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.

Given the importance of both RIG-I and PKR in EBOV's evasion of the host antiviral response (Cardenas et al., 2006; Feng et al., 2007; Leung et al., 2010; Luthra et al., 2013; Schumann et al., 2009) , and reports that NTZ can weakly induce auto-phosphorylation of PKR (Ashiru et al., 2014; Elazar et al., 2009) , we next sought to determine the role of these molecules in NTZ's antiviral activity using the VSV model system. Because traditional short hairpin RNA (shRNA)-based methods of gene knockdown, including in A549 cells, are associated with both non-specific type I IFN induction by the shRNA itself, and reduced shRNA activity in response to type I IFN stimulation (Machitani et al., 2017) , we chose to knockdown RIG-I or PKR expression in A549 cells by targeting dead(d)Cas9-KRAB to their genes' promoters (see Transparent Methods and Figure S1 for gRNA target sequences and Figure 3B for protein knockdown efficiency). RIG-I depletion ( at any NTZ concentration tested (50% inhibition with 4.8 mM NTZ in CRISPR-Ctrl A549 cells versus 4.7 mM NTZ in CRISPR-PKR A549 cells) ( Figure 3D , right panel), indicating that NTZ's suppression of VSV replication is not dependent on PKR.