Sodium Polyoxotungstate Inhibits the Replication of Influenza Virus by Blocking the Nuclear Import of vRNP

Abstract

Both pandemic and seasonal influenza are major health concerns, causing significant mortality and morbidity. Current influenza drugs primarily target viral neuraminidase and RNA polymerase, which are prone to drug resistance. Polyoxometalates (POMs) are metal cation clusters bridged by oxide anions. They have exhibited potent anti-tumor, antiviral, and antibacterial effects. They have remarkable activity against various DNA and RNA viruses, including human immunodeficiency virus, herpes simplex virus, hepatitis B and C viruses, dengue virus, and influenza virus. In this study, we have identified sodium polyoxotungstate (POM-1) from an ion channel inhibitor library. In vitro, POM-1 has been demonstrated to have potent antiviral activity against H1N1, H3N2, and oseltamivir-resistant H1N1 strains. POM-1 can cause virion aggregation during adsorption, as well as endocytosis. However, the aggregation is reversible; it does not interfere with virus adsorption and endocytosis. Our results suggest that POM-1 exerts its antiviral activity by inhibiting the nuclear import of viral ribonucleoprotein (vRNP). This distinct mechanism of action, combined with its wide range of efficacy, positions POM-1 as a promising therapeutic candidate for influenza treatment and warrants further investigation.

Keywords: antiviral; influenza virus; nuclear import of vRNP; sodium metatungstate; virion aggregation.

Figure 1

POM-1 inhibits influenza virus replication in vitro. (A) A549 and MDCK cells were infected with the PR8 virus at a multiplicity of infection (MOI) of 0.1 and 0.02, respectively, and serially diluted POM-1 was added during infection. After 48 h of incubation at 37 °C, supernatants were collected, the inhibitory effects of POM-1 on virus replication were determined based on the reduction in NA activities using NA activity assay. Cell viability was determined by Cell Titer-Glo assay after 48 h of exposure to POM-1 at the indicated concentrations. (B) The production of infectious virions in supernatants after treatment with serially diluted POM-1 was determined by TCID₅₀ assay. ND is not detectable. (C) A549 and MDCK cells were infected with 0.1 or 0.02 MOI of H1N1, H3N2, or oseltamivir-resistant H1N1 virus (H1N1(Ose)) and treated with various concentrations of POM-1. At 24 hpi, the cells were fixed with 4% PFA, and the influenza virus NP expression was detected by IFA using a monoclonal NP antibody and imaged using a Nikon Eclipse Ti2 inverted microscope. (D) The fluorescence intensities were quantified using ImageJ software (version 7.04) and normalized to virus control. Values are expressed as the means ± SEM of three independent experiments.

Figure 2

POM-1 inhibits influenza virus replication in the early stage of its life cycle. (A) A549 cells were infected with the PR8 virus at an MOI of 0.5, and 50 μM POM-1 was added at the indicated times. At 10 hpi, the cells were fixed and stained with anti-NP antibody and Alexa Fluor 488-labeled secondary antibody, followed by 4′,6-diamidino-2-phenylindole (DAPI) staining. The virus replication level, represented by the expression of virus NP, was determined by IFA and visualized under a Nikon Eclipse Ti2 inverted microscope. (B) The virus replication level was quantified by calculating the ratios of infected cells per field and normalizing them to virus control. Values are expressed as the means ± SEM of three independent experiments. The statistical significance of the results is indicated by * p < 0.05, **** p < 0.0001. ns, not significant when p > 0.05. (C) A549 cells were precooled at 4 °C for 30 min before infection with PR8 (MOI = 1) at 4 °C for 1 h. For pretreatment of POM-1 during viral attachment (−60–0 min) at 4 °C, and treatment of POM-1 (100 µM) during which POM-1 was added at different times post-virus infection, the whole-cell lysates were collected at 8 hpi, and the viral NP protein levels were determined by Western blotting. (D) In the virucidal assay, the PR8 virus solutions were incubated with different concentrations of POM-1 at 37 °C for 1 h, followed by being diluted and titrated using the TCID₅₀ assay. For controls, the virus- and medium-containing compounds were separately incubated at 37 °C for 1 h. Then, they were diluted and mixed before titration. Values are expressed as the means ± SEM of three independent experiments.

Figure 3

POM-1 causes virion aggregation during adsorption to host cells and blocks the nuclear import of vRNP. (A) A549 cells were precooled at 4 °C for 30 min, then infected with PR8 at an MOI of 10 in the absence or presence of POM-1 at indicated concentrations, and incubated at 4 °C for 1 h. The cells were fixed with 4% PFA, NP was fluorescently stained (green) using IFA, and the nucleus was DAPI-stained (blue) and imaged using a Leica SP8 confocal microscope. (B) After viral adsorption (MOI = 0.5) at 4 °C for 1 h in the absence or the presence of POM-1 at indicated concentrations, A549 cells were lysed immediately, and NP protein levels were determined by Western blotting. (C) The A549 cells were attached by PR8 virus (MOI = 2) for 1 h in the absence or presence of 100 µM POM-1 at 4 °C. Then, cells were fixed, dehydrated, coated, and imaged with a scanning electron microscope. The rectangular line-selected area is enlarged and displayed on the right, while arrows point out scattered virions, and the aggregated virions are indicated by an oval dotted line. (D) A549 cells were infected with PR8 at an MOI 1 and treated with 100 µM POM-1 at 37 °C until fixed at 1, 3, 5, and 7 hpi., respectively. Viral NP (green) and nuclei (blue) were visualized using confocal microscopy. (E) The endocytosis in (D) was determined by counting the number of NP fluorescent foci in the cytoplasm per cell. The numbers of counted cells at 1 hpi for the virus control group and POM-1-treated group were 35 and 34, respectively, and for 3 hpi, they were 38 and 39. (F) Infected cells containing NP nuclei were considered the import of vRNP, while the percentage of cells with infected nuclei in (D) was calculated. The number of cells counted for the virus control group and POM-1-treated group at 3 hpi were 95 and 91, respectively, while at 5 hpi, they were 73 and 106. (G) The total fluorescence intensities per field in (D) were quantified using ImageJ software and normalized to virus control at 1 hpi. Values are expressed as the means ± SEM of three independent experiments. The statistical significance of the results is indicated by **** p < 0.0001; ns, not significant.

Figure 3

POM-1 causes virion aggregation during adsorption to host cells and blocks the nuclear import of vRNP. (A) A549 cells were precooled at 4 °C for 30 min, then infected with PR8 at an MOI of 10 in the absence or presence of POM-1 at indicated concentrations, and incubated at 4 °C for 1 h. The cells were fixed with 4% PFA, NP was fluorescently stained (green) using IFA, and the nucleus was DAPI-stained (blue) and imaged using a Leica SP8 confocal microscope. (B) After viral adsorption (MOI = 0.5) at 4 °C for 1 h in the absence or the presence of POM-1 at indicated concentrations, A549 cells were lysed immediately, and NP protein levels were determined by Western blotting. (C) The A549 cells were attached by PR8 virus (MOI = 2) for 1 h in the absence or presence of 100 µM POM-1 at 4 °C. Then, cells were fixed, dehydrated, coated, and imaged with a scanning electron microscope. The rectangular line-selected area is enlarged and displayed on the right, while arrows point out scattered virions, and the aggregated virions are indicated by an oval dotted line. (D) A549 cells were infected with PR8 at an MOI 1 and treated with 100 µM POM-1 at 37 °C until fixed at 1, 3, 5, and 7 hpi., respectively. Viral NP (green) and nuclei (blue) were visualized using confocal microscopy. (E) The endocytosis in (D) was determined by counting the number of NP fluorescent foci in the cytoplasm per cell. The numbers of counted cells at 1 hpi for the virus control group and POM-1-treated group were 35 and 34, respectively, and for 3 hpi, they were 38 and 39. (F) Infected cells containing NP nuclei were considered the import of vRNP, while the percentage of cells with infected nuclei in (D) was calculated. The number of cells counted for the virus control group and POM-1-treated group at 3 hpi were 95 and 91, respectively, while at 5 hpi, they were 73 and 106. (G) The total fluorescence intensities per field in (D) were quantified using ImageJ software and normalized to virus control at 1 hpi. Values are expressed as the means ± SEM of three independent experiments. The statistical significance of the results is indicated by **** p < 0.0001; ns, not significant.

Figure 4

The aggregation of virions caused by POM-1 during adsorption is reversible if POM-1 is removed. (A) A549 cells were infected with PR8 and treated with 100 µM POM-1 at 4 °C for 1 h. After removing the supernatants, the cells were washed twice with PBS before being cultured in a fresh medium at 37 °C. Infected cells (MOI = 5) were fixed at different time points post-infection, and NP was fluorescently stained (green) using IFA, while the nucleus was stained (blue) with DAPI. Imaging was performed using a Leica SP8 confocal microscope. (B) The endocytosis in (A) was determined by quantifying the relative fluorescent intensities of NP foci in cytoplasm per cell. The number of counted cells at 2 hpi for virus control group and POM-1-treated group were 36 and 46, respectively. (C) Infected nuclei containing NP were considered imports of vRNP, while the percent of cells with infected nuclei in (A) were calculated per field. The number of counted cells at 4 hpi for the virus control group and POM-1-treated group were 79 and 79, respectively. Values are expressed as the means ± SEM of three independent experiments. The statistical significance of the results is indicated by * p < 0.05; ns, not significant. (D) A549 cells were treated as described in (A), except that the infected cells (MOI = 1) were lysed, and NP protein levels at different times post-infection were determined by Western blotting.

Figure 5

POM-1 causes virion aggregation in the cytoplasm and hinders the nuclear import of vRNP. (A) A549 cells were precooled at 4 °C for 30 min and infected with PR8 for 1 h. Subsequently, cells were washed, followed by the addition of medium containing POM-1 (100 µM) at 0 hpi and incubated at 37 °C. At different time points post-infection, infected cells (MOI = 5) were fixed and visualized using IFA staining for NP (green), while the nucleus was stained with DAPI (blue). (B) A549 cells were treated as described in (A) except for the used MOI of 1. Infected nuclei containing NP were considered imports of vRNP, while the percentage of cells with infected nuclei was calculated per field. The number of counted cells at 4 hpi for the virus control group and POM-1-treated group were 79 and 90, respectively, and for 6 hpi, they were 117 and 82. Values are expressed as the means ± SEM of three independent experiments. The statistical significance of the results is indicated by **** p < 0.0001. (C) A549 cells were treated as described in (A), except that lysed infected cells (MOI = 1) were subjected to Western blotting to determine NP protein levels. (D) A549 cells were precooled and infected with PR8 at 4 °C for 1 h. Then, the cells were washed twice with PBS and cultured at 37 °C in the presence of POM-1 (100 µM) for an additional hour; subsequently, they were washed again with PBS and cultured in a fresh DMEM medium. At different times post-infection, infected cells (MOI = 5) were fixed and visualized using IFA staining for NP protein expression (green), while the nucleus was stained with DAPI (blue). (E) A549 cells underwent treatment as described in (D), except that lysed infected cells (MOI = 1) were subjected to Western blotting analysis to determine NP protein levels.

Figure 6

POM-1 does not impact the release of influenza virions. (A,B) In the neuraminidase inhibition assay, the PR8 virus was incubated with serially diluted POM-1 or oseltamivir at 37 °C for 1 h. Subsequently, the mixtures were incubated with MUNANA at 37 °C for 1 h, and the inhibition rate was calculated based on the fluorescence intensity measured using a microplate reader. The data are presented as means ± SEM from three independent experiments. (C) A549 cells were infected with PR8 at an MOI of 0.5 for 6 h, followed by the replacement of a culture medium with DMEM containing different concentrations of POM-1. At 10 hpi, supernatants were collected and assessed for infectious virion production using a TCID₅₀ assay after dialysis to remove POM-1 or without dialysis. Data are presented as means ± SEM from three independent experiments. (D) Western blotting was performed to analyze viral NP levels in collected supernatants and total lysates in infected cells.