Fig 2: In a non-washed in vitro infection system, IFNβ-ACE2 exhibited enhanced antiviral activity compared to IFN-β alone, ACE2 alone, or the unlinked combination. NL63 was incubated for 1 h at 4 °C with either IFNβ-ACE2, sACE2(18-611), sACE2(18-740), recombinant IFN-β, IFN-β (Peprotech), or the unlinked combination of sACE2(18-611) and IFN-β. In contrast to experiments shown in Figure 3 and Figure 4, we omitted the virus-washing step. The NL63 + protein mixtures were added to Vero E6-TMPRSS2-T2A-ACE2 cells in a 96-well plate. The cells were harvested after a 2-day incubation at 33 °C and stained with LIVE/DEAD Fixable Blue Dead Cell Stain. Cells were surface-stained with FITC-conjugated mouse anti-human ACE2, were fixed and permeabilized, and then were intracellularly stained with AF647-conjugated rabbit anti-NL63 nucleocapsid antibody. Cells were then analyzed for viral infection by flow cytometry. Cells gated as viable, single, and live cells (parent gate) were subgated to define nucleocapsid+ and ACE2high subsets. Shown (A,D) are representative dot plots showing percentages of the nucleocapsid+ subset at the 1 pM concentration. Shown (B,F) are the percentages of the nucleocapsid+ subset for each group over concentrations ranging from 100 fM to 1 μM. Bar graph (C) shows mean percentage values of nucleocapsid+ cells at the 1 pM concentration. Shown (E) are representative dot plots showing percentages of the ACE2high subset at the 1 pM concentration. Shown (G) are the percentages of ACE2high subset for each group over concentrations ranging from 100 fM to 1 μM. Each data point represents the mean value (n = 2), and error bars represent SD. Statistical significance was analyzed by (C) one-way ANOVA with the Dunnett multiple comparisons test or (F,G) two-way ANOVA with Tukey’s multiple comparisons test comparing the unlinked combination of IFN-β and ACE2 treatment groups to the IFNβ-ACE2 treatment group at each concentration (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001). These data are representative of three independent experiments.

Fig 3: IFNβ-ACE2 exhibited virus-specific targeting in accordance with viral host receptor specificity. The NL63 or 229E viruses were incubated at 4 °C with either IFNβ-ACE2, sACE2(18-611), IFN-β, or the unlinked combination of IFN-β and sACE2(18-611). After a 1 h incubation, the virus + protein mixtures were washed, and the retentate containing virion–protein complexes was used for infection of Vero E6-TMPRSS2-T2A-ACE2 or A549 cells, respectively, in a 96-well plate (A,B). After the washing step, IFNβ-ACE2 or the unlinked combination of IFN-β and sACE2(18-611) were directly added to designated groups (B). Alternatively, the virus + protein mixtures were not subjected to a virus-washing step and the mixtures were used for infection of the respective host cells (C–E). The cells were harvested after a 2-day incubation and stained with LIVE/DEAD Fixable Blue Dead Cell Stain. After fixation and permeabilization, Vero E6-TMPRSS2-T2A-ACE2 cells were stained with AF647-conjugated rabbit anti-NL63 nucleocapsid antibody and A549 cells were stained with AF647-conjugated rabbit anti-229E nucleocapsid antibody. Cells were then analyzed for viral infection by flow cytometry. Cells were gated on viable, single, and live cells before subgating on nucleocapsid+ cells. Shown (A) are the percentages of nucleocapsid+ cells for each treatment group normalized to the ‘no protein’ control group (1 nM concentrations). (B) Bar graph shows mean percentages of nucleocapsid+ 229E-infected cells when proteins were or were not added after the washing step (1 nM concentrations). Shown (C) are representative dot plots including percentages of nucleocapsid+ 229E-infected cells (1 nM concentrations). Shown (D) are the percentages of nucleocapsid+ cells for each treatment group normalized to the ‘no protein’ group for each virus (1 μM concentrations). Shown (E) are the percentages of nucleocapsid+ 229E-infected cells for each treatment group at designated concentrations (100 fM to 100 nM). Each data point represents the mean value (n = 2), and error bars represent SD. Statistical significance was analyzed by use of two-way ANOVA with Tukey’s multiple comparisons test comparing the IFN-β and/or ACE2 treatment groups to the IFNβ-ACE2 treatment group at each concentration (unless otherwise noted in the figure) (ns nonsignificant, **** p < 0.0001). Experiments shown are representative of three independent experiments.

Fig 4: The sACE2 domain of IFNβ-ACE2 targeted IFN-β to the surface of NL63. NL63 was incubated at 4 °C with designated concentrations of IFNβ-ACE2, sACE2(18-611), recombinant IFN-β, or IFN-β (Peprotech). After a 1 h incubation, NL63 was washed of any unbound protein using 300kD centrifugal filters. NL63-protein complexes were then added to Vero E6-TMPRSS2-T2A-ACE2 cultures (100 μL) in a 96-well plate. The cells were harvested after a 2-day incubation at 33 °C, stained with LIVE/DEAD Fixable Blue Dead Cell Stain, and then surface-labeled with FITC-conjugated anti-human ACE2 and PE-conjugated anti-human TMPRSS2. After fixation and permeabilization, cells were stained with AF647-conjugated rabbit anti-NL63 nucleocapsid antibody. Cells were then analyzed for viral infection by flow cytometry. Cells were gated on viable, single, and live cells (parental gate) before subgating on nucleocapsid+, ACE2high, or TMPRSS2high cells. Shown are representative dot plots ((A,D,I), x-axis = FSC-A as in (A)) when NL63 was incubated with 1 nM IFNβ-ACE2 or controls. The IFNβ-ACE2 versus sACE2(18-611) groups were compared based on percentages of nucleocapsid+ cells (B), percentages of ACE2high cells (E), MFI of anti-ACE2 staining (F), percentages of TMPRSS2high cells (J), and MFI of anti-TMRSS2 staining (K). The IFNβ-ACE2 versus IFN-β groups were compared based on percentages of nucleocapsid cells (C), percentages of ACE2high cells (G), MFI of anti-ACE2 staining (H), percentages of TMPRSS2high cells (L), and MFI of anti-TMPRSS2 staining (M). Cell percentages were calculated by dividing events in the positive subgate by the parental gate, and MFI values represent all events in the parental gate. Each data point represents the mean value (n = 2), and error bars represent SD. Statistical significance comparing IFN-β and ACE2 treatment groups to the IFNβ-ACE2 treatment group at each concentration was analyzed by use of two-way ANOVA with Tukey’s multiple comparisons test (** p < 0.01, *** p < 0.001, **** p < 0.0001). Statistical significance comparing each protein group at each concentration to blank was analyzed by use of two-way ANOVA with Tukey’s multiple comparisons test (° p < 0.05, °° p < 0.01, °°° p < 0.001, °°°° p < 0.0001). These data are representative of three independent experiments.