Fig. 3. SARS-CoV-2 Omicron variant spike enters cells less efficiently by TMPRSS2-dependent plasma membrane fusion.
a, PV entry in airway organoids, Calu-3 lung cells, gall bladded organoids, H1299 lung cells, HeLa ACE2-overexpressing cells and HEK293 ACE2-overexpressing cells. Black, WT Wuhan-Hu-1 D614G; orange, Delta/B.1.617.2; grey, Omicron/BA.1. Data are mean ± s.e.m. of n = 2–4 technical replicates. RLU, relative light unit; RT, reverse transcriptase. Statistical analysis was performed using two-sided unpaired Student’s t-tests; *P < 0.05, **P < 0.01, ****P < 0.0001. Data are representative of three independent experiments. b, ACE2 and TMPRSS2 mRNA transcripts in the indicated cell types and organoids as measured using qPCR. Samples were run as n = 4 technical replicates. The centre line shows the median, the box limits show the interquartile range and the whiskers denote the range. Data are representative of two independent experiments. c, Entry of PV expressing spike in HEK293T cells transduced to overexpress ACE2 and either depleted for TMPRSS2 (A2ΔT2) or overexpressing TMPRSS2 (A2T2). d, Entry of PV expressing spike in HEK293T cells with endogenous (−) or overexpressed TMPRSS2 (T2). For c and d, data are the mean ± s.e.m. of n = 4 technical replicates, representative of three independent experiments. Statistical analysis was performed using two-sided unpaired Student’s t-tests; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. e, An illustration of two cell entry pathways that are known to be used by SARS-CoV-2. The schematic was created using BioRender.com. f, Titration of inhibitors in A549-ACE2/TMPRSS2 cells using PV expressing Delta (orange) or Omicron (grey) spike in the presence of the indicated doses of Camostat or E64d, then analysed after 48 h. The percentage inhibition was calculated relative to the maximum luminescence signal for each condition. For each variant and dilution, data are mean ± s.e.m. of experiments conducted in duplicate. Statistical analysis was performed using two-sided unpaired t-tests; ****P < 0.0001. g, Titration of inhibitors in A549-ACE2/TMPRSS2-based luminescent reporter cells using live virus. Cells were infected at a multiplicity of infection (m.o.i.) of 0.01 with Delta or Omicron variants in the presence of the indicated doses of Camostat or E64d, and then analysed after 24 h. The percentage inhibition was calculated relative to the maximum luminescence signal for each condition. For each variant and dilution, data are mean ± s.e.m. of experiments conducted in triplicate. Data in f and g are representative of two independent experiments. h, ACE2 and TMPRSS2 mRNA expression determined using qPCR in human lung tissue (four pieces of tissue each from upper (main bronchus) and lower (lung parenchyma) airways). Each data point is the mean of n = 2 technical replicates. The centre line shows the median of biological replicates (n = 4 for each lung region), the box limits show the interquartile range and the whiskers denote the range. Statistical analysis was performed using two-sided unpaired t-tests. No adjustments were made for multiple comparisons.