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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

Model for potential binding of RK424 to NP.In silico docking analysis was used to predict potential binding sites for RK424 on NP. The configuration with the highest binding energy was visualized using PyMol. (A) Crystal structure of NP. The nuclear export signal (NES) (yellow: amino acid (aa) 256–266), RNA binding grove (orange: aa 1–180), and dimer interface (purple: aa 482–489) are shown on the surface representation. The small pocket is highlighted by red circles. (B) Close-up of the NP small pocket. An electrostatic surface representation of the potential binding site on NP. Amino acids are colored blue (R162), pink (S165), red (L264), and green (Y487). (C) Purified wild-type NP-Flag (WT) and purified mutant NP-Flag (Mut) proteins harboring alanine substitutions at all four potential binding sites (162 165, 264, and 487) were added to RK424 cross-linked affinity beads or uncross-linked beads. Isolated NP-Flag (WT) and mutant NP-Flag (Mut) proteins were run in 10% SDS-PAGE gels and purity was checked by Coomassie Brilliant Blue (CBB) staining (upper panel). The binding of NP to RK424 beads was detected by western blotting with an anti-Flag MAb (lower panel). The positions of the NP-Flag proteins are indicated. Three independent experiments were performed and one representative result is shown. (D) Structure-activity relationship (SAR) analysis of RK424. The in vitro antiviral activity (IC50) and cell toxicity (CC50) of four different structural compounds derived from RK424 were evaluated in a plaque assay and in a WST-1 assay based on MDCK cells. Data are expressed as the mean ± SD of three samples in each of three independent experiments.
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ppat.1005062.g004: Model for potential binding of RK424 to NP.In silico docking analysis was used to predict potential binding sites for RK424 on NP. The configuration with the highest binding energy was visualized using PyMol. (A) Crystal structure of NP. The nuclear export signal (NES) (yellow: amino acid (aa) 256–266), RNA binding grove (orange: aa 1–180), and dimer interface (purple: aa 482–489) are shown on the surface representation. The small pocket is highlighted by red circles. (B) Close-up of the NP small pocket. An electrostatic surface representation of the potential binding site on NP. Amino acids are colored blue (R162), pink (S165), red (L264), and green (Y487). (C) Purified wild-type NP-Flag (WT) and purified mutant NP-Flag (Mut) proteins harboring alanine substitutions at all four potential binding sites (162 165, 264, and 487) were added to RK424 cross-linked affinity beads or uncross-linked beads. Isolated NP-Flag (WT) and mutant NP-Flag (Mut) proteins were run in 10% SDS-PAGE gels and purity was checked by Coomassie Brilliant Blue (CBB) staining (upper panel). The binding of NP to RK424 beads was detected by western blotting with an anti-Flag MAb (lower panel). The positions of the NP-Flag proteins are indicated. Three independent experiments were performed and one representative result is shown. (D) Structure-activity relationship (SAR) analysis of RK424. The in vitro antiviral activity (IC50) and cell toxicity (CC50) of four different structural compounds derived from RK424 were evaluated in a plaque assay and in a WST-1 assay based on MDCK cells. Data are expressed as the mean ± SD of three samples in each of three independent experiments.

Mentions: To further examine how RK424 inhibits NP function, we used in silico docking analysis to create a potential model for the binding of RK424 to NP. To establish unbiased predictive virtual docking models, we obtained the crystal structure of monomeric influenza A/WSN/1933 (H1N1) NP from the Protein Data Bank (PDB) and performed docking studies using AutoDock molecular modeling simulation software [31]. Three potential binding sites were identified; however, the potential binding models showed that the interaction with the highest binding free energy (ΔG) occupied binding site 1 (S6 Fig). Moreover, binding site 1 was surrounded by three functionally important domains: the RNA binding groove (orange) [11], the NP dimer interface (purple), [12] and NES3 (yellow) [18] (Fig 4A and S6 Fig, front side). Therefore, we focused on the binding model based on binding site 1. The interaction map predicted six different configurations for binding site 1, revealing that four amino acid residues (R162, S165, L264, and Y487) were predominantly involved in the interaction with RK424 (S7 Fig); the amino acid residues within binding sites 2 and 3 that were predicted to interact with RK424 did not correlate with any known NP functions (S8 Fig). RK424 occupied a small pocket on NP; configuration 0 had the best fitting score (ΔG of −8.03 kcal/mol) (Fig 4B and S7 Fig).


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)

Model for potential binding of RK424 to NP.In silico docking analysis was used to predict potential binding sites for RK424 on NP. The configuration with the highest binding energy was visualized using PyMol. (A) Crystal structure of NP. The nuclear export signal (NES) (yellow: amino acid (aa) 256–266), RNA binding grove (orange: aa 1–180), and dimer interface (purple: aa 482–489) are shown on the surface representation. The small pocket is highlighted by red circles. (B) Close-up of the NP small pocket. An electrostatic surface representation of the potential binding site on NP. Amino acids are colored blue (R162), pink (S165), red (L264), and green (Y487). (C) Purified wild-type NP-Flag (WT) and purified mutant NP-Flag (Mut) proteins harboring alanine substitutions at all four potential binding sites (162 165, 264, and 487) were added to RK424 cross-linked affinity beads or uncross-linked beads. Isolated NP-Flag (WT) and mutant NP-Flag (Mut) proteins were run in 10% SDS-PAGE gels and purity was checked by Coomassie Brilliant Blue (CBB) staining (upper panel). The binding of NP to RK424 beads was detected by western blotting with an anti-Flag MAb (lower panel). The positions of the NP-Flag proteins are indicated. Three independent experiments were performed and one representative result is shown. (D) Structure-activity relationship (SAR) analysis of RK424. The in vitro antiviral activity (IC50) and cell toxicity (CC50) of four different structural compounds derived from RK424 were evaluated in a plaque assay and in a WST-1 assay based on MDCK cells. Data are expressed as the mean ± SD of three samples in each of three independent experiments.
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Related In: Results  -  Collection

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

ppat.1005062.g004: Model for potential binding of RK424 to NP.In silico docking analysis was used to predict potential binding sites for RK424 on NP. The configuration with the highest binding energy was visualized using PyMol. (A) Crystal structure of NP. The nuclear export signal (NES) (yellow: amino acid (aa) 256–266), RNA binding grove (orange: aa 1–180), and dimer interface (purple: aa 482–489) are shown on the surface representation. The small pocket is highlighted by red circles. (B) Close-up of the NP small pocket. An electrostatic surface representation of the potential binding site on NP. Amino acids are colored blue (R162), pink (S165), red (L264), and green (Y487). (C) Purified wild-type NP-Flag (WT) and purified mutant NP-Flag (Mut) proteins harboring alanine substitutions at all four potential binding sites (162 165, 264, and 487) were added to RK424 cross-linked affinity beads or uncross-linked beads. Isolated NP-Flag (WT) and mutant NP-Flag (Mut) proteins were run in 10% SDS-PAGE gels and purity was checked by Coomassie Brilliant Blue (CBB) staining (upper panel). The binding of NP to RK424 beads was detected by western blotting with an anti-Flag MAb (lower panel). The positions of the NP-Flag proteins are indicated. Three independent experiments were performed and one representative result is shown. (D) Structure-activity relationship (SAR) analysis of RK424. The in vitro antiviral activity (IC50) and cell toxicity (CC50) of four different structural compounds derived from RK424 were evaluated in a plaque assay and in a WST-1 assay based on MDCK cells. Data are expressed as the mean ± SD of three samples in each of three independent experiments.
Mentions: To further examine how RK424 inhibits NP function, we used in silico docking analysis to create a potential model for the binding of RK424 to NP. To establish unbiased predictive virtual docking models, we obtained the crystal structure of monomeric influenza A/WSN/1933 (H1N1) NP from the Protein Data Bank (PDB) and performed docking studies using AutoDock molecular modeling simulation software [31]. Three potential binding sites were identified; however, the potential binding models showed that the interaction with the highest binding free energy (ΔG) occupied binding site 1 (S6 Fig). Moreover, binding site 1 was surrounded by three functionally important domains: the RNA binding groove (orange) [11], the NP dimer interface (purple), [12] and NES3 (yellow) [18] (Fig 4A and S6 Fig, front side). Therefore, we focused on the binding model based on binding site 1. The interaction map predicted six different configurations for binding site 1, revealing that four amino acid residues (R162, S165, L264, and Y487) were predominantly involved in the interaction with RK424 (S7 Fig); the amino acid residues within binding sites 2 and 3 that were predicted to interact with RK424 did not correlate with any known NP functions (S8 Fig). RK424 occupied a small pocket on NP; configuration 0 had the best fitting score (ΔG of −8.03 kcal/mol) (Fig 4B and S7 Fig).

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