title: Quantitative analysis of influenza virus RNP interaction with RNA cap structures and comparison to human cap binding protein eIF4E.
abstract: Influenza virus polymerase uses capped RNA primers for transcription initiation in infected cells. This unique mechanism involves the specific binding of the polymerase to capped mRNA precursors in the nucleus of infected cells. These host RNAs are then cleaved by a polymerase associated endonuclease at a position 10-15 nucleotides downstream of the cap structure. The resulting capped RNA oligonucleotides function as primers for transcription initiation. The viral cap binding site has previously been mapped to the PB2 subunit of the trimeric influenza polymerase complex. We have established a quantitative assay system for the analysis of cap interaction with PB2 as part of the native, viral ribonucleoprotein complex (RNP) using a specific UV cross-linking approach. Cap binding was not affected by the RNase pretreatment of the capped RNA substrate and cap binding was not inhibited by excess uncapped RNA, indicating that under the assay conditions, the majority of the binding energy was contributed by the interaction with the cap structure. Binding to 7-methyl-GTP was found to involve synergistic interaction with 7-methyl guanosine and triphosphate binding subsites. A similar mode of interaction with 7-methyl-GTP was found for human cap binding protein eIF4E. However, the potency of 7-methyl-GTP for cap binding inhibition was 200-fold stronger with eIF4E and had a higher contribution from the triphosphate moiety as compared to influenza RNP. Due to this difference in cap subsite interaction, it was possible to identify novel cap analogues, which selectively interact with influenza virus, but not human cap binding protein.
authors.affiliation: Roche Discovery Welwyn, Welwyn Garden City, UK.
authors.forename: Lisa
authors.initials: L
authors.lastname: Hooker
authors.forename: Rachel
authors.initials: R
authors.lastname: Sully
authors.forename: Balraj
authors.initials: B
authors.lastname: Handa
authors.forename: Naomi
authors.initials: N
authors.lastname: Ono
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Koyano
authors.forename: Klaus
authors.initials: K
authors.lastname: Klumpp
mesh_terms: D001483:Base Sequence; D003432:Cross-Linking Reagents; D004720:Endonucleases / Q000378:metabolism; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D009980:Influenza A virus / Q000378:metabolism* / Q000502:physiology; D012314:RNA Cap Analogs / Q000235:genetics / Q000378:metabolism; D039381:RNA Cap-Binding Proteins / Q000378:metabolism; D012315:RNA Caps / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D014466:Ultraviolet Rays; D014764:Viral Proteins / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D003432:Cross-Linking Reagents; D039561:Eukaryotic Initiation Factor-4E; D012314:RNA Cap Analogs; D039381:RNA Cap-Binding Proteins; D012315:RNA Caps; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D014764:Viral Proteins; D004720:Endonucleases