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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 viral transcription, replication, and translation.(A) HEK293T cells were transfected with pCAGGS expression plasmids encoding PB2, PB1, PA, NP, and vNP-luc in the absence or presence of RK424 (0.2, 0.5, and 2 μM). The effect of vRNA transcription was then evaluated by measuring luciferase activity at 48 h post-transfection. Data are expressed as the mean ± SD of three samples in each of three independent experiments. *;p<0.005 and **;p<0.001. (B) MDCK cells were infected with A/WSN/1933 (H1N1) at an MOI of 5 in the absence or presence of RK424 (0.2 and 2 μM) and then fixed at 6 h post-infection. The cells were probed with Quasar 670-labeled probes against the PB2 segment (magenta) and nuclei were stained with Hoechst (blue). Two independent experiments were performed and one representative result is shown. (C) Virus-infected cells were treated with RK424 (0.2 μM and 2 μM) for 18 h and the cell lysates subjected to western blotting with anti-WSN virus serum and an anti-β-actin monoclonal antibody (MAb). Bands representing HA, NP, NA, M1, M2, and actin are indicated. Oseltamivir phosphate (Os) was used as the negative control. Two independent experiments were performed and one representative result is shown.
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ppat.1005062.g002: Effect of RK424 on viral transcription, replication, and translation.(A) HEK293T cells were transfected with pCAGGS expression plasmids encoding PB2, PB1, PA, NP, and vNP-luc in the absence or presence of RK424 (0.2, 0.5, and 2 μM). The effect of vRNA transcription was then evaluated by measuring luciferase activity at 48 h post-transfection. Data are expressed as the mean ± SD of three samples in each of three independent experiments. *;p<0.005 and **;p<0.001. (B) MDCK cells were infected with A/WSN/1933 (H1N1) at an MOI of 5 in the absence or presence of RK424 (0.2 and 2 μM) and then fixed at 6 h post-infection. The cells were probed with Quasar 670-labeled probes against the PB2 segment (magenta) and nuclei were stained with Hoechst (blue). Two independent experiments were performed and one representative result is shown. (C) Virus-infected cells were treated with RK424 (0.2 μM and 2 μM) for 18 h and the cell lysates subjected to western blotting with anti-WSN virus serum and an anti-β-actin monoclonal antibody (MAb). Bands representing HA, NP, NA, M1, M2, and actin are indicated. Oseltamivir phosphate (Os) was used as the negative control. Two independent experiments were performed and one representative result is shown.

Mentions: In contrast to treatment with oseltamivir phosphate (which inhibits the budding step of the viral life cycle), treatment with RK424 led to a reduction in the number, but not the size, of viral plaques (S3 Fig) and it was effective against the oseltamivir resistant virus, A/Tex/131/E119V (H3N2) (Fig 1B) [28]. Furthermore, RK424 did not interfere with viral replication when added to MDCK cells prior to virus inoculation (S4 Fig) and exhibited a prominent antiviral effect at an early stage in the virus life cycle (S5 Fig). These results indicate that the RK424 may target the post-entry and early replication step of the viral life cycle. To examine this further, we first explored the possibility that RK424 affects viral genome replication and/or transcription, both of which occur during the early stages in viral life cycle. To examine the effect of RK424 on vRNP activity (vRNP regulates viral replication and transcription), we performed a mini-genome assay using five expression plasmids harboring influenza A/WSN/1933 (H1N1): PB2/pCAGGS, PB1/pCAGGS, PA/pCAGGS and NP/pCAGGS, which control viral genome replication and transcription, and the luciferase-containing plasmid vNP-luc/pHH21, which encodes a viral-like genome in the absence or presence of RK424. We found that RK424 caused a notable and dose-dependent reduction in luciferase activity (Fig 2A) compared with control (dimethyl sulfoxide (DMSO)) treatment. By contrast, treatment with 10 μM of oseltamivir phosphate did not significantly inhibit luciferase activity.


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 viral transcription, replication, and translation.(A) HEK293T cells were transfected with pCAGGS expression plasmids encoding PB2, PB1, PA, NP, and vNP-luc in the absence or presence of RK424 (0.2, 0.5, and 2 μM). The effect of vRNA transcription was then evaluated by measuring luciferase activity at 48 h post-transfection. Data are expressed as the mean ± SD of three samples in each of three independent experiments. *;p<0.005 and **;p<0.001. (B) MDCK cells were infected with A/WSN/1933 (H1N1) at an MOI of 5 in the absence or presence of RK424 (0.2 and 2 μM) and then fixed at 6 h post-infection. The cells were probed with Quasar 670-labeled probes against the PB2 segment (magenta) and nuclei were stained with Hoechst (blue). Two independent experiments were performed and one representative result is shown. (C) Virus-infected cells were treated with RK424 (0.2 μM and 2 μM) for 18 h and the cell lysates subjected to western blotting with anti-WSN virus serum and an anti-β-actin monoclonal antibody (MAb). Bands representing HA, NP, NA, M1, M2, and actin are indicated. Oseltamivir phosphate (Os) was used as the negative control. Two independent experiments were performed and one representative result is shown.
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Related In: Results  -  Collection

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ppat.1005062.g002: Effect of RK424 on viral transcription, replication, and translation.(A) HEK293T cells were transfected with pCAGGS expression plasmids encoding PB2, PB1, PA, NP, and vNP-luc in the absence or presence of RK424 (0.2, 0.5, and 2 μM). The effect of vRNA transcription was then evaluated by measuring luciferase activity at 48 h post-transfection. Data are expressed as the mean ± SD of three samples in each of three independent experiments. *;p<0.005 and **;p<0.001. (B) MDCK cells were infected with A/WSN/1933 (H1N1) at an MOI of 5 in the absence or presence of RK424 (0.2 and 2 μM) and then fixed at 6 h post-infection. The cells were probed with Quasar 670-labeled probes against the PB2 segment (magenta) and nuclei were stained with Hoechst (blue). Two independent experiments were performed and one representative result is shown. (C) Virus-infected cells were treated with RK424 (0.2 μM and 2 μM) for 18 h and the cell lysates subjected to western blotting with anti-WSN virus serum and an anti-β-actin monoclonal antibody (MAb). Bands representing HA, NP, NA, M1, M2, and actin are indicated. Oseltamivir phosphate (Os) was used as the negative control. Two independent experiments were performed and one representative result is shown.
Mentions: In contrast to treatment with oseltamivir phosphate (which inhibits the budding step of the viral life cycle), treatment with RK424 led to a reduction in the number, but not the size, of viral plaques (S3 Fig) and it was effective against the oseltamivir resistant virus, A/Tex/131/E119V (H3N2) (Fig 1B) [28]. Furthermore, RK424 did not interfere with viral replication when added to MDCK cells prior to virus inoculation (S4 Fig) and exhibited a prominent antiviral effect at an early stage in the virus life cycle (S5 Fig). These results indicate that the RK424 may target the post-entry and early replication step of the viral life cycle. To examine this further, we first explored the possibility that RK424 affects viral genome replication and/or transcription, both of which occur during the early stages in viral life cycle. To examine the effect of RK424 on vRNP activity (vRNP regulates viral replication and transcription), we performed a mini-genome assay using five expression plasmids harboring influenza A/WSN/1933 (H1N1): PB2/pCAGGS, PB1/pCAGGS, PA/pCAGGS and NP/pCAGGS, which control viral genome replication and transcription, and the luciferase-containing plasmid vNP-luc/pHH21, which encodes a viral-like genome in the absence or presence of RK424. We found that RK424 caused a notable and dose-dependent reduction in luciferase activity (Fig 2A) compared with control (dimethyl sulfoxide (DMSO)) treatment. By contrast, treatment with 10 μM of oseltamivir phosphate did not significantly inhibit luciferase activity.

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