Figure 3. Mutation of the human IFITM3 endocytosis motif converts IFITM3 into an enhancer of SARS‐CoV‐2 infections.

HEK293T‐ACE2‐GFP stable cells were transiently transfected with human IFITM plasmids or vector control for 24 h.

1. Flow cytometry gating strategy for analyzing cells with high ACE2‐GFP expression and IFITM expression.
2. Flow cytometry histograms showing expression of each of the IFITM constructs, and the gating as indicated in (A) and by the dotted line, which was used for examining percent infections in IFITM‐expressing cells.
3. Transfected HEK293T‐ACE2‐GFP cells were infected with SARS‐CoV‐2 (MOI 1) for 24 h and analyzed for percent infection by flow cytometry. Representative example plots are shown for each transfection condition.
4. Graph depicts normalized infection percentage measurements from 2 to 3 independent experiments, each performed in triplicate, from cells infected as in (C). Bars represent averages with individual data points shown as circles. Error bars represent SD. ^# P < 0.05 compared to vector control by ANOVA followed by Tukey’s multiple comparisons test.
5. Representative example flow cytometry histograms of ACE2‐GFP from non‐infected samples.
6. Normalized measurements of ACE2‐GFP mean fluorescence intensity (MFI) of non‐infected samples from three separate experiments. Bars represent averages with individual data points shown as circles. Error bars represent SD. Statistical significance (P < 0.5 by ANOVA followed by Tukey’s multiple comparisons test) was not observed for comparisons between any samples.
7. Confocal microscopy of HEK293T cells transfected with empty vector, hIFITM3, hIFITM3‐Y20A, or hIFITM3‐L23Q. Green, anti‐IFITM3; blue, DAPI. White arrows highlight plasma membrane localization of IFITM3‐Y20A and IFITM3‐L23Q. Scale bars represent 10 µm.

Source data are available online for this figure.