Fig 2: Therapeutic RDV but not LPV/RTV-IFNb diminishes signs of ALI.a Representative images of the histological features of acute lung injury 6 dpi comparing a mock-infected mouse to the therapeutic treatment groups described in Figs. 5 and 6. Symbols identifying example features of disease are indicated in the figure. b American Thoracic Society Lung Injury Score derived as described in Fig. 3. The numbers of animals per group quantitated: vehicle RDV N = 7, RDV N = 7, vehicle LPV/RTV-IFNb N = 9, LPV/RTV-IFNb low N = 7, LPV/RTV-IFNb high N = 8. c Diffuse alveolar damage score quantitating the degree of cellular sloughing, necrosis, and breakdown of barrier epithelium and vascular leakage. For both b and c, scores were blindly assessed in three random high power (×60) fields of diseased lung tissue sections. d Quantitation of cleaved caspase-3 antigen staining in lung tissue sections from studies described in Figs. 5–7. Cleaved caspase-3 is a marker of cell death. The numbers of animals per group quantitated for all groups was N = 5/group. For the box and whisker plots, the boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range. For b–d, asterisks indicate statistical significance by one-way ANOVA and Kruskal–Wallis multiple comparison test.

Fig 3: RDV and IFNb have superior antiviral activity to LPV and RTV.Graphs depict mean % inhibition of MERS-CoV replication (left Y-axis) and % cytotoxicity (right Y-axis) of antivirals. Calu-3 cells were infected in sextuplicate with MERS-CoV nanoluciferase (nLUC) at a multiplicity of infection (MOI) of 0.08 in the presence of a dose response of drug for 48 h, after which replication was measured through quantitation of MERS-CoV–expressed nLUC. Cytotoxicity was measured in similarly treated but uninfected cultures via Cell-Titer-Glo assay. Representative data are shown from four independent experiments.

Fig 4: Therapeutic RDV reduces replication and pathology.Percent starting weight of 10–12-week-old female Ces1c-/- hDPP4 mice infected with 5E + 04 pfu MERS M35C4 and treated with a subcutaneous vehicle for RDV (N = 13) or remdesivir (RDV, 25 mg/kg, N = 14) BID beginning 1 dpi or b vehicle for LPV/RTV-IFNb (N = 15), LPV/RTV-IFNb low (N = 16) or LPV/RTV-IFNb high (N = 16) beginning 1 dpi. Oral vehicle or lopinavir/ritonavir (160/40 mg/kg) was administered orally once daily. IFNb low (1x human equivalent dose of 1.6 MIU/kg) and high (25x human equivalent dose of 40 MIU/kg) or PBS vehicle were administered via subcutaneous injection every other day. Asterisks indicate statistical differences by two-way ANOVA with Tukey’s multiple comparison test. c Lung hemorrhage 6 dpi for all animals in a, b scored on a scale of 0–4, where 0 is a normal pink healthy lung and 4 is a diffusely discolored dark red lung. d MERS-CoV lung titer 6 dpi in mice as described in a, b. Asterisks indicate statistical significance (N group described in a and b, P < 0.05) by one-way ANOVA with Kruskal–Wallis test for (c, d). Data for a–d are compiled from two independent experiments. For the box and whisker plots, the boxes encompass the 25th to 75th percentile, the line is at the median, while the whiskers represent the range. e Representative photomicrographs of MERS-CoV antigen (brown) and hematoxylin stained nuclei (blue) in mouse lung tissue sections from 6 dpi. The black bar is 100 µM.

Fig 5: Loss of ZBP1 attenuates IFN response and promotes crisis bypass.a, Line chart showing growth curves analysis of IMR90E6E7 or WI38SV40LT that have bypassed senescence expressing control sgRNAs (sgLUC, sgGFP) or three individual sgRNAs targeting ZBP1 (sgZBP1#1, sgZBP1#2, sgZBP1#3). Three independent experiments were performed. b, Crystal violet viability assay of IMR90E6E7 or WI38SV40LT as in a. Cells were seeded at day 30 and fixed at day 40. One experiment was performed. c, Immunoblotting of IMR90E6E7 or WI38SV40LT as in a. Protein extracts were collected at day 30 when control cells (sgLUC, sgGFP) entered crisis and upregulated ZBP1. GAPDH loading control. Two independent experiments were performed. d, Line chart showing IncuCyte analysis of cell death of IMR90E6E7 or WI38SV40LT as in a, measured in real time by Cytotox green incorporation at day 30 of the experiment. Bars represent mean ± s.e.m. from technical replicate. One-way ANOVA, *** p < 0.001. Two independent experiments were performed. e, Scatter plots with bars showing RT-qPCR analysis of IFNB1 in IMR90E6E7 or WI38SV40LT as in a. RNA extracts were collected at day 30 of the experiment. Expression levels were normalized to control cells with sgLUC. Bars represent mean ± s.d. of technical replicates. n: number of technical replicates. One-way ANOVA, ns: not significant, *** p < 0.001. Two independent experiments were performed. f, Scatter plots with bars showing ELISA assays of IFN-ß levels secreted by IMR90E6E7 or WI38SV40LT as in a. Culture supernatant were collected at day 30 of the experiment. Bars represent mean ± s.d. of technical replicates. n: number of technical replicates. One-way ANOVA, ns: not significant, *** p < 0.001. One experiment was performed. Abbreviations: GFP: Green fluorescent protein; LUC: luciferase; PDs: population doublings. Source data