The conserved QTQTX motif in the SARS-CoV-2 spike protein is dispensable for cleavage and lung cell entry of the emerging variant BA.3.2

Figures

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  Fig 1. Naturally occurring and engineered deletions in the QTQTX motif. (A) Deletions in the QTQTX motif observed in two individuals infected with the BA.3.2.2 variant in Australia. (B) Engineered deletions in the QTQTX motif analyzed within the present study. (C) Protein structure of the SARS-CoV-2 S protein (B.1, based on PDB:6XR8 [16]) with the S1/S2 loop highlighted (left) and S1/S2 loop structures predicted by homology modeling (right). The amino acid numbering is based on B.1 S protein wild type.

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  Fig 2. The QTQTX motif is dispensable for cleavage of the BA.3.2.2 S protein. (A) Equal volumes of pseudoviruses bearing the indicated S proteins were subjected to SDS-PAGE, followed by immunoblot analysis to assess S protein incorporation into viral particles and processing at the S1/S2 cleavage site. Immunodetection was performed using an antibody specific for the SARS-CoV-2 S protein and a peroxidase-conjugated secondary antibody. Detection of vesicular stomatitis virus matrix protein (VSV-M) served as a loading control. A representative immunoblot is shown; the results were confirmed in two additional independent biological replicates. (B) Band intensities of the S0 and S2 species were quantified by densitometry using ImageJ. For each sample, the combined S protein signal (S0 + S2) was set to 100%, and the relative abundance of S0 and S2 is shown as the mean of three independent biological replicates. Error bars represent the standard error of the mean (SEM). Statistical significance was assessed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (not significant [ns], P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

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  Fig 3. Deletion of the QTQTX motif does not reduce cell-cell and virus-cell fusion driven by the BA.3.2.2 S protein. (A) 293T effector cells co-expressing the beta-galactosidase alpha fragment and the indicated S proteins or Nipah virus fusion- and glycoprotein (NiV-F+G), or no viral glycoprotein (control effector cells), were co-cultured with 293T target cells coexpressing the beta-galactosidase omega fragment and either ACE2 or ACE2 and TMPRSS2. Efficiency of cell-cell fusion was determined by quantification of beta-galactosidase activity in cell lysates, and data were normalized against the signals obtained upon co-culture of target cells with control effector cells expressing no viral glycoprotein. The mean of four biological replicates (each conducted with four technical replicates) is shown. Error bars represent the SEM. (B) Pseudoviruses bearing the indicated S proteins were used to infect different target cell lines. Entry efficiency was determined 16 h post-infection by quantification of pseudovirus-expressed firefly luciferase in cell lysates. The results were normalized against signals obtained for pseudoviruses equipped with B.1 S protein (set as 1). Data show the mean of…

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  Fig 4. Calu-3 cell entry is Camostat-sensitive. (A) Vero and Calu-3 target cells were preincubated for 30 min with the indicated inhibitors, followed by inoculation with pseudoviruses bearing the indicated S proteins and further incubation in the presence of the inhibitor for 16 h. Entry efficiency was determined by quantification of pseudovirus-expressed firefly luciferase in cell lysates and normalized against signals obtained in the absence of inhibitor (set as 100%). Data show the mean of three biological replicates performed with four technical replicates; error bars indicate SEM. Statistical significance was assessed by two-way ANOVA with Tukey’s multiple comparison test (ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (B) The experiment was conducted as described for panel A with the exception that fixed concentrations of inhibitor were used (12.5 µM MDL28170; 12.5 µM Camostat) and particles bearing wild-type (WT) or mutant S proteins were investigated. Data show the mean of three biological replicates performed with four technical replicates; error bars indicate the SEM. Statistical significance was assessed by one-way ANOVA with Tukey’s multiple comparison test (ns, P…