (H3N2) and six genes of A/WSN/33 (H1N1), kindly provided by Mikhail Matrosovich, designated as H3N2), recombinant H7N7/SC35M (reverse genetics system kindly provided by Jürgen Stech) and 2009 H1N1 isolate A/Hamburg/5/09 (H1N1pdm). Human IAVs were propagated in MDCK(II) cells in infection medium (DMEM supplemented with 0.1% bovine serum albumin (BSA), glutamine, and antibiotics) containing 1 µg/mL tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma), H7N7/SC35M was propagated in MDCK(II) cells in infection medium without the addition of TPCK-trypsin. MERS-CoV (kindly provided by Lucie Sauerhering, Institute of Virology, Marburg) was propagated in Calu-3 cells in DMEM supplemented with 3% FCS, penicillin, streptomycin, and glutamine (DMEM+++). Cell supernatants were cleared from cell debris by low-speed centrifugation and stored as virus stocks at −80°C or in liquid nitrogen (MERS-CoV). Experiments with MERS-CoV were conducted under BSL4 conditions.
Plasmids
pcDNA3.1(+) plasmids encoding TMPRSS2 or TMPRSS2(S441A) with a C-terminal 3 x FLAG epitope and ACE2, ACE2-∆CD∆TMD, ACE2-∆PD, or ACE2-∆Glyco7 (with amino acid substitutions N53Q, N90Q, N103Q, N322Q, N432Q, N546Q, N690Q) with a HA-tag were commercially obtained from GeneART. pcDNA3.1(+) encoding TMPRSS2(R255Q)−3x FLAG and ACE2-∆CD-HA, respectively, were generated by site-directed mutagenesis using the Q5 Site-Directed Mutagenesis Kit (New England biolabs) according to the supplier’s protocol. All primer sequences are available upon request.
PPMO
Phosphorodiamidate morpholino oligomers (PMO) were synthesized at Gene Tools, LLC (Philomath, OR, USA). The PMO sequences (5′ to 3′) targeted to ACE2 are as follows: ACE2-AUG (
GGAAGAGCTTGACATCGTCCCCTGT), ACE2-UTR (
TCACATCCACTGATGACTTTCCCT), ACE2-e5i5 (
GTACAGTTCCTTGCTTACCTCTTCA), and scramble (
CCTCTTACCTCAGTTACAATTTATA). The cell-penetrating peptide (RXR)
4 (R = arginine and X = 6-aminohexanoic acid) was covalently conjugated to the 3′ ends of each PMO through a non-cleavable linker, to produce peptide-PMO (PPMO), by methods previously described (
67).
Recombinant enzymes, protease substrates and inhibitors
Recombinant soluble human TMPRSS2 (aa 109–492; mutation SRQSR255 → DDDDK255 to avoid autocatalytic activation during overexpression and to facilitate controlled zymogen activation by enterokinase) was generated in insect cells using materials and the protocol provided by Fraser et al. (
68). Briefly, the TMPRSS2 expression plasmid was transformed into DH10 Bac cells. Baculovirus DNA was transfected into ExpiSf9 cells according to the manufacturer’s protocol (Thermo Fisher Scientific). For protein production, 1 L of ExpiSf9 culture was infected with the P2 virus, and cells were harvested when viability dropped below 80%. The culture medium was incubated with nickel affinity resin. The resin was washed with PBS and eluted with 0.5M imidazole in PBS, pH 7.5. Protein was dialyzed overnight against 2 L of dialysis buffer (25 mM Tris pH 8.0, 72 mM NaCl, 2 mM CaCl
2) in the presence of enterokinase (New England Biolabs). The cleaved protein was further purified on a Hiload 26/600 Superdex 75 column (Cytiva). Protein was flash-frozen and stored at −80°C until use. Recombinant soluble human ACE2 (aa 18–740) was purchased from Abcam (ab273297). Fluorogenic TMPRSS2 substrate Boc-Gln-Ala-Arg-7-amino-4-methyl coumarin (AMC) was used. ACE2 activity was measured using the Angiotensin II Converting Enzyme (ACE2) Activity Assay Kit (Abcam, ab273297) according to the supplier’s protocol. ACE2 peptide inhibitor DX600 (
37) was purchased from Cayman Chemical.
Antibodies
The following antibodies were used in this study: anti-ACE2 (Abcam, ab108252), anti-beta-actin (Abcam, ab6276), anti-H1N1pdm (Sino Biological, 11085-T62), anti-H7 (Sino Biological, 40103-RP01), anti-H3 (Sino Biological, GTX 127363), anti-HA protein tag (Sigma Aldrich, H6908), anti-FLAG (Sigma Aldrich, F7425), and anti MERS-CoV-Spike-S2 (Sino Biological, 40070-T62). Species-specific horseradish-peroxidase-conjugated antibodies were purchased from DAKO.
Measurement of protease activities
To measure TMPRSS2 activity, recombinant TMPRSS2 (final assay concentration 0.12 nM) was mixed with recombinant ACE2 (0.8 µU) prior to the addition of 25 µM fluorogenic substrate Boc-Gln-Ala-Arg-AMC in 50 mM Tris HCl pH 8.0 containing 154 mM NaCl. Protease activity was measured over 1 h at room temperature (RT) or at 37°C using a BioTek Synergy H1 Plate Reader at 350/380 nm excitation and 460 nm emission. When indicated, ACE2 inhibitor DX600 (10 µM in assay) was added.
To measure ACE2 activity, the ACE2 Activity Assay Kit (Abcam, ab273297) was used according to the supplier’s protocol. Recombinant ACE2 (0.8 µU) was mixed with recombinant TMPRSS2 (0.12 nM) in the absence or presence of DX600 (10 µM) prior to the addition of a quenched fluorogenic ACE2 substrate. The cleavage rate of the substrate was measured over 1 h at RT in relative fluorescence units at 320 nm excitation and 420 nm emission. All protease activity measurements were performed in two to three independent experiments performed in duplicate or triplicate.
Cell viability assay
Confluent Calu-3 cells were treated with 25 µM of the respective PPMO in infection medium or infection medium containing 5% EtOH for 24 h at 37°C and 5% CO2. Cell viability was determined using the CellTiter-Glo Kit according to the manufacturer’s manual.
IAV infection of cells for multicycle viral replication and HA cleavage analysis
Infection experiments and PPMO treatment of Calu-3 cells were performed using an infection medium. For analysis of multicycle viral replication Calu-3 cells were seeded in 24-well plates and grown to near-confluence. Cells were incubated with 25 µM of PPMO in the infection medium for 24 h or remained untreated. The cells were then inoculated with the virus at a low MOI of 0.001 (virus growth kinetics) or an MOI of 1 (analysis of HA cleavage) in fresh infection medium without PPMO for 1 h, washed with PBS, and incubated in infection medium without PPMO for 72 h. At 16, 24, 48, and 72 h p.i., supernatants were collected, and viral titers were determined by plaque assay in MDCK(II) cells with Avicel overlay as described previously (
47). Cells were subjected to SDS-PAGE and western blot as described below.
Caco-2 cells were seeded in 24-well plates and grown to 80% confluence. The cells were transfected with either plasmids encoding wild-type ACE2 or empty vector using Lipofectamin2000 (ThermoFisher) according to the manufacturer’s instructions. At 24 h post-transfection, the cells were washed once with PBS and then inoculated with the virus at a low MOI of 0.001 in the infection medium for 1 h. The cells were washed with PBS and incubated with fresh infection medium for 72 h. At 24 h and 48 h p.i., supernatants were collected, and viral titers were determined by plaque assay in MDCK(II) as described above. The cells were harvested after 72 h and subjected to SDS-PAGE and western blot as described below.
MERS-CoV infection of cells and analysis of S cleavage
PPMO treatment of Calu-3 cells 24 h prior to infection was performed as described above. The cells were then inoculated with MERS-CoV at an MOI of 0.001 under serum- and BSA-free conditions with DMEM supplemented with glutamine and penicillin/streptomycin only (DMEM++) for 1 h. Subsequently, the cells were thoroughly washed with PBS and further incubated in DMEM+++. The supernatant was sampled at indicated time points and subjected to TCID50 titration as described below to determine viral titers. At 72 h p.i., infected cells were harvested for analysis of S cleavage via SDS-PAGE and western blot as described below.
TCID50 titration
Calu-3 cells were cultivated to confluency in 96-well plates before the medium was changed to DMEM+++. Supernatants taken from infected cells at indicated time points p.i. were serially diluted from 5
−1 to 5
−11 in quadruplets directly on the cells. After 3–4 days, when CPE was clearly visible, viral titers were calculated with the Spearman and Kaerber algorithm (
69).
SDS-PAGE and western blot analysis
Cells were washed with PBS, lysed using CellLytic M Cell Lysis Reagent (Sigma-Aldrich) for 30 min on ice, and cleared from cell debris by centrifugation. Lysates were supplemented with reducing SDS-PAGE sample buffer and heated at 95°C for 10 min. Samples produced under BSL4 conditions were heated to 100°C for 10 min twice. Proteins were subjected to SDS-PAGE (12% or 8% gel), transferred to a polyvinylidene difluoride (PVDF) membrane (GE Healthcare), and detected by incubation with primary antibodies and species-specific peroxidase-conjugated secondary antibodies. Proteins were visualized using the ChemiDoc XRS + system and Image Lab software (Bio-Rad). Using Image Lab Software (Bio-Rad), detected protein bands were relatively quantified. ACE2 bands in cell lysates expressing wt ACE2 only were set as 100% and used for the relative quantification. The relative quantifications of ACE2 +ACE2 cyto in input samples of the Co-IP were added together.
Co-immunoprecipitation assay
HEK293 cells seeded in 60 mm cell culture dishes were transfected with plasmids encoding wild-type ACE2 or the respective mutant jointly with TMPRSS2 or inactive mutant encoding plasmids or empty vector using Lipofectamin2000 (ThermoFisher) according to the manufacturer’s instructions. The plasmids were transfected at a ratio of 1:2 TMPRSS2 versus ACE2 plasmid. Cells were incubated at 37°C and 5% CO2 for 48 h. Cells were then harvested and lysed in 400 µL NP40-buffer [50 mM Tris, 150 nM NaCl, 1% Nonidet P-40, 1 mM PMSF, and 1× Protease Inhibitor Cocktail (Sigma-Aldrich)] per 60 mm cell culture dish. Lysates were precleared with 100 µL Protein-A-Sepharose (Sigma-Aldrich) for 30 min at 4°C with end-over-end rotation. The Protein-A-Sepharose was discarded, and the protein concentration of the lysates was determined using the Pierce BCA Protein Assay Kit (ThermoFisher). Lysates containing 40 µg protein were left untreated for input control. Lysates containing 1 mg protein were used for co-immunoprecipitation analysis with 30 µL of anti-HA tag agarose (Sigma-Aldrich) or ANTI FLAG M2-Affinity gel (Sigma Aldrich) incubated overnight at 4°C with end-to-end rotation. After incubation, the precipitates were washed three times with ice-cold NP-40 buffer, and bound proteins were eluted by adding SDS sample buffer + β-mercaptoethanol and boiling for 10 min. Precipitates and lysates were analyzed by SDS-PAGE and western blot using HA-tag and FLAG-tag-specific antibodies, respectively.
ACKNOWLEDGMENTS
We thank Diana Kruhl for excellent technical assistance, Lucie Sauerhering for providing the MERS-CoV, and the BSL-4 team for supporting work in the BSL4 laboratory.
This project has been funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) Project-ID 197785619—SFB 1021 (to E.B.-F.) and Project-ID 284237345—KFO309 (to E.B.-F.), State of Hesse LOEWE Center DRUID (to E.B.-F. and T.S.), and Federal funds from the National Institute of Allergy and Infectious Diseases, National Institutes of Health, Department of Health and Human Services, under Contract No. 75N93022C00036 (to A.DR. and B.S.). M.R.H., and E.B.-F. conceived the study and designed the experiments.
M.R.H., A.-L.R., M.S., D.B., A.H., A.DR., L.C.S., and E.B.-F. performed and analyzed the experiments. A.DR. and B.S. provided recombinant TMPRSS2. D.A.S. and H.M.M. designed and provided PPMO. B.S., T.S., and E.B.-F. provided funding for the studies. E.B.-F. wrote the manuscript. All authors contributed to the editing of the manuscript.