Antiviral Activity of Nordihydroguaiaretic Acid and Its Derivative Tetra- O-Methyl Nordihydroguaiaretic Acid against West Nile Virus and Zika Virus

Abstract

Flaviviruses are positive-strand RNA viruses distributed all over the world that infect millions of people every year and for which no specific antiviral agents have been approved. These viruses include the mosquito-borne West Nile virus (WNV), which is responsible for outbreaks of meningitis and encephalitis. Considering that nordihydroguaiaretic acid (NDGA) has been previously shown to inhibit the multiplication of the related dengue virus and hepatitis C virus, we have evaluated the effect of NDGA, and its methylated derivative tetra-O-methyl nordihydroguaiaretic acid (M₄N), on the infection of WNV. Both compounds inhibited the infection of WNV, likely by impairing viral replication. Since flavivirus multiplication is highly dependent on host cell lipid metabolism, the antiviral effect of NDGA has been previously related to its ability to disturb the lipid metabolism, probably by interfering with the sterol regulatory element-binding proteins (SREBP) pathway. Remarkably, we observed that other structurally unrelated inhibitors of the SREBP pathway, such as PF-429242 and fatostatin, also reduced WNV multiplication, supporting that the SREBP pathway may constitute a druggable target suitable for antiviral intervention against flavivirus infection. Moreover, treatment with NDGA, M₄N, PF-429242, and fatostatin also inhibited the multiplication of the mosquito-borne flavivirus Zika virus (ZIKV), which has been recently associated with birth defects (microcephaly) and neurological disorders. Our results point to SREBP inhibitors, such as NDGA and M₄N, as potential candidates for further antiviral development against medically relevant flaviviruses.

Keywords: West Nile virus; Zika virus; antiviral agents; flavivirus; lipid.

FIG 1

NDGA and its synthetic derivative M₄N inhibit WNV infection. (A) Chemical structure of NDGA and M₄N. R corresponds to OH in NDGA or OCH₃ in M₄N. (B and C) Reduction of WNV infection in Vero cells treated with NDGA (B) or M₄N (C). Cells were infected with WNV lineage 1 strain NY99 (MOI of 1 PFU/cell), and virus yield in culture supernatant was determined by plaque assay at 24 h p.i. (D and E) Inhibition of WNV lineage 2 infection in Vero cells treated with NDGA (D) or M₄N (E). Cells were infected with WNV SRB-Novi Sad/12 (MOI of 1 PFU/cell), and virus yield in culture supernatant was determined by plaque assay at 24 h p.i. (F and G) Evaluation of the cytotoxicity of NDGA (F) and M₄N (G) on Vero cells by determination of ATP content 24 h posttreatment. Data are presented as means ± SDs (n = 3 to 6). Statistically significant differences are indicated. *, P < 0.05; **, P < 0.005.

FIG 2

Evaluation of the direct effect of NDGA and M₄N on the infectivity of WNV. WNV NY99 (∼1.5 × 10⁹ PFU) was treated with NDGA (A) or M₄N (B) for 1 h at 37°C in culture medium. Then, the infectivity in each sample was determined by plaque assay. Data are presented as means ± SDs (n = 4). Statistically significant differences are indicated. **, P < 0.005.

FIG 3

NDGA and M₄N inhibit WNV replication. (A and B) Reduction of genome-containing particles in culture supernatant of Vero cells treated with NDGA (A) or M₄N (B). Cells were infected with WNV NY99 (MOI of 1 PFU/cell), and the amount of genome-containing particles in culture supernatant was determined by quantitative RT-PCR at 24 h p.i. (C and D) Amount of cell-associated viral RNA in cell cultures infected with WNV and treated with NDGA (C) or M₄N (D) as described in A and B. The amount of cell-associated RNA in each sample was normalized by quantification of rRNA 18S. (E) Visualization of intracellular dsRNA accumulation in cells infected with WNV NY99 (MOI of 10 PFU/cell) and treated with 35 μM NDGA or 35 μM M₄N. Infected cells treated with drug vehicle (DMSO) were included as a control. Cells were fixed and processed for immunofluorescence using J2 monoclonal antibody to dsRNA and a secondary antibody coupled to Alexa Fluor 488 (green). Nuclei were stained with To-Pro-3 (blue). Bar, 10 μm. (F) Quantification of the fluorescence intensity of dsRNA in cells infected with WNV and treated with 35 μM NDGA or M₄N as shown in E (n = 89, 36, and 39 cells analyzed for DMSO, NDGA, and M₄N, respectively). (G) Western blot analysis of E glycoprotein expression in cells treated with NDGA or M₄N. Vero cells were infected, or not, with WNV NY99 (MOI of 1 PFU/cell) and treated with different concentrations of NDGA or M₄N. Membrane was retested against a β-actin antibody as a control for protein loading. (H) RSP release into the culture medium by HeLa3-WNV cells treated (4 h) with 35 μM NDGA, 35 μM M₄N, or 5 μg/ml BFA was analyzed by enzyme-linked immunodot assay using a monoclonal antibody against E glycoprotein. The graph displays the quantification of the amount of RSPs released. For each drug, the cellular ATP content is also depicted as an indicator of the cell viability upon drug treatment. Data are presented as means ± SDs (n = 3 to 6). Statistically significant differences are indicated. **, P < 0.005.

FIG 4

Different inhibitors of the SREBP pathway reduce WNV infection. (A) Chemical structure of the SREBP pathway inhibitors tested: resveratrol, PF-429242, and fatostatin. The structure of zileuton, a 5-LOX inhibitor, is also depicted. (B to E) Quantification of WNV infectious particle production in Vero cells infected with WNV NY99 (MOI of 1 PFU/cell) and treated with resveratrol (B), PF-429242 (C), fatostatin (D), or zileuton (E). Virus yield in culture supernatant was determined by plaque assay at 24 h p.i. (F to I) Evaluation of the cytotoxicity of resveratrol (F), PF-429242 (G), fatostatin (H), and zileuton (I) on Vero cells by determination of ATP content 24 h posttreatment. Data are presented as means ± SDs (n = 4). Statistically significant differences are indicated. **, P < 0.005.

FIG 5

Reduction of ZIKV infectious particle production in Vero cells treated with NDGA, M₄N, fatostatin, or PF-429242. Cells were infected with ZIKV PA259459 (MOI of 1 PFU/cell), treated with 35 μM of each drug, or not (DMSO), and the virus yield in culture supernatant was determined by plaque assay at 24 h p.i. Data are presented as means ± SDs (n = 3). Statistically significant differences between control (DMSO) and drug-treated cells are indicated. **, P < 0.005.