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

Figures

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  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.

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  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…

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  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.