title: A Novel Antiviral Target Structure Involved in the RNA Binding, Dimerization, and Nuclear Export Functions of the Influenza A Virus Nucleoprotein.
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.
authors.affiliation: Viral Infectious Diseases Unit, RIKEN, Wako, Saitama, Japan.
authors.forename: Michinori
authors.initials: M
authors.lastname: Kakisaka
authors.affiliation: Viral Infectious Diseases Unit, RIKEN, Wako, Saitama, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Sasaki
authors.affiliation: Viral Infectious Diseases Unit, RIKEN, Wako, Saitama, Japan; Computational Biology Research Center (CBRC), National Institute of Advanced Industrial Science and Technology (AIST), Koto-ku, Tokyo, Japan.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Yamada
authors.affiliation: Chemical Biology Group, RIKEN CSRS, Wako, Saitama, Japan.
authors.forename: Yasumitsu
authors.initials: Y
authors.lastname: Kondoh
authors.affiliation: Influenza and Prion Disease Research Center, National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan.
authors.forename: Hirokazu
authors.initials: H
authors.lastname: Hikono
authors.affiliation: Chemical Biology Group, RIKEN CSRS, Wako, Saitama, Japan.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Osada
authors.affiliation: Computational Biology Research Center (CBRC), National Institute of Advanced Industrial Science and Technology (AIST), Koto-ku, Tokyo, Japan.
authors.forename: Kentaro
authors.initials: K
authors.lastname: Tomii
authors.affiliation: Influenza and Prion Disease Research Center, National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan.
authors.forename: Takehiko
authors.initials: T
authors.lastname: Saito
authors.affiliation: Viral Infectious Diseases Unit, RIKEN, Wako, Saitama, Japan.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Aida
mesh_terms: D021581:Active Transport, Cell Nucleus / Q000187:drug effects; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D006801:Humans; D009980:Influenza A virus / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D019590:Nucleocapsid Proteins; D055503:Protein Multimerization; D012367:RNA, Viral / Q000187:drug effects / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014758:Viral Core Proteins / Q000378:metabolism*
chemical_list: D000998:Antiviral Agents; C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins