INTRODUCTION
The coronavirus disease 2019 (COVID-19) disease spectrum is caused by severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2), which was first identified in patients with pneumonia of unknown origin in the city of Wuhan, China (
1). While first characterized as a pneumonia, COVID-19 probably affects a number of organ systems (
2–4). SARS-CoV-2 was shown to use the angiotensin-converting enzyme 2 (ACE2) receptor, which was previously described as the receptor for the closely related severe acute respiratory syndrome-related coronavirus (SARS-CoV) (
5), for the infection of human cells (
1,
6,
7). For the proteolytic activation of the viral spike protein, a prerequisite for the fusion activity of coronaviruses (reviewed in reference
8), transmembrane protease serine 2 (TMPRSS2) (
7,
9), as well as the related TMPRSS4 (
2), was reported to be of critical importance. In addition, TMPRSS2 was demonstrated to colocalize with the ACE2 receptor (
10) and therefore may be biologically particularly relevant. Depending on the cell type, SARS-CoV-2 spike (SARS2-S)-driven entry can also occur through endocytotic pathways where virus-cell fusion is most likely activated by cathepsins (
7). Another study reported that several members of the TMPRSS family can activate SARS2-S-mediated membrane fusion (
11). The proposed mechanisms for spike priming and initiation of fusion therefore require further clarification, e.g., whether serine protease activity is required under all circumstances or whether fusion can also occur without the action of serine proteases, when these proteases act on the spike protein, and whether there are differences between cell-cell and cell-particle fusions.
It was recently discovered that the polybasic S1/S2 cleavage site of SARS2-S is required for efficient infection of lung-derived cells and promotes the formation of syncytia (
12). Understanding syncytium formation may be important, as large syncytial elements are reported to constitute a hallmark of COVID-19-associated pathology (
13). Nevertheless, the exact contribution of the two known proteolytic priming sites to cell-cell fusion and their protease usage are not entirely clear. To address these questions, we mutated the S1/S2 site as well as the S2 cleavage (S2′) site, we assessed the effects of proteolytic activation by using inhibitors of TMPRSS2 and other proteases, and we analyzed the effects of different levels of protease and receptor expression on SARS-CoV spike (SARS1-S) and SARS2-S fusion activity.
TMPRSS2, which is expressed in airway cells (
14), may be amenable to specific inhibition by bromhexine (
15), a molecule normally used as an expectorant that thins phlegm, eases coughing, and is widely known as a popular over-the-counter medication, which would make its repurposing for COVID-19 particularly attractive. For these or additional reasons, bromhexine is now being tested in at least three clinical trials (NCT04355026, NCT04273763, NCT04340349) for efficacy against COVID-19. We therefore tested the effect of the TMPRSS2 inhibitor bromhexine on spike-mediated cell-cell fusion and SARS2-S-driven cell entry and compared its potency to that of the serine protease inhibitor camostat. We also included ambroxol, an active metabolite of bromhexine, in our studies (
16). Ambroxol has often replaced bromhexine as an over-the-counter medication, and its structural similarity to bromhexine may hint at potential inhibitory effects toward TMPRSS2. Ambroxol may also exhibit weak but broad antiviral activity, as it was shown to reduce the occurrence of respiratory infections (
17) and to inhibit proteolytic activation of influenza virus by triggering the release of antiviral factors (
18), and it is used to treat acute respiratory distress syndrome in adults and antenatally in infants (
19,
20). Further, two recent preprints, one describing modulation of the ACE2-SARS2-S interaction by both bromhexine and ambroxol (
21) and the other reporting weak inhibitory activity of ambroxol against SARS-CoV-2 replication (
22) in Vero E6 cells, point at a potential utility of these molecules in the therapy of COVID-19.
DISCUSSION
We have established a two-hybrid-based protocol for measuring spike-mediated cell-cell fusion that allows for the quantitation of cell-cell fusion by luciferase activity and visualization of syncytia by GFP fluorescence. Our finding that SARS1-S-mediated and SARS2-S-mediated fusion activity is activated by the ACE2 receptor is in accordance with published data (
11), whereas our finding that SARS2-S-mediated cell-cell fusion is relatively more restricted by ACE2 expression and less by proteolytic activation than SARS1-S-mediated fusion is novel. This is because SARS-CoV-2 can efficiently utilize metalloproteases for activation of cell-cell fusion. Further, we have faithfully established the S2′ site of SARS2-S as the target for TMPRSS2-mediated activation through generation of a mutant that is defective for TMPRSS2 activation but otherwise fully functional.
In our system, TMPRSS2 coexpression on ACE2-expressing target cells was not required for SARS2-S-mediated fusion of ACE2-overexpressing 293T cells, comparable with the results of Ou et al., in whose study ACE2 expression alone was also sufficient to induce cell-cell fusion without the addition of exogenous protease (
11), which was corroborated by a very recent report (
40). Furthermore, we did not observe any effect on SARS2-S-mediated fusion activity upon inhibition of TMPRSS2 on target cells by the serine protease inhibitor camostat when ACE2 was present. Together, these results imply that proteolytic activation by TMPRSS2 may not be a limiting factor for cell-cell fusion in 293T cells. A recent report demonstrated that upon cotransfection of spike, ACE2, and TMPRSS2, TMPRSS2 accelerates fusion. The size of the resulting syncytia showed a TMPRSS2 dependency only within the first 12 h but was independent after 24 h in that report (
41), which is compatible with our observations of efficient cell-cell-fusion without TMPRSS2 in 293T cells.
While SARS1-S-mediated cell-cell fusion was also weakly activated when ACE2 was expressed alone, activation was much higher in the presence of TMPRSS2, indicating stronger dependence of SARS1-S on TMPRSS2, compatible with the monobasic S1/S2 cleavage site in the SARS-CoV spike protein. Surprisingly, we even observed maximal activation with overexpression of only TMPRSS2, indicating that SARS1-S-mediated cell-cell fusion is mostly protease and not ACE2 driven. In line with this observation, SARS1-S-mediated cell-cell fusion was clearly sensitive to camostat, which reversed the TMPRSS2-mediated activation (
Fig. 3A).
Interestingly, mutational ablation of the S1/S2 cleavage site of SARS2-S rendered the mutated spike protein sensitive to inhibition by camostat in the presence of ACE2 and TMPRSS2 (
Fig. 3A), suggesting that TMPRSS2 or a related protease is required for processing at the S2′ site to reach full activation when the S1/S2 site is not cleaved. In addition, in the absence of recombinantly expressed TMPRSS2, SARS2-S1/S2-mut was clearly impaired with regard to fusion activity (
Fig. 2A). Conversely, mutation of the S2′ priming site abrogated any effects of TMPRSS2 on SARS2-S-mediated fusion, e.g., when TMPRSS2 alone was provided by means of recombinant expression (
Fig. 3A and
6A) or when TMPRSS2 was inhibited by camostat (
Fig. 6E). It should be noted that the SARS2-S S2′ mutants were still fusogenic in the presence of high levels of the ACE2 receptor (
Fig. 2A,
3A, and
6A and
E), in the case of the GH and HH S2′ mutants even at moderate ACE2 levels, and in the absence of TMPRSS2 with activity similar to that of the wild type (
Fig. 6A). The S2′ GH mutant was also efficiently incorporated (
Fig. 7D) and able to drive the infection of pseudotyped lentiviral particles (
Fig. 7E). With wt SARS2-S or SARS2-S1/S2-mut, but not with the SARS2-S S2′ mutants, recombinant expression of TMPRSS2 led to low but detectable fusion activity (
Fig. 2A and
6A). Collectively, these findings identify the S2′ site as the primary target of TMPRSS2 for fusion activation.
Another observation was that the S2′-AA mutant, as observed in
Fig. 1C and
6C, exhibited drastically reduced surface expression. In fact, a similar incorporation defect has been described in the literature for SARS-CoV (
26). Whatever the reason for this defect, we were able to overcome it completely by replacing the S2′ motif KR with the amino acids GH, which restored surface expression (
Fig. 6C), processing into S1 and S2 subunits (
Fig. 6B), and particle incorporation (
Fig. 7D). The reasons for this phenomenon are unclear. Charge reversal of S2′ from KR to EE was definitely detrimental to activity, indicating that solubility may not be the critical point. As histidine may carry a positive charge depending on the local environment, our findings might hint at a requirement for at least one positive charge at this position.
Our results clearly demonstrate that cleavage at the S1/S2 site alone is not sufficient for fusion activity in the presence of ACE2 and requires additional processing at S2′ or another site. This has been established for particle entry (
12), but it was not entirely clear for cell-cell fusion, as the precleaved spike was clearly fusogenic also in conditions without exogenous protease activity in several reports (
11,
12,
25,
41), which may have been interpreted as a cell-cell fusion-ready state after S1/S2 cleavage. While our initial attempts to block the fusion activity of wt SARS2-S and the S1/S2 mutant in the presence of ACE2 receptor but without TMPRSS2 were relatively unsuccessful, treatment with the metalloprotease inhibitor batimastat reduced fusion by both the wt (
Fig. 5A) and the S1/S2 mutant (
Fig. 5C), as well as fusion by the S2′ mutants (
Fig. 6E). These findings indicate that metalloproteases can activate SARS2-S and that this activation occurs at least in part independently of the S1/S2 site and of the S2′ site, as both mutants were still batimastat sensitive. On the other hand, the S1/S2 mutant was clearly less active in the absence of TMPRSS2, indicating that matrix metalloproteases activate more efficiently when the S1/S2 site is present. These findings are in line with a very recent report describing similar observations using different inhibitors (
40).
As SARS2-S did not require TMPRSS2 on target cells for robust cell-cell fusion, our attempts to test the impact of bromhexine as a specific inhibitor of TMPRSS2 on SARS2-S-mediated fusion activity were somewhat artificial. Nevertheless, SARS1-S-mediated fusion was clearly enhanced by TMPRSS2, as was fusion by SARS2-S1/S2-mut, and both were inhibited by camostat but not by bromhexine. Therefore, our finding that bromhexine specifically enhanced the fusion of 293T cells in the presence of SARS2-S, ACE2, and TMPRSS2 is something that we cannot explain easily. According to our results, the bromhexine-mediated enhancement was specific for SARS2-S and was not seen with VSV-G as a fusion effector (
Fig. 3C), nor did we observe significant effects with the SARS2-S mutants or SARS1-S (
Fig. 3A). We observed some inhibition of SARS1-S-mediated fusion in the presence of 50 μM ambroxol (
Fig. 3A) and also with SARS2-S with longer incubation times (
Fig. 3C), which may hint at some activity of this substance against TMPRSS2, which would fit with the observation of an atypical autoproteolytic fragment of TMPRSS2 in the presence of ambroxol. The observation of the paradoxical effect of bromhexine in the presence of TMPRSS2 suggests that bromhexine somehow modulates proteolytic processing. It is at the moment not clear by what mechanism of action bromhexine modulates TMPRSS2 activity, if it does so, and we therefore cannot exclude the possibility that processing of some substrates is actually enhanced or altered instead of inhibited, as reported for several substrates (
15,
42). Recently, another study also reported the lack of an inhibitory activity of bromhexine against TMPRSS2 (
29). The activity of bromhexine against TMPRSS2-mediated receptor shedding, which may also explain our observations, was not observed, unlike with camostat, which increased ACE2 expression levels in the presence of TMPRSS2 (
Fig. 3B and
5B). This may explain the slight increase, even if not always statistically significant, in fusion activity that we observed in some experiments with SARS2-S in the presence of camostat when ACE2 and TMPRSS2 were coexpressed (
Fig. 3A and
5A).
Compared to another study (
43), our fusion assay yielded slightly different results, with SARS2-S-mediated fusion appearing less dependent on activation by TMPRSS2. This may be due to differences in the protocol. The study by Yamamoto et al. (
43) allowed only for very short contact times of 4 h and used nonadherent 293T FT cells, whereas we cocultured the cells for a longer time, which allows for extended contact between cells and may enable the action of matrix metalloproteases. Our finding that TMPRSS2 is not required for fusion is in line with several reports making the same observation (
11,
12,
25,
40,
41,
44). In general, we observed a higher fusion activity with our SARS2-S1/S2-mut spike mutant than was observed with furin cleavage site mutants in previous studies (
12,
25), but we observed this only when TMPRSS2 was recombinantly overexpressed together with ACE2. When only ACE2 or only TMPRSS2 was recombinantly expressed, SARS2-S1/S2-mut fusion activity was strongly impaired (
Fig. 2A). It should be noted, that we left the loop intact and replaced only the basic residues with alanine in our mutant, whereas other groups deleted the loop structure, which may result in a less flexible conformation. Nevertheless, our mutational approach for ablating the furin cleavage site clearly rendered the spike protein more dependent on additional serine protease activity by recombinantly expressed TMPRSS2. This proteolytic activity was directed toward the S2′ site, as SARS2-S1/S2-mut fusion activity was dependent on TMPRSS2 and was significantly inhibited by camostat (
Fig. 3A) in the presence of TMPRSS2.
Taken together, our results actually reconcile several seemingly conflicting observations by other groups. The strong reduction in fusion activity by mutation of the S1/S2 site observed in one study using Vero cells (
12) is reflected in our experimental conditions with only TMPRSS2 and endogenous levels of ACE2 expression, whereas our findings of more or less normal fusion activity under conditions of high-level ACE2 and TMPRSS2 expression are similar to the findings of another group with ACE2-overexpressing cells and the addition of trypsin or human airway trypsin-like protease (HAT) (
25).
Overall, we propose that the dependence on S1/S2 cleavage, the activity of TMPRSS2 or a related protease, and receptor expression are to a certain degree interdependent and that one factor can at least partially compensate for another; e.g., more extensive proteolytic activation at S2′ can render the spike more fusogenic even with lower receptor levels, which was particularly observed for SARS1-S and to a lesser degree for SARS2-S (
Fig. 2). Similarly, batimastat-sensitive metalloproteases can activate SARS2-S for cell-cell fusion (
Fig. 5A). This is partially dependent on the S1/S2 site, as SARS2-S1/S2-mut was still impaired in the absence of TMPRSS2 but completely independent of the S2′ site, as demonstrated by the full fusion activity of the SARS2-S2′-GH spike mutant on ACE2-expressing 293T cells (
Fig. 6A).
According to our results, the requirements for cell-cell fusion and virus-cell fusion differ: additional TMPRSS2 activity drastically enhanced pseudotype entry into transfected 293T cells (
Fig. 7A) but was not needed for cell-cell fusion with identically transfected 293T cells (
Fig. 2A and
3A). In addition, the matrix metalloprotease inhibitor batimastat did not affect particle entry in the presence of the TMPRSS2 inhibitor camostat, indicating that matrix metalloproteases can activate cell-cell fusion but not particle-cell fusion (
Fig. 7C and
E), at least not in our experimental system. Similar observations were previously made for SARS-CoV (
33). The interpretation of these results is complicated by the ability of virus particles to enter cells both through direct membrane fusion or an endocytotic pathway and by different prepriming states of viral spike proteins, depending on proteolytic activity in the producer cell (
45). As activation of the spike protein is expected to differ between organ systems depending on the presence of different proteolytic activities, these processes ultimately need to be studied in appropriate tissue systems or animal models. It is tempting to speculate that, relative to SARS-CoV, more relaxed requirements for cell-cell fusion with regard to proteolytic activation contribute to the broad organ tropism and neuroinvasion by SARS-CoV-2, as well as the clinically observed formation of extended syncytia (
13). Irrespective of the role of cell-cell fusion in COVID-19, in light of the observed paradoxical activation of cell-cell fusion by bromhexine and its lack of inhibitory activity against the entry of SARS2-S-pseudotyped lentiviruses on TMPRSS2-expressing cells, we at the moment caution against clinical use of bromhexine for the treatment or prophylaxis of COVID-19, at least at high concentrations that aim at the inhibition of TMPRSS2. A recent, small randomized trial showed promising results for bromhexine at 8 mg three times per day combined with hydroxychloroquine (
46), which should result in bromhexine plasma concentrations in the range of 0.1 μM (
47). We are fairly confident to postulate that these favorable results are unlikely due to the inhibition of TMPRSS2, although we cannot fully exclude the possibility of extremely weak activity. This view is supported by a recent study that found no effect of bromhexine on TMPRSS2 activity (
29). More likely, favorable patient outcomes are attributable to the beneficial effects of bromhexine or its main metabolite ambroxol on lung function, general defense mechanisms against airway infections, and inflammatory responses (
16–19,
48). Another recent study by Olaleye et al. (
21) specifically analyzed the effects of bromhexine and ambroxol on the interaction of ACE2 with the SARS-CoV-2 spike receptor binding domain (RBD) and reported a very peculiar behavior of these substances, which in part may explain the paradoxical results of our fusion assays and would support a beneficial effect of low-dose bromhexine, which is converted to ambroxol
in vivo. While ambroxol weakly inhibited the ACE2-RBD interaction up to a 100 μM concentration, bromhexine exhibited a biphasic behavior and was weakly inhibitory below 10 μM but increased ACE2-RBD binding at higher concentrations in that study. Both substances were reported to weakly inhibit SARS-CoV-2-mediated cytopathic effect (CPE) in culture (
22), and ambroxol was also shown to moderately impact the replication of SARS-CoV-2 in that report (
22), albeit on Vero cells and not lung cells. Our results suggest that ambroxol can weakly inhibit spike-driven entry of lentiviral pseudotypes into Calu-3 cells at high but potentially attainable concentrations (
Fig. 8B), and our experiments with authentic SARS-CoV-2 on Calu-3 cells (
Fig. 8C and
D) demonstrated a trend toward inhibition of replication by both ambroxol and bromhexine, with bromhexine possibly being slightly more potent but also more toxic (
Fig. 4C and
8E). Thus, the specificity of bromhexine-mediated inhibition is questionable. In sum, it seems likely that ambroxol acts weakly on TMPRSS2, which would explain its modest but significant effect on the TMPRSS2-mediated activation of SARS1-S-mediated fusion (
Fig. 3A). It should be noted that replication of the authentic virus can be influenced at numerous points, not necessarily only during entry, and that effects can be amplified over several replication cycles. Of course, compared to the potency of camostat, the effect of both substances is marginal. Nevertheless, ambroxol can be administered in high doses of 1 g and more intravenously (
19) or orally (
49) and reportedly accumulates strongly in lung tissue (
50). Thus, ambroxol, which exhibited a trend toward the inhibition of SARS2-S-mediated entry and fusion in several assays without enhancing effects, as was observed with bromhexine at high concentrations, may represent an interesting option for supportive therapy at higher dosages, in particular as it is a proven therapeutic for antenatal respiratory distress syndrome (
51) and has shown efficacy in the treatment of radiation-induced lung injury (
48).
MATERIALS AND METHODS
Cell culture.
All cell lines in this study were incubated at 37°C and 5% CO
2. 293T cells (a kind gift from Vladan Rankovic and originally purchased from the ATCC, Göttingen, Germany) and Calu-3 cells (a kind gift from Stefan Ludwig) were cultured in Dulbecco's modified Eagle medium (DMEM), high glucose, GlutaMAX, 25 mM HEPES (Thermo Fisher Scientific) supplemented with 10% fetal calf serum (FCS) (Thermo Fisher Scientific) and 50 μg/ml gentamicin (PAN Biotech). For Calu-3 cells, 1 mM sodium-pyruvate (Thermo Fisher Scientific) was added. For seeding and subculturing of cells, the medium was removed, and the cells were washed with phosphate-buffered saline (PBS; PAN-Biotech) and detached with trypsin (PAN-Biotech). All transfections were performed using polyethylenimine (PEI; Polysciences) in a 1:3 ratio (μg DNA/μg PEI) mixed in Opti-MEM. The cell viability assay with Calu-3 cells (
Fig. 8E) was performed as described previously (
7); unlike with the other assays in this series of experiments, bromhexine and ambroxol were used in the form of commercial cough suppressants (Krewel Meuselbach bromhexine at 12 mg/ml and Mucosolvan at 30 mg/5 ml; Sanofi-Aventis).
Plasmids.
Expression plasmids for pQCXIPBL-hTMPRSS2 (
52), pCG1-SARS-2-S_humanized (
7), pCG1-ACE2 (
7), and pCG1-SARS S (
53) are described elsewhere. For generation of pVAX1-SARS2-S, the codon-optimized sequence encoding the spike protein of SARS-CoV-2 was amplified by PCR and cloned into the pVAX1 backbone. psPAX2 and pMD2.G were a gift from Didier Trono (Addgene plasmid numbers 12260 and 12259), and pLenti CMV GFP Neo (657-2) was a gift from Eric Campeau and Paul Kaufman (Addgene plasmid number 17447). The expression plasmids SARS2-S2′-AA, SARS2-S1/S2-mut, and SARS2-D614G were generated from humanized pCG1_SL-Cov_Wuhan-S_SARS2-S by PCR-based mutation of the SARS2-S S1/S2 and the S2′ cleavage site using around-the-horn PCR mutagenesis with S7 fusion PCR (Biozym) or Phusion PCR, T4 PNK, and Quick ligase (all from New England Biolabs) and using the following primers: S1-S2 AAAA mut for V2 (
CTGCCTCTGTGGCCAGCCAGAGCATC), S1-S2 AAAA mut rev V2 (
CAGCGGCGGGGCTGTTTGTCTGTGTCTG), S2 to AA mut_Forward (
GCCAGCTTCATCGAGGACCTGCTG), S2 to AA mut_Reverse (
AGCGCTGGGCTTGCTAGGATCGG), SARS2S R815 H for (
CACAGCTTCATCGAGGACCTGCTG), SARS2S K814H rev (
GTGGCTGGGCTTGCTAGGATCGG), SARS2S R815E for (
GAGAGCTTCATCGAGGACCTGCTG), SARS2S K814E rev (
CTCGCTGGGCTTGCTAGGATCGG), SARS2S R815E for (
GAGAGCTTCATCGAGGACCTGCTG), SARS2S R815S for (
AGCAGCTTCATCGAGGACCTGCTG), SARS2S K814G rev (
TCCGCTGGGCTTGCTAGGATCGG), D614G for aroundthehorn (
GCGTGAACTGTACCGAAGTGCC), and D614G rev aroundthehorn (
CCTGGTACAGCACTGCCACCTG). Sequence integrity was verified by sequencing of the coding region. Plasmid pCG1-SARS2-S_S2′mut contains a silent G-to-T mutation in the codon for leucine 441.
Expression plasmids pVAX1-SARS2-S_S2′-GH, pVAX1-SARS2-S1/S2-mut, and pVAX1-SARS2-S_D614G were generated from pVAX1-SARS2-S by PCR-based mutation in a similar manner.
The Gal4-Luc reporter plasmid encoding firefly luciferase under the control of an activator sequence that binds the Gal4 transcription factor has been described elsewhere (
33). The Gal4 DNA binding domain VP16 fusion plasmid corresponds to GenBank identifier
X85976. The TurboGFP-luciferase fusion reporter gene was constructed using Gibson Assembly master mix (New England Biolabs) to insert the TurboGFP open reading frame with a Ser-Gly-Ser-Gly linker in front of the Met codon of the luciferase open reading frame. Before assembly, the two fragments were generated using Phusion PCR (New England Biolabs) by amplifying the TurboGFP open reading frame from the vector pGIPZ (Thermo Scientific Open Biosystems), using the primers TurboGFP for Gal4Luc before ATG ov (
GGTACTGTTGGTAAAATGGAGAGCGACGAGAGC) and TurboGFP rev (
TTCTTCACCGGCATCTGCATC), and the Gal4-Luc backbone by amplification with primer Gal4Luc before ATG rev (
TTTACCAACAGTACCGGAATGC) and primer Luc for SGSG TurboGFP overhang (
GATGCAGATGCCGGTGAAGAAAGCGGTAGCGGTATGGAAGACGCCAAAAACATAAAG).
The pLenti-CMV-TurboGFP-luciferase fusion reporter gene was constructed using Gibson Assembly master mix (New England Biolabs) to exchange the insert in pLenti-CMV-BLAST-EphA7-Strep (described elsewhere [
54]) with the TurboGFP-luciferase open reading frame without the Strep tag; the two fragments were generated using CloneAmp HiFi PCR premix (TaKaRa Bio) by amplifying the TurboGFP-Luc open reading frame from the vector Gal4-TurboGFP-Luc using the primers GA_TurboGFP-Luc_pLentiBlast-StrepOneOv_For (
ACAAAAAAGCAGGCTCCACCATGGAGAGCGACGAGAGC) and GA_TurboGFP-Luc_pLentiBlast-StrepOneOv_Rev (
TGTGGATGGCTCCAAGCGCTTTACAATTTGGACTTTCCGCC), and the pLenti-CMV-BLAST-EphA7-Strep backbone by amplification with primer pLenti attB1 rev at ATG (
CATGGTGGAGCCTGCTTTTTTGTAC) and OneStrep for (
AGCGCTTGGAGCCATCCAC).
Western blotting.
Protein expression was analyzed by polyacrylamide gel electrophoresis on 8% to 16% precast gradient gels (Thermo) and Western blotting using antibodies to ACE2 (AF933; R&D Systems), the c-Myc epitope (clone 9E10; Santa Cruz Biotechnology), SARS spike (NB100-56578; Novus Biologicals), HIV-1 Gag p24 (clone 749140; R&D), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase; GenScript) in NETT-G (150 mM NaCl, 5 mM EDTA, 50 mM Tris, 0.05% Triton X-100, 0.25% gelatin, pH 7.5) and donkey anti-mouse horseradish peroxidase (HRP)-coupled (Dianova), goat anti-rabbit HRP-coupled (Life Technologies), or rabbit anti-goat HRP-coupled (Proteintech) secondary antibody in 5% dry milk powder in PBS with 0.05% Tween 20. Imaging was performed using the Immobilon Forte substrate (Merck) on an INTAS ECL ChemoCam system.
Flow cytometry.
293T cells were transfected with the respective spike expression constructs. On day 2 posttransfection, the cells were harvested by gentle pipetting in PBS and were fixed in 2% methanol-free formaldehyde in PBS for 15 min. The cells were then washed once in PBS and then incubated in 10% FCS in PBS for 30 min to block nonspecific binding. The cells were then incubated in either convalescent-phase serum at a 1:1,000 dilution or soluble ACE2-Fc fusion protein at 2 ng/μl, both described elsewhere (
55), for 1 h in 10% FCS in PBS, followed by one wash in a large volume of PBS and then incubation with Alexa 647-coupled anti-human secondary antibody (Thermo Fisher Scientific) at 1:200 in 10% FCS in PBS. The RRV gHΔ21-27-Fc fusion protein, which was used as a control protein, was generated from RRV 26-95 gH-Fc (
56) by deletion of the codons for amino acids 21 to 27, which are important for receptor binding (
27), and was produced analogously to the gH-Fc protein in the study of Hahn and Desrosiers (
56). The cells were then washed once in a large volume of PBS and postfixed in 2% paraformaldehyde (PFA) in PBS before analysis on an LSRII flow cytometer (BD Biosciences). Data were analyzed using Flowing software (version 2.5) and GraphPad Prism, version 6, for Windows (GraphPad Software). COVID-19 convalescent-phase serum was collected previously (
55) in accordance with ethical requirements (ethics committee UK Erlangen, license number AZ. 174_20 B).
Fusion assay.
293T target cells were seeded in a 48-well plate at 50,000 cells/well and transfected with Vp16-Gal4 (
Fig. 3C) or the Gal4-TurboGFP-luciferase expression plasmid (Gal4-TurboGFP-Luc in all other experiments) as well as expression plasmids for ACE2 and TMPRSS2, as indicated in the figure legends. In case only ACE2 or TMPRSS2 was transfected, the missing amount of DNA was replaced by an empty vector. 293T effector cells were seeded in a 10-cm dish at 70 to 80% confluence and transfected with either the Vp16-Gal4 (all experiments except
Fig. 3C) or Gal4-luciferase (
Fig. 3C) expression plasmid as well as expression plasmids for SARS2-S, SARS2-S1/S2-mut, SARS2-S2′-AA, SARS2-S2′-GH, SARS2-S2′-HH, SARS2-S2′-EE, SARS2-S2′-ES, SARS1-S, VSV glycoproteins, or pcDNA6/V5-HisA (Thermo). For effector cell preincubation experiments, the medium of effector cells was changed to bromhexine hydrochloride (Merck), ambroxol hydrochloride (Merck), camostat mesylate (Tocris), batimastat (Merck), AEBSF (Merck), EDTA (Merck), EGTA (Merck), 100× animal-free cocktail set V (Calbiochem; Merck), or decanoyl-RVKR-CMK (Merck) containing medium at a final concentration 6 h after transfection. Twenty-four hours after transfection, target cells were preincubated with bromhexine hydrochloride (Merck), ambroxol hydrochloride (Merck), camostat mesylate (Tocris), batimastat (Merck), AEBSF (Merck), EDTA (Merck), EGTA (Merck), or decanoyl-RVKR-CMK (Merck) for 30 min at the concentrations indicated in the figure legends. Effector cells were then added to the target cells in a 1:1 ratio, reaching the final inhibitor concentration. After 24 to 48 h, GFP fluorescence was detected using a Vert.A1 fluorescence microscope and ZEN software (Zeiss), and luciferase activity was analyzed using the PromoKine firefly luciferase kit or Beetle-Juice luciferase assay (PJK Biotech) according to the manufacturer’s instructions and a BioTek Synergy 2 plate reader. Statistical analysis was performed using GraphPad Prism, version 6, for Windows (GraphPad Software).
Production of lentiviral and pseudoparticles and pseudoparticle infection experiments.
Lentiviral pseudoparticles were produced by transfecting 293T cells with expression plasmids for psPAX2, pLenti-CMV-GFP, or pLenti-CMV-TurboGFP-luciferase and either SARS2-S variants (pVAX1-SARS2-S_S2′-GH, pVAX1-SARS2-S1/S2-mut, and pVAX1-SARS2-S_D614G) or VSV-G (pMD2.G; Addgene number 12259). The cell culture supernatants were harvested 24 to 72 h posttransfection, followed by the addition of fresh medium, and again after 48 to 72 h. The supernatants were passed through a 0.45-μm cellulose acetate (CA) filter, and the SARS2-S pseudoparticles were concentrated via low-speed centrifugation at 4°C for 16 h at 4,200 × g. For detection of particle incorporation, the virus supernatant was further concentrated by centrifugation at 4°C for 2 h at 21,000 × g on 5% OptiPrep (Merck), the supernatant was removed, and the pellet was resuspended and subjected to Western blot analysis. The SARS-CoV-2 spike and VSV-G lentiviral pseudoparticles were used to transduce 293T cells transfected with TMPRSS2 and ACE2 expression plasmids or Calu-3 cells. Forty-eight hours after transfection with control or ACE2 and TMPRSS2 expression plasmids, the pseudoparticles were added to cells preincubated with the inhibitors bromhexine hydrochloride (Merck), ambroxol hydrochloride (Merck), camostat mesylate (Tocris), batimastat (Merck), AEBSF (Merck), and E64-d (Biomol) for 30 min at twice the concentration indicated in the figure legends, and the final concentration was reached after the addition of the inoculum. Cells transduced with pLenti-CMV-GFP pseudoparticles were harvested 48 h after transduction using trypsin. Bald particles from 293T cells that had been transfected with an empty vector instead of glycoprotein expression plasmids and the lentiviral packaging system were used as background control for normalization. Trypsin activity was inhibited by adding 5% FCS in PBS, and after being washed with PBS, the cells were fixed with 4% formaldehyde (Roth) in PBS. The percentages of GFP-positive cells were determined using a LSRII flow cytometer, and at least 10,000 cells were analyzed. Cells transduced with pLenti-CMV-TurboGFP-luciferase pseudoparticles were lysed after 48 h with luciferase lysis buffer (Promega) and detected using the Beetle-Juice luciferase assay according to manufacturer’s instructions and a BioTek Synergy 2 plate reader. Statistical analysis was performed using GraphPad Prism 6.
SARS-CoV-2 infections.
Primary SARS-CoV-2 isolate ER-PR2 was a kind gift from Klaus Überla, Erlangen, Germany, and was originally isolated on Vero cells. The virus stock was then grown on Calu-3 cells in DMEM plus 2% FCS plus penicillin/streptomycin. The virus-containing supernatant was harvested after CPE was clearly visible, and the supernatant was cleared by low-speed centrifugation at 1,200 rpm for 10 min before passage through a 0.2-μm syringe filter (Mini-Sart; Sartorius). Virus stocks were aliquoted in 200-μl aliquots and stored at −150°C. Infectivity was determined by the method of Reed and Muench (
57) at 10
6.1 50% tissue culture infectious doses (TCID
50s)/ml. Calu-3 cells were seeded 1 day (first experiment) or 2 days (other two experiments) before infection, and approximately 100,000 Calu-3 cells were infected at an MOI of approximately 0.002 in a 96-well plate in triplicates. The cells were preincubated with the respective inhibitors in 50 μl at twice the concentration for ∼1.5 h, and the virus was then added in 50 μl medium. Total RNA from the cells and the culture supernatant was harvested 20 h (experiment 3) and 24 h (experiments 1 and 2) postinfection.
RNA isolation, cDNA synthesis, and RT-qPCR.
RNA was isolated using the Direct-zol RNA Miniprep Plus kit (Zymo) according to the manufacturer’s instructions. For quantification of viral RNA in infected cultures, the cells and cellular supernatant in a volume of 100 μl were lysed and inactivated by addition of 300 μl TRI reagent (Zymo). RT-qPCR of viral genomes was performed using the N1 CDC primer set from IDT (2019-nCoV_N1-F, GACCCCAAAATCAGCGAAAT, and 2019-nCoV_N1-R, TCTGGTTACTGCCAGTTGAATCTG, both at 500 nM, and 2019-nCoV_N1-P, FAM-ACCCCGCATTACGTTTGGTGGACC-BHQ1 [where FAM is 6-carboxyfluorescein and BHQ1 is black hole quencher 1] at 125 nM) and the SensiFAST Probe Hi-ROX one-step kit (Bioline) according to the manufacturer’s instructions in a 20-μl reaction mixture with a 5-μl sample. All RT-qPCRs were performed in technical duplicates on a StepOne Plus (Thermo) real-time cycler. PCR conditions were 45°C for 10 min, 95°C for 2 min, and then 45 cycles of 95°C for 5 s followed by 55°C for 20 s. To determine the PCR efficiency across the whole dynamic range, a 7-step 10-fold dilution series with the H2O-treated SARS-CoV-2-infected Calu-3 cell sample was performed. These data points with the undiluted sample set to 1 was approximated by an exponential function using Microsoft Excel 2020. The measured PCR efficiency was additionally fitted by multiplication with a constant factor to match our RNA standard (Charité, Berlin, Germany), which was available only at 50, 500, and 5,000 copies, which confirmed our approach but was not used for relative quantification. Fit was performed by minimizing the sum of the squared relative deviations from the standard concentrations with an exactness of two digits.
For quantification of cellular TMPRSS2 and GAPDH expression, cDNA synthesis and qPCR were performed according to the manufacturer’s instructions using the SensiFAST cDNA kit and SensiFAST SYBR qPCR kit (both from Bioline). The qPCR was run on a StepOnePlus real-time PCR cycler (Thermo) and analyzed using the StepOne software, which was also used to calculate ΔΔCT values and error estimates for TMPRSS2 expression. TMPRSS2 mRNA was detected using primer set Hs.PT.58.39408998 (IDT) (forward primer GTCAAGGACGAAGACCATGT, reverse primer TGCCAAAGCTTACAGACCAG). GAPDH mRNA was detected using primers GAPDH_Hs-Mm_s (CTTTGGTATCGTGGAAGGACTC) and GAPDH_Hs-Mm_as (GTAGAGGCAGGGATGATGTTC). Amplifications with a CT above 35 and nonmatching melting curve were scored as not detected.