Nitazoxanide Inhibits Human Norovirus Replication and Synergizes with Ribavirin by Activation of Cellular Antiviral Response

TZD robustly stimulated the expression of IRF-1 and several other ISGs in a HuNV replicon model and intestinal models of Caco-2 cells and primary organoids. (A) Fold changes of ISGs induced by NTZ (10 μg/ml), TIZ (10 μg/ml), or IFN-α (1,000 IU/ml) in HG23 cells. After 2 days of treatment, the levels of ISG mRNA were quantified by qRT-PCR and normalized to those of human GAPDH. The results are expressed as fold changes compared to the vehicle control (DMSO; n = 3 independent experiments, each in duplicate). (B) Western blot analysis of IRF-1 protein expression in response to NTZ (10 μg/ml), TIZ (10 μg/ml), or IFN-α (1,000 IU/ml). The data are representative of the results of three independent experiments. (C) Naive Huh7 cells were treated with NTZ (10 μg/ml), TIZ (10 μg/ml), IFN-α (1,000 IU/ml), or vehicle control. After 2 days of treatment, relative levels of ISG mRNA were quantified by qRT-PCR (n = 3 independent experiments, each in duplicate). (D) Morphology of 3D human primary intestinal organoids in Matrigel. (E) Relative levels of ISG RNA were quantified by qRT-PCR after 2 days of treatment with NTZ (10 μg/ml) or IFN-α (1,000 IU/ml) in Caco-2 cells (n = 3 independent experiments, each in duplicate). (C) Relative levels of ISG RNA in human intestinal organoids after treatment with NTZ (10 μg/ml) or IFN-α (1,000 IU/ml) for 2 days (n = 3 independent experiments, each in duplicate). The data are presented as means and SEM (*, P < 0.05; **, P < 0.01).

IRF-1 mediated TZD-triggered inhibition of HuNV replication. (A) To overexpress (OE) IRF-1, HG23 cells were transduced with lentiviral IRF-1-expressing vector or Fluc-expressing vector (control). After 2 days of transduction, the levels of IRF-1 expression were detected by qRT-PCR (n = 3 independent experiments, each in duplicate) and Western blotting (n = 3 independent experiments). IFN-α (1,000 IU/ml) was used as a positive control. (B) Overexpression of IRF-1 potently inhibited HuNV replication (n = 3 independent experiments, each in duplicate). (C) To further evaluate the role of basal IRF-1 in HuNV replication, HG23 cells were transduced with lentiviral shRNA vector targeting IRF-1. A scrambled vector was used as a nontargeting control. Successful knockdown of IRF-1 was confirmed by qRT-PCR (n = 3 independent experiments, each in duplicate) and Western blotting (n = 3 independent experiments). (D) Knockdown of IRF-1 had minor effects on HuNV replication (n = 3 independent experiments, each in duplicate). (E) IRF-1 shRNA and nontargeting control cells were mock treated or treated with the indicated concentrations of TZD. After 2 days of treatment, the levels of HuNV RNA were quantified by qRT-PCR and compared to those of the respective control (n = 3 independent experiments, each in duplicate). The data are presented as means and SEM (**, P < 0.01; ***, P < 0.001; ns, not significant).

Stimulation of antiviral response by TZD was independent of the JAK-STAT pathway. (A) HG23 cells were treated with vehicle only (DMSO; control), NTZ (10 μg/ml), TIZ (10 μg/ml), or IFN-α (100 IU/ml) alone or in combination with CP-690550 (1,000 ng/ml). After 2 days of treatment, HuNV replication was quantified by qRT-PCR (n = 3 independent experiments, each in duplicate). (B and C) The expression of IRF-1 and other ISGs, including PKR, ISG15, and MDA5, was quantified by qRT-PCR (n = 3 independent experiments, each in duplicate). (D) A STAT1 KO clone was established from Huh7 cells expressing STAT1 sgRNAs. Successful KO of STAT1 was confirmed by Western blotting. (E) To further confirm successful KO of STAT1, Huh7 control and STAT1 KO cells were treated with IFN-α (1,000 IU/ml) for 2 days. The levels of STAT1 and STAT2 RNA were evaluated by qRT-PCR (n = 3 independent experiments, each in duplicate). KO of STAT1 abolished the induction of STAT1, but not STAT2, after IFN-α treatment. (F and G) Huh7 control and STAT1 KO cells were mock treated (DMSO; control) or treated with NTZ (10 μg/ml), TIZ (10 μg/ml), or IFN-α (1,000 IU/ml). After 2 days of treatment, the expression levels of IRF-1, MDA5, Mx1, DDX60, ISG15, and IFIT1 were detected by qRT-PCR. The results were first normalized to human GAPDH and then compared to control cells (n = 3 independent experiments, each in duplicate). The data are presented as means and SEM (*, P < 0.05; **, P < 0.01; ns, not significant).

TZD worked additively with ribavirin to inhibit HuNV replication after short-term treatment. (A) HG23 cells were treated with various concentrations of NTZ alone, ribavirin alone, or the two in combination for 2 days. (Left) Antiviral activities were determined by qRT-PCR (n = 3 independent experiments, each in duplicate). (Right) To further explore the drug-drug interaction, the antiviral results were analyzed with a mathematical model. The 3D surface plot shown represents the difference (within a 95% confidence interval [CI]) between actual experimental effects and the theoretical additive effect of the combination at various concentrations of the two compounds. (B) Combination of TIZ with ribavirin. The error bars represent SEM.

Long-term combination of NTZ with ribavirin synergistically inhibited HuNV replication and completely depleted HuNV replicons from host cells. (A) HG23 cells were incubated with NTZ alone, ribavirin alone, or the two drugs in combination. After 2 days of culture, the cells were passaged to fresh drug-containing medium for another 4 days of incubation. After 6 days, the cells were passaged with another round of 4 days of treatment. At the end of each treatment, the HG23 cells were harvested and analyzed for levels of HuNV RNA (n = 3 independent experiments, each in duplicate). (B) Synergy analysis. The antiviral results after 10 days of treatment were analyzed with MacSynergy. (C) Rebound assay. After 10 days of treatment, HG23 cells were plated into a 48-well plate (2 × 10⁵ cells per well) containing 250 μl medium with 1 mg/ml G418. After 5 days of culture, the cell colonies were stained and visualized using an inverted light microscope. The data presented are representative of 3 independent experiments. The data are presented as means ± SEM (**, P < 0.01).

Combining TIZ with ribavirin synergistically inhibited HuNV replication. (A) Combination of TIZ with ribavirin showed greater efficacy against HuNV replication than the individual drugs (n = 3 independent experiments, each in duplicate). (B) Synergy analysis. (C) Rebound assay after long-term treatment. The data are presented as means ± SEM (**, P < 0.01).