title: Sequence in the influenza A virus nucleoprotein required for viral polymerase binding and RNA synthesis.
abstract: Many proposed mechanisms for influenza A viral RNA synthesis include an interaction of the nucleoprotein (NP) with the viral polymerase. To identify an NP sequence required for this interaction, we used the cryoelectron microscopic structure of an influenza virus miniribonucleoprotein as a guide for choosing promising surface-exposed sequences. We show that three amino acids (R204, W207, and R208) located in a loop at the top of the head domain of NP are required for functional interaction with the viral polymerase. Quantitative reverse transcription-PCR (RT-PCR) measurements of RNAs synthesized in minigenome assays established that each of these NP amino acids is required for viral RNA synthesis. The mutation of these three amino acids does not affect nuclear localization or RNA-binding and oligomerization activities of NP. In vitro binding experiments with purified virus polymerase and NPs established that these three amino acids are required for NP binding to the viral polymerase.
authors.affiliation: Department of Molecular Genetics and Microbiology, Institute for Cell and Molecular Biology, University of Texas at Austin, Texas, USA.
authors.forename: Jesper K
authors.initials: JK
authors.lastname: Marklund
authors.forename: Qiaozhen
authors.initials: Q
authors.lastname: Ye
authors.forename: Jinhui
authors.initials: J
authors.lastname: Dong
authors.forename: Yizhi Jane
authors.initials: YJ
authors.lastname: Tao
authors.forename: Robert M
authors.initials: RM
authors.lastname: Krug
mesh_terms: D002460:Cell Line; D020285:Cryoelectron Microscopy; D006801:Humans; D009980:Influenza A virus / Q000201:enzymology / Q000502:physiology* / Q000648:ultrastructure; D046911:Macromolecular Substances / Q000648:ultrastructure; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D019590:Nucleocapsid Proteins; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D012367:RNA, Viral / Q000096:biosynthesis*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D060888:Real-Time Polymerase Chain Reaction; D014758:Viral Core Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D014779:Virus Replication
chemical_list: D046911:Macromolecular Substances; D050505:Mutant Proteins; C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins; D012324:RNA-Dependent RNA Polymerase