Structural and Biochemical Analyses on the RNA-dependent RNA Polymerase of Influenza Virus for Development of Novel Anti-influenza Agents.
The PA, PB1, and PB2 subunits, components of the RNA-dependent RNA polymerase of influenza A virus, and the nucleoprotein (NP) interact with the genomic RNA of influenza viruses and form ribonucleoproteins. Especially, the PB2 subunit binds to the host RNA cap [7-methylguanosine triphosphate (m7GTP)] and supports the endonuclease activity of PA to "snatch" the cap from host pre-mRNAs. In this study, we describe a novel Val/Arg/Gly (VRG) site in the PB2 cap-binding domain, which is necessary for 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, m7GTP also inhibited this interaction, suggesting that the same active pocket is capable of interacting with acetyl-CoA and m7GTP. 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 2 or 3 amino acid residues of the VRG site to alanine significantly reduced PB2's binding ability 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. I will also discuss some novel functions of NP in this review.
Faculty of Pharmaceutical Sciences, Tokushima Bunri University.
Dai
D
Hatakeyama
D000595:Amino Acid Sequence / Q000235:genetics; D000998:Antiviral Agents; D055808:Drug Discovery; D006801:Humans; D009980:Influenza A virus / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D007251:Influenza, Human / Q000188:drug therapy*; D009154:Mutation; D009698:Nucleoproteins / Q000737:chemistry / Q000502:physiology; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry* / Q000502:physiology; D012261:Ribonucleoproteins / Q000737:chemistry; D014779:Virus Replication
D000998:Antiviral Agents; D009698:Nucleoproteins; D012367:RNA, Viral; D012261:Ribonucleoproteins; D012324:RNA-Dependent RNA Polymerase