title: Biophysical characterization of sites of host adaptive mutation in the influenza A virus RNA polymerase PB2 RNA-binding domain.
abstract: Influenza RNA polymerase is composed of three subunits, PA, PB1, and PB2, which interact with each other for transcription and replication of the viral RNA genome in the nucleus of infected cells. PB2 RNA-binding 627-domain (residues 535-693), located in the C-terminus, presents a highly basic surface around residue lysine 627 and has been proposed to interact with viral or cellular factors, resulting in host adaptation. However, the function of this domain is not yet characterized in detail. In this study, we identified RNA-binding activity and RNA-binding surfaces in both the N-terminal and basic C-terminal regions of PB2 627-domain using NMR experiments. Through mutagenesis studies, we confirmed which residues directly interact with RNA and mapped their locations on the RNA-binding surface. In addition, by luciferase activity assays, we showed that influenza virus polymerase activity may correlate with the interaction between PB2 and RNA. Representative host adaptive mutations (residues 591 and 627) were found to be located on the RNA-binding surface and were confirmed to directly interact with RNA and to affect polymerase activity. From these results, we suggest that influenza virus polymerase activity may be regulated through the interaction between PB2 627-domain and RNA and that consequently host adaptation of the virus may be influenced.
authors.affiliation: Department of Chemistry, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Kyungeun
authors.initials: K
authors.lastname: Lim
authors.affiliation: Virus Research and Testing Group, KRICT, Sinseongno, Yuseong-gu, Daejeon 305-600, Republic of Korea.
authors.forename: Meehyein
authors.initials: M
authors.lastname: Kim
authors.affiliation: Department of Chemistry, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Mi-Kyung
authors.initials: MK
authors.lastname: Lee
authors.affiliation: Department of Chemistry, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Junsang
authors.initials: J
authors.lastname: Ko
authors.affiliation: Department of Chemistry, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea.
authors.forename: Sungwoo
authors.initials: S
authors.lastname: Hong
authors.affiliation: Department of Chemistry, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 305-701, Republic of Korea. Electronic address: byongseok.choi@kaist.ac.kr.
authors.forename: Byong-Seok
authors.initials: BS
authors.lastname: Choi
mesh_terms: D000595:Amino Acid Sequence / Q000235:genetics; D019943:Amino Acid Substitution; D055592:Biophysical Phenomena; D006801:Humans; D009980:Influenza A virus / Q000235:genetics* / Q000472:pathogenicity; D016296:Mutagenesis; D009154:Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics; D012313:RNA / Q000737:chemistry* / Q000235:genetics; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism
chemical_list: C057027:PB2 protein, Influenzavirus A; D016601:RNA-Binding Proteins; D014764:Viral Proteins; D012313:RNA; D012324:RNA-Dependent RNA Polymerase
keywords: Host adaptive mutation; Influenza RNA polymerase; Nuclear magnetic resonance; PB2 subunit; RNA-binding activity