title: Metal ion catalysis of RNA cleavage by the influenza virus endonuclease.
abstract: The influenza virus RNA-dependent RNA polymerase protein complex contains an associated RNA endonuclease activity, which cleaves host mRNA precursors in the cell nucleus at defined positions 9-15 nucleotides downstream of the cap structure. This reaction provides capped oligoribonucleotides, which function as primers for the initiation of viral mRNA synthesis. The endonuclease reaction is dependent on the presence of divalent metal ions. We have used a number of divalent and trivalent metal ions alone and in combination to probe the mechanism of RNA cleavage by the influenza virus endonuclease. Virus-specific cleavage was observed with various metal ions, and maximum cleavage activity was obtained with 100 microM Mn2+ or 100 microM Co2+. This activity was about 2-fold higher than that observed with Mg2+ at the optimal concentration of 1 mM. Activity dependence on metal ion concentration was cooperative with Hill coefficients close to or larger than 2. Synergistic activation of cleavage activity was observed with combinations of different metal ions at varying concentrations. These results support a two-metal ion mechanism of RNA cleavage for the influenza virus cap-dependent endonuclease. The findings are also consistent with a structural model of the polymerase, in which the specific endonuclease active site is spatially separated from the nucleotidyl transferase active site of the polymerase module.
authors.affiliation: Roche Discovery Welwyn, Welwyn Garden City, Herts, U.K.
authors.forename: L
authors.initials: L
authors.lastname: Doan
authors.forename: B
authors.initials: B
authors.lastname: Handa
authors.forename: N A
authors.initials: NA
authors.lastname: Roberts
authors.forename: K
authors.initials: K
authors.lastname: Klumpp
mesh_terms: D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002413:Cations, Divalent; D004357:Drug Synergism; D004789:Enzyme Activation; D006868:Hydrolysis; D009980:Influenza A virus / Q000201:enzymology*; D008274:Magnesium / Q000737:chemistry / Q000378:metabolism; D008345:Manganese / Q000737:chemistry / Q000378:metabolism; D008670:Metals / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D011817:Rabbits
chemical_list: D002413:Cations, Divalent; D008670:Metals; D012367:RNA, Viral; D008345:Manganese; D012324:RNA-Dependent RNA Polymerase; D008274:Magnesium