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. 2023 Jul 28;9:78. doi: 10.1038/s41421-023-00581-9

Fig. 4. Residue 372 of S proteins modulates viral infectivity and stability.

Fig. 4

a Transduction of 293 cells expressing various bat ACE2s by WT or T372A (or T368A) mutant S pseudovirions. b, c WT and T372A (or T368A) mutant S pseudovirions, BANAL-20-52 (b), BANAL-20-236 (c), were pre-treated with indicated concentration of trypsin (0.625, 1.25, 2.5, and 5 μg/mL) at pH 5.5 for 10 min and the remaining infectivity was assayed on 293/hACE2 cells. d, e WT and T372A (or T368A) mutant S pseudovirions, BANAL-20-52 (d), BANAL-20-236 (e), were pre-treated with 2.5 μg/mL trypsin for various time points and the remaining infectivity was assayed on 293/hACE2 cells. f, g Western blot analysis of pseudovirions, BANAL-20-52 (f), BANAL-20-236 (g), treated with different concentrations of trypsin (0.313, 0.625, 1.25, 2.5, and 5 μg/mL) for 10 min using anti-SARS-CoV-2 RBD antibodies. h, i Detection of association of cleaved S1 subunits with pseudovirions, BANAL-20-52 (h), BANAL-20-236 (i), by western blot analysis, after different concentrations of trypsin (0.625, 1.25, 2.5, and 5 μg/mL) treatment at pH 5.5 for 10 min. Trypsin-treated pseudovirions were pelleted by centrifugation to separate the supernatants and pellets, and the cleaved S1 subunits in the supernatants and pellets were separated in a 10% SDS-PAGE and detected using rabbit polyclonal anti-SARS-CoV-2 RBD antibodies. Data are represented as mean ± SD from at least triplicates. P-values in a–e are calculated by unpaired two-sided Student’s t-test. *P < 0.05; **P < 0.01; ns, P > 0.05.