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A Novel Antiviral Target Structure Involved in the RNA Binding, Dimerization, and Nuclear Export Functions of the Influenza A Virus Nucleoprotein.

Kakisaka M, Sasaki Y, Yamada K, Kondoh Y, Hikono H, Osada H, Tomii K, Saito T, Aida Y - PLoS Pathog. (2015)

Bottom Line: The accuracy of this binding model was confirmed in a NP-RK424 binding assay incorporating photo-cross-linked RK424 affinity beads and in a plaque assay evaluating the structure-activity relationship of RK424.In addition, in vitro nuclear export assays confirmed that RK424 inhibited nuclear export of NP.Furthermore, we found that the NP pocket has a surface structure different from that of the pocket in host molecules.

View Article: PubMed Central - PubMed

Affiliation: Viral Infectious Diseases Unit, RIKEN, Wako, Saitama, Japan.

ABSTRACT
Developing antiviral therapies for influenza A virus (IAV) infection is an ongoing process because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. The ideal strategy is to develop drugs that target well-conserved, functionally restricted, and unique surface structures without affecting host cell function. We recently identified the antiviral compound, RK424, by screening a library of 50,000 compounds using cell-based infection assays. RK424 showed potent antiviral activity against many different subtypes of IAV in vitro and partially protected mice from a lethal dose of A/WSN/1933 (H1N1) virus in vivo. Here, we show that RK424 inhibits viral ribonucleoprotein complex (vRNP) activity, causing the viral nucleoprotein (NP) to accumulate in the cell nucleus. In silico docking analysis revealed that RK424 bound to a small pocket in the viral NP. This pocket was surrounded by three functionally important domains: the RNA binding groove, the NP dimer interface, and nuclear export signal (NES) 3, indicating that it may be involved in the RNA binding, oligomerization, and nuclear export functions of NP. The accuracy of this binding model was confirmed in a NP-RK424 binding assay incorporating photo-cross-linked RK424 affinity beads and in a plaque assay evaluating the structure-activity relationship of RK424. Surface plasmon resonance (SPR) and pull-down assays showed that RK424 inhibited both the NP-RNA and NP-NP interactions, whereas size exclusion chromatography showed that RK424 disrupted viral RNA-induced NP oligomerization. In addition, in vitro nuclear export assays confirmed that RK424 inhibited nuclear export of NP. The amino acid residues comprising the NP pocket play a crucial role in viral replication and are highly conserved in more than 7,000 NP sequences from avian, human, and swine influenza viruses. Furthermore, we found that the NP pocket has a surface structure different from that of the pocket in host molecules. Taken together, these results describe a promising new approach to developing influenza virus drugs that target a novel pocket structure within NP.

No MeSH data available.


Related in: MedlinePlus

Effect of RK424 on cytoplasmic localization of NP.(A) HeLa cells were infected with A/WSN/1933 (H1N1) virus at an MOI of 10 in the absence or presence of 0.5 and 2 μM of RK424 for 6 h. Subcellular localization of NP was observed by indirect immunofluorescence staining with anti-NP MAb under a confocal laser scanning microscope. Three independent experiments were performed and one representative result is shown. (B) HeLa cells were transfected for 48 h with NP/pCAGGS plasmid in the absence or presence of RK424 (0.5μM and 2 μM). The subcellular localization of NP was observed as described for virus-infected cells. Three independent experiments were performed and one representative result is shown. (C) The percentage of cells showing nuclear localization (black bar) or cytoplasm localization (white bar) of NP was calculated by counting 500 cells per sample. Oseltamivir phosphate (Os) was used as a negative control. Data are expressed as the mean ± SD in each of three independent experiments.
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ppat.1005062.g003: Effect of RK424 on cytoplasmic localization of NP.(A) HeLa cells were infected with A/WSN/1933 (H1N1) virus at an MOI of 10 in the absence or presence of 0.5 and 2 μM of RK424 for 6 h. Subcellular localization of NP was observed by indirect immunofluorescence staining with anti-NP MAb under a confocal laser scanning microscope. Three independent experiments were performed and one representative result is shown. (B) HeLa cells were transfected for 48 h with NP/pCAGGS plasmid in the absence or presence of RK424 (0.5μM and 2 μM). The subcellular localization of NP was observed as described for virus-infected cells. Three independent experiments were performed and one representative result is shown. (C) The percentage of cells showing nuclear localization (black bar) or cytoplasm localization (white bar) of NP was calculated by counting 500 cells per sample. Oseltamivir phosphate (Os) was used as a negative control. Data are expressed as the mean ± SD in each of three independent experiments.

Mentions: NP is the major component of the vRNP complex [30]. Therefore, to further investigate the effects of RK424 on the nuclear import and export of the vRNP complex in virus-infected cells, we examined NP localization by fluorescence microscopy. We found that NP localized in both the nucleus and cytoplasm at 6 h post-infection in HeLa cells treated with either DMSO (control) or 10 μM of oseltamivir phosphate. By contrast, treatment with RK424 (0.5 or 2 μM) led to nuclear rather than cytoplasmic localization of NP, indicating that the vRNP complex could enter the nucleus but could not be exported to the cytoplasm (Fig 3A). Importantly, the effects of RK424 in virus-infected HeLa cells were also observed in HeLa cells transiently expressing NP (Fig 3B). The cytoplasmic distribution of NP was dependent upon the dose of RK424. As shown in Fig 3C, the cytoplasmic distribution of NP in HeLa cells transiently expressing NP in the absence and presence of 0.5, 2, and 10 μM of RK424 was 75%, 77%, 57%, and 30%, respectively. This shows that RK424 interferes with the nuclear export of NP in the absence of other viral components. Taken together, these results suggest that RK 424 may exert its antiviral effects by inhibiting the function of NP.


A Novel Antiviral Target Structure Involved in the RNA Binding, Dimerization, and Nuclear Export Functions of the Influenza A Virus Nucleoprotein.

Kakisaka M, Sasaki Y, Yamada K, Kondoh Y, Hikono H, Osada H, Tomii K, Saito T, Aida Y - PLoS Pathog. (2015)

Effect of RK424 on cytoplasmic localization of NP.(A) HeLa cells were infected with A/WSN/1933 (H1N1) virus at an MOI of 10 in the absence or presence of 0.5 and 2 μM of RK424 for 6 h. Subcellular localization of NP was observed by indirect immunofluorescence staining with anti-NP MAb under a confocal laser scanning microscope. Three independent experiments were performed and one representative result is shown. (B) HeLa cells were transfected for 48 h with NP/pCAGGS plasmid in the absence or presence of RK424 (0.5μM and 2 μM). The subcellular localization of NP was observed as described for virus-infected cells. Three independent experiments were performed and one representative result is shown. (C) The percentage of cells showing nuclear localization (black bar) or cytoplasm localization (white bar) of NP was calculated by counting 500 cells per sample. Oseltamivir phosphate (Os) was used as a negative control. Data are expressed as the mean ± SD in each of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4519322&req=5

ppat.1005062.g003: Effect of RK424 on cytoplasmic localization of NP.(A) HeLa cells were infected with A/WSN/1933 (H1N1) virus at an MOI of 10 in the absence or presence of 0.5 and 2 μM of RK424 for 6 h. Subcellular localization of NP was observed by indirect immunofluorescence staining with anti-NP MAb under a confocal laser scanning microscope. Three independent experiments were performed and one representative result is shown. (B) HeLa cells were transfected for 48 h with NP/pCAGGS plasmid in the absence or presence of RK424 (0.5μM and 2 μM). The subcellular localization of NP was observed as described for virus-infected cells. Three independent experiments were performed and one representative result is shown. (C) The percentage of cells showing nuclear localization (black bar) or cytoplasm localization (white bar) of NP was calculated by counting 500 cells per sample. Oseltamivir phosphate (Os) was used as a negative control. Data are expressed as the mean ± SD in each of three independent experiments.
Mentions: NP is the major component of the vRNP complex [30]. Therefore, to further investigate the effects of RK424 on the nuclear import and export of the vRNP complex in virus-infected cells, we examined NP localization by fluorescence microscopy. We found that NP localized in both the nucleus and cytoplasm at 6 h post-infection in HeLa cells treated with either DMSO (control) or 10 μM of oseltamivir phosphate. By contrast, treatment with RK424 (0.5 or 2 μM) led to nuclear rather than cytoplasmic localization of NP, indicating that the vRNP complex could enter the nucleus but could not be exported to the cytoplasm (Fig 3A). Importantly, the effects of RK424 in virus-infected HeLa cells were also observed in HeLa cells transiently expressing NP (Fig 3B). The cytoplasmic distribution of NP was dependent upon the dose of RK424. As shown in Fig 3C, the cytoplasmic distribution of NP in HeLa cells transiently expressing NP in the absence and presence of 0.5, 2, and 10 μM of RK424 was 75%, 77%, 57%, and 30%, respectively. This shows that RK424 interferes with the nuclear export of NP in the absence of other viral components. Taken together, these results suggest that RK 424 may exert its antiviral effects by inhibiting the function of NP.

Bottom Line: The accuracy of this binding model was confirmed in a NP-RK424 binding assay incorporating photo-cross-linked RK424 affinity beads and in a plaque assay evaluating the structure-activity relationship of RK424.In addition, in vitro nuclear export assays confirmed that RK424 inhibited nuclear export of NP.Furthermore, we found that the NP pocket has a surface structure different from that of the pocket in host molecules.

View Article: PubMed Central - PubMed

Affiliation: Viral Infectious Diseases Unit, RIKEN, Wako, Saitama, Japan.

ABSTRACT
Developing antiviral therapies for influenza A virus (IAV) infection is an ongoing process because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. The ideal strategy is to develop drugs that target well-conserved, functionally restricted, and unique surface structures without affecting host cell function. We recently identified the antiviral compound, RK424, by screening a library of 50,000 compounds using cell-based infection assays. RK424 showed potent antiviral activity against many different subtypes of IAV in vitro and partially protected mice from a lethal dose of A/WSN/1933 (H1N1) virus in vivo. Here, we show that RK424 inhibits viral ribonucleoprotein complex (vRNP) activity, causing the viral nucleoprotein (NP) to accumulate in the cell nucleus. In silico docking analysis revealed that RK424 bound to a small pocket in the viral NP. This pocket was surrounded by three functionally important domains: the RNA binding groove, the NP dimer interface, and nuclear export signal (NES) 3, indicating that it may be involved in the RNA binding, oligomerization, and nuclear export functions of NP. The accuracy of this binding model was confirmed in a NP-RK424 binding assay incorporating photo-cross-linked RK424 affinity beads and in a plaque assay evaluating the structure-activity relationship of RK424. Surface plasmon resonance (SPR) and pull-down assays showed that RK424 inhibited both the NP-RNA and NP-NP interactions, whereas size exclusion chromatography showed that RK424 disrupted viral RNA-induced NP oligomerization. In addition, in vitro nuclear export assays confirmed that RK424 inhibited nuclear export of NP. The amino acid residues comprising the NP pocket play a crucial role in viral replication and are highly conserved in more than 7,000 NP sequences from avian, human, and swine influenza viruses. Furthermore, we found that the NP pocket has a surface structure different from that of the pocket in host molecules. Taken together, these results describe a promising new approach to developing influenza virus drugs that target a novel pocket structure within NP.

No MeSH data available.


Related in: MedlinePlus