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 determine if RIG-I, PKR, or GADD34 were targets of NTZ and required for its inhibitory effects upon EBOV, we next pretreated the CRISPR/dCas9-KRAB nonspecific control cells and the CRISPR/dCas9-RIG-I-KD, PKR-KD, or GADD34-KD A549 cell lines with 0 or 20 mM NTZ for 4 h and then infected with EBOV Kikwit at an MOI of 0.01. As shown in Figure 4B (left), there was a 10.5-fold reduction (>1 log change) A B Figure 4 . NTZ Requires PKR and RIG-I, but Not GADD34, for Full Inhibition of EBOV Replication in A549 Cells (A) Knockdown of RIG-I and PKR in A549 cells augments EBOV replication, whereas knockdown of GADD34 has a more modest effect on EBOV growth. Cells were mock-treated or treated with 20 mM NTZ for 4 h and infected with EBOV at MOI of 0.01 in the presence of pretreatment NTZ supernatants. Supernatants were harvested at 1, 48, or 72 h, clarified, and EBOV plaque-forming unit (PFU) was calculated by plaque assay. Titers are displayed on linear (left) and log (right) scales for clarity. **p < 0.01, ***p < 0.001. (B) Compared with control A549 cells, NTZ inhibition of EBOV replication is impaired in RIG-I-KD and PKR-KD A549 cells but is not affected by GADD34 knockdown. Cells were mock-treated or treated with 20 mM NTZ for 4 h and infected with EBOV at MOI of 0.01 in the presence of pretreatment NTZ supernatants. Supernatants were harvested at 1, 48, or 72 h, clarified, and EBOV PFU was calculated by plaque assay. Titers are displayed on linear (left) and log (right) scales for clarity.