Fig 1.
Trypsin and TMPRSS2 cleave SARS S at the same site but process different SARS S glycoforms.
(A) 293 T cells were cotransfected with expression plasmids for SARS S and decreasing amounts TMPRSS2 encoding plasmid and treated with trypsin or PBS. Subsequently, SARS S cleavage was analyzed by Western blot employing anti-V5 antibody for S protein detection. Detection of β-actin served as loading control. Similar results were obtained in three independent experiments. The black filled triangle indicates the SARS S cleavage product generated by trypsin, while the product of SARS S processing by TMPRSS2 is marked by the white filled triangle. (B) The experiment was carried out as in (A). However, cells were treated with PNGaseF to remove all N-linked glycans before Western blot analysis. The asterisk indicates deglycosylated SARS S cleavage products. The results were confirmed in five independent experiments.
Fig 2.
R667 but not R797 is required for SARS S cleavage by trypsin and TMPRSS2.
Plasmids encoding SARS S wt or the indicated SARS S mutants were cotransfected with TMPRSS2 plasmid or empty plasmid into 293T cells. Subsequently, the cells were treated with trypsin or PBS and SARS S cleavage was analyzed by Western blot employing anti-V5 antibody. Detection of β-actin served as loading control. The results were confirmed in two to four separate experiments. The SARS S cleavage products generated by trypsin and TMPRSS2 are indicated by black and white filled triangles, respectively.
Fig 3.
Mutations K543A/R544A and R563A/K566A interfere with SARS S trafficking through the constitutive secretory pathway.
(A) COS-7 cells were cotransfected with expression plasmids for SARS S wt or the indicated mutants and with a plasmid encoding YFP fused to a plasma membrane anchor (red color). After staining for SARS S with V5 antibody and anti-mouse Alexa Fluor 647 secondary antibody (green color) the cells were analyzed by confocal microscopy. Similar results were obtained in a second independent experiment. (B) The experiment was carried out as described for panel A, but GFP fused to a Golgi marker (red color) was used instead of YFP with plasma membrane anchor. The results were confirmed in a second separate experiment. For optimal visualization, colors were manually assigned with LSM Pascal 5 software version 3 and do not correspond to the actual emission spectra of YFP, GFP and Alexa Fluor 647.
Fig 4.
Mutations R667A, T678S and R797N are compatible with robust S protein-driven cell entry.
(A) 293T cells transfected to express MLV particles pseudotyped with SARS S wt or the indicated SARS S mutants and the corresponding supernatants were analyzed for expression of S protein (employing anti-V5 antibody) and MLV capsid protein (employing anti-p30 antibody) by Western blot. Cells transfected to express S protein alone or transfected with empty plasmid were used as controls. Similar results were obtained in two independent experiments. (B) 293T target cells transfected with empty plasmid (control) or ACE2 plasmid were transduced with equal volumes of pseudotypes bearing the indicated viral glycoproteins. Transduction efficiency was quantified by measuring luciferase activities in cell lysates. Results were normalized for SARS S-driven transduction of ACE2 expressing cells, which was set as 1-fold. The average of three to eight independent experiments is shown. Error bars indicate standard error of the mean (SEM).
Fig 5.
R797 but not R667 is required for SARS S activation by TMPRSS2.
293T target cells transfected with ACE2 plasmid or cotransfected with ACE2 and TMPRSS2 plasmid were pretreated with DMSO or the cathepsin B/L inhibitor MDL 28170 for 1 h before addition of equal volumes of pseudotypes bearing the indicated glycoproteins. Cellular entry was quantified by measurement of luciferase activity in cell lysates. Results were normalized for transduction of ACE2+, TMPRSS2- cells in the absence of inhibitor, which was set as 1-fold. The average of three to six independent experiments is shown (R667A, n = 3; SARS S wt and VSV-G, n = 4; R797N, n = 6). Error bars indicate SEM. Statistical significance was assessed using one-tailed student’s t-est.