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

Abstract

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.

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.

Fig 2. Raccoon dog ACE2 supports entry driven by the S proteins of diverse sarbecoviruses.

A) Binding of soluble human ACE2 to S protein expressing cells. 293T cells transiently expressing the indicated S proteins (or no S protein) were first incubated with soluble ACE2 containing a C-terminal Fc-tag (derived from human immunoglobulin G; solACE2-Fc) and subsequently incubated with an AlexaFluor-488-coupled secondary antibody, before solACE2-Fc binding was analyzed by flow cytometry (see S3 Fig for details on the gating strategy). Presented are the average (mean) mean fluorescence intensity (MFI) data from five biological replicates (each conducted with single samples). Signals obtained from control transfected cells (no S protein expression) that were incubated with solACE2-Fc and secondary antibody were used to determine the background (grey area). Error bars indicate the standard error of the mean (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, ***). B) Receptor activity of ACE2 orthologues. BHK-21 cells transiently expressing the indicated ACE2 orthologues (or empty vector) were inoculated with pseudotyped particles bearing the indicated S proteins (or no S protein). S-protein driven cell entry was analyzed by measuring the activity of virus-encoded firefly luciferase in the cell lysate at 16-18h post inoculation. Presented are the average (mean) data from three biological replicates (each conducted with four technical replicates) in which cell entry was normalized against that measured for particles bearing no S protein (set as 1). 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, ***). C) Heat map for the data presented in panel B. Entry into cells expressing ACE2 orthologues was normalized against entry into cells expressing human ACE2 (set as 1).

Fig 3. Trypsin treatment can allow for ACE2-independent cell entry.

A) S protein driven cell entry in the presence and absence of trypsin. Particles bearing the indicated S proteins (or no S protein) were preincubated with or without trypsin (50 μg/ml for 30 min at 37°C) before being added to the respective cell lines. S-protein driven cell entry was analyzed by measuring the activity of virus-encoded firefly luciferase in the cell lysate at 16-18h post inoculation. Presented are the average (mean) data from three biological replicates (each conducted with four technical replicates) in which cell entry was normalized against that measured for particles bearing no S protein (set as 1). 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, ***). B) Trypsin treatment of viral particles but not target cells promotes entry. Vero cells or pseudotyped particles bearing the indicated S proteins were pre-incubated with trypsin (50 μg/ml for 30 min at 37°C) and subsequently trypsin inhibitor (200 μg/ml for 10 min at 37°C) as indicated. The pseudotyped particles were added to the cells. S-protein-driven cell entry was analyzed and data presented as described for panel A. Presented are the average (mean) data of three biological replicates, each performed with four technical replicates. Error bars show 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) Multiple bat sarbecovirus spike proteins can employ an ACE2-independent entry pathway following exposure to trypsin. Particles bearing the indicated S proteins were incubated with trypsin (50 μg/ml for 30 min at 37°C) before addition onto 293T wildtype (293T WT) and 293T ACE2 knockout cells (293T KO-ACE2). S-protein-driven cell entry was analyzed and data presented as described for panel A. Presented are the average (mean) data of three biological replicates, each performed with four technical replicates. Error bars show 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, ***). Of note, entry of particles bearing VSV-G in the presence of trypsin has not been analyzed (n.t. = not tested).

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, ***).

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, ***).

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, ***).

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, ***).

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, ***).

Fig 9. Antibodies induced by quadruple vaccination neutralize particles bearing diverse sarbecovirus S proteins.

A) Particles bearing the indicated S proteins were preincubated with a 1:25 dilution of plasma from convalescent patients, individuals vaccinated two times with BNT162b2 (BNT/BNT), three times with ChAdOx1-S and BNT162b2 (AZ/BNT/BNT), and four times, including a bivalent, BA.5-adapted booster, before being added to A549-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 of in the absence of plasma. Presented are the combined data for 9–10 plasma per group. Please see S3 Table for detailed information on the plasma samples. B) 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). Subsequently, the particles were incubated with a fixed 1:25 dilution of plasma from quadruple vaccinated donors (30 min at 37°C) and 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 of in the absence of plasma. Presented are the combined data for 10 plasma (three technical replicates). Statistical significance was assessed by Mann-Whitney test (p > 0.05, not significant [ns]; p ≤ 0.05, *; p ≤ 0.01, **; p ≤ 0.001, ***). Please see S3 Table for detailed information on the plasma samples tested.