Extended Data Fig. 6: Binding of HKU and SARS-CoV-2 spikes to TMPRSS2 or ACE2. a. Binding of the indicated recombinant spikes to 293 T cells expressing TMPRSS2.

Cells were transfected with WT or mutant TMPRSS2 and incubated or not overnight with 10 µM of Camostat. The spikes were then incubated for 0.5 h and their binding was revealed with streptavidin-647 and measured by flow cytometry. The % of cells binding to TMPRSS2 was quantified. Data are mean ± SD of 3 (HKU1A) or 4 (HKU1B, SARS-CoV-2) independent experiments. Two Way ANOVA with Dunnett’s multiple comparisons compared to control cells with or without Camostat. Exact p-values: HKU1A: TMPRSS2 WT-: *0.029, TMPRSS2 WT + :*** < 0.0001, TMPRSS2 R255Q-: **0.0010, TMPRSS2 R255Q + : **0,0013, TMPRSS2 S441A-: ***0,0001, TMPRSS2 S441A + : ***<0.0001. HKU1B: ***<0.0001. b. Binding of the indicated recombinant spikes to 293 T cells expressing ACE2. Cells were transfected with ACE2. The spikes were then incubated for 0.5 h and their binding was revealed with streptavidin-647 and measured by flow cytometry. The % of cells binding to ACE2 was quantified. Data are mean of 2 independent experiments. c. Binding of the indicated soluble spikes on immobilized ACE2 measured by ELISA. d. Binding of S441A TMPRSS2 to HKU1A, HKU1B or SARS-CoV-2 RBD measured by BLI. The response was measured at the indicated concentrations of spikes. Left: HKU1A. Middle: HKU1B. Right: SARS-CoV-2. One representative experiment of 4 is shown. e. Determination of the affinity of HKU1A and B RBD for TMPRSS2 using the steady state method. Circles: Experimental values. Black: Fitting of the experimental data f. Binding of ACE2 to SARS-CoV-2 or HKU1B RBD quantified by BLI, at different concentrations of spikes. g. Binding of S441A TMPRSS2 to HKU1B mutants. Response was measured by BLI at different concentrations of spikes. Left: HKU1B RBD mutant W515A. B: HKU1B RBD mutant R517A. h. Determination of the affinity of HKU1B-R517A RBD for TMPRSS2 using the steady state method. Circles: Experimental values. Black: Fitting of the experimental data. i. Cell surface levels of WT and mutant HKU1 spikes. 293 T were transfected with the indicated WT or mutant HKU1A or B spikes, expression was measured by flow cytometry after 24 h, using the anti-spike mAb10.