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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 the NP-RNA and NP-NP interactions, and on NP oligomerization.(A) Sensorgram showing the interaction between NP and RNA in the absence or presence of RK424. Two independent experiments were performed and one representative result is shown. (B) Quantification of RNA binding activity. (C) Purified NP-HA proteins were incubated with RK424 for 1 h followed by incubation with FLAG agarose beads coupled to mRFP-Flag (mRFP-Flag beads) or NP-mRFP-Flag proteins (NP-mRFP-Flag beads). Binding of NP-HA to mRFP-Flag or NP-mRFP-Flag beads and the input NP-HA were detected by western blotting with an anti-HA MAb. The amount of protein bound to FLAG agarose beads was detected with an anti-Flag MAb (loading control). Three independent experiments were performed and one representative result is shown. (D) Pull-down assay to examine NP binding activity. (E) Purified NP proteins were pre-incubated with RK424 followed by incubation with vRNA synthesized by in vitro transcription. The incubated samples were injected onto a Superdex 200 Increase size exclusion column. The integrated peaks between and elution volume of 8 mL and 9 mL were evaluated to measure the oligomerization rate of NP. Two independent experiments were performed and one representative result is shown. (F) Oligomerization rate of NP as assessed by size exclusion chromatography.
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ppat.1005062.g005: Effect of RK424 on the NP-RNA and NP-NP interactions, and on NP oligomerization.(A) Sensorgram showing the interaction between NP and RNA in the absence or presence of RK424. Two independent experiments were performed and one representative result is shown. (B) Quantification of RNA binding activity. (C) Purified NP-HA proteins were incubated with RK424 for 1 h followed by incubation with FLAG agarose beads coupled to mRFP-Flag (mRFP-Flag beads) or NP-mRFP-Flag proteins (NP-mRFP-Flag beads). Binding of NP-HA to mRFP-Flag or NP-mRFP-Flag beads and the input NP-HA were detected by western blotting with an anti-HA MAb. The amount of protein bound to FLAG agarose beads was detected with an anti-Flag MAb (loading control). Three independent experiments were performed and one representative result is shown. (D) Pull-down assay to examine NP binding activity. (E) Purified NP proteins were pre-incubated with RK424 followed by incubation with vRNA synthesized by in vitro transcription. The incubated samples were injected onto a Superdex 200 Increase size exclusion column. The integrated peaks between and elution volume of 8 mL and 9 mL were evaluated to measure the oligomerization rate of NP. Two independent experiments were performed and one representative result is shown. (F) Oligomerization rate of NP as assessed by size exclusion chromatography.

Mentions: The four amino acids residues that play a potential role in RK424 binding to NP reside within different functional domains. These domains are involved in (i) oligomerization of NP via NP-RNA and NP-NP interactions, and (ii) nuclear export of NP. We first used surface plasmon resonance (SPR; Biacore) to examine the effect of inhibiting the NP-RNA interaction. A 24-mer RNA oligomer was biotinylated at the 5’-end (to facilitate immobilization) and methylated at the 2’-OH of ribose (to provide nuclease resistance) was immobilized on an SA sensor chip. We chose a 24-mer because studies show that this length approximates the binding distance between NP and vRNA [33,34,35,36]. Samples containing RK424 (10 μM) showed a reduced binding response to RNA. This reduction was dependent upon the dose of RK424. The RNA binding observed for treated samples was almost 50% less than that observed for control samples (Fig 5A and 5B). Notably, RK424 affected the rate of association without affecting the rate of disassociation. This suggests that RK424 interferes with the binding of NP to RNA.


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 the NP-RNA and NP-NP interactions, and on NP oligomerization.(A) Sensorgram showing the interaction between NP and RNA in the absence or presence of RK424. Two independent experiments were performed and one representative result is shown. (B) Quantification of RNA binding activity. (C) Purified NP-HA proteins were incubated with RK424 for 1 h followed by incubation with FLAG agarose beads coupled to mRFP-Flag (mRFP-Flag beads) or NP-mRFP-Flag proteins (NP-mRFP-Flag beads). Binding of NP-HA to mRFP-Flag or NP-mRFP-Flag beads and the input NP-HA were detected by western blotting with an anti-HA MAb. The amount of protein bound to FLAG agarose beads was detected with an anti-Flag MAb (loading control). Three independent experiments were performed and one representative result is shown. (D) Pull-down assay to examine NP binding activity. (E) Purified NP proteins were pre-incubated with RK424 followed by incubation with vRNA synthesized by in vitro transcription. The incubated samples were injected onto a Superdex 200 Increase size exclusion column. The integrated peaks between and elution volume of 8 mL and 9 mL were evaluated to measure the oligomerization rate of NP. Two independent experiments were performed and one representative result is shown. (F) Oligomerization rate of NP as assessed by size exclusion chromatography.
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ppat.1005062.g005: Effect of RK424 on the NP-RNA and NP-NP interactions, and on NP oligomerization.(A) Sensorgram showing the interaction between NP and RNA in the absence or presence of RK424. Two independent experiments were performed and one representative result is shown. (B) Quantification of RNA binding activity. (C) Purified NP-HA proteins were incubated with RK424 for 1 h followed by incubation with FLAG agarose beads coupled to mRFP-Flag (mRFP-Flag beads) or NP-mRFP-Flag proteins (NP-mRFP-Flag beads). Binding of NP-HA to mRFP-Flag or NP-mRFP-Flag beads and the input NP-HA were detected by western blotting with an anti-HA MAb. The amount of protein bound to FLAG agarose beads was detected with an anti-Flag MAb (loading control). Three independent experiments were performed and one representative result is shown. (D) Pull-down assay to examine NP binding activity. (E) Purified NP proteins were pre-incubated with RK424 followed by incubation with vRNA synthesized by in vitro transcription. The incubated samples were injected onto a Superdex 200 Increase size exclusion column. The integrated peaks between and elution volume of 8 mL and 9 mL were evaluated to measure the oligomerization rate of NP. Two independent experiments were performed and one representative result is shown. (F) Oligomerization rate of NP as assessed by size exclusion chromatography.
Mentions: The four amino acids residues that play a potential role in RK424 binding to NP reside within different functional domains. These domains are involved in (i) oligomerization of NP via NP-RNA and NP-NP interactions, and (ii) nuclear export of NP. We first used surface plasmon resonance (SPR; Biacore) to examine the effect of inhibiting the NP-RNA interaction. A 24-mer RNA oligomer was biotinylated at the 5’-end (to facilitate immobilization) and methylated at the 2’-OH of ribose (to provide nuclease resistance) was immobilized on an SA sensor chip. We chose a 24-mer because studies show that this length approximates the binding distance between NP and vRNA [33,34,35,36]. Samples containing RK424 (10 μM) showed a reduced binding response to RNA. This reduction was dependent upon the dose of RK424. The RNA binding observed for treated samples was almost 50% less than that observed for control samples (Fig 5A and 5B). Notably, RK424 affected the rate of association without affecting the rate of disassociation. This suggests that RK424 interferes with the binding of NP to RNA.

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