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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

Production of mutant viruses and conservation of amino acids at RK424 and nucleozin binding sites.(A) In vitro selection of escape viruses in the presence of serial dilutions of RK424 and nucleozin. RK424 concentration range: 1st; 0.1–2 μM, 2nd; 0.2–3 μM, 3rd; 0.3–3μM and 4th; 0.4–3 μM. Nucleozin concentration range: 1st; 0.1–2 uM, 2nd; 0.3–3 μM, 3rd; 1–5μM and 4th; 3–10 μM. The highest concentrations of compound that elicited CPE at each passage are listed. (B) Plaque titration of escape viruses in each passage. Values represent the mean ± SD of three independent experiments. The symbol (*) indicates statistically significant differences in mean viral titer (PFU/mL) between RK424 passaged virus and nucleozin passaged virus at each passage number.*;p<0.001. (C) Viral titer of RK424 3rd passaged virus and nucleozin 4th passaged virus stocks normalized to absolute virion numbers (based on hemagglutination units; HAU). Values represent the mean ± SD of three independent experiments. *;p<0.005. (D) Sensitivity of RK424- and nucleozin-selected viruses to DMSO (left well), 10 μM nucleozin (middle well) and 10 μM RK424 (right well). Two independent experiments were performed and one representative result is shown. (E) Models showing the binding sites on NP targeted by RK424 and nucleozin. (F) Production of recombinant viruses harboring R162A, S165A, L264A, Y487A, Y52H, and Y289H mutations in NP. HEK293T/MDCK cells were transfected with plasmids expressing four viral proteins (PB2, PB1, PA, and NP) and eight vRNAs (PB2, PB1, PA, HA, NP, NA, M, and NS). Plasmids expressing mutant NP proteins and genomes were used as a substitute for the WT plasmid when generating the R162A, S165A, L264A, Y487A, Y52H, and Y289H NP mutant viruses. After 72 h of transfection, supernatants were harvested and used in plaque assays on MDCK cells. Data are expressed as the mean ± SD of three independent experiments. (G) The 50% inhibitory concentration (IC50) of RK424 and nucleozin against rWSN and Y289H rWSN viruses were evaluated in a plaque assay in the absence or presence of RK424 and nucleozin (0–100 μM). Data are expressed as the mean + SD of three samples in each of three independent experiments. (H) Conservation of RK424 binding sites (R162, S165, L264, and Y487) and nucleozin binding sites (Y52 and Y289) in human, avian, and swine influenza A viruses. Perl script was used to analyze 7683 NP sequences derived from human, avian, and swine influenza A viruses.
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ppat.1005062.g007: Production of mutant viruses and conservation of amino acids at RK424 and nucleozin binding sites.(A) In vitro selection of escape viruses in the presence of serial dilutions of RK424 and nucleozin. RK424 concentration range: 1st; 0.1–2 μM, 2nd; 0.2–3 μM, 3rd; 0.3–3μM and 4th; 0.4–3 μM. Nucleozin concentration range: 1st; 0.1–2 uM, 2nd; 0.3–3 μM, 3rd; 1–5μM and 4th; 3–10 μM. The highest concentrations of compound that elicited CPE at each passage are listed. (B) Plaque titration of escape viruses in each passage. Values represent the mean ± SD of three independent experiments. The symbol (*) indicates statistically significant differences in mean viral titer (PFU/mL) between RK424 passaged virus and nucleozin passaged virus at each passage number.*;p<0.001. (C) Viral titer of RK424 3rd passaged virus and nucleozin 4th passaged virus stocks normalized to absolute virion numbers (based on hemagglutination units; HAU). Values represent the mean ± SD of three independent experiments. *;p<0.005. (D) Sensitivity of RK424- and nucleozin-selected viruses to DMSO (left well), 10 μM nucleozin (middle well) and 10 μM RK424 (right well). Two independent experiments were performed and one representative result is shown. (E) Models showing the binding sites on NP targeted by RK424 and nucleozin. (F) Production of recombinant viruses harboring R162A, S165A, L264A, Y487A, Y52H, and Y289H mutations in NP. HEK293T/MDCK cells were transfected with plasmids expressing four viral proteins (PB2, PB1, PA, and NP) and eight vRNAs (PB2, PB1, PA, HA, NP, NA, M, and NS). Plasmids expressing mutant NP proteins and genomes were used as a substitute for the WT plasmid when generating the R162A, S165A, L264A, Y487A, Y52H, and Y289H NP mutant viruses. After 72 h of transfection, supernatants were harvested and used in plaque assays on MDCK cells. Data are expressed as the mean ± SD of three independent experiments. (G) The 50% inhibitory concentration (IC50) of RK424 and nucleozin against rWSN and Y289H rWSN viruses were evaluated in a plaque assay in the absence or presence of RK424 and nucleozin (0–100 μM). Data are expressed as the mean + SD of three samples in each of three independent experiments. (H) Conservation of RK424 binding sites (R162, S165, L264, and Y487) and nucleozin binding sites (Y52 and Y289) in human, avian, and swine influenza A viruses. Perl script was used to analyze 7683 NP sequences derived from human, avian, and swine influenza A viruses.

Mentions: Finally, to demonstrate the accuracy of our docking model and confirm that RK424 binds to the binding pocket within NP, we selected escape mutant viruses by passaging an IAV in the presence of RK424. In this experiment, we selected nucleozin as the positive control, because nucleozin exerts a potent antiviral effect via its ability to cross-link two NP molecules [25]. The structures of the NP inhibitors are listed in S11 Fig. The selection was carried out for a total of four passages with serially increasing concentrations of RK424 and nucleozin. In the cells passaged with RK424, CPE was observed until 3rd passage, but no CPE occurred at the 4th passage. On the other hand, as the number of passages increased, the concentration of nuclozin at which CPE occurred was up to 10 μM by the 4th passage (Fig 7A). Furthermore, viral replication imposed by nucleozin treatment increased as the number of passages increased but viral replication imposed by RK424 treatment decreased (Fig 7B).These results suggest that RK424 makes viral infected cells produce more propagation-deficient virions than fully infectious virions. To confirm this notion, we evaluated the infectivity of RK424 3rd passage virus by comparing the viral titer of this virus normalized to absolute virion numbers (estimated by hemagglutination units; HAU) with that of the initial WT viral titer used in the selection of the escape mutant. The titer of the RK424 3rd passage virus was 1.41×103 PFU/mL per HAU, which was lower than that of the WT (2.19×105 PFU/mL per HAU) (Fig 7C). These results indicate that the number of propagation-deficient virions produced by RK424 3rd passage virus had increased. After four passages, we confirmed sensitivity of RK424- and nucleozin-selected viruses to 10 μM RK424 and nucleozin (Fig 7D). Nucloezin 4th passage virus exhibited resistance to 10 μM nucleozin but not RK424. However, RK424 3rd passage virus was susceptible to both 10 μM RK424 and nucleozin. These results showed that nucleozin-resistant viruses appeared after four passages with nucleozin but RK424 3rd passage viruses were unable to acquire resistance to RK424. Sequence analysis revealed that nucleozin 4th passage virus had three amino acid mutations (Y52H, Y289H and Y313S) which were previously reported to be result in resistance to nucleozin [25], but no amino acid mutation was detected in RK424 3rd passage virus, which also support the result of sensitivity of RK424- and nucleozin-selected viruses to RK424 and nucleozin.


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)

Production of mutant viruses and conservation of amino acids at RK424 and nucleozin binding sites.(A) In vitro selection of escape viruses in the presence of serial dilutions of RK424 and nucleozin. RK424 concentration range: 1st; 0.1–2 μM, 2nd; 0.2–3 μM, 3rd; 0.3–3μM and 4th; 0.4–3 μM. Nucleozin concentration range: 1st; 0.1–2 uM, 2nd; 0.3–3 μM, 3rd; 1–5μM and 4th; 3–10 μM. The highest concentrations of compound that elicited CPE at each passage are listed. (B) Plaque titration of escape viruses in each passage. Values represent the mean ± SD of three independent experiments. The symbol (*) indicates statistically significant differences in mean viral titer (PFU/mL) between RK424 passaged virus and nucleozin passaged virus at each passage number.*;p<0.001. (C) Viral titer of RK424 3rd passaged virus and nucleozin 4th passaged virus stocks normalized to absolute virion numbers (based on hemagglutination units; HAU). Values represent the mean ± SD of three independent experiments. *;p<0.005. (D) Sensitivity of RK424- and nucleozin-selected viruses to DMSO (left well), 10 μM nucleozin (middle well) and 10 μM RK424 (right well). Two independent experiments were performed and one representative result is shown. (E) Models showing the binding sites on NP targeted by RK424 and nucleozin. (F) Production of recombinant viruses harboring R162A, S165A, L264A, Y487A, Y52H, and Y289H mutations in NP. HEK293T/MDCK cells were transfected with plasmids expressing four viral proteins (PB2, PB1, PA, and NP) and eight vRNAs (PB2, PB1, PA, HA, NP, NA, M, and NS). Plasmids expressing mutant NP proteins and genomes were used as a substitute for the WT plasmid when generating the R162A, S165A, L264A, Y487A, Y52H, and Y289H NP mutant viruses. After 72 h of transfection, supernatants were harvested and used in plaque assays on MDCK cells. Data are expressed as the mean ± SD of three independent experiments. (G) The 50% inhibitory concentration (IC50) of RK424 and nucleozin against rWSN and Y289H rWSN viruses were evaluated in a plaque assay in the absence or presence of RK424 and nucleozin (0–100 μM). Data are expressed as the mean + SD of three samples in each of three independent experiments. (H) Conservation of RK424 binding sites (R162, S165, L264, and Y487) and nucleozin binding sites (Y52 and Y289) in human, avian, and swine influenza A viruses. Perl script was used to analyze 7683 NP sequences derived from human, avian, and swine influenza A viruses.
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ppat.1005062.g007: Production of mutant viruses and conservation of amino acids at RK424 and nucleozin binding sites.(A) In vitro selection of escape viruses in the presence of serial dilutions of RK424 and nucleozin. RK424 concentration range: 1st; 0.1–2 μM, 2nd; 0.2–3 μM, 3rd; 0.3–3μM and 4th; 0.4–3 μM. Nucleozin concentration range: 1st; 0.1–2 uM, 2nd; 0.3–3 μM, 3rd; 1–5μM and 4th; 3–10 μM. The highest concentrations of compound that elicited CPE at each passage are listed. (B) Plaque titration of escape viruses in each passage. Values represent the mean ± SD of three independent experiments. The symbol (*) indicates statistically significant differences in mean viral titer (PFU/mL) between RK424 passaged virus and nucleozin passaged virus at each passage number.*;p<0.001. (C) Viral titer of RK424 3rd passaged virus and nucleozin 4th passaged virus stocks normalized to absolute virion numbers (based on hemagglutination units; HAU). Values represent the mean ± SD of three independent experiments. *;p<0.005. (D) Sensitivity of RK424- and nucleozin-selected viruses to DMSO (left well), 10 μM nucleozin (middle well) and 10 μM RK424 (right well). Two independent experiments were performed and one representative result is shown. (E) Models showing the binding sites on NP targeted by RK424 and nucleozin. (F) Production of recombinant viruses harboring R162A, S165A, L264A, Y487A, Y52H, and Y289H mutations in NP. HEK293T/MDCK cells were transfected with plasmids expressing four viral proteins (PB2, PB1, PA, and NP) and eight vRNAs (PB2, PB1, PA, HA, NP, NA, M, and NS). Plasmids expressing mutant NP proteins and genomes were used as a substitute for the WT plasmid when generating the R162A, S165A, L264A, Y487A, Y52H, and Y289H NP mutant viruses. After 72 h of transfection, supernatants were harvested and used in plaque assays on MDCK cells. Data are expressed as the mean ± SD of three independent experiments. (G) The 50% inhibitory concentration (IC50) of RK424 and nucleozin against rWSN and Y289H rWSN viruses were evaluated in a plaque assay in the absence or presence of RK424 and nucleozin (0–100 μM). Data are expressed as the mean + SD of three samples in each of three independent experiments. (H) Conservation of RK424 binding sites (R162, S165, L264, and Y487) and nucleozin binding sites (Y52 and Y289) in human, avian, and swine influenza A viruses. Perl script was used to analyze 7683 NP sequences derived from human, avian, and swine influenza A viruses.
Mentions: Finally, to demonstrate the accuracy of our docking model and confirm that RK424 binds to the binding pocket within NP, we selected escape mutant viruses by passaging an IAV in the presence of RK424. In this experiment, we selected nucleozin as the positive control, because nucleozin exerts a potent antiviral effect via its ability to cross-link two NP molecules [25]. The structures of the NP inhibitors are listed in S11 Fig. The selection was carried out for a total of four passages with serially increasing concentrations of RK424 and nucleozin. In the cells passaged with RK424, CPE was observed until 3rd passage, but no CPE occurred at the 4th passage. On the other hand, as the number of passages increased, the concentration of nuclozin at which CPE occurred was up to 10 μM by the 4th passage (Fig 7A). Furthermore, viral replication imposed by nucleozin treatment increased as the number of passages increased but viral replication imposed by RK424 treatment decreased (Fig 7B).These results suggest that RK424 makes viral infected cells produce more propagation-deficient virions than fully infectious virions. To confirm this notion, we evaluated the infectivity of RK424 3rd passage virus by comparing the viral titer of this virus normalized to absolute virion numbers (estimated by hemagglutination units; HAU) with that of the initial WT viral titer used in the selection of the escape mutant. The titer of the RK424 3rd passage virus was 1.41×103 PFU/mL per HAU, which was lower than that of the WT (2.19×105 PFU/mL per HAU) (Fig 7C). These results indicate that the number of propagation-deficient virions produced by RK424 3rd passage virus had increased. After four passages, we confirmed sensitivity of RK424- and nucleozin-selected viruses to 10 μM RK424 and nucleozin (Fig 7D). Nucloezin 4th passage virus exhibited resistance to 10 μM nucleozin but not RK424. However, RK424 3rd passage virus was susceptible to both 10 μM RK424 and nucleozin. These results showed that nucleozin-resistant viruses appeared after four passages with nucleozin but RK424 3rd passage viruses were unable to acquire resistance to RK424. Sequence analysis revealed that nucleozin 4th passage virus had three amino acid mutations (Y52H, Y289H and Y313S) which were previously reported to be result in resistance to nucleozin [25], but no amino acid mutation was detected in RK424 3rd passage virus, which also support the result of sensitivity of RK424- and nucleozin-selected viruses to RK424 and nucleozin.

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