Abstract
Although numerous sarbecoviruses have been identified in bats, but most lack the ability to infect human cells. Some barriers limit coronavirus zoonosis, including susceptibility to host proteases. Here, we investigated whether exogenous protease treatment can circumvent host restrictions in two severe acute respiratory syndrome (SARS)-related bat coronaviruses. We found that the spike proteins of RaTG13 and Khosta-2, which are sarbecoviruses obtained from horseshoe bats in China and Russia, respectively, facilitated the ACE2-mediated entry of pseudotyped viruses into VeroE6/TMPRSS2 cells following elastase treatment. In contrast, trypsin and thermolysin exhibited no effects. Elastase-enhanced infectivity correlated with increased fusogenicity driven by the cleavage of spike proteins. This process was TMPRSS2-dependent and was inhibited by nafamostat, a TMPRSS2 inhibitor. Additionally, mutation of residue 809 within the S2 subunit of the RaTG13 spike protein (S809D) impaired elastase-induced cleavage and infectivity. Hence, proteolytic processing of the spike protein serves as a restriction to RaTG13 and Khosta-2 infections, which can be overcome by elastase. This suggests that elastase secreted in inflamed tissues during viral infection may increase the zoonotic potential of sarbecoviruses by facilitating human cell entry.
Similar content being viewed by others
Introduction
Many emerging and re-emerging infectious diseases are caused by viruses that occur naturally in non-human vertebrates. If introduced into humans, these viruses can cause disease outbreaks, epidemics, or pandemics. In the last two decades, coronaviruses (CoVs) of likely bat origin, such as the severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have emerged and caused widespread epidemics in humans1,2,3. However, the animal origins and zoonotic transmission cycles of important CoVs that cause human outbreaks are not yet fully understood.
Currently, there are no well-documented cases of direct spillover infections from bats to humans by CoVs; however, this is likely due to inadequate surveillance rather than the absence of spillovers4. Researchers have reported that bat sarbecoviruses, including RaTG13 and Khosta-2, isolated from China and Russia, can bind to human angiotensin-converting enzyme 2 (ACE2) and enter cells, similar to SARS-CoV-25,6. Furthermore, the Khosta-2 spike protein is resistant to both SARS-CoV-2 monoclonal antibodies and serum from individuals vaccinated against SARS-CoV-26. The low cross-reactivity of Khosta-2 RBD can be because it shares approximately 60% similarity with the SARS-CoV-2 RBD at the amino acid level. The emergence of these new bat sarbecoviruses may pose a threat to the current SARS-CoV-2 vaccine strategies6, especially when considering the similarity between RaTG13, Khosta-2, and SARS-CoV-2, and the devastation caused by SARS-CoV-2. To assess the risks associated with these viruses, it is necessary to identify the factors that control cross-species infections.
In addition to binding to host receptors, successful infection requires several consecutive steps, including entry, replication, evasion of host innate immunity, and budding. Host proteases activate (cleave) the viral spike (S) protein to enable entry, and this process may be as important as host receptor binding in determining the zoonotic potential of the virus7,8. Coronavirus S proteins have multiple cleavage sites for host proteases, which are cleaved at different stages of the cell infection cycle9,10. Cell surface proteases, such as transmembrane serine protease 2 (TMPRSS2) and trypsin-like proteases, are involved in cleaving S proteins after viral attachment, whereas lysosomal proteases, such as cathepsins, cleave S proteins after viral endocytosis. In contrast, furin, a membrane-bound protease present in the Golgi apparatus, may be involved in cleaving S protein during biosynthesis11. Extracellular proteases, such as trypsin and elastase, may also be involved in the activation of human CoVs12. Several studies have reported that these proteases enhance the infectivity of SARS-CoV and SARS-CoV-213,14,15. The distribution and activity of these proteases vary across cell types and physiological conditions, thereby influencing coronavirus tissue tropism and entry into host cells15,16,17,18. In the absence of exogenous proteases, only MERS-CoV S proteins mediated pseudotyped virus entry into human cells, whereas the S protein of bat coronavirus HKU4 did not19. However, upon trypsin treatment, HKU4 S proteins efficiently mediated pseudotyped virus entry into human cells. Thus, host cellular proteases were established as a host range determinant for HKU419. Determining whether host proteases activate the S proteins of bat CoVs is essential for predicting potential changes in host range and tissue tropism.
In this study, we aimed to examine the effects of three proteases (trypsin, thermolysin, and elastase) on S protein-mediated cell entry of the bat SARS-like CoVs RaTG13 and Khosta-2. We also aimed to assess the S protein proteolysis and its impact on membrane fusion to delineate the distinct effects of these proteases.
Results
Elastase, but not trypsin or thermolysin, mediated cell entry of pseudotyped bat SARS-like CoVs
We used a pseudotyped system with the S protein of bat SARS-like CoVs to investigate whether trypsin, thermolysin, and elastase mediate host cell entry. Trypsin, thermolysin, and elastase were selected as representative proteases that cover a broad range of cleavage specificities relevant to coronavirus spike activation: trypsin cleaves at basic residues and mimics TMPRSS2 activity10; thermolysin preferentially cleaves at hydrophobic residues20; and elastase, secreted by neutrophils during inflammation, may be relevant to in vivo airway infection conditions13. RaTG13 and Khosta-2 S protein-expressing pseudotyped viruses exhibited minimal infectivity in VeroE6/TMPRSS2 cells. However, elastase treatment of pseudotyped viruses facilitated RaTG13 and Khosta-2 S protein-mediated cell entry into these cells. In contrast, trypsin and thermolysin treatments did not result in S protein-mediated cell entry (Fig. 1A, B). Next, to determine whether elastase acts on viral particles or host cells, we treated cells with elastase before or after pseudotyped virus inoculation, which did not facilitate the entry of the virus. In contrast, elastase treatment of the viral particles facilitated RaTG13 and Khosta-2 S protein-mediated entry (Fig. 1C, D). The effect of elastase on RaTG13 and Khosta-2 virus particles was consistent with the results of a previous study that assessed the effect of trypsin treatment on Rs4874 and Rs7327 (clade 1a) or Rs4081 and Rs4237 (clade 2) virus particles, which belong to different clades21. These results suggested that elastase acted on viral particles rather than on host cells to facilitate cell entry mediated by bat SARS-like coronavirus S protein.
Elastase facilitates RaTG13 and Khosta-2 S protein-mediated cell entry Elastase, but not trypsin or thermolysin, facilitates cell entry of pseudotyped RaTG13 (A) and Khosta-2 (B) viruses. Particles bearing S proteins were preincubated for 5 min at room temperature with or without each protease and then added to VeroE6/TMPRSS2 cells. S protein-mediated cell entry was analyzed by measuring the activity of virus-encoded luciferase in the cell lysate at 3 days post inoculation. Experiments were independently repeated three times, and similar results were observed. Data from a representative experiment are shown (mean ± SD). Statistical analysis was performed using one-way ANOVA and subsequent Dunnett’s test. **** indicates p < 0.0001, ns: no significance. (C, D) Elastase treatment of viral particles but not target cells facilitates entry, nor does it facilitate post inoculation. VeroE6/TMPRSS2 cells or pseudotyped particles bearing RaTG13 (panel C) or Khosta-2 (panel D) S proteins were treated with elastase (final concentration: 50 µg/mL). In the “Pre” condition, cells were treated with elastase for 5 min at room temperature before virus inoculation. In the “Simultaneous” condition, pseudotyped virus particles were pretreated with elastase and immediately added to the cells. In the “Post” condition, cells were first inoculated with virus and then treated with elastase 24 h later under the same conditions. S protein-mediated cell entry was analyzed as described in panels (A, B). Experiments were independently repeated three times, and similar results were observed. Data from a representative experiment are shown (mean ± SD). Statistical analysis was performed using the Student’s t-test or Welch’s t-test. * indicates p < 0.05, **** indicates p < 0.0001, ns: no significance. Trypsin and elastase treatments facilitate cell entry of pseudotyped SARS-CoV-2, as shown in panels (E) and (F), respectively. Protease treatments were performed in the same manner as described in panels (A and B). Experiments were independently repeated twice, and similar results were observed. Data from a representative experiment are shown (mean ± SD). Statistical analysis was performed using one-way ANOVA and subsequent Dunnett’s test. * indicates p < 0.05, ** indicates p < 0.01, **** indicates p < 0.0001, ns: no significance. (G) Elastase treatment has no effect on VSV-G-mediated cell entry. Particles bearing the VSV-G were pre-incubated for 5 min with or without elastase and then added to VeroE6/TMPRSS2 cells. The experiments were independently repeated twice, and similar results were observed. Data from a representative experiment are shown (mean ± SD). Statistical analysis was performed using one-way ANOVA and subsequent Dunnett’s test. * indicates p < 0.05, **** indicates p < 0.0001, ns: no significance.
Given that trypsin is known to facilitate SARS-CoV-2 S protein-dependent cell entry15, we investigated whether the entry-enhancing effect of elastase is unique to bat SARS-like CoVs. For this, we assessed S protein-dependent cell entry using pseudotyped SARS-CoV-2 treated with each protease. Both trypsin and elastase facilitated SARS-CoV-2 S protein-mediated cell entry by approximately two-fold (Fig. 1E, F). These findings indicate that the protease-mediated enhancement is not specific to bat-derived spike proteins, although the magnitude of the effect depends on the specific S protein. RaTG13 and Khosta-2 S protein-mediated entry was assessed at reduced fetal bovine serum (FBS), concentrations (1 and 2%), but trypsin and thermolysin did not mediate entry into cells (Fig. S1A and B). Finally, we confirmed that this enhancement was specific to bat SARS-like coronavirus S proteins, as elastase treatment significantly and dose-dependently decreased the entry efficiency of vesicular stomatitis virus G (VSV-G) pseudotyped viruses (Fig. 1G).
Elastase cleaved the RaTG13 S protein
A distinctive feature of the SARS-CoV-2 S protein is the insertion of a four-amino acid (PRRA) sequence at the junction between the S1/S2 sites, which sensitizes the S protein to furin-mediated cleavage2. In contrast, the TMPRSS2 cleavage site was conserved in both RaTG13 and Khosta-2 S proteins, but these proteins lacked furin-sensitized insertions at the S1/S2 junction (Fig. 2A). To investigate the potential role of elastase in the cleavage of bat S proteins to facilitate cell entry, we examined whether trypsin, thermolysin, or elastase induced cleavage. Since SARS-CoV-2 spike antibodies failed to detect the Khosta-2 S protein due to the low amino acid sequence similarity between the SARS-CoV-2 and Khosta-2 S proteins, we analyzed the cleavage pattern of the RaTG13 S protein in pseudotyped viruses.
Elastase cleaves the RaTG13 S protein at the S2 and S2′ site. (A) Alignment of the S1/S2 loop and the adjacent to the S2′ region sequences of SARS-CoV-2, RaTG13 and Khosta-2 S proteins. (B, C) Cleavage of S proteins by three proteases. Particles pseudotyped with and RaTG13 (panel B) and SARS-CoV-2 (panel C) S protein were incubated with the highest tested concentrations of three proteases for 5 min at 37 ℃ and S protein expression analyzed by immunoblot with SARS-CoV-2 spike antibody. The experiments were independently repeated twice, and similar results were observed. The spike antibodies detected bands corresponding to the uncleaved S protein (S0), the S2 subunit (S2), and S2 subunit cleaved at the S2′ site (S2′) and additional S2 cleavage fragments (S2*). Original blots are presented in Fig. S9.
The pseudotyped virus was concentrated, and the RaTG13 S protein was incubated with trypsin, thermolysin, and elastase and then analyzed by immunoblotting. Trypsin treatment reduced the uncleaved S0 protein signal and produced a small S2-derived fragment. In contrast, the elastase treatment reduced the S0 protein signal and increased the signal corresponding to the S2 subunit. Thermolysin treatment did not result in a large degree of cleavage, as indicated by the prominent signal for the S0 protein (Fig. 2B, top). In SARS-CoV-2, the S1/S2 cleavage site can be cleaved by host furin and trypsin. Moreover, trypsin has been shown to cleave the S protein at the S1/S2 site10,15. We investigated whether the RaTG13 S protein is cleaved at the S1/S2 site by trypsin, thermolysin, or elastase using a Cleaved SARS-CoV-2 S protein (Ser686) antibody, which recognizes the spike protein only when it is cleaved at the S1/S2 cleavage site (at Ser686). Trypsin treatment detected a signal corresponding to the S2 subunit cleaved at the S1/S2 cleavage site, whereas thermolysin and elastase did not produce any detectable signals, indicating that elastase did not cleave the S1/S2 cleavage site (Fig. 2B, middle). Although the cleaved-S2 antibody (anti-Ser686) did not detect any signal after elastase treatment, the 1A9 antibody detected an S2 fragment of similar size, suggesting that elastase cleaves near the S1/S2 boundary but not exactly at R685/S686. The proteolytic cleavage event within the S2 subunit of the SARS-CoV S protein is responsible for subsequent membrane fusion between the viral and host membranes11,22, and this cleavage is mediated by TMPRSS223,24. Additional cleavage at the S2′ site is critical for generating the fusion peptide during the entry of the virus. Therefore, we examined the cleavage of the S2 and S2′ fragments of the RaTG13 S protein by these proteases. Trypsin treatment resulted in a small S2-derived fragment of approximately 48 kDa, while elastase treatment produced bands corresponding to S2 and S2′ fragments in the 75–100 kDa range. In contrast, the thermolysin treatment primarily yielded a prominent signal for the uncleaved S0 protein (Fig. 2B, bottom). Next, we analyzed the protease-dependent cleavage pattern of the SARS-CoV-2 S protein to compare it with the RaTG13 S protein. Consistent with previous reports21, the SARS-CoV-2 S protein was substantially cleaved even in the absence of exogenous protease treatment, as evidenced by a clear increase in the signal corresponding to the S2 subunit (Fig. 2C, top). Furthermore, in the absence of protease treatment, a distinct S2 band was detected using a Cleaved SARS-CoV-2 S protein (Ser686) antibody, confirming efficient cleavage at the S1/S2 site due to the presence of a furin cleavage site (Fig. 2C, middle). Similar to the RaTG13 S protein, trypsin treatment led to the appearance of a small S2-derived fragment of approximately 48 kDa. In contrast, thermolysin and elastase treatment did not produce any detectable bands (Fig. 2C, bottom). These findings suggest that elastase-mediated proteolytic cleavage generates an S2′ fragment that may facilitate RaTG13 S protein-mediated cell entry.
Elastase facilitated cell–cell fusion mediated by RaTG13 and Khosta-2 S proteins
RaTG13 S protein was cleaved by both trypsin and elastase; however, the cleavage sites differed, as evidenced by the generation of distinct S2-derived proteolytic fragments To determine whether these differences in cleavage sites affected spike protein function, we assessed cell–cell fusion between S protein-expressing 293 T cells pretreated with each protease and ACE2-expressing VeroE6/TMPRSS2 cells (Fig. 3A). Elastase treatment promoted cell–cell fusion mediated by RaTG13 and Khosta-2 S proteins, whereas trypsin and thermolysin did not (Fig. 3B, C). This is similar to the findings of a previous study that papain treatment of SARS-CoV and Rs4081 S proteins generates multiple small S2-derived fragments (≤ 50 kDa) but does not enhance S protein-mediated cell entry in pseudotyped virus assays21. Together, our findings suggest that elastase-mediated proteolytic cleavage within the S2 subunit may trigger membrane fusion between the viral and host membranes, thereby facilitating the entry of the virus into cells.
Elastase facilitates RaTG13 and Khosta-2 S protein-mediated cell–cell fusion (A) Schematics of the cell–cell fusion assay are presented in the panel (A). (B) Spike- and EGFP-transfected HEK293 T cells were suspended, pre-incubated for 5 min with each protease (final concentration: 100 µg/mL), and overlaid onto a monolayer of VeroE6/TMPRSS2 cells. After 3 h, the GFP-positive fused cells were photographed (panel B). Scale bar = 100 μm. (C) The green signal area of GFP-positive fused cells, as in (B), was quantified using ImageJ, and the results are shown as a bar graph. The experiments were independently repeated twice, and similar results were observed. Data from a representative experiment are shown (n = 5). Statistical analysis was performed using one-way ANOVA and subsequent Dunnett’s test. *** indicates p < 0.001, **** indicates p < 0.0001, ns: no significance.
Elastase did not facilitate RaTG13 and Khosta-2 S protein-mediated cell entry in Vero cells
RaTG13 and Khosta-2 spikes did not contain the PRRA sequence at the S1/S2 site; however, the cleavage site of TMPRSS2 was conserved (Fig. 2A). Notably, previous study has shown that pseudotyped viruses carrying the RaTG13 spike with an inserted PRRA sequence exhibit a significant loss of infectivity via pangolin and horseshoe bat ACE225. Factors other than PRRA insertion have also been implicated in the efficient cleavage of the S protein. In SARS-CoV-2, TMPRSS2 facilitates cell–cell fusion in the absence of furin-mediated cleavage at the S1/S2 site, and high TMPRSS2 expression is required to cleave S2′ and activate the fusion mechanism for efficient entry26,27. To assess the role of TMPRSS2 in elastase-mediated enhancement of viral infectivity, we examined the entry efficiency of RaTG13 and Khosta-2 pseudotyped viruses into Vero cells, which have low expression of TMPRSS2. In contrast to the results observed in VeroE6/TMPRSS2 cells, elastase treatment did not enhance RaTG13- or Khosta-2 S protein-mediated entry into Vero cells (Fig. 4A, B). These results suggest that TMPRSS2 plays an essential role in the elastase-mediated enhancement of infectivity of pseudotyped bat SARS-like CoVs.
Elastase does not facilitate RaTG13 and Khosta-2 S protein-mediated cell entry in Vero cells Elastase does not mediate cell entry of pseudotyped RaTG13 (A) and Khosta-2 (B) viruses in Vero cells. Pseudotyped viruses were pre-incubated for 5 min with or without trypsin or elastase and then added to Vero cells. S protein-mediated cell entry was analyzed by measuring the activity of virus-encoded luciferase in the cell lysate at 3 days post inoculation. The experiments were independently repeated three times, and similar results were observed. Data from a representative experiment are shown (mean ± SD). Statistical analysis was performed using one-way ANOVA and subsequent Dunnett’s test. ns: no significance.
Elastase and TMPRSS2 synergistically facilitated RaTG13 and Khosta-2 S protein-mediated syncytium formation and cell–cell fusion
In CoVs, cell entry is the first and critical step for successful viral infection and can be divided into two sequential steps: cell binding and membrane fusion, with membrane fusion relying on mechanical factors, such as host proteases9,28. Many studies have reported that host proteases promote membrane fusion13,14,15,29,30. However, the synergistic effects of host proteases on bat SARS-like coronavirus S protein-mediated cell entry remain unclear. Further, we investigated whether the presence of elastase and TMPRSS2 had a synergistic effect on the function of the bat SARS-like coronavirus spike. We treated RaTG13- or Khosta-2 S protein-expressing Vero cells and VeroE6/TMPRSS2 cells with elastase and then assessed S protein-mediated syncytium formation and cell–cell fusion. Interestingly, RaTG13- and Khosta-2 S protein-mediated syncytium formation was facilitated by elastase treatment in VeroE6/TMPRSS2 cells, but not in Vero cells (Fig. 5A, B). Furthermore, we investigated cell–cell fusion using the split green fluorescent protein (GFP) technique31,32. When GFP 1–10- and S protein-expressing HEK293T cells were fused with GFP11-, ACE2-, and TMPRSS2-expressing HEK293T cells, the GFP signal was restored in the fused cells. Similar to the results for syncytium formation, neither elastase nor TMPRSS2 alone facilitated cell–cell fusion, whereas the presence of both elastase and TMPRSS2 facilitated cell–cell fusion (Fig. 5C, D). Next, we investigated whether elastase can function as a trigger for membrane fusion. Cells expressing the S protein were overlaid with cells co-expressing ACE2 and TMPRSS2, followed by post-overlay treatment with elastase. RaTG13 and Khosta-2 S protein-mediated cell–cell fusion was enhanced even when elastase was applied after the overlay. Notably, this fusion activity was further increased in the presence of TMPRSS2 (Fig. S2 A and B). To elucidate the functional roles of elastase and TMPRSS2 in membrane fusion, we performed a cell–cell fusion assay using trypsin or elastase alone, as well as sequential treatment with both proteases. Trypsin was used as a surrogate for TMPRSS2. Fusion activity was higher with elastase alone than with trypsin alone. Sequential treatment with both proteases also resulted in greater fusion activity than trypsin alone; however, no clear difference was observed between the two treatment orders (Fig. S3A and B). Our results suggest that both elastase and TMPRSS2 are required for the fusogenic activity of bat SARS-like CoV S proteins.
Elastase and TMPRSS2 synergistically facilitate RaTG13 and Khosta-2 S protein-mediated syncytium formation and cell–cell fusion (A) Syncytium formation by elastase-treated RaTG13 or Khosta-2 S proteins. Each S protein was co-expressed with EGFP in Vero or VeroE6/TMPRSS2 cells. S protein-expressing cells were treated with elastase (final concentration: 100 µg/mL) for 5 min at 37℃, washed, and then observed by a fluorescent microscope 24 h later. Syncytium formation was visualized by the green signal from GFP. Large green cells indicate syncytium formation. Scale bar = 100 μm. (B) The green signal area of GFP-positive fused cells, as in A, were quantified using ImageJ, and the results are shown as a bar graph. The experiments were independently repeated three times, and similar results were observed. Data from a representative experiment are shown (n = 12–15). Statistical analysis was performed using Student’s t-test or Welch’s t-test. ** indicates p < 0.01, **** indicates p < 0.0001, ns: no significance. (C) Cell–cell fusion assay was performed using the split-GFP system. As effector cells, HEK293T cells were co-transfected with RaTG13 or Khosta-2 spike, split GFP 1–10 and DsRed. As target cells, HEK293T cells were co-transfected with ACE2, split GFP11 and DsRed. To investigate the effect of TMPRSS2, target cells were additionally transfected with TMPRSS2. After two days, effector cells were treated with elastase (final concentration: 100 µg/mL) for 30 min at 37 °C, washed and then the effector cell suspensions were mixed with target cells to generate fusion cells, and the reconstituted GFP signals were detected. After 24 h, the GFP-positive fused cells were photographed (panel C). (D) GFP-positive fused cells, as in (C), were quantified by flow cytometry. The experiments were independently repeated twice, and similar results were observed. Data from a representative experiment are shown (mean ± SD). Statistical analysis was performed using one-way ANOVA and subsequent Dunnett’s test. **** indicates p < 0.0001, ns: no significance.
Nafamostat and anti-ACE2 inhibited RaTG13 and Khosta-2 S protein-mediated cell entry
Our results indicate that TMPRSS2 is required for the elastase-mediated enhancement of bat SARS-like coronavirus infectivity. The TMPRSS2 inhibitor nafamostat mesylate (NM) inhibits SARS-CoV-2 S protein-mediated cell entry more efficiently than camostat mesylate and further blocks infection by authentic SARS-CoV-233,34. Therefore, we investigated the inhibitory effect of NM on elastase-mediated enhancement of bat SARS-like coronavirus infectivity. We used decanoyl-RVKR-chloromethylketone (CMK), a furin inhibitor, as a control. NM abolished the infectivity of RaTG13 and Khosta-2 at a low concentration of 0.1 μM, but CMK reduced the infectivity by about 60% at 10 μM (Fig. 6A, B). These results demonstrate that TMPRSS2 was required for elastase-mediated enhancement of the infectivity of bat SARS-like coronavirus, and suggest that NM treatment is also effective against cell entry of bat SARS-like coronavirus mediated by elastase.
Nafamostat and anti-ACE2 inhibits RaTG13 and Khosta-2 S protein-mediated cell entry by elastase NM efficiently inhibits RaTG13 (A) and Khosta-2 (B) S protein-mediated cell entry by elastase and TMPRSS2. Particles bearing S proteins were preincubated with elastase and CMK or NM at the indicated concentrations for 5 min at room temperature and then added to VeroE6/TMPRSS2 cells. Anti-ACE2 efficiently inhibit RaTG13 (C) and Khosta-2 (D) S protein-mediated cell entry by elastase and TMPRSS2. VeroE6/TMPRSS2 cells were preincubated with anti-ACE2 antibody for 1 h at 37 °C, and then the pseudotyped virus was preincubated with elastase (final concentration: 50 µg/mL) for 5 min at room temperature before being added to the VeroE6/TMPRSS2 cells. S protein-mediated cell entry was analyzed by measuring the activity of virus-encoded luciferase in the cell lysate at 3 days post inoculation. The experiments were independently repeated three times, and similar results were observed. Data from a representative experiment are shown (mean ± SD). Statistical analysis was performed using one-way ANOVA and subsequent Dunnett’s test. *** indicates p < 0.001, **** indicates p < 0.0001, ns: no significance.
RaTG13 and Khosta-2 S proteins facilitate entry into host cells by binding to hACE2 5,6,35,36, and hACE2 is 95% identical to the Africa green monkey ACE2 (Fig. S4A). Next, we investigated whether the promotion of cell entry by elastase and TMPRSS2 is inhibited by anti-ACE2. We incubated VeroE6/TMPRSS2 cells, a cell line derived from African green monkeys, with an anti-human ACE2 antibody and subsequently infected the cells with pseudotyped viruses under elastase treatment. Blocking ACE2 efficiently suppressed RaTG13 and Khosta-2 S protein-mediated cell entry promoted by elastase and TMPRSS2 (Fig. 6C, D). Similar to previous studies examining ACE2 from humans and other species5,6,35,36, elastase-activated RaTG13 and Khosta-2 spike proteins were shown to enter host cells via ACE2.
Mutation of Ser809 impaired RaTG13 S protein-mediated cell entry by elastase
Our results indicated that elastase-mediated activation of bat CoVs RaTG13 and Khosta-2 requires a cleavage event adjacent to the S2′ region. The elastase cleavage site at S2′ position in SARS-CoV S protein has been identified as the residue threonine (Thr) 79514. We analyzed the amino acid sequences surrounding the S2′ region of the SARS-CoV-2 and RaTG13 S proteins and found that residue 809 (Ser-809) of the RaTG13 S protein likely corresponds to Thr-795 of the SARS-CoV S protein (Fig. 7A). To assess the functional significance of residue 809, we generated a mutant RaTG13 S protein (S809D) by substituting serine with aspartic acid (Asp). First, we analyzed the expression levels of the S proteins of these amino acid mutants and found that they were comparable between the S809D mutant and the wild-type (Fig. 7B). Next, we generated pseudotyped viruses using the S809D-RaTG13 S protein and investigated the effects of elastase on infectivity. As a result, the infectivity of the wild-type RaTG13 S protein was enhanced by elastase, whereas the enhancement of infectivity was impaired in S809D-RaTG13 S protein (Fig. 7C). Interestingly, there was no difference in infectivity enhancement between the wild-type and S813D mutant S proteins in SARS-CoV-2 (Fig. 7C). We further investigated whether elastase treatment affected the cleavage of these amino acid mutant S proteins. MLV-based pseudotyped viruses were concentrated and S809D-RaTG13 or wild-type RaTG13 S proteins were incubated with elastase and analyzed by immunoblotting. Elastase treatment led to a reduction in the signals for the S809D and wild-type S0 proteins, whereas the signals corresponding to the S2 subunit (S2, S2′, S2*) were significantly reduced in the S809D mutant S protein (Fig. 7D, E). Consistent with a previous study21, the exception was the SARS-CoV-2 S protein, which was efficiently cleaved in the absence of elastase owing to the presence of a unique furin cleavage site. The ratio of S0 spike to S2 subunit (S2, S2′, S2*) signals was similar for S813D and wild-type SARS-CoV-2 S protein (Fig. S5A and B). Mutation of the Ser-809 residue in the bat coronavirus S protein reduced elastase-dependent proteolysis and host cell entry of RaTG13, although these processes were not completely inhibited, suggesting that this residue is involved in the elastase-dependent entry mechanism.
Mutation of Ser809 impairs RaTG13 S protein-mediated cell entry by elastase Elastase-enhanced cell entry is impaired in the S809D-RaTG13 S protein. (A) Alignment of the adjacent to the S2′ region sequences of SARS-CoV, SARS-CoV-2 and RaTG13 S proteins. (B) HEK293T cells were transfected with plasmids encoding the wild-type (WT)-RaTG13 S protein or the S809D mutant RaTG13 S protein. For comparison, HEK293T cells were transfected with WT-SARS-CoV-2 S protein or S813D mutant SARS-CoV-2 S protein. Two days after transfection, the cells were lysed using RIPA buffer, and the lysates were analyzed the expression levels of the RaTG13 S protein by Western blotting. The experiments were independently repeated twice, and similar results were observed. Original blots are presented in Fig. S10 (upper panel). (C) Pseudotyped viruses bearing WT-RaTG13 S protein or S809D mutant RaTG13 S protein were preincubated with elastase (final concentration: 50 µg/mL) for 5 min at room temperature and then added to VeroE6/TMPRSS cells. S protein-mediated cell entry was analyzed by measuring the activity of virus-encoded luciferase in the cell lysate at 3 days post inoculation. The experiments were independently repeated three times, and similar results were observed. Data from a representative experiment are shown (mean ± SD). Statistical analysis was performed using two-way ANOVA and subsequent Tukey’s test. * indicates p < 0.05, **** indicates p < 0.0001, ns: no significance. (D) Particles pseudotyped with WT-RaTG13 S protein or S809D mutant RaTG13 S protein were incubated with elastase (final concentration: 50 µg/mL) for 5 min at 37 ℃ and S protein expression analyzed by immunoblot with SARS-CoV-2 spike antibody. The spike antibody detected bands corresponding to the uncleaved S protein (S0), the S2 subunit (S2), and S2 subunit cleaved at the S2′ and S2* site (S2′, S2*). Original blots are presented in Fig. S10 (lower panel). (E) The S2 + S2′ + S2*/S0 ratio, as in (D), was quantified using ImageJ software. The experiments were independently repeated three times, and similar results were observed. Data from three experiments are shown (mean ± SD). Statistical analysis was performed using two-way ANOVA and subsequent Tukey’s test. ** indicates p < 0.01, *** indicates p < 0.001, ns: no significance.
Discussion
Previous studies have emphasized that processing of the viral S protein by host proteases plays a crucial role in crossing the species barrier7,8,19,37. It has been hypothesized that even bat CoVs lacking a furin-sensitive sequence can acquire the ability to infect humans if they are susceptible to cleavage by trypsin or other proteases38.
Here, we investigated host cell entry, S protein cleavage patterns, and membrane fusion of pseudotyped bat SARS-like CoVs and assessed whether mutations in the S2 subunit affect these characteristics. Elastase and TMPRSS2 synergistically facilitated RaTG13 and Khosta-2 S protein-mediated cell entry. In contrast, thermolysin did not enhance cell entry, consistent with the results of previous studies on SARS-CoV39. The incompatibility of host proteases with viral S proteins blocks the entry of the virus13,19,40. Our results also suggest that incompatibility of trypsin and thermolysin with RaTG13 and Khosta-2 S proteins does not facilitate cell entry. Thus, the availability of appropriate proteases can regulate the entry of bat CoVs into host cells.
Previous studies demonstrated that elastase cleaves the SARS-CoV S protein at specific sites within the S2 domain, enhancing viral infectivity. Notably, even when endosomal entry is blocked by bafilomycin, elastase treatment can rescue infection of SARS-CoV particles bound at the cell surface, indicating that elastase-mediated cleavage can enable membrane fusion and viral entry independently of the endosomal (cathepsin-dependent) pathway13,14. In addition, neutrophil elastase is predicted to cleave the S1/S2 site of SARS-CoV-2 not at S686, the furin cleavage site, but rather at A688 or S689 and that of SARS-CoV at the S66441. The elastase cleavage sites of SARS-CoV-2 S protein at A688 or S689 are identical to the A684 and S685 of RaTG13 S protein (Fig. S4B). Consistently, we showed that elastase cleaved the RaTG13 S protein at the S2 and S2′ portions detected at 100 to 75 kDa (Fig. 7D). Since anti-Ser686 antibody did not detect the S2 fragment of RaTG13 S protein at the S1/S2 cleavage site R685/S686 (Fig. 2B, middle), elastase-induced S2 proteolysis of RaTG13 S protein may not be identical to furin-induced cleavage. Regarding other proteases, trypsin and furin can cleave MERS-CoV and SARS-CoV-2 S proteins at the same site10,15,30,42 and trypsin cleaved the RaTG13 S protein at the S1/S2 cleavage site (at R685/S686 in SARS-CoV-2), whereas trypsin also caused much small fragment derived from S2. Most recent study reported that elastase induces S2 fragment of another bat SARS-related coronavirus Rs4081 S protein and enhances pseudotyped virus infection, while papain treatment generated multiple small S2-derived fragments of SARS-CoV and Rs4081 S proteins and did not enhance S protein-mediated cell entry in pseudotyped virus assays21. Analogously, we suppose that the trypsin-mediated cleavage of the RaTG13 S protein may be excessively extensive, resulting in its inactivation. Furthermore, we generate 3D structural model of SARS-CoV, Khosta-2 and Rs4081 S proteins with AlphaFold server43, and a structural comparison of the S proteins of suggests that their putative S1/S2 cleavage sites look a similar conformation and may be susceptible to elastase (Fig. S6).
In addition, our findings indicate that elastase may play a dual role in promoting spike-dependent membrane fusion. The enhancement of RaTG13- and Khosta-2 S protein-mediated cell–cell fusion following post-overlay elastase treatment suggests that elastase may function not only as a priming protease that converts the spike into a fusion-competent form, but also as a triggering protease that cleaves the spike after ACE2 engagement to directly initiate membrane fusion (Fig. S2). To further elucidate the respective roles of elastase and TMPRSS2 in spike-dependent membrane fusion, we performed comparative experiments using trypsin as a surrogate for TMPRSS2. Fusion activity was higher with elastase alone than with trypsin alone for both RaTG13 and Khosta-2 spikes, suggesting that elastase may act as a more efficient fusion trigger than TMPRSS2. However, under sequential treatment with both proteases, we were unable to determine whether cleavage by either protease in a specific order plays a decisive role in triggering fusion (Fig. S3). Given that trypsin possesses distinct biochemical properties, structural features, and physiological context compared to TMPRSS2, it may not fully replicate the spatiotemporal regulation required for TMPRSS2-dependent spike proteolytic activation. Therefore, these results should be interpreted with caution.
The S2′ cleavage site at R815/S816 of SARS-CoV-2 generates functional fusion peptide element and is required for S protein-mediated syncytium formation44. Elastase-1 prefers Pro residue at P2 position, and Thr-795 of the SARS-CoV S protein S2′ region is a predicated elastase cleavage site important for elastase-triggered infectivity and T795D mutation abrogated elastase-enhanced infectivity of SARS-CoV S protein14. Our findings highlight the importance of residue Ser-809 of the RaTG13 S protein, which is located at a position similar to Thr-795 of the SARS-CoV S protein (Fig. S7). The cell entry activity and S2 proteolysis of the wild-type RaTG13 S protein by elastase was impaired in the S809D-RaTG13 S protein. Combined with the findings of previous studies, our observations suggest that elastase with ability to generate S2′ fragments can perform subsequent membrane fusion abilities. Moreover, TMPRSS2 inhibitor assay, syncytium formation assays and cell–cell fusion assays demonstrated that elastase-mediated cell entry of RaTG13 and Khosta-2 requires TMPRSS2, emphasizing the synergistic action between elastase and TMPRSS2. However, it has also been reported that elastase treatment reduces SARS-CoV-2 infectivity by degrading both the viral S protein and ACE245,46. Using VeroE6/TMPRSS2 cells and an MLV-based pseudotyped virus, we found that elastase treatment increased SARS-CoV-2 S protein-mediated infection efficiency by approximately two-fold; however, the enhancement was small, and SARS-CoV-2 S proteins showed little sensitivity to elastase (Figs. 1F and 7C, and S8). These findings suggest that the effect of elastase on viral infection, whether inhibitory or enhancing, depends on the specific S protein type, the balance between non-specific degradation of the S protein and ACE2, and proper activation of the S protein. If the nonspecific degradation activity of elastase is excessively strong, its inhibitory effect on infection may predominate.
The infectivity of bat CoVs in humans has been extensively studied, particularly in terms of the binding affinity between RBD of the bat S protein and hACE2, and RaTG13 and Khosta-2 S proteins can bind human ACE2 to enter host cells5,6,35,36. In this study, we verified that elastase-enhanced cell entry of RaTG13 and Khosta-2 S proteins employed ACE2 as a functional receptor. However, RBD-hACE2 affinity alone is insufficient to confer infectivity. For instance, the RaTG13 S protein binds hACE2 with relatively low efficiency and does not exhibit significant infectivity47. Structural analyses of the S proteins from RaTG13 and other bat CoVs have revealed that many prefusion conformations favor a more compact “closed” state, and the RBD adopts a “down” state, rendering it less accessible for ACE2 binding48,49,50. As protease-mediated cleavage is involved in the structural transitions and dynamics of the S protein51,52,53, our findings, suggest that elastase-mediated cleavage of the RaTG13 S protein, which possesses an S1/S2 site sequence that is less susceptible to furin cleavage, may increase structural instability. This destabilization by elastase may, in turn, promote further activation by TMPRSS2, thereby enhancing viral infectivity.
The role of elastase in enabling bat CoVs to enter host cells is significant for spillover as elastase is produced by inflammatory cells in the lungs during viral infection, which can potentially facilitate infection13. Elastase is produced in high concentrations by neutrophils in pulmonary inflammatory cells during viral infection54. Alpha-1 antitrypsin (AAT) deficiency, which is common in European and North American populations55,56, delays the inhibition of neutrophil elastase, consequently enhancing activation of the D614G S protein and facilitating host cell entry of the D614G SARS-CoV-2 variant57. Our findings suggest that bat CoVs may pose a spillover risk in conditions such as acute lung injury and AAT deficiency. In such cases, elastase inhibitors and AAT supplementation may be effective countermeasures.
Collectively, our study provides new insights into the potential mechanisms by which SARS-like bat CoVs adapt to host cells during interspecies transmission events under conditions where elastase is available. Our observations can be used to evaluate the zoonotic disease potential of bat CoVs and to prevent and control their spread of bat CoVs to humans.
Limitations of the study
In the present study, we demonstrated that elastase facilitates RaTG13 and Khosta-2 S protein-mediated cell entry by proteolytic activation of S proteins and subsequent enhancement of their membrane fusion ability. We used pseudotyped bat coronaviruses that mimic the entry process of the virus. Therefore, it is unclear whether elastase facilitates the replication process of bat CoVs, and the effects of elastase on authentic viruses need to be examined. Next, we observed that the MLV-based pseudovirus system had very low transduction efficiency in cell lines that endogenously express TMPRSS2, such as Calu-3 and Caco-2 cells (data not shown). Therefore, to fully evaluate the role of elastase under these conditions, further studies using an alternative pseudotyped virus system or authentic virus infection in these physiologically relevant cells are warranted. Furthermore, most experimental studies have used porcine pancreatic elastase but have considered its actual impact on humans. Although both neutrophil elastase and porcine elastase recognize valine and alanine at the P1 position, porcine elastase shows a higher preference for methionine and glycine at this position58. This needs to be verified using human neutrophil elastase.
Methods
Cell culture and plasmids
HEK293T (ATCC; CRL-3216), Vero (ATCC; CCL-81) and VeroE6/TMPRSS2 (JCRB; JCRB1819) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 7.5% (v/v) FBS and kanamycin (50 µg/mL) in 5% CO2 and 95% air at 37 °C. Trypsin, thermolysin, and elastase were purchased from FUJIFILM Wako Pure Chemical Corporation (Cat# 201–19181, 207–08333, 058–05361, respectively). These proteases were dissolved in DMEM supplemented with 7.5% (v/v) FBS and kanamycin at the indicated concentrations. The plasmid pUC57-2019-nCoV-S (human) containing synthetic cDNA for expressing the human codon-optimized SARS-CoV-2 S protein was purchased from GenScript (https://www.genscript.com) and cloned into the expression plasmid pcDNA3.1 (Table S1). The mutant spike cDNA was synthesized using GenScript59,60.
Pseudotyped RaTG13 and Khosta-2 production and purification
The retrovirus-based pseudotyped RaTG13 and Khosta-2 viruses were prepared as previously described61. Briefly, HEK293T cells were seeded in a T-25 flask at a density of 2 × 106 cells one day before transfection, and the next day, cells were co-transfected with RaTG13 or Khosta-2 spike-expressing plasmids containing phCMV-Gag-Pol 5349 and reporter pTG-Luc126 plasmids62 using the PEIpro® transfection reagent (Cat# 101000017, Polyplus Transfection, New York, NY). Cell supernatants containing pseudotyped viruses were collected three days after transfection, filtered using a 0.45-μm filter, and stored at − 80 °C.
Luciferase assay for pseudotyped virus infection
Vero and VeroE6/TMPRSS2 cells were seeded in 96-well white plates at a density of 2–2.5 × 104 cells and cultured for one day. After one day of culture, for three protease pretreatments, pseudotyped viruses were preincubated with medium containing each protease at the indicated concentration (DMEM containing 7.5% FBS) for 5 min at room temperature and then added to wells. After three days, the medium was removed. Cells were washed once with phosphate buffered saline (PBS) and subsequently lysed using a luciferase assay reagent (PicaGene MelioraStar-LT Luminescence Reagent, TOYO B-NET Co., Ltd., Tokyo, Japan). Luminescence signals were measured using an EnVision multilabel plate reader (PerkinElmer, 2104–0020, Waltham, MA, USA). Transduction was performed in triplicate in each experiment, and reproducibility was confirmed by at least two independent biological experiments.
Immunoblotting
The supernatant containing pseudotyped viruses was mixed at a 3:1 (v/v) ratio with Retro-X™ Concentrator (Cat# 631455, Clontech) and incubated overnight at 4 °C. The next day, retroviral particles were concentrated by centrifugation at 1500×g for 45 min at 4 °C. Supernatants were removed, and pellets were resuspended in RIPA buffer (Cat# 16488-34, Nacalai Tesque Inc., Kyoto, Japan) and stored at − 80 °C until further use. To assess SARS-CoV-2 and RaTG13 S protein cleavage by the three proteases, the resuspended pellet and the residual volume were vortexed and divided equally into four different tubes. Trypsin, thermolysin, and elastase (50 µg/mL, 10 µg/mL, and 50 µg/mL, respectively) were added to the tubes, which were then incubated at 37 °C for 5 min. After incubation, the 2 × SDS-sample buffer was added and boiled for 10 min at 96 °C. This was followed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and immunoblotting. Immunoblotting was performed with the standard method using the following antibodies: anti-SARS-CoV-2 Spike (1A9, GTX632604, mouse monoclonal antibody, GeneTex), anti-SARS-CoV-2 Spike S2 (40590-T62, rabbit polyclonal antibody, Sino Biological), Cleaved SARS-CoV-2 Spike (Ser686) (84534, rabbit polyclonal antibody, Cell Signaling Technology), anti-GAPDH (3H12, M171–3, mouse monoclonal antibody, Medical & Biological Laboratories), goat anti-mouse IgG antibody conjugated with horseradish peroxidase (Cat# SA00001-1, Proteintech Group), donkey anti-rabbit IgG antibody conjugated with horseradish peroxidase (Cat# NA934, Cytiva). Immunoblot signals were developed using EzWestLumi plus (ATTO) and recorded using an ImageQuant LAS 4000mini image analyzer (GE Healthcare, Japan).
Cell–cell fusion assay
To evaluate the ability of the three proteases to facilitate bat coronavirus S protein-mediated cell–cell fusion, the assay was performed as previously described59. Briefly, HEK293T cells were seeded in a 12-well plate (standard F, Cat# 83.3921, SARSTEDT) at a density of 2 × 105 cells and cultured for one day. They were then co-transfected with enhanced green fluorescent protein (EGFP) and RaTG13 or Khosta-2 S protein-expressing plasmids. Cells were collected 48 h after transfection and treated with these proteases at a final concentration of 100 μg/mL at 37 °C for 5 min. Thereafter, the cells were layered on VeroE6/TMPRSS2 cells and co-cultured at 37 °C for 3 h. After incubation, five fields were randomly selected from each well and images were captured using a fluorescence microscope (BZ-9000 microscope, Keyence, Osaka, Japan). Images were analyzed using ImageJ software (Rasband, W.S., ImageJ, U.S. National Institutes of Health, Bethesda, MD, USA) to quantify the GFP area. HEK293T cells co-transfected with split GFP1-10 and RaTG13 or Khosta-2 S protein-expressing plasmids were used as effector cells. Target cells were HEK293T cells co-transfected with split GFP11, TMPRSS2, and hACE2-expressing plasmids. After 48 h of transfection, three experimental conditions were applied. In the first condition, to assess whether cell–cell fusion depends on elastase and TMPRSS2, effector cells were treated with elastase at a final concentration of 100 μg/mL at 37 °C for 30 min prior to overlay. After treatment, the cells were washed with PBS and co-cultured with target cells for 24 h. In the second condition, to determine whether elastase functions as a fusion trigger, effector and target cells were overlaid and co-cultured for 3 h, followed by treatment with elastase (100 μg/mL, 37 °C, 3 min). Elastase was then removed, the medium was replaced, and the cells were co-cultured for a total of 24 h following the initiation of the overlay. In the third condition, the roles of TMPRSS2 and elastase—specifically as priming or triggering proteases—were evaluated. Trypsin was used to mimic the function of TMPRSS210. Effector cells were sequentially treated with trypsin followed by elastase or elastase followed by trypsin (each at 100 μg/mL, 37 °C, 5 min) prior to overlay. After PBS washing, the cells were overlaid onto target cells and co-cultured for 24 h. GFP-positive fused cells were quantified by flow cytometry (FACSLyric™, BD Biosciences, Osaka, Japan). The flow cytometry data were analyzed using FlowJo version 10 (TreeStar, Ashland, OR, USA).
Syncytium formation assay
RaTG13 and Khosta-2 S protein-mediated syncytium formation assays were performed as previously described60,61. Briefly, Vero and VeroE6/TMPRSS2 cells were seeded in a 24-well plate (standard F, Cat# 83.3922, SARSTEDT) at a density of 4 × 104 cells/well and co-transfected with RaTG13 or Khosta-2 spike-expressing plasmids containing EGFP after one day of culture. One day after transfection, the cells were treated with elastase (100 µg/mL) for 5 min at 37 °C. After washing with PBS and incubating for 24 h, the cells were observed under a BZ-9000 microscope. The percentage of GFP-positive fused cells was quantified using ImageJ software. At least 10 fields per well were randomly selected for each experiment, and reproducibility was confirmed in three independent biological experiments.
Inhibition assay by protease inhibitors and anti-ACE2 antibody
VeroE6/TMPRSS2 cells were seeded in a 96-well white plate at a density of 2–2.5 × 104 cells and cultured for one day. To test protease inhibitors, pseudotyped viruses were preincubated with medium (DMEM with 7.5% FBS and 50 µg/mL elastase) containing the indicated concentrations of each protease inhibitor (Nafamostat Mesylate: Cat# N0959, Tokyo Chemical Industry Co., Ltd., Tokyo, Japan, Decanoyl-RVKR-CMK: Cat# 3501, Tocris Bioscience, Bristol, U.K) at room temperature for 5 min and then added to the wells. For the anti-ACE2 blocking assay, target cells were preincubated with 20 μg/mL anti-ACE2 antibody (Cat# AF933, goat, R&D Systems) at 37 °C for one hour before inoculating with pseudotyped viruses as described above63,64. After 3 days, luciferase assays were performed as described above. Cell entry assays were performed in triplicate for each experiment, and reproducibility was confirmed by three independent biological experiments.
Molecular graphics and analyses
Molecular graphics and structural analyses were conducted using UCSF ChimeraX, a software developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (https://www.rbvi.ucsf.edu/chimerax)65. The spike protein sequences analyzed included those from SARS-CoV (UniProt: P59594, SPIKE_SARS), Rs4081 (UniProt: A0A2D1PX44_SARS), RaTG13 (PDB: 7CN4), and Khosta-2 (UniProt: A0A8E6HRK4_9BETC). For SARS-CoV (UniProt: P59594, SPIKE_SARS), Rs4081 (UniProt: A0A2D1PX44_SARS), and Khosta-2 (UniProt: A0A8E6HRK4_9BETC), three-dimensional structural models were generated using the AlphaFold server (https://alphafoldserver.com)43, as experimentally determined PDB structures were not available for these proteins.
Statistical analyses
Statistical analyses were performed using GraphPad Prism software (version 9.4.1). Data are expressed as mean and standard deviation (SD). Comparisons of the means of the two groups were evaluated using the Student’s t-test or Welch’s t-test. Differences between the means of three or more groups were evaluated using one-way or two-way ANOVA. Multiple comparisons were performed using the Dunnett’s or Tukey’s multiple comparison tests. Differences between groups were considered significant if the p values were < 0.05.
Data availability
The data supporting the findings of this study are available in this published article and its supporting Information, and additional data can be obtained from the corresponding author upon reasonable request.
References
Memish, Z. A. et al. Middle east respiratory syndrome coronavirus in bats, Saudi Arabia. Emerg. Infect. Dis. 19, 1819–1823 (2013).
Andersen, K. G., Rambaut, A., Lipkin, W. I., Holmes, E. C. & Garry, R. F. The proximal origin of SARS-CoV-2. Nat. Med. 26, 450–452 (2020).
Li, W. et al. Bats are natural reservoirs of SARS-like coronaviruses. Science 310, 676–679 (2005).
Ruiz-Aravena, M. et al. Ecology, evolution and spillover of coronaviruses from bats. Nat. Rev. Microbiol. 20, 299–314 (2022).
Liu, K. et al. Binding and molecular basis of the bat coronavirus RaTG13 virus to ACE2 in humans and other species. Cell 184, 3438–3451 (2021).
Seifert, S. N. et al. An ACE2-dependent sarbecovirus in Russian bats is resistant to SARS-CoV-2 vaccines. PLoS Pathog. https://doi.org/10.1371/journal.ppat.1010828 (2022).
Letko, M., Marzi, A. & Munster, V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses. Nat. Microbiol. 5, 562–569 (2020).
Menachery, V. D. et al. Trypsin treatment unlocks barrier for zoonotic bat coronavirus infection. J. Virol. https://doi.org/10.1128/jvi.01774-19 (2020).
Millet, J. K. & Whittaker, G. R. Host cell proteases: Critical determinants of coronavirus tropism and pathogenesis. Virus Res. 202, 120–134 (2015).
Zabiegala, A., Kim, Y. & Chang, K. O. Roles of host proteases in the entry of SARS-CoV-2. Anim. Dis. https://doi.org/10.1186/s44149-023-00075-x (2023).
Belouzard, S., Chu, V. C. & Whittaker, G. R. Activation of the SARS coronavirus spike protein via sequential proteolytic cleavage at two distinct sites. Proc. Natl. Acad. Sci. U.S.A. 106, 5871–5876.
Luan, B., Huynh, T., Cheng, X., Lan, G. & Wang, H. R. Targeting proteases for treating COVID-19. J. Proteome Res. 19, 4316–4326 (2020).
Matsuyama, S., Ujike, M., Morikawa, S., Tashiro, M. & Taguchi, F. Protease-mediated enhancement of severe acute respiratory syndrome coronavirus infection. Proc. Natl. Acad. Sci. U.S.A. 102, 12543–12547 (2005).
Belouzard, S., Madu, I. & Whittaker, G. R. Elastase-mediated activation of the severe acute respiratory syndrome coronavirus spike protein at discrete sites within the S2 domain. J. Biol. Chem. 285, 22758–22763 (2010).
Kim, Y. et al. Trypsin enhances SARS-CoV-2 infection by facilitating viral entry. Arch. Virol. 167, 441–458 (2022).
Barlan, A. et al. Receptor variation and susceptibility to middle east respiratory syndrome coronavirus infection. J. Virol. 88, 4953–4961 (2014).
Zheng, Y. et al. Lysosomal proteases are a determinant of coronavirus tropism. J. Virol. https://doi.org/10.1128/jvi.01504-18 (2018).
Hou, Y. J. et al. SARS-CoV-2 reverse genetics reveals a variable infection gradient in the respiratory tract. Cell 182, 429–446 (2020).
Yang, Y. et al. Receptor usage and cell entry of bat coronavirus HKU4 provide insight into bat-to-human transmission of MERS coronavirus. Proc. Natl. Acad. Sci. U.S.A. 111, 12516–12521 (2014).
De Kreij, A. et al. The effect of changing the hydrophobic S1′subsite of thermolysin-like proteases on substrate specificity. Eur. J. Biochem. 268, 4985–4991 (2001).
Zhang, L. et al. ACE2-independent sarbecovirus cell entry can be supported by TMPRSS2-related enzymes and can reduce sensitivity to antibody-mediated neutralization. PLoS Pathog. https://doi.org/10.1371/journal.ppat.1012653 (2024).
Matsuyama, S. & Taguchi, F. Two-step conformational changes in a coronavirus envelope glycoprotein mediated by receptor binding and proteolysis. J. Virol. 83, 11133–11141 (2009).
Glowacka, I. et al. Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response. J. Virol. 85, 4122–4134 (2011).
Matsuyama, S. et al. Efficient activation of the severe acute respiratory syndrome coronavirus spike protein by the transmembrane protease TMPRSS2. J. Virol. 84, 12658–12664 (2010).
Liu, S. et al. The PRRA insert at the S1/S2 site modulates cellular tropism of SARS-CoV-2 and ACE2 usage by the closely related bat RaTG13. J. Virol. https://doi.org/10.1128/jvi.01751-20 (2021).
Essalmani, R. et al. Distinctive roles of furin and TMPRSS2 in SARS-CoV-2 infectivity. J. Virol. https://doi.org/10.1128/jvi.00128-22 (2022).
Winstone, H. et al. The polybasic cleavage site in SARS-CoV-2 spike modulates viral sensitivity to type I interferon and IFITM2. J. Virol. https://doi.org/10.1128/jvi.02422-20 (2021).
Cheng, Y. R., Li, X., Zhao, X. & Lin, H. Cell entry of animal coronaviruses. Viruses https://doi.org/10.3390/v13101977 (2021).
Koch, J. et al. TMPRSS2 expression dictates the entry route used by SARS-CoV-2 to infect host cells. EMBO J. https://doi.org/10.15252/embj.2021107821 (2021).
Hoffmann, M., Kleine-Weber, H. & Pöhlmann, S. A multibasic cleavage site in the spike protein of SARS-CoV-2 is essential for infection of human lung cells. Mol. Cell. 78, 779–784 (2020).
Kodaka, M. et al. A new cell-based assay to evaluate myogenesis in mouse myoblast C2C12 cells. Exp. Cell Res. 336, 171–181 (2015).
Feng, S. et al. Improved split fluorescent proteins for endogenous protein labeling. Nat. Commun. https://doi.org/10.1038/s41467-017-00494-8 (2017).
Hoffmann, M. et al. Nafamostat mesylate blocks activation of SARS-CoV-2: New treatment option for COVID-19. Antimicrob. Agents Chemother. https://doi.org/10.1128/aac.00754-20 (2020).
Li, K., Meyerholz, D. K., Bartlett, J. A. & McCray, P. B. The tmprss2 inhibitor nafamostat reduces sars-cov-2 pulmonary infection in mouse models of covid-19. MBio https://doi.org/10.1128/mbio.00970-21 (2021).
Roelle, S. M., Shukla, N., Pham, A. T., Bruchez, A. M. & Matreyek, K. A. Expanded ACE2 dependencies of diverse SARS-like coronavirus receptor binding domains. PLoS Biol. https://doi.org/10.1371/journal.pbio.3001738 (2022).
Starr, T. N. et al. ACE2 binding is an ancestral and evolvable trait of sarbecoviruses. Nature 603, 913–918 (2022).
Yang, Y. et al. Two mutations were critical for bat-to-human transmission of middle east respiratory syndrome coronavirus. J. Virol. 89, 9119–9123 (2015).
Barrett, C. T. et al. Effect of clinical isolate or cleavage site mutations in the SARS-CoV-2 spike protein on protein stability, cleavage, and cell–cell fusion. J. Biol. Chem. https://doi.org/10.1016/j.jbc.2021.100902 (2021).
Simmons, G. et al. Different host cell proteases activate the SARS-coronavirus spike-protein for cell-cell and virus-cell fusion. Virology 413, 265–274 (2011).
Kam, Y. W. et al. Cleavage of the SARS coronavirus spike glycoprotein by airway proteases enhances virus entry into human bronchial epithelial cells in vitro. PLoS ONE https://doi.org/10.1371/journal.pone.0007870 (2009).
Mustafa, Z., Zhanapiya, A., Kalbacher, H. & Burster, T. Neutrophil elastase and proteinase 3 cleavage sites are adjacent to the polybasic sequence within the proteolytic sensitive activation loop of the SARS-CoV-2 spike protein. ACS Omega 6, 7181–7185 (2021).
Jaimes, J. A., Millet, J. K. & Whittaker, G. R. Proteolytic cleavage of the SARS-CoV-2 spike protein and the role of the novel S1/S2 site. IScience. https://doi.org/10.1016/j.isci.2020.101212 (2020).
Abramson, J. et al. Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature 630, 493–500 (2024).
Yu, S. et al. SARS-CoV-2 spike engagement of ACE2 primes S2′site cleavage and fusion initiation. Proc. Natl. Acad. Sci. U.S.A. https://doi.org/10.1073/pnas.2111199119 (2022).
Leborgne, N. G. F. et al. Neutrophil proteases are protective against SARS-CoV-2 by degrading the spike protein and dampening virus-mediated inflammation. JCI Insight. https://doi.org/10.1172/jci.insight.174133 (2024).
Kummarapurugu, A. B. et al. Neutrophil elastase decreases SARS-CoV-2 spike protein binding to human bronchial epithelia by clipping ACE-2 ectodomain from the epithelial surface. J. Biol. Chem. https://doi.org/10.1016/j.jbc.2023.104820 (2023).
Zech, F. et al. Spike residue 403 affects binding of coronavirus spikes to human ACE2. Nat. Commun. https://doi.org/10.1038/s41467-021-27180-0 (2021).
Wrobel, A. G. et al. SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects. Nat. Struct. Mol. Biol. 27, 763–767 (2020).
Wrobel, A. G. et al. Structure and binding properties of Pangolin-CoV spike glycoprotein inform the evolution of SARS-CoV-2. Nat. Commun. https://doi.org/10.1038/s41467-021-21006-9 (2021).
Ou, X. et al. Host susceptibility and structural and immunological insight of S proteins of two SARS-CoV-2 closely related bat coronaviruses. Cell Discov. https://doi.org/10.1038/s41421-023-00581-9 (2023).
Zhang, L. et al. SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity. Nat. Commun. https://doi.org/10.1038/s41467-020-19808-4 (2020).
Gobeil, S. M. C. et al. D614G mutation alters SARS-CoV-2 spike conformation and enhances protease cleavage at the S1/S2 junction. Cell Rep. https://doi.org/10.1016/j.celrep.2020.108630 (2021).
Qiao, S. & Wang, X. Structural determinants of spike infectivity in bat SARS-like coronaviruses RsSHC014 and WIV1. J. Virol. https://doi.org/10.1128/jvi.00342-24 (2024).
Kawabata, K., Hagio, T. & Matsuoka, S. The role of neutrophil elastase in acute lung injury. Eur. J. Pharmacol. 451, 1–10 (2002).
Crowther, D. C. et al. Practical genetics: Alpha-1-antitrypsin deficiency and the serpinopathies. Eur. J. Hum. Genet. 12, 167–172 (2004).
de Serres, F. J., Blanco, I. & Fernández-Bustillo, E. Ethnic differences in alpha-1 antitrypsin deficiency in the United States of America. Ther. Adv. Respir. Dis. 4, 63–70 (2010).
Bhattacharyya, C. et al. SARS-CoV-2 mutation 614G creates an elastase cleavage site enhancing its spread in high AAT-deficient regions. Infect. Genet. Evol. https://doi.org/10.1016/j.meegid.2021.104760 (2021).
Boros, E. et al. Overlapping specificity of duplicated human pancreatic elastase 3 isoforms and archetypal porcine elastase 1provides clues to evolution of digestive enzymes. J. Biol. Chem. 292, 2690–2702 (2017).
Inoue, T. et al. Overcoming antibody-resistant SARS-CoV-2 variants with bispecific antibodies constructed using non-neutralizing antibodies. IScience. https://doi.org/10.1016/j.isci.2024.109363 (2024).
Yamamoto, Y. et al. SARS-CoV-2 spike protein mutation at cysteine-488 impairs its golgi localization and intracellular S1/S2 processing. Int. J. Mol. Sci. https://doi.org/10.3390/ijms232415834 (2022).
Murae, M. et al. The function of SARS-CoV-2 spike protein is impaired by disulfide-bond disruption with mutation at cysteine-488 and by thiol-reactive N-acetyl-cysteine and glutathione. Biochem. Biophys. Res. Commun. 597, 30–36 (2022).
Rafique, S., Idrees, M., Ali, A., Sahibzada, K. I. & Iqbal, M. Generation of infectious HCV pseudo typed particles and its utilization for studying the role of CD81 & SRBI receptors in HCV infection. Mol. Biol. Rep. 41, 3813–3819 (2014).
Hoffmann, M. et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181, 271–280 (2020).
Guo, H. et al. ACE2-independent bat sarbecovirus entry and replication in human and bat cells. MBio https://doi.org/10.1128/mbio.02566-22 (2022).
Meng, E. C. et al. UCSF ChimeraX: Tools for structure building and analysis. Protein Sci. https://doi.org/10.1002/pro.4792 (2023).
Acknowledgements
This study was supported by on-campus grant in TUS, funded by donation from “Account for Donations to Develop Vaccine and Medicine to Treat COVID-19” which was established by Sumitomo Mitsui Trust Bank, Limited, and research grant from the Japan Agency for Medical Research and Development (AMED), JSPS KAKENHI Grant (JP22K15284) to Y.Y. Molecular graphics and analyses performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases.
Author information
Authors and Affiliations
Contributions
Conceptualization, Y.Y.; Investigation, Y.Y., T.I., N.S., M.F., K.S., and K.N.; Resources, T.O., Y.T., T. W., and M.F.; Writing—original draft preparation, Y.Y. and K.N.; Writing—review and editing, Y.Y. and K.N.; Funding acquisition, Y.Y. and K.N.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
About this article
Cite this article
Yamamoto, Y., Inoue, T., Sugiyama, N. et al. Synergistic activation of bat SARS-like coronaviruses spike protein by elastase and TMPRSS2. Sci Rep 15, 26469 (2025). https://doi.org/10.1038/s41598-025-11600-y
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-025-11600-y
