As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants with increased transmissibility and pathology. In addition to this entrenched diversity, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriSΔ variant, originally identified as a viral subpopulation from SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike an eight amino-acid deletion encompassing a furin recognition motif and S1/S2 cleavage site. We elucidate the structure, function and molecular dynamics of this spike providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity of this SARS-CoV-2 variant. Our results reveal long-range allosteric communication between functional domains that differ in the wild-type and the deletion variant and support a view of SARS-CoV-2 probing multiple evolutionary trajectories in distinct cell types within the same infected host.
SARS-CoV-2 spike (S) glycoprotein prominently differs from other betacoronavirus S proteins in the insertion of a furin cleavage site in the S1/S2 junction site1. The S trimer glycoprotein is responsible for binding to the ACE2 receptor and for viral cell entry after cleavage at the S1/S2 junction and S2´ sites2. Critical to this process is proteolytic processing of S by host cell proteases3. After intracellular cleavage at the S1/S2 junction by a furin-like protease to produce the S1 and S2 subunits, S gets destabilized and can be further primed by cleavage at the S2´ site by host serine proteases on the plasma membrane such as TMPRSS24,5 or the endosomal cysteine proteases cathepsin B/L6. S1 comprises the N-terminal domain (NTD), the receptor-binding domain (RBD), and the SD1 and SD2 domains7,8. S2 contains the S2´ cleavage site, the fusion peptide, a fusion peptide proximal region (FPPR), a HR1 heptad repeat, a central helix and a connector domain followed by a HR2 heptad repeat, the transmembrane domain and the C-terminal cytoplasmic domain7,8. Receptor binding destabilizes S, allowing S2´ cleavage, leading to shedding of S1 while S2 reorganizes to mediate fusion of viral and cellular membranes, enabling entry of SARS-CoV-2 into the host cells9. The furin cleavage site is a four amino acid motif located on a solvent-exposed flexible loop of S7. Furin-cleaved S was shown to open more efficiently suggesting an increased binding to human ACE2 than uncleaved S10. The furin cleavage site thus contributes substantially to the high infectivity of SARS-CoV-2, adding to the lethality of the virus.
After the growth of a low passage isolate of SARS-CoV-2 from February 2020 in the African green monkey kidney cell line Vero E6, a cell line routinely used to propagate viruses from clinical isolates, we discovered a virus subpopulation with an S variant (termed here BriSΔ) exhibiting an in-frame 8 amino acid deletion encompassing the furin recognition motif and S1/S2 cleavage site (amino acids 679-687 NSPRRARSV, replaced by I)11. Subsequently, further deletion variants abrogating S1/S2 cleavage were identified after viral passaging in cell culture12,13,14 and at low frequency in clinical samples, attenuating infection in animal models15,16,17,18. Moreover, deleting only PRRA in S by reverse genetics resulted in a recombinant ΔPRRA SARS-CoV-2 which exhibited increased infectivity and viral titer in Vero E6 cells, but a 10-fold reduced viral titer in Calu-3 2B4 lung epithelial carcinoma cells compared to the wild-type (WT) virus19, indicating the acquisition of a furin cleavage site increased SARS-CoV-2 fitness for replication in respiratory cells.
Here, we dissect the structure, dynamics and mechanism of the BriSΔ deletion variant S we identified, to gain insight into how diversification of the virus by elimination of a loop-region comprising the furin recognition motif and S1/S2 cleavage site impacts viral cell tropism, infectivity, spike protein stability and receptor binding, revealing molecular communication between functional regions within the spike glycoprotein allowing SARS-CoV-2 to evolve intra-host diversity in distinct cell types.
BriSΔ variant and wild-type SARS-CoV-2 clonal isolation
Direct RNA sequence analysis of a virus stock of SARS-CoV-2 isolate hCoV-19/England/02/2020, produced by a single passage in Vero E6 cells, revealed the presence of the WT SARS-CoV-2 and the BriSΔ variant (Fig. 1a). To obtain homogenous virus populations, the mixed virus stock was subjected to two rounds of limiting dilution in Vero E6 and human Caco-2 cells (Supplementary Fig. 1). Nanopore direct RNA sequencing confirmed that the limiting dilution yielded WT SARS-CoV-2 from Caco-2 cells. In contrast, BriSΔ was selected for in Vero E6 cells (Fig. 1a) as expected11. The differences in the infectivity of the WT and BriSΔ viruses were then compared on Vero E6, Vero E6/TMPRSS2, Caco-2, Caco-2-ACE2 and Calu-3 cells using a range of virus dilutions for infection (Fig. 1b–f). The starting virus volumes for the infections were based on equal viral genome copy numbers as determined by qRT-PCR (equating to a starting multiplicity of infection (MOI) of 10 for the WT virus, based on the Vero E6 cell titer) rather than MOI values. Although viral genome copy numbers do not necessarily reflect virus infectivity, viral growth assays on Vero E6 and Caco-2-ACE2 cells using MOIs determined on either Vero E6 or Caco-2-ACE2 cells showed that the infectivity of the two viruses differed, depending on the cell type used to determine the MOI (Supplementary Fig. 2). The percentage of virus-infected cells for the five different cell lines was analyzed 18 hours after virus infection, before multiple rounds of virus replication. In Vero E6 cells, half-maximal infection was achieved with an ~6-fold higher dilution of BriSΔ as compared to WT virus (Fig. 1b). Overexpression of TMPRSS2 protease in Vero E6 cells5 resulted in a substantially higher infection efficiency for both viruses; close to 100% of cells were infected with an up to 16-fold dilution of WT virus and an up to 64-fold dilution of BriSΔ virus (Fig. 1c). Thus, the lack of the TMPRSS2 protease contributes to, but is not the only reason why the BriSΔ variant infects Vero E6 cells better than WT virus. Differences in the route of cell entry either via fusion at the plasma membrane or receptor-mediated endocytosis20,21,22 may account for this result. Interestingly our results using Vero E6/TMPRSS2 cells are similar to those of Zhu et al18 comparing the replication of WT SARS-CoV-2 and a virus (Sdel) containing a 7 amino acid deletion encompassing the furin cleavage site but differ from those based on a competition assay between the WT and ΔPRRA viruses, which infected Vero E6/TMPRSS2 cells equally well19. Even though we selected WT SARS-CoV-2 from Caco-2 cells by serial dilution, BriSΔ infected Caco-2 cells better than the WT at high virus titers; 25% versus 10% infected cells were observed with the starting dilutions of BriSΔ and WT, respectively (Fig. 1d). Overexpression of the ACE2 receptor in Caco-2 cells led to 70% infection of cells up to 32-fold dilution of WT, whereas only about 35% of cells could be infected by the BriSΔ variant at the same dilution (Fig. 1e). Thus, both the WT and BriSΔ viruses infect Caco-2 cells better when ACE-2 expression was increased, but the improvement for WT virus was substantially higher. Calu-3 lung cells were infected about 2-fold better by WT virus for all dilutions except the starting dilutions (Fig. 1f), corroborating the contribution of the furin site in SARS-CoV-2 S to improved infection of lung cells19. Differences in the maximal level of infection of the different cell lines at 18 hours after virus infection were observed, most likely due to either difference in the expression levels of ACE2 and cellular proteases required for virus entry or intrinsic cellular factors restricting initial viral replication22.
Fig. 1: hCoV-19/England/02/2020 derived SARS-CoV-2 BriS∆ variant.
a Depth of read across S glycoprotein gene at furin cleavage site is shown for three different stocks of SARS-CoV-2 isolate hCoV-19/England/02/2020. Horizontal blue lines indicate read depth. The original stock of virus (top) evidences sharp decline in read depth corresponding to in-frame deletion of the furin cleavage site and indicative of a mixed population of viruses. The middle panel shows the sequencing depth at same region for a virus stock that has been isolated by growth on human Caco-2 cells and purified by limiting dilution. The bottom panel shows the equivalent sequencing data for a stock of the SARS-CoV-2 BriSΔ variant grown on Vero E6 cells and purified by limiting dilution. b–f SARS-CoV-2 infection assays: Approximately equal amounts of the WT virus and BriSΔ virus based on genome amounts (estimated by qRT-PCR) were diluted (2-fold dilution series starting with neat virus) and used to infect Vero E6, Vero E6/TMPRSS2, Caco-2, Caco-2-ACE2, and Calu-3 cells. At 18 h after infection, cells were fixed, stained with an anti-N antibody and the % of cells infected was determined by immunofluorescence microscopy. Data (b–f) are presented as mean values ±SD. n = 3 biological replicates. g WT virus and BriSΔ virus were used to infect Vero E6/TMPRSS2 cells in the presence of a range of dilutions of a commercial antibody against SARS-CoV-2 RBD. Cells were infected with equal amounts of infectious virus (based on cell infectivity). At 18 h after infection, cells were fixed and stained with an anti-N antibody and the % of cells infected was determined by immunofluorescence microscopy. Data are presented as mean values ±SD. n = 2 biological replicates. Source data for graphs shown in panels b–g are provided as a Source Data file.
Next, we tested neutralization of the WT and BriSΔ viruses. No difference was found in neutralization of the two viruses when Vero E6/TMPRSS2 and Vero E6 cells were infected with equal amounts of infectious virus based on cell infectivity, in the presence of a commercial antibody binding the RBD (Fig. 1g) or human serum from a convalescent COVID-19 patient (Supplementary Fig. 3), respectively, indicating that both virus species were neutralized with equal potency by the antibodies.
Cryo-EM structure of BriSΔ glycoprotein
To understand the structural impact of the deletion of the furin cleavage site on SARS-CoV-2 S architecture, we produced the BriSΔ spike by MultiBac/insect cell expression23. We purified the glycoprotein by affinity purification and size exclusion chromatography (Supplementary Fig. 4a, b) We used the peak fraction from SEC for negative stain EM quality control (Supplementary Fig. 4c) and cryogenic electron microscopy (cryo-EM) (Supplementary Fig. 5 and Supplementary Table 1). We determined the BriSΔ structure without applying symmetry (C1) at 3.0 Å resolution (Supplementary Fig. 6a). In our analysis, all BriSΔ particles exhibited the locked conformation of the S trimer we had described previously23. After applying 3-fold symmetry (C3) we obtained a 2.8 Å cryo-EM map (Fig. 2 and Supplementary Fig. 6b). In this compact locked S conformation, the receptor-binding motif (RBM) is buried inside the RBD trimer obstructing ACE2 receptor binding (Supplementary Fig. 7). – Previously, we discovered a free fatty acid (FFA) binding pocket in the locked structure of SARS-CoV-2 S, and identified a small molecule tightly bound in the pocket, with the molecular mass of linoleic acid (LA) as determined by electron-spray ionization mass-spectroscopy (ESI-MS)23, a feature subsequently corroborated in coronavirus S from pangolin24. Subsequently, similar density was also identified in other S structures (PDBIDs 6ZB5, 7JJI, 6ZGI, 6ZGE, 6XR8, 6ZP2, and 7DF3. In the locked BriSΔ structure, all three pockets are again occupied by a small molecule (Fig. 2a, b). We chose a method orthogonal to ESI-MS, namely hydrophilic interaction liquid chromatography followed by tandem mass spectrometry (HILIC-MS-MS) and highly purified LA as a calibration standard, to analyze our BriSΔ glycoprotein samples. Our HILIC-MS-MS analysis provides unambiguous, complementary evidence that the small molecule is indeed LA. In our structure, LA is bound in a bi-partite binding pocket where one RBD provides a hydrophobic ‘greasy’ tube to accommodate the hydrocarbon tail of LA, while residues R408 and Q409 of the adjacent RBD provide a polar lid coordinating the carboxy head group of LA (Fig. 2b). In the BriSΔ C1 structure, we identified virtually identical tube-shaped densities in all three RBD domains (Supplementary Fig. 7), indicating high occupancy of all three pockets. Using masked 3D classification, we scrutinized the data set for potential heterogeneity in LA binding and found that at least 95% of the RBDs were LA-bound in our structure (Supplementary Fig. 8). Our previous ESI-MS results and the present HILIC-MS-MS results thus are consistent and together identify the small molecule bound in the FFA-pocket unambiguously as the essential free fatty acid LA.
Fig. 2: Cryo-EM structure of BriS∆ glycoprotein.
a Top view cartoon representation with trimer subunits colored yellow, green and blue, LA shown as orange spheres. b Composite LA-binding pocket formed by adjacent RBDs (yellow and blue). EM density is shown as gray-colored mesh; LA ligand (orange) in sticks and balls representation c Selected-reaction monitoring mass chromatogram of hydrophilic interaction liquid chromatography (HILIC) coupled tandem mass spectrometry analysis for 10 ng/mL LA analytical standard (gray) and BriSΔ protein preparation (black). Source data are provided as a Source Data file. d Side view of BriSΔ trimer with boxes for the close-up views in panels e–k. e Disulfide bond between Cys336 and Cys361 in the RBD. f Cys840 forms a disulfide bond with Cys851 and stabilizes the fusion peptide proximal region (FPPR). g H-bond cluster involving R1039 cation-π interaction on F1042 and forming a salt bridge to E1031. h BriSΔ K986 and V987. K986 sidechain EM density indicates flexibility. i BriSΔ shortened loop devoid of furin and S1/S2 proteolytic sites modeled as a poly-alanine chain in the C1 structure. k R634 cation- π interaction to Y837 in the FPPR of neighboring polypeptide chain.
We scrutinized our BriSΔ structure and compared it with previously determined S structures for conserved stabilizing features (Fig. 2). Disulfide bonds are known to play a crucial role in stabilizing the S trimer and individual domains. Five out of 14 annotated disulfide bonds in S25 stabilize the RBD including the disulfide bond linking C336 and C361 (Fig. 2d, e). Three arginine R1039 residues, one each from the three polypeptide chains in the S trimer, form a hydrogen bond cluster (Fig. 2g). In this cluster, the arginine residues are symmetrically arranged around the central trimer axis with short-range contacts of 4.65 Å present between the carbon atoms of the guanidino groups. The guanidinium planes stack in a parallel manner on top of the aromatic plane of the juxtaposed F1042, and a salt bridge is formed to E1031 of the adjacent S polypeptide chain (Fig. 2g). R1039, E1031, and F1042 are conserved in all human coronaviruses, highlighting their central importance. In the vicinity, a disulfide bond is formed by conserved residues C1032 and C1043, arranging E1031 and F1042 at the required distance and in the proper conformation to stabilize the R1039-mediated interaction (not shown). Opening of the RBDs was shown previously to induce an asymmetry in the trimer structure that breaks this H-bond cluster8,26. LA binding in the FFA-pockets induces conformational changes to the residues surrounding the FFA-binding pocket in the RBD and beyond, including the NTD, SD2, and the fusion peptide proximal region (FFPR). Re-organization of SD2 in the locked structure results in a stabilization of the region around R634 (Fig. 2k). This arginine residue is stabilized by π-stacking on Y837 and thus connects to the FPPR of the neighboring subunit. Such π-stacking interactions were observed in a previous structure that had an intact furin site and unassigned density in the FFA-binding pocket10. R634 stacking to Y837 is additionally stabilized by a hydrophobic interaction (Fig. 2k). Fixed in a rigid position through these interactions, C840 can form a disulfide bond to C851 which additionally stabilizes the FPPR (Fig. 2f). This disulfide bond-mediated stabilization of the FPPR has been described in a cryo-EM structure of full-length S protein comprising the native transmembrane domain9. We scrutinized S structures in the protein data bank (PDB) and the sandwiched R634 appears to be a hall mark of the locked conformation. In contrast, S structures in the closed, but not locked, conformation show no π-stacking interaction of R634. Instead, residues 620–640, as well as parts of the FPPR, are disordered, and residues Y636 and R634 adopt different conformation, underscoring a functional link between the locked conformation, the sandwiched R634 and the FFA-binding pocket.
Our BriSΔ construct harbors WT residues K986 and V987, which often are mutated to prolines to stabilize S in a prefusion state. In BriSΔ, the valine fits well into the density while the lysine sidechain appears to be somewhat flexible (Fig. 2h). Importantly, BriSΔ lacks 8 amino acids including the furin cleavage site and the S1/S2 cleavage site located on a flexible loop. This loop is now shorter due to the deletion and thus more rigid, as evidenced by density in the C1 map which allowed to build a poly-alanine chain (Fig. 2i).
N-glycosylation of BriSΔ is comparable to previous S structures (Supplementary Table 2). Interestingly, WT residues S673, T676, T678, and S680 close to the furin site are all candidates for O-glycosylation which is dependent on proline P68127. It was shown that O-glycosylation of these residues negatively affects furin cleavage, contributing to S stability and infectivity27. P681 and S680 are lacking in BriSΔ, and P681 is mutated in other lineages, including the B.1.1.7 variant that emerged in Kent, UK28 and rapidly spread globally. The enzymes responsible for O-glycosylation (GALNTs) are expressed differently depending on cell type29, and the absence of P681 in BriSΔ could thus contribute to the observed dominance of this variant in certain cell types. Indeed, it was shown that mutation of P681 to R, which is present in several variants of concerns including the recent rapidly spreading Indian ‘delta’ variant, increases viral fusion30.
Functional analysis of BriSΔ
The cryo-EM structure of BriSΔ evidenced exclusively particles in the locked conformation in which RBM binding to ACE2 is obstructed (Fig. 2 and Supplementary Figs. 5–7). In cell-based assays, however, BriSΔ virus remains infectious (Fig. 1). To address this apparent discrepancy, we biochemically analyzed the interaction of a range of S proteins with the ACE2 receptor. We compared BriSΔ binding to ACE2 with S protein lacking the RBM, S protein where the furin site residues are replaced by an alanine (SRRAR->A), uncleaved WT S (SRRAR) and furin-cleaved WT S (SRRAR*) (Fig. 3a and Supplementary Table 3). ACE2-binding ELISA (performed using a surrogate virus neutralization test kit (sVNT)) indicated that all S proteins efficiently bind ACE2, except the S protein lacking the RBM used as a control (Fig. 3b). For the other S proteins, half-maximal binding was observed between 64 and 128 nM S protein in the assay. We determined the dissociation constant (KD) of BriSΔ and ACE2 by surface plasmon resonance (SPR) with biotinylated ACE2 immobilized on a streptavidin-coated chip (Fig. 3c and Supplementary Fig. 9). The binding of BriSΔ (KD = 2.5 nM) to ACE2 is not significantly different as compared to SRRAR->A (1.4 nM)23. In agreement, the maximal RU values, indicating mass deposited on the ACE2-coated chip, were virtually identical for BriSΔ, SRRAR->A and uncleaved WT S (Fig. 2d and Supplementary Fig. 9a, b). The partially cleaved WT S yielded lower RUmax signals, possibly due to partial dissociation of the S trimer and binding of the smaller S1 fragment to ACE2. Our biochemical assays thus establish that the BriSΔ trimer can adopt an open, ACE2-binding competent conformation, which however was not observed on the cryo-EM grids. This finding is in agreement with our results that the BriSΔ variant and WT virus can be neutralized efficiently with similar amounts of a commercial monoclonal antibody recognizing the RBD (Fig. 1g), and with human convalescent sera (Supplementary Fig. 3). Also, similar observations were made in a recent study that identified a fully closed Cryo-EM structure of S with no significant change in ACE2-binding affinity31.