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ACE2-independent sarbecovirus cell entry can be supported by TMPRSS2-related enzymes and can reduce sensitivity to antibody-mediated neutralization [1]

['Lu Zhang', 'Infection Biology Unit', 'German Primate Center Leibniz Institute For Primate Research', 'Göttingen', 'Faculty Of Biology', 'Psychology', 'Georg-August-University Göttingen', 'Hsiu-Hsin Cheng', 'Nadine Krüger', 'Platform Infection Models']

Date: 2024-12

The COVID-19 pandemic, caused by SARS-CoV-2, demonstrated that zoonotic transmission of animal sarbecoviruses threatens human health but the determinants of transmission are incompletely understood. Here, we show that most spike (S) proteins of horseshoe bat and Malayan pangolin sarbecoviruses employ ACE2 for entry, with human and raccoon dog ACE2 exhibiting broad receptor activity. The insertion of a multibasic cleavage site into the S proteins increased entry into human lung cells driven by most S proteins tested, suggesting that acquisition of a multibasic cleavage site might increase infectivity of diverse animal sarbecoviruses for the human respiratory tract. In contrast, two bat sarbecovirus S proteins drove cell entry in an ACE2-independent, trypsin-dependent fashion and several ACE2-dependent S proteins could switch to the ACE2-independent entry pathway when exposed to trypsin. Several TMPRSS2-related cellular proteases but not the insertion of a multibasic cleavage site into the S protein allowed for ACE2-independent entry in the absence of trypsin and may support viral spread in the respiratory tract. Finally, the pan-sarbecovirus antibody S2H97 enhanced cell entry driven by two S proteins and this effect was reversed by trypsin while trypsin protected entry driven by a third S protein from neutralization by S2H97. Similarly, plasma from quadruple vaccinated individuals neutralized entry driven by all S proteins studied, and availability of the ACE2-independent, trypsin-dependent pathway reduced neutralization sensitivity. In sum, our study reports a pathway for entry into human cells that is ACE2-independent, can be supported by TMPRSS2-related proteases and may be associated with antibody evasion.

Bats host a wide range of coronaviruses, including those related to SARS-CoV-1 and SARS-CoV-2 (subgenus Sarbecovirus), but their ability to infect human cells remains poorly understood. Identifying sarbecoviruses with zoonotic potential and understanding the factors controlling their entry into human cells are crucial for risk assessment and antiviral development. In this study, we examined how bat sarbecovirus spike proteins facilitate entry into human cells and identified key host factors involved. Using pseudovirus particles, we show that several bat sarbecovirus spike proteins use human and animal ACE2 receptors for entry. Additionally, we confirm that some spike proteins utilize an ACE2-independent entry pathway requiring trypsin treatment and demonstrate that this process is controlled by the spike protein receptor binding domain. Furthermore, we reveal that a subset of ACE2-using bat sarbecovirus spike proteins can switch to ACE2-independent entry following trypsin exposure and show that certain human type II transmembrane serine proteases can substitute for trypsin, enabling ACE2-independent entry. Finally, we demonstrate that repeated COVID-19 vaccination generates cross-neutralizing activity against bat sarbecoviruses, though ACE2-independent entry reduces neutralization sensitivity.

Competing interests: SP and MH conducted contract research (testing of vaccinee plasma for neutralizing activity against SARS-CoV-2) for Valneva unrelated to this work. GMNB served as advisor for Moderna and SP served as advisor for BioNTech, unrelated to this work. MSW. received funding from Sartorius AG (Göttingen, Germany) from GRIFOLS SA (Barcelona, Spain), Sphingotec (Henningsdorf, Germany), Inflammatix (Sunnyvale, CA, USA) and the German Research Foundation (Bonn, Germany) unrelated to this work. MSW is in the advisory board of Amomed (Wien, Austria) and Gilead Science Inc. (Foster City, CA, USA). All other authors declare no competing interests.

Funding: This work was supported by the European Union project UNDINE (101057100 to SP), the EU Hera project DURABLE (101102733 to CD), the COVID-19-Research Network Lower Saxony (COFONI) through funding from the Ministry of Science and Culture of Lower Saxony in Germany (14-76103-184, projects 7FF22, 6FF22, and 10FF22 to SP; project 4LZF23 to GMNB), the German Research Foundation (Deutsche Forschungsgemeinschaft, DFG; PO 716/11-1 to SP), the German Center for Infection Research (grant no 80018019238 to GMNB), the European Regional Development Funds Defeat Corona (ZW7-8515131 to GMNB) and Getting AIR (ZW7-85151373 to GMNB), the China Scholarship Council (202006270031 to LZ), the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung; 01KI2043 and NaFoUniMedCovid19-COVIM: 01KX2021 to H-MJ; project DZIF [8040701710 and 8064701703] to CD), the Bavarian State Ministry for Science and the Arts and DFG (through the research training groups RTG1660 and TRR130 to H-MJ), the Bayerische Forschungsstiftung (Project CORAd to H-MJ), and the Kastner Foundation (to H-MJ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Here, examining a panel of bat and pangolin sarbecovirus S proteins, we found that multiple S proteins utilized human ACE2 for entry and that, among animal ACE2 orthologues, raccoon dog ACE2 exhibited the broadest receptor activity. We confirm that certain S proteins mediate ACE2-independent, trypsin-dependent entry and that this process is controlled by the RBD. Furthermore, we found that expression of certain type II transmembrane serine proteases (TTSPs) in particle-producing cells, analogous to trypsin treatment, allowed for ACE2-independent entry into human cells. In addition, we discovered that antibodies from quadruple vaccinated individuals neutralized entry driven by all S proteins studied, suggesting that COVID-19 vaccines might also offer some protection against diverse animal sarbecoviruses. Finally, we obtained evidence that ACE2-independent, trypsin-dependent entry can modulate neutralization by the pan sarbecovirus antibody S2H97 in a spike-dependent fashion and allows for partial antibody evasion in the context of plasma from COVID-19 vaccines.

The subgenus Sarbecovirus within the genus Betacoronavirus contains a single species, severe acute respiratory syndrome-related coronavirus. This species comprises SARS-CoV-2 and more than 100 related viruses that have been identified in bats and pangolins. A subset of these viruses can use angiotensin-converting enzyme 2 (ACE2) for entry into human and animal cells [ 19 – 29 ]. However, it is not fully clear whether certain animal species can be identified as potential reservoirs or intermediate hosts for animal sarbecoviruses based on exceptionally broad receptor activity of their ACE2 orthologues. Recent studies provided evidence that the exposure of certain sarbecovirus S proteins to trypsin can facilitate ACE2-independent viral entry into human cells, a process that is determined by the receptor binding domain (RBD), and that equipping these S proteins with a multibasic cleavage site, a major virulence determinant of SARS-CoV-2 [ 16 , 30 ], is insufficient for trypsin-independent entry [ 21 , 31 – 33 ]. However, it has not been resolved whether cellular proteases other than trypsin can cleave and activate trypsin-dependent sarbecovirus S proteins for host cell entry. Finally, it is incompletely understood whether antibodies elicited by multiple COVID-19 vaccinations neutralize a broad spectrum of animal sarbecoviruses and it is unknown how usage of the ACE2-independent pathway impacts susceptibility to antibody-mediated neutralization.

Trimers of the coronavirus spike protein (S) are incorporated into the viral envelope and facilitate viral entry into target cells. For this, the surface unit, S1, of S protein monomers binds to cellular receptors, ACE2, in case of SARS-CoV-1 and SARS-CoV-2 [ 8 , 12 , 13 ], while the S2 subunit facilitates fusion of the viral and a target cell membrane, allowing delivery of the viral genetic information into the host cell cytoplasm, the site of coronavirus replication. Cleavage of the S protein by host cell proteases at the S1/S2 site (located at the S1/S2 interface) and the S2’ site (located within the S2 subunit) is essential for membrane fusion and can be facilitated by the lysosomal cysteine protease cathepsin L or the cell surface serine protease TMPRSS2 [ 12 , 14 ] with the latter being essential for lung cell infection and pathogenesis [ 14 – 17 ]. Finally, protease and receptor usage are major determinants of coronavirus cell and species tropism and are thus in the focus of many current research efforts [ 18 ].

The zoonotic transmission of animal coronaviruses of the genus Betacoronavirus to humans can present a major health threat. Thus, the transmission of SARS-CoV-1 from bats to humans via raccoon dogs and other intermediate hosts in 2002 resulted in the SARS epidemic that claimed roughly 800 lives [ 1 – 3 ]. In 2012, a new, severe respiratory disease, Middle East respiratory syndrome (MERS), emerged in Saudi Arabia and was found to be caused by a novel coronavirus, MERS-CoV, which is transmitted from dromedary camels to humans and causes fatal diseases in roughly 30% of the afflicted patients [ 4 , 5 ]. Finally, the emergence of SARS-CoV-2 in the human population in the winter season of 2019 in Hubei province, China, resulted in the COVID-19 pandemic that has claimed 18 million lives in the first two years alone [ 6 – 8 ]. Although emergence of SARS-CoV-2 from a research laboratory has been suggested, a constantly increasing amount of evidence indicates that the virus was transmitted from animals to humans, likely at the Huanan Seafood market, in Wuhan, China [ 9 – 11 ]. Thus, several betacoronaviruses from animals have zoonotic and pandemic potential and identifying which determinants control their ability to infect human cells will be instrumental for risk assessment and for devising antiviral strategies.

Results

The RBMs of RBD clade 2 and 3 sarbecovirus S proteins display major structural differences compared to RBD clade 1a and 1b RBMs due to sequence variations in two surface exposed loops The alignment of the amino acid sequence of 184 sarbecovirus S proteins revealed clustering into 5 clusters and 14 S proteins, representing all clusters except cluster 5, were selected for detailed analyses (Figs 1A and S1). Generally, sarbecoviruses are grouped into 4 different clades based on the presence and size of two loops within the RBD [34–36]. Viruses belonging to cluster 1 and 3 possess intact loop structures and are grouped into RBD clade 1 while viruses belonging to cluster 2, 4 or 5 exhibit shortened or missing loop structures and are assigned to clades 2, 3 and 4, respectively. Furthermore, clade 1 viruses are subdivided into clade 1a viruses (SARS-CoV-1-related viruses) and clade 1b viruses (SARS-CoV-2-related viruses). Structural studies had previously determined that the SARS-CoV-1 S (SARS-1-S) and SARS-CoV-2 S (SARS-2-S) receptor binding motifs (RBM), which are located within the RBD and make direct contact with ACE2, exhibit a similar structure [37]. The predicted structures of the RBDs of bat sarbecovirus clade 1 S proteins were similar among each other and comparable to that of the RBD of SARS-1-S, the prototypic clade 1a S protein (Figs 1B and S2). Similar findings were made for the structures of clade 1b RBDs, including the RBD of the SARS-2-S (Fig 1B). In contrast, loop 1 in the RBD was largely absent from clade 2, clade 3 and clade 4 S proteins (Figs 1B, 1C and S2) and some clade 2 S proteins contained a shortened loop 2 (Figs 1B, 1C and S2). Thus, the RBDs of the S proteins selected for analysis likely exhibit similar structures but two surface exposed loops are partially or largely absent from clade 2 and 3 S proteins, due to clade-specific sequence variations in the S gene, which may impact receptor interactions. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Alignment of S protein sequences and structural predictions. A) Phylogenetic analysis of human and animal sarbecoviruses. The sarbecoviruses were grouped into five clades, indicated by different colors, based on the full spike sequences. The sarbecoviruses functionally analyzed in the present study are indicated in grey boxes. (See S1 Fig for more details). B) Structure of RBD. The structure of RBDs was predicted based on homology modeling using SARS-2-S RBD as template. Two loops involved in ACE2 interactions are highlighted (See S2 Fig for more details), RBD-based clades are indicated. C) Schematic overview of the spike (S) protein domain structure (upper panel) and alignment of the RBM sequences of the S proteins analyzed in panel A. The ACE2 interacting residues of SARS-1-S and SARS-2-S are marked in blue (lower panel). “*” indicates conserved amino acid residues, “-”indicates gaps. The S proteins under study are indicated by circles. Abbreviations: NTD = N-terminal domain; RBD = receptor-binding domain; TD = transmembrane domain; S1/S2 and S2’ = cleavage sites in the S protein. https://doi.org/10.1371/journal.ppat.1012653.g001

The S proteins of clade 2 bat sarbecoviruses Rs4237 and Rs4081 mediate trypsin-dependent entry into human cells We next asked whether lack of proteolytic activation of the Rs4237 and Rs4081 S proteins was responsible for lack of cell entry. To address this possibility, we preincubated S protein-bearing particles with trypsin before addition to target cells. Trypsin treatment modulated S protein-driven entry in a cell line- and S protein-dependent manner. For most clade 1a S proteins, including the S proteins of Rs7327, Rs4231, RsSHC014, trypsin treatment either had no impact or increased entry efficiency (Fig 3A). For instance, entry of Rs4231 S protein into Calu-3 and Caco-2 cells was markedly increased by trypsin pre-treatment although this effect was not observed with 293T-ACE2 cells. For clade 1b S proteins of the pangolin sarbecoviruses cDNA8 and P5L trypsin treatment reduced entry efficiency or did not change entry efficiency for most cell lines except Calu-3, Calu-3-ACE2 and Caco-2 (Fig 3A). Interestingly, among the clade 2 S proteins, RS4237 and Rs4081, that were unable to mediate cell entry in the absence of trypsin, trypsin pre-treatment allowed for Rs4081 S protein-driven entry into all cell lines studied (Fig 3A) with bat-derived MyDauLu/47 cells being the only exception (S4B Fig). Similarly, trypsin pre-treatment allowed for Rs4237 S protein-driven entry into most cell lines studied, except for Calu-3 cell lines (Fig 3A) and most bat-derived cell lines studied (S4B Fig). In sum, availability of an appropriate protease can limit sarbecovirus entry into human cells and this limitation can be overcome by trypsin treatment, in keeping with published data [21,31,38,39]. We next investigated whether trypsin promoted viral entry by acting on viral particles or on target cells. For this, cells, particles or particles and cells were preincubated with trypsin followed by addition of a trypsin inhibitor and mixing of particles and cells. Treatment of target cells with trypsin had no effect on entry driven by VSV-G or any of the sarbecovirus S proteins studied (Fig 3B). In contrast, pretreatment of particles with trypsin allowed for entry driven by the Rs4081 and Rs4237 S proteins and augmented entry driven by Rs4874 and Rs7327 but not SARS-CoV-1 and SARS-CoV-2 S proteins (Fig 3B). Finally, augmentation of viral entry by trypsin treatment of particles was not further increased when both particles and target cells were preincubated with trypsin (Fig 3B), indicating that trypsin acts on viral particles rather than target cells to promote entry driven by a subgroup of sarbecovirus S proteins.

Trypsin-dependent cell entry driven by the S proteins of Rs4237 and Rs4081 is ACE2-independent The finding that trypsin-promoted entry driven by the Rs4081and Rs4237 S proteins did not correlate with ACE2 expression suggested that these S proteins might mediate entry in an ACE2-independent manner. In order to investigate this possibility, we mock treated or pre-treated particles with trypsin and employed them for infection of 293T WT cells, which express endogenous ACE2, or for infection of 293T KO-ACE2 cells, in which ACE2 expression was knocked-out via CRISPR/Cas9. Knock out of ACE2 abrogated entry driven by all S proteins studied and for clade 1b S proteins, including SARS-2-S, this defect was not rescued by trypsin treatment of viral particles (Fig 3C). In contrast, trypsin treatment of particles bearing several RBD clade 1a S proteins, including that from LYRa11, trypsin treatment allowed for entry into ACE2-KO cells (Fig 3C). Furthermore, clade 2 S proteins, Rs4081and Rs4237, failed to enter 293T WT and 293T ACE2-KO cells in the absence of trypsin but entered both cell lines in the presence of trypsin (Fig 3C) and similar results were obtained when trypsin-mediated evasion of an anti-ACE2 antibody was studied (S4C Fig). Collectively, these results demonstrate that Rs4081 and Rs4237 S protein engage a receptor other than ACE2 for host cell entry and that trypsin treatment can confer partial ACE2-independence to entry driven by other S proteins, including LYRa11, RsSHC014, Rs4231, Rs4874 and Rs7327.

Trypsin cleaves sarbecovirus S proteins We next investigated whether trypsin treatment resulted in S protein cleavage and how much trypsin was needed for S protein cleavage and S protein-driven entry. For analysis of cleavage, S protein bearing VSV particles were incubated with 0.5, 5 and 50 μg/ml of trypsin and then analyzed by immunoblot. All S proteins were largely uncleaved in the absence of trypsin, as documented by prominent signals for the uncleaved S0 protein, with exception of SARS-2-S, which was efficiently cleaved in the absence of trypsin due to the presence of a unique furin cleavage site (Fig 4A). The addition of trypsin led to the cleavage of all S proteins studied, as indicated by a reduction in signals for the S0 protein and an increase in signals corresponding to the S2 subunit (Fig 4A). For some of the S proteins additional signals were observed in the presence of 50 μg/ml trypsin, which likely corresponded to the S2’ fragment (produced upon cleavage of the S protein at the S2’ site) and cleavage products thereof (Fig 4A). Thus, all S proteins studied were cleaved by trypsin, although with different efficiencies, resulting in a concentration-dependent disappearance of S0 and appearance of the S2’ fragment and S2’ sub-fragments. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Trypsin cleaves the S proteins of diverse sarbecoviruses. A) Cleavage of S proteins by trypsin. Particles pseudotyped with the indicated S proteins were incubated with the indicated concentrations of trypsin for 30 min at 37°C and S protein expression analyzed by immunoblot with SARS-CoV-2 S2 antibody. VSV-M served as loading control. Similar results were obtained in two separate experiments. Bands corresponding to uncleaved S proteins (S0), the S2 subunit (S2), S2 subunit cleaved at the S2’ site (S2’) and additional S2 cleavage fragments (S2*) are indicated and were determined based on their respective molecular weight. B) Modulation of S protein driven entry by trypsin is concentration-dependent. Particles pseudotyped with the indicated S proteins were treated with the indicated concentrations of trypsin for 30 min at 37°C before addition to Vero cells. The efficiency of S protein-driven cell entry was determined by measuring the activity of virus-encoded firefly luciferase in cell lysates at 16-18h post inoculation. Results for S protein bearing particles were normalized against those obtained for particles bearing no S protein (set as 1). The average (mean) data of three biological replicates is presented, each performed with four technical replicates. Error bars show the SEM. Statistical significance was assessed by two-tailed Student’s t-tests (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). https://doi.org/10.1371/journal.ppat.1012653.g004 We next analyzed concentration-dependence of trypsin-dependent S protein-driven cell entry by pre-incubation of pseudotyped particles with increasing amounts of trypsin. For this, we chose 200 μg/ml trypsin as maximal concentration, considering that concentrations of roughly 150 μg/ml are present in the human intestine [40]. S proteins that did not exhibit augmented cell entry activity upon exposure to 50 μg/ml trypsin (Fig 3A), including SARS-1-S and SARS-2-S, were also not appreciably stimulated for augmented cell entry when a higher concentration of trypsin was used (Fig 4B). In contrast, S proteins that mediated increased entry upon exposure to 50 μg/ml trypsin, including RsSHC014 and RS7327 S proteins, were slightly more active in the presence of 200 μg/ml and this group included the clade 2 S proteins of Rs4081 and Rs4237, which allowed for cell entry only upon trypsin-treatment (Fig 4B). Importantly, trypsin-treatment did not increase the ability of the S proteins to bind to ACE2 (S5 Fig). Collectively, we found that 50 and 200 μg/ml trypsin robustly increased or allowed for cell entry activity of several animal sarbecovirus S proteins and these protease concentrations are likely attained in the intestine, which is believed to be a major target for sarbecovirus infection in bats [41–43]. On a more general level, our findings suggest that lack of proteolytic activation of the viral S protein might impede host cell entry of Rs4237 and Rs4081.

Thermolysin and elastase cleave Rs4081 S protein at the S1/S2 site and confer infectivity to Rs4081 S protein-bearing particles We next investigated whether secreted proteases other than trypsin can promote entry driven by the clade 2 S protein from Rs4081. For this, we first analyzed the effect of thermolysin, papain and elastase on cell entry. Thermolysin is a bacterial protease, while papain is a protease produced in plants, and thermolysin has been used previously to characterize coronavirus S proteins [44]. Elastase promotes inflammation and plays a role in several lung pathologies, likely including COVID-19 [45,46]. Immunoblot analyses revealed that trypsin, thermolysin and elastase cleaved both SARS-1-S and Rs4081-S at the S1/S2 site, resulting in production of the S2 fragment (Fig 5A). In contrast, papain digest of SARS-1-S and Rs4081-S resulted in several S2-derived fragments, suggesting multiple papain cleavage sites in the S2 subunit (Fig 5A). PPT PowerPoint slide

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TIFF original image Download: Fig 5. Elastase and type II transmembrane serine proteases can activate the otherwise trypsin-dependent Rs4081 S protein. A) Analysis S protein cleavage. Particles pseudotyped with SARS-1-S or Rs4081 S protein (or no S protein) were incubated with the indicated proteases (at highest concentration used for panel B, 20 min incubation) and S protein expression was analyzed by immunoblot using an antibody directed against the S2 subunit of SARS-2-S. VSV-M served as loading control. Similar results were obtained in two separate experiments. Bands corresponding to uncleaved S proteins (S0), the S2 subunit (S2) and the S2 subunit cleaved at the S2’ site (S2’) are indicated and were determined based on their respective molecular weight. B) Impact of proteases on cell entry. Particles pseudotyped with SARS-1-S or Rs4081 S protein were treated with the indicated concentrations of trypsin, thermolysin, papain or elastase for 30 min at 37°C before addition to Vero cells. The efficiency of S protein-driven cell entry was determined by measuring the activity of virus-encoded firefly luciferase in cell lysates at 16-18h post inoculation. Results for S protein bearing particles were normalized against those obtained for particles bearing no S protein (set as 1). The average (mean) data of three biological replicates are presented, each performed with four technical replicates. Error bars indicate SEM. Statistical significance was assessed by two-tailed Student’s t-tests (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). C) Expression of type II transmembrane serine proteases (TTSPs). 293T cells were transiently transfected with plasmids encoding the indicated proteases with a c-myc antigenic tag or empty plasmid and cell lysates were harvested at 48 h after transfection. Cell lysates were analyzed by immunoblot for protease expression using c-myc antibody. Detection of ACTB served as loading control. Similar results were obtained in two separate experiments. D) Expression of TTSPs on target cells does not allow for entry driven by the trypsin-dependent Rs4081 S protein. 293T cells transiently expressing the indicated TTSPs of furin were Mock treated or treated with ammonium chloride to block cathepsin L-dependent endo/lysosomal entry and inoculated with pseudotypes bearing SARS-1-S, Rs4081-S or VSV-G. Alternatively, particles were treated with trypsin (50 μg/ml for 30 min at 37°C) and added to mock treated cells. S-protein-driven cell entry was analyzed by and data presented as described for panel B. The average (mean) data of three biological replicates are presented, each performed with four technical replicates. Error bars show the SEM. Statistical significance was assessed by two-tailed Student’s t-tests (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). E) S protein cleavage by TTSPs. Particles pseudotyped with SARS-1-S or Rs4081 S proteins (or no S protein) were produced in 293T cells coexpressing the indicated TTSPs or furin. Alternatively, particles were treated with the indicated concentrations of trypsin for 30 min. S protein expression was analyzed by immunoblot using an antibody directed against the S2 subunit of SARS-2-S. VSV-M served as loading control. Similar results were obtained in two separate experiments. Bands corresponding to uncleaved S proteins (S0), the S2 subunit (S2) and the S2 subunit cleaved at the S2’ site (S2’) are indicated and were determined based on their respective molecular weight. F) Coexpression of TTSPs in particle producing cells can activate the Rs4081 S protein. Particles bearing SARS-1-S or Rs4081 S protein and produced in 293T cells expressing the indicated TTSPs or furin were added to Vero cells. S-protein-driven cell entry was analyzed by and data presented as described for panel B. The average (mean) data of three biological replicates are presented, each performed with four technical replicates. Error bars show the SEM. Statistical significance was assessed by two-tailed Student’s t-tests (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). https://doi.org/10.1371/journal.ppat.1012653.g005 Analyses of S protein pseudotyped particles revealed that none of the proteases tested augmented entry driven by SARS-1-S protein and trypsin and thermolysin treatment even reduced particle infectivity (Fig 5B). In contrast, trypsin, thermolysin and elastase allowed for cell entry driven by the Rs4081 S protein in a concentration-dependent manner while papain had no effect (Fig 5B). In sum, Rs4081 S protein can employ elastase, which is expressed in the lung by neutrophils and alveolar macrophages, instead of trypsin for entry into human cells.

TMPRSS11A, TMPRSS11D and TMPRSS11E cleave coexpressed Rs4081 S protein at the S1/S2 site and confer infectivity to Rs4081 S protein bearing particles TMPRSS2 and other TTSPs are expressed in the lung and/or gastrointestinal tract and cleave and activate diverse coronavirus S proteins [12,47,48]. Therefore, we examined whether directed expression of TMPRSS2, TMPRSS11A, TMPRSS11D, TMPRSS11E or TMPRSS13 results in S protein cleavage and promotes entry driven by the Rs4081 S protein. In addition, we analyzed the effect of the expression of furin, which cleaves SARS-2-S at the S1/S2 site in the constitutive secretory pathway of infected cells [14]. All proteases examined were efficiently expressed in transfected 293T cells (Fig 5C) and their expression in target cells rescued SARS-1-S but not VSV-G-driven entry from inhibition by ammonium chloride, as expected (Fig 5D). In contrast, protease expression in target cells did not allow for Rs4081 S protein-driven entry (Fig 5D). Therefore, we analyzed whether protease expression in particle-producing cells modulates S protein cleavage and particle infectivity. Expression of TMPRSS11A, TMPRSS11E and furin in SARS-1-S pseudotyped particles producing cells as well as trypsin-treatment slightly improved generation of the S2 fragment (which results from cleavage at the S1/S2 site) (Fig 5E, left panel). Further, TMPRSS11D expression strongly increased production of the S2 fragment and the S2’ fragment (which results from cleavage at the S2’ site) while TMPRSS2 and TMPRSS13 expression and trypsin treatment only augmented production of the S2’ fragment and decreased production of the S2 fragment (Fig 5E). Finally, similar findings were made for the Rs4081 S protein, although exposure to 5 and particularly 50 μg/ml trypsin resulted in processing of the S2’ fragment into smaller fragments (Fig 5E). Expression of TTSPs or furin in particle producing cells or trypsin treatment of particles did not augment cell entry driven by SARS-1-S (Fig 5F). In contrast, expression of TMPRSS11A in Rs4081-S particle producing cells increased particle infectivity with similar efficiency as trypsin treatment of particles (Fig 5F). Expression of TMPRSS11D and TMPRSS11E also augmented particle infectivity but with reduced efficiency as compared to TMPRSS11A while expression of TMPRSS2, TMPRSS13 and furin had no effect (Fig 5F). Thus, Rs4081 S protein is cleaved by TMPRSS11A, TMPRSS11D and TMPRSS11E at the S1/S2 site upon protease coexpression and cleavage confers infectivity to Rs4081 S protein-bearing particles.

Insertion of a multibasic cleavage site increases lung cell infection in a spike-specific fashion The SARS-CoV-2 S protein but none of the other S proteins studied harbors a multibasic cleavage site at the S1/S2 loop (Fig 6A). The S protein is cleaved at this site by furin and cleavage is essential for robust lung cell entry [14]. Therefore, we tested whether insertion of the multibasic cleavage site of SARS-2-S in the other S proteins analyzed here increased lung cell entry. The presence of a multibasic cleavage site was compatible with robust expression and particle incorporation of S proteins (Fig 6B) and resulted in efficient proteolytic processing of all S proteins studied (Fig 6B). Notably, the presence of a multibasic cleavage site invariably reduced entry into 293T-ACE2 cells (Fig 6C), which depends on the activity of the S protein activating endo/lysosomal protease cathepsin L. In contrast, the multibasic cleavage site either had no effect or, for the majority of S proteins tested, augmented entry into Calu-3-ACE2 lung cells, with enhancement of entry driven by the S proteins of SARS-CoV-2, RaTG13 and LYRa11 being particularly prominent (Fig 6C). Finally, the presence of a multibasic cleavage site was not sufficient to allow for trypsin-independent 293T-ACE2 or Calu-3-ACE2 cell entry driven by Rs4237 and Rs4081 S proteins (Fig 6C). Thus, a multibasic cleavage site may promote lung cell entry of diverse animal sarbecoviruses but fails to allow for cell entry driven by the S proteins of Rs4237 and Rs4081. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Insertion of a multibasic cleavage site into sarbecovirus S proteins universally increases lung cell entry but does not allow for trypsin-independent entry by RS4081 and Rs4237 S proteins. A) Alignment of the S1/S2 loop sequences of the indicated S proteins. Amino acid residues were color coded on the basis of biochemical properties. Asterisks indicate conserved residues. B) Analysis of S protein cleavage. Particles pseudotyped with the indicated S proteins were subjected to immunoblot analysis, using an antibody directed against the S2 subunit of SARS-2-S. Black and red indicate uncleaved precursor respective S (S0) and S2, respectively. Detection of VSV-M served as a loading control. Shown is a representative immunoblot from three independent experiments. C) Impact of the multibasic cleavage site on S protein-driven entry. Particles bearing the indicated S proteins (or no S protein) were added to 293T-ACE2 or Calu-3-ACE2 cells. The efficiency of S protein-driven cell entry was determined by measuring the activity of virus-encoded firefly luciferase in cell lysates at 16-18h post inoculation. Results for S protein bearing particles were normalized against those obtained for particles bearing no S protein (set as 1). Presented are the average (mean) data of three biological replicates, each performed with four technical replicates. Error bars indicate SEM. Statistical significance was assessed by two-tailed Student’s t-tests (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). https://doi.org/10.1371/journal.ppat.1012653.g006

The receptor binding domain is a determinant of trypsin-dependent entry of Rs4081 Our studies had so far revealed that Rs4081 and Rs4237 S proteins facilitated entry into human cells only upon pre-cleavage by trypsin or certain other soluble or membrane-bound proteases. However, which determinants in the S protein controlled trypsin-dependent entry was unclear. To address this question, we constructed chimeras between SARS-1-S, which facilitates entry in a trypsin-independent fashion, and the Rs4081 S protein, which facilitates entry in a trypsin-dependent fashion. Specifically, we exchanged the S1 subunit between these S proteins or the N-terminal domain (NTD), receptor binding domain (RBD), the domain harboring the S1/S2 and S2’ cleavage sites (priming domain, PD), or NTD jointly with RBD (Fig 7A and 7B). All chimeric S proteins were efficiently and comparably incorporated into VSV particles (Fig 7C). Introduction of the NTD or PD from Rs4081-S into SARS-1-S was compatible with robust entry into Vero and Caco-2 cells although entry driven by the S protein with PD from the Rs4081 S protein was reduced as compared to WT S protein, and trypsin did not increase entry efficiency (Fig 7D). In contrast, SARS-1-S chimeras harboring the S1 subunit, RBD or NTD+RBD of the Rs4081 S protein mediated entry only upon trypsin treatment. Trypsin-dependent entry mediated by SARS-1-S with the S1 subunit of Rs4081 spike was robust, although not as efficient as entry driven by WT SARS-1-S in the absence of trypsin, while trypsin-dependent entry driven by the SARS-1-S chimera harboring the Rs4081 RBD or NTD+RBD was inefficient (Fig 7D). Finally, the reverse observations were made for Rs4081 S protein harboring domains of SARS-1-S. Entry remained trypsin-dependent when the NTD or PD of the SARS-CoV-1 S protein were introduced into Rs4081 S protein while introduction of the S1 subunit, RBD or NTD+RBD allowed for trypsin-independent entry (Fig 7D). In sum, these results show that the RBD is a major determinant of trypsin-dependent entry but also suggest the domains outside the RBD might contribute to this phenotype. PPT PowerPoint slide

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TIFF original image Download: Fig 7. The RBD is the key determinant of trypsin-dependent entry. A) Overview of the chimeric SARS-1-S and Rs4081 S proteins analyzed. The sequences of the S1/S2 and S2’ cleavage sites are indicated, asterisk indicate conserved amino acids. B) The domains exchanged between SARS-1-S and Rs4081 S proteins are color coded in the context of the S protein monomer. C) Expression of chimeric S proteins. Particles pseudotyped with the indicated S protein were subjected to immunoblot analysis, using anti an antibody directed against the S2 subunit of SARS-2-S. Detection of VSV-M served as loading control. Similar results were obtained in two separate experiments. D) Cell entry of driven by chimeric S proteins. Particles bearing the indicated S proteins (or no S protein) were treated with trypsin (50 μg/ml for 30 min at 37°C) before addition to Vero or Caco-2 cells. The efficiency of S protein-driven cell entry was determined by measuring the activity of virus-encoded firefly luciferase in cell lysates at 16-18h post inoculation. Results for S protein bearing particles were normalized against those obtained for particles bearing no S protein (set as 1). Presented are the average (mean) data of three biological replicates, each performed with four technical replicates. Error bars indicate SEM. Statistical significance was assessed by two-tailed Student’s t-tests (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). https://doi.org/10.1371/journal.ppat.1012653.g007

Trypsin treatment can modulate sarbecovirus neutralization by antibody S2H97 The antibody S2H97 binds to a cryptic epitope within the RBD and recognizes the S proteins of sarbecoviruses from all clades [35]. The antibody neutralizes particles bearing the S proteins from diverse sarbecoviruses in cell culture and efficiently suppresses SARS-CoV-2 amplification in the lung of experimentally infected hamsters [35]. Thus, S2H97 and related antibodies could be useful for pandemic preparedness. We investigated whether S2H97 neutralizes particles bearing the S proteins analyzed here and determined whether trypsin treatment modulates neutralization sensitivity. We found that S2H97 neutralized particles bearing clade 1b S proteins in a concentration-dependent fashion and irrespective of whether the particles had been pretreated with trypsin while clade 2 S proteins mediated entry only in the presence of trypsin, as expected, and entry was inhibited by S2H97 (Fig 8). In contrast, S2H97 had differential effects on entry driven by clade 1a S proteins. Particles harboring SARS-1-S, Rs4231-S, WIV1-S or Rs4874-S were not efficiently neutralized, irrespective of the presence of trypsin. Entry driven by the S protein from LYRa11 and Rs7327 was augmented by S2H97 in the absence of trypsin while moderate but concentration-dependent neutralization was measured in the presence of trypsin (Fig 8). Finally, trypsin treatment protected particles bearing the RsSHC014 S protein from neutralization by S2H97. These results suggest that S2H97, and potentially related RBD antibodies, might efficiently neutralize clade 1b and 2 but not clade 1a sarbecoviruses and might even augment cell entry of the latter, suggesting limited suitability for pandemic preparedness. Furthermore, our findings indicate that trypsin treatment can alter susceptibility of sarbecoviruses to antibody-mediated neutralization. PPT PowerPoint slide

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TIFF original image Download: Fig 8. Trypsin treatment modulates sarbecovirus neutralization by the pan-sarbecovirus monoclonal antibody S2H97. Particles bearing the indicated S proteins were preincubated with or without trypsin (50 μg/ml for 30 min at 37°C) and subsequently trypsin inhibitor (200 μg/ml for 10 min at 37°C). Thereafter, the particles were incubated with different concentrations of the pan-sarbecovirus monoclonal antibody S2H97 (30 min at 37°C) before being added to Vero-ACE2-TMPRSS2 cells. S protein-driven cell entry was analyzed by measuring the activity of virus-encoded firefly luciferase in cell lysates at 16-18h post inoculation and normalized to entry in the absence of antibody. Presented are the average (mean) data of three biological replicates, each performed with four technical replicates. Error bars indicate SEM. Statistical significance was assessed by two-way analysis of variance with Sidak’s multiple comparisons test (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). https://doi.org/10.1371/journal.ppat.1012653.g008

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[1] Url: https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1012653

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