Skip to main content
. 2023 Oct 11;24(12):e57224. doi: 10.15252/embr.202357224

Figure 5. SARS‐CoV‐2 ORF3a redistributes tetherin away from the biosynthetic pathway via defective retrograde trafficking.

Figure 5

  1. A miniscreen was performed to analyse the ability of other SARS‐CoV‐2 ORFs to downregulate surface tetherin. HeLa cells were transiently transfected with Strep‐tagged plasmids encoding: ORF3a‐Strep, ORF6‐Strep, ORF7a‐Strep, Strep‐ORF7b, ORF8‐Strep, ORF9b‐Strep, Strep‐ORF9c or ORF10‐Strep. 48 h post transfection, cells were stained for surface tetherin. Two biological replicates were performed. Representative data shown.
  2. ORF3a‐Strep transfected cells displayed intracellular tetherin accumulation. Tetherin accumulated in ORF3a‐Strep positive compartments. Representative confocal immunofluorescence microscopy image of fixed HeLa cells transiently transfected with SARS‐CoV‐2 ORF3a‐Strep. Cells were fixed and stained with anti‐Strep (green) and anti‐tetherin (red) antibodies and DAPI (blue). Non‐transfected cells are shown with asterisks.
  3. HeLa cells were transiently transfected with SARS‐CoV‐2 ORF3a‐Strep. Mock and transfected cells were lysed 48 h post transfection and analysed by western blot. Blots were analysed using anti‐tetherin, anti‐Strep and anti‐EF2 (loading control) antibodies.
  4. Confocal immunofluorescence microscopy was performed on mock or ORF3a‐Strep transiently transfected HeLa cells to analyse tetherin overlap with TGN46. Cells were fixed and stained using anti‐TGN46 (green), anti‐tetherin (red) antibodies, and DAPI (blue). Representative images are shown.
  5. Colocalisation analysis was performed to quantify the Mander's overlap coefficient of tetherin overlapping TGN46. At least 20 cells per condition from three biological replicates were analysed. Individual data points are plotted with mean and standard deviation. Two‐tailed, unpaired t‐tests were performed (****P < 0.0001).
  6. Confocal immunofluorescence microscopy was performed on mock or ORF3a‐Strep transiently transfected HeLa cells to analyse tetherin overlap with the lysosomal marker, LAMP1. Cells were fixed and stained using anti‐LAMP1 (green), anti‐tetherin (red) antibodies, and DAPI (blue). Representative images are shown.
  7. Colocalisation analysis was performed to quantify the Mander's overlap coefficient of tetherin overlapping LAMP1. At least 30 cells per condition from three biological replicates were analysed. Individual data points are plotted with mean and standard deviation. Two‐tailed, unpaired t‐tests were performed (****P < 0.0001).
  8. Antibody uptake experiments were performed to investigate the fate of endocytosed tetherin. Transient transfections were performed 48 h prior to uptake experiments. Anti‐tetherin antibodies were bound to live cells on ice for 30 min before a 2 h chase at 37°C. Cells were fixed and immunolabelled using anti‐TGN46 (green), anti‐Strep (white), secondary anti‐rabbit555 (red) antibodies, and DAPI (blue). Representative images are shown.
  9. Colocalisation analysis was performed to quantify the Mander's overlap coefficient of endocytosed anti‐tetherin overlapping TGN46. At least 26 cells per condition from three biological replicates were analysed. Individual data points are plotted with mean and standard deviation. Two‐tailed, unpaired t‐tests were performed (****P < 0.0001).

Source data are available online for this figure.