title: A novel functional site in the PB2 subunit of influenza A virus essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication.
abstract: The PA, PB1, and PB2 subunits, components of the RNA-dependent RNA polymerase of influenza A virus, are essential for viral transcription and replication. The PB2 subunit binds to the host RNA cap (7-methylguanosine triphosphate (m(7)GTP)) and supports the endonuclease activity of PA to "snatch" the cap from host pre-mRNAs. However, the structure of PB2 is not fully understood, and the functional sites remain unknown. In this study, we describe a novel Val/Arg/Gly (VRG) site in the PB2 cap-binding domain, which is involved in interaction with acetyl-CoA found in eukaryotic histone acetyltransferases (HATs). In vitro experiments revealed that the recombinant PB2 cap-binding domain that includes the VRG site interacts with acetyl-CoA; moreover, it was found that this interaction could be blocked by CoA and various HAT inhibitors. Interestingly, m(7)GTP also inhibited this interaction, suggesting that the same active pocket is capable of interacting with acetyl-CoA and m(7)GTP. To elucidate the importance of the VRG site on PB2 function and viral replication, we constructed a PB2 recombinant protein and recombinant viruses including several patterns of amino acid mutations in the VRG site. Substitutions of the valine and arginine residues or of all 3 residues of the VRG site to alanine significantly reduced the binding ability of PB2 to acetyl-CoA and its RNA polymerase activity. Recombinant viruses containing the same mutations could not be replicated in cultured cells. These results indicate that the PB2 VRG sequence is a functional site that is essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication.
authors: From the Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan.
authors: Dai
authors: D
authors: Hatakeyama
authors: From the Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan.
authors: Masaki
authors: M
authors: Shoji
authors: the Department of Microbiology and Immunology, Division of Virology, Institute of Medical Science, and.
authors: Seiya
authors: S
authors: Yamayoshi
authors: From the Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan.
authors: Takenori
authors: T
authors: Hirota
authors: From the Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan.
authors: Monami
authors: M
authors: Nagae
authors: From the Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan.
authors: Shin
authors: S
authors: Yanagisawa
authors: the Department of Microbiology and Immunology, Division of Virology, Institute of Medical Science, and PRESTO, Japan Science and Technology Agency, Saitama 332-0012, Japan, and.
authors: Masahiro
authors: M
authors: Nakano
authors: From the Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan.
authors: Naho
authors: N
authors: Ohmi
authors: the Department of Microbiology and Immunology, Division of Virology, Institute of Medical Science, and PRESTO, Japan Science and Technology Agency, Saitama 332-0012, Japan, and.
authors: Takeshi
authors: T
authors: Noda
authors: the Department of Microbiology and Immunology, Division of Virology, Institute of Medical Science, and the Department of Special Pathogens, International Research Center for Infectious Diseases, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan, the Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin 53711.
authors: Yoshihiro
authors: Y
authors: Kawaoka
authors: From the Laboratory of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima 770-8514, Japan, kuzuhara@ph.bunri-u.ac.jp.
authors: Takashi
authors: T
authors: Kuzuhara
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism*; D000123:Acetyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D004285:Dogs; D005057:Eukaryotic Cells / Q000201:enzymology; D006801:Humans; D053122:Influenza A Virus, H3N2 Subtype / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D061985:Madin Darby Canine Kidney Cells; D062105:Molecular Docking Simulation; D008969:Molecular Sequence Data; D009154:Mutation; D011387:Prokaryotic Cells / Q000201:enzymology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012315:RNA Caps / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: C057027:PB2 protein, Influenzavirus A; D012315:RNA Caps; D014764:Viral Proteins; D000105:Acetyl Coenzyme A; D000123:Acetyltransferases; D012324:RNA-Dependent RNA Polymerase
keywords: Acetyl-coenzyme A (Acetyl-CoA); Influenza Virus; Negative-strand RNA Virus; RNA Polymerase; Recombinant Protein Expression