title: Using mutagenesis to explore conserved residues in the RNA-binding groove of influenza A virus nucleoprotein for antiviral drug development.
abstract: Nucleoprotein (NP) is the most abundant type of RNA-binding viral protein in influenza A virus-infected cells and is necessary for viral RNA transcription and replication. Recent studies demonstrated that influenza NP is a valid target for antiviral drug development. The surface of the groove, covered with numerous conserved residues between the head and body domains of influenza A NP, plays a crucial role in RNA binding. To explore the mechanism by which NP binds RNA, we performed a series of site-directed mutagenesis in the RNA-binding groove, followed by surface plasmon resonance (SPR), to characterize the interactions between RNA and NP. Furthermore, a role of Y148 in NP stability and NP-RNA binding was evaluated. The aromatic residue of Y148 was found to stack with a nucleotide base. By interrupting the stacking interaction between Y148 and an RNA base, we identified an influenza virus NP inhibitor, (E, E)-1,7-bis(4-hydroxy-3-methoxyphenyl) -1,6-heptadiene-3,5-dione; this inhibitor reduced the NP's RNA-binding affinity and hindered viral replication. Our findings will be useful for the development of new drugs that disrupt the interaction between RNA and viral NP in the influenza virus.
authors.affiliation: National Chung Hsing University, Department of Life Science, Taichung, 40227, Taiwan.
authors.forename: Chia-Lin
authors.initials: CL
authors.lastname: Liu
authors.affiliation: National Health Research Institutes, Institute of Biotechnology and Pharmaceutical Research, Miaoli, 35053, Taiwan.
authors.forename: Hui-Chen
authors.initials: HC
authors.lastname: Hung
authors.affiliation: National Chung Hsing University, Institute of Genomics and Bioinformatics, Taichung, 40227, Taiwan.
authors.forename: Shou-Chen
authors.initials: SC
authors.lastname: Lo
authors.affiliation: National Chung Hsing University, Department of Life Science, Taichung, 40227, Taiwan.
authors.forename: Ching-Hui
authors.initials: CH
authors.lastname: Chiang
authors.affiliation: National Health Research Institutes, Institute of Biotechnology and Pharmaceutical Research, Miaoli, 35053, Taiwan.
authors.forename: I-Jung
authors.initials: IJ
authors.lastname: Chen
authors.affiliation: National Health Research Institutes, Institute of Biotechnology and Pharmaceutical Research, Miaoli, 35053, Taiwan.
authors.forename: John T-A
authors.initials: JT
authors.lastname: Hsu
authors.affiliation: National Chung Hsing University, Department of Life Science, Taichung, 40227, Taiwan.|National Chung Hsing University, Institute of Genomics and Bioinformatics, Taichung, 40227, Taiwan.|National Chung Hsing University, Institute of Biotechnology, Taichung, 40227, Taiwan.
authors.forename: Ming-Hon
authors.initials: MH
authors.lastname: Hou
mesh_terms: D000072283:A549 Cells; D000998:Antiviral Agents / Q000737:chemistry / Q000494:pharmacology*; D003474:Curcumin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D006801:Humans; D009980:Influenza A virus / Q000187:drug effects* / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D019590:Nucleocapsid Proteins; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D000071376:RNA-Binding Motifs; D016601:RNA-Binding Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D014758:Viral Core Proteins / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism
chemical_list: C000613419:1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione; D000998:Antiviral Agents; C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins; D012313:RNA; D003474:Curcumin