The FDA-Approved Oral Drug Nitazoxanide Amplifies Host Antiviral Responses and Inhibits Ebola Virus

Jasenosky, Luke D.; Cadena, Cristhian; Mire, Chad E.; Borisevich, Viktoriya; Haridas, Viraga; Ranjbar, Shahin; Nambu, Aya; Bavari, Sina; Soloveva, Veronica; Sadukhan, Supriya; Cassell, Gail H.; Geisbert, Thomas W.; Hur, Sun; Goldfeld, Anne E.

To investigate whether GADD34 participates in NTZ's inhibitory effect on VSV, we knocked down GADD34 in A549 cells using the CRISPR/dCas9-KRAB strategy described above (see Transparent Methods and Figures S1 and 3B) and infected these GADD34-depleted A549 cells ( Figure 3F , left panel) with the VSV-Luc reporter virus. GADD34 depletion resulted in a significant increase in VSV infection/growth ( Figure 3F , right panel, 0 mM NTZ data point), similar to previously reported findings with VSV and GADD34 À/À mouse embryonic fibroblasts (Minami et al., 2007) . Furthermore, GADD34 depletion reduced VSV sensitivity to NTZ (50% inhibition at 4.8 mM NTZ in CRISPR-Ctrl A549 cells versus at 6.9 mM NTZ in GADD34-KD A549 cells) Figure 3 . NTZ Strongly Inhibits VSV Infection/Replication in A549 Cells: Depletion of RIG-I and GADD34, but Not PKR, Impairs Anti-VSV Activity of NTZ (A) Left panel: A549 cells were treated with NTZ at the indicated concentrations for 4 h and were then infected with a VSV firefly luciferase reporter (Cureton et al., 2012) . After 16 h, cells were harvested and firefly luciferase activity was measured by luminescence assay. Means and standard deviations from three independent experiments performed in duplicate are shown. Right panel: A549 cells were treated with vehicle or NTZ at the indicated concentrations for 18 h. Cells were harvested, exposed to 1 mM propidium iodide for 5 min, and subjected to flow cytometry. ( Figure 3F , right panel), indicating that NTZ's anti-VSV activity also depends on its ability to induce the expression of GADD34 in addition to its enhancement of RIG-I function. Taken together, although RIG-I, PKR, and GADD34, play a role in host inhibition of VSV, only RIG-I and GADD34, and not PKR, are involved in NTZ-mediated repression of this virus.