Impact of SARS-CoV-2 Spike Mutations on Its Activation by TMPRSS2 and the Alternative TMPRSS13 Protease

Abstract

The continuous emergence of new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) urges better understanding of the functional motifs in the spike (S) protein and their tolerance to mutations. Here, we focused on the S2' motif, which, during virus entry, requires cleavage by a host cell protease to release the fusion peptide. Though belonging to an immunogenic region, the SARS-CoV-2 S2' motif (811-KPSKR-815) has shown hardly any variation, with its three basic (K/R) residues being >99.99% conserved thus far. By creating a series of mutant pseudoviruses bearing the spikes of Wuhan-Hu-1, its G614 mutant or the Delta and Omicron variants, we show that residue K₈₁₄ (preceding the scissile R₈₁₅) is dispensable for TMPRSS2 yet favored by the alternative TMPRSS13 protease. Activation by TMPRSS13 was drastically reduced when the SARS-CoV-2 S2' motif was swapped with that of the low pathogenic 229E coronavirus (685-RVAGR-689), and also, the reverse effect was seen. This swap had no impact on recognition by TMPRSS2. In the Middle East respiratory syndrome coronavirus (MERS-CoV) spike, introducing a dibasic scissile motif was easily accepted by TMPRSS13 but less so by TMPRSS2, confirming that TMPRSS13 favors a sequence rich in K/R residues. Pseudovirus entry experiments in Calu-3 cells confirmed that the S2' mutations have minor impact on TMPRSS2. Our findings are the first to demonstrate which S2' residues are important for SARS-CoV-2 spike activation by these two airway proteases, with TMPRSS2 being more tolerant to variation than TMPRSS13. This preemptive insight will help to estimate the impact of S2' motif changes as they appear in new SARS-CoV-2 variants. IMPORTANCE Since its introduction in humans, SARS-CoV-2 is evolving with frequent appearance of new variants. The surveillance would benefit from proactive characterization of the functional motifs in the spike (S) protein, the most variable viral factor. This is linked to immune evasion but also influences spike functioning. Remarkably, though located in a strongly immunogenic region, the S2' cleavage motif has, thus far, remained highly conserved. This suggests that its sequence is critical for spike activation by airway proteases. To investigate this, we assessed how pseudovirus entry is affected by changes in the S2' motif. We demonstrate that TMPRSS2 readily accepts variations in this motif, whereas the alternative TMPRSS13 protease is more fastidious. The Wuhan-Hu-1, G614, Delta and Omicron spikes showed no difference in this regard. Being the first in its kind, our study will help to assess the impact of S2' variations as soon as they are detected during variant surveillance.

Keywords: SARS-CoV-2; TMPRSS13; TMPRSS2; cleavage; human coronavirus 229E; mutation; protease; spike protein.

FIG 1

The S2′ motif of SARS-CoV-2 is rich in basic residues and, so far, highly conserved. (A) The S2′ motif is identical in the Wuhan-Hu-1 strain and G614, Delta, and Omicron variants of SARS-CoV-2 and conserved in SARS-CoV-1. Its KR motif is not present in MERS-S and 229E-S, while the scissile R (underlined) is shared by all spikes. Boldface indicates basic (R or K) residues. FP, fusion peptide. The alignment further shows the sequence differences in the S1/S2 and D614 regions (note that amino acid differences outside these regions are not covered). The frame at the bottom shows the P5-P1 and P1′-P2′ designation of the S2′ cleavage motif. (B) To determine the conservation rate of the P5 to P2′ residues, a variation analysis was performed on 10,480,461 SARS-2-S sequences submitted to the GISAID database between 10 January 2020 and 29 April 2022. The y axis (different scales in the various panels) shows the number of sequences carrying the specified amino acid, with the consensus residue marked in bold on the x axis.

FIG 2

Impact of S2′ site changes on TMPRSS2- or TMPRSS13-mediated activation of pseudovirus entry. (A) Experimental setup. (B) Spike protein levels and cleavage state in S-pseudotyped virions. The particles were produced in HEK293T cells, pelleted, and subjected to western blot analysis with anti-V5 antibody recognizing the S0 and S2 forms (top) and anti-MLV gag antibody (bottom). Open arrowheads, full-length (S0) spike; hatched arrowheads, cleaved forms resulting from S1/S2 processing (SARS-2-S and MERS-S) or less specific cleavage (229E-S). The graphs below the images show the quantitative data based on band intensities, obtained for two batches of pseudoparticles. Circles, sum of all S bands (normalized to the band of MLV-gag), expressed relative to Wuhan-Hu-1 WT for SARS-2-S and the respective WT for MERS-S and 229E-S; bars, percent cleaved spike, calculated as 100 − [S0]/[sum of all S bands]. (C) Entry of WT and mutant S2′ S-pseudotyped viruses into HEK293T cells transfected with TMPRSS2 or TMPRSS13. The bars show the factor activation, i.e., luminescence signal relative to the condition receiving empty instead of protease plasmid. Pooled data from four experiments, each performed in five or six replicates, are shown. *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (nested t test, two tailed). ns, not significant. (D) Entry activation in TMPRSS2/TMPRSS13-cotransfected HEK293T cells, for the S2′-WT and -Mut3 forms of G614, Delta, and Omicron SARS-2-S pseudoviruses. ns, not significant (P > 0.05) (E). Expression of TMPRSS2 and TMPRSS13 following transfection in HEK293T cells. The two Flag-tagged proteases were produced at similar protein levels, as assessed by dot blot assay with anti-Flag antibody.

FIG 3

Impact of the S2′ motif changes on SARS-2-S pseudovirus entry in Calu-3 cells, in which TMPRSS2 is the activating protease. (A) Entry efficiency of the four S2′-WT viruses, expressed relative to Wuhan-Hu-1 pseudovirus. (B) Entry of the S2′-mutants, expressed relative to the corresponding WT. (A and B) Pooled data from three experiments, each performed in 6 replicates. (C) Knocking down TMPRSS2 in Calu-3 cells caused a significant reduction in the entry efficiency of Wuhan-Hu-1 pseudovirus (S2′-WT), while knockdown of TMPRSS13 had no effect. Mean results of two experiments, each performed in three to four replicates, are shown. **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001 (nested t test, two-tailed). ns, not significant.