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. 2022 May 19;13:883597. doi: 10.3389/fmicb.2022.883597

Figure 2.

Figure 2

Expression of ORF8 shows the downregulation and insufficient S1/S2 processing of the spike protein in a dose-dependent manner. (A) Spike protein immunostaining of Spike pseudovirus-packaging cells was performed. One representative result is shown, and the yellow color indicates the spike signal. EV: empty vector. (B) Immunoblots of Spike pseudovirus-packaging cells were obtained with antibodies specific to the spike protein and FLAG tag to visualize the protein level of the spike protein and ORF8. An antibody specific to Hsp90 served as a loading control. The scatter plot shows the relative intensity of the signals quantified by the ImageJ software from three independent experiments. The cleaved S1 and S2 fragments (between 100 to 130 kDa) were selected and determined as “S1 + S2 spike protein” and all fragments visualized above 100 kDa (containing the cleaved S1/S2 fragments and the full-length spike protein) were all selected and evaluated as “total spike protein” for quantification in this figure. In order to visualize the spike signal in ORF8-transfected samples together with EV-transfected samples in the same blot, the intensity of S1/S2 fragment of EV-transfected might reach plateau level then the real intensity might be underestimated. So the true efficiency of S1/S2 cleavage in EV-transfected cells might be much higher than the data shown here. (C) Immunoblots of cells transfected with ORF8 or ORF3a in combination with different spike variants. Here, an antibody specific to the FLAG tag indicates the protein levels of ORF8 and ORF3a. WT: Spike obtained from the original Wuhan strain; D614G: WT spike protein with the D614G mutation; D614G/N501Y: WT spike protein with the D614G and N501Y mutations. (D) Immunoblots of HEK293T cells transfected with different amounts of ORF8-expressing plasmids were obtained 3 days post-transfection. Here, an antibody against actin served as the loading control. Empty: empty vector does not encode ORF8. (E) FACs analyses of surface spike protein were performed using HEK293T cells cotransfected with spike-encoding plasmid together with pLAS2-based plasmid carrying ORF8 or empty vector at 1–9 ratio at 3 days post-transfection. The data are shown as the means ± SDs (error bars). Here, the paired Student’s t-test was used, and p < 0.05 indicates a statistically significant difference; *p < 0.05, **p < 0.01, ***p < 0.001. The scale bar in each figure represents 100 μm.