Fig 1.
Isolation of SARS-CoV-2 S gene mutants.
(A) Schematic representation of SARS-CoV-2 S protein. Full-length S protein is cleaved into S1 and S2 proteins at the S1/S2 cleavage site. Functional domains (RBD, receptor binding domain; RBM, receptor binding motif) are highlighted. (B) Multiple amino acid sequence alignments focused on the S1/S2 cleavage site of wild type (WT) and isolated mutant viruses (del1, del2, del3 and R685H). Amino acid substitutions and deletions are shown as gray boxes, and the polybasic cleavage motif (RARR) at the S1/S2 cleavage site is highlighted in red. A red arrowhead indicates S1/S2 cleavage site. (C) Plaque formation for SARS-CoV-2 WT and isolated mutants grown in Vero-TMPRSS2 cells on 6 well plates. (D) Detection of virus S and N proteins in Vero-TMPRSS2 cells infected with WT or isolated mutants. The full-length S and cleaved S2 proteins are indicated by closed and open arrowheads, respectively.
Fig 2.
Infection and growth of SARS-CoV-2 S gene mutants in different cell lines.
(A) Vero, Vero-TMPRSS2, Caco-2 and Calu-3 cells were inoculated with SARS-CoV-2 WT or S gene mutants at a multiplicity of infection (MOI) of 1. At 24 h post infection, cells were stained with anti-SARS-CoV-2 S antibody (green) and Hoechst 33342 nuclear dye (blue); scale bars, 50 μm. White arrowheads indicate cell syncytia. (B) Cells were inoculated with SARS-CoV-2 WT or S gene mutants at an MOI of 0.1. Culture supernatants were harvested at 24, 48 and 72 h after inoculation. Virus titration was performed by plaque assay. The values shown are mean ± standard deviation (SD) of triplicate samples. One-way analysis of variance with Dunnett's test was used to determine the statistical significance of the differences in virus titer between the WT and S gene mutants at 72 h after inoculation. *p < 0.05, **p < 0.01.
Fig 3.
Infection and growth of SARS-CoV-2 S gene mutants in 293T and 293T-ACE2 cells.
(A) 293T and 293T-ACE2 cells were inoculated with SARS-CoV-2 WT or S gene mutants at an MOI of 1. At 24 h after inoculation, cells were stained with anti-SARS-CoV-2 S antibody (green) and Hoechst 33342 nuclear dye (blue); scale bars, 50 μm. (B) Cells were infected with SARS-CoV-2 WT or S gene mutants at an MOI of 0.1. Culture supernatants were harvested at 24, 48 and 72 h after inoculation and titration of infectious virus was determined by plaque assay. The values shown are means ± SD of triplicate samples. One-way analysis of variance with Dunnett's test was used to determine the statistical significance of the differences in virus titer between the WT and S gene mutants at 72 h after inoculation. **p < 0.01.
Fig 4.
Impact of biochemical inhibitors on cellular entry of SARS-CoV-2 S gene mutants.
(A) Vero-TMPRSS2 cells were infected with SARS-CoV-2 WT or del2 mutant in the presence of varying concentrations of the TMPRSS2 inhibitor, camostat, or the cathepsin B/L inhibitor, E-64d, for 1h. At 6 h post-inoculation, the relative levels of viral N protein RNA were evaluated quantitatively by qRT-PCR. (B) Vero-TMPRSS2 and Vero cells were infected with SARS-CoV-2 WT or S gene mutants in the presence of 50 μM camostat and/or 25 μM E-64d for 1 h. At 6 h post-inoculation, the relative levels of viral N protein RNA were quantified by qRT-PCR. Cellular β-actin mRNA levels were used as reference controls. The values shown are mean ± SD of triplicate samples. One-way analysis of variance with Dunnett's test was used to determine the statistical significance between the responses to treatment with inhibitors and the no-treatment controls; *p < 0.05, **p < 0.01, ***p < 0.001.
Table 1.
Impact of antivirals against WT and S gene mutants of SARS-CoV-2 in Vero-TMPRSS2 cells.
Fig 5.
Frequencies of S gene mutants detected during SARS-CoV-2 propagation.
SARS-CoV-2 was serially passaged in (A) Vero-TMPRSS2, Vero cells or (B) Calu-3, Caco-2 and 293T-ACE2 cells, each with three biological replicates. Nucleotide sequence diversity at viral S1/S2 cleavage site was determined by deep-sequencing.