title: Endonuclease substrate selectivity characterized with full-length PA of influenza A virus polymerase.
abstract: The influenza A polymerase is a heterotrimer which transcribes viral mRNAs and replicates the viral genome. To initiate synthesis of mRNA, the polymerase binds a host pre-mRNA and cleaves a short primer downstream of the 5' end cap structure. The N-terminal domain of PA has been demonstrated to have endonuclease activity in vitro. Here we sought to better understand the biochemical nature of the PA endonuclease by developing an improved assay using full-length PA protein. This full-length protein is active against both RNA and DNA in a cap-independent manner and can use several different divalent cations as cofactors, which affects the secondary structure of the full-length PA. Our in vitro assay was also able to demonstrate the minimal substrate size and sequence selectivity of the PA protein, which is crucial information for inhibitor design. Finally, we confirmed the observed endonuclease activity of the full-length PA with a FRET-based assay.
authors: Department of Microbiology and Immunology, University of Rochester, Rochester, NY 14642, USA.
authors: Erin
authors: E
authors: Noble
authors: Andrew
authors: A
authors: Cox
authors: Jerome
authors: J
authors: Deval
authors: Baek
authors: B
authors: Kim
mesh_terms: D001681:Biological Assay; D002413:Cations, Divalent; D003067:Coenzymes / Q000378:metabolism; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D009980:Influenza A virus / Q000201:enzymology* / Q000235:genetics; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D055503:Protein Multimerization; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012315:RNA Caps / Q000737:chemistry* / Q000235:genetics; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002413:Cations, Divalent; D003067:Coenzymes; C061290:PA protein, influenza viruses; D012315:RNA Caps; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D008345:Manganese; D012324:RNA-Dependent RNA Polymerase; D004720:Endonucleases; D008274:Magnesium