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.

Although three previous reports have suggested that ISGs induced by NTZ or its metabolite TIZ play an antiviral role, a direct functional impact of NTZ or TIZ upon any of these genes with respect to inhibition of viral growth, has not been shown. For example, one report described increased average expression of a panel of 26 ISGs in peripheral blood mononuclear cells (PBMCs) treated with a very low concentration (0.1 mM) of NTZ for an unspecified time, with no functional demonstration of how any of these individual ISGs affected replication of a virus in NTZ-treated cells (Petersen et al., 2016) . Another article reported that treatment of human monocyte-derived macrophages (MDM) with NTZ for 48 h induced the expression of the ISGs tetherin and APOBEC3A/G, and although the authors associated this with NTZ inhibition of HIV in MDMs, no functional demonstration was provided showing that these molecules played any role in NTZ's anti-HIV activity (Gekonge et al., 2015) . A third study reported that TIZ treatment of human PBMCs for 7 days induced a set of 14 IFNs and ISGs approximately 2-fold when compared with untreated PBMCs, with a slightly greater increase, on average, observed in the context of HIV infection (including Mx1, 1.8to 2.9-fold respectively); however, again, no functional links between the ISGs identified and NTZ inhibition of HIV replication were made, and no mechanism for this response was investigated (Trabattoni et al., 2016) . In contrast to the above studies, through both biochemical and gene knockdown approaches we have demonstrated that NTZ treatment results in broad amplification of RLR pathway components that are primary drivers of type I IFN production and ISG induction and amplifies type I IFN signaling. Furthermore, we have functionally linked RIG-I and PKR to NTZ's antiviral activity against EBOV, and functionally linked RIG-I and GADD34 to NTZ's activity against VSV using CRISPR/dCas9-KRAB-mediated knockdown of these molecules in host cells.