title: A yeast-based genetic system for functional analysis of viral mRNA capping enzymes.
abstract: Virus-encoded mRNA capping enzymes are attractive targets for antiviral therapy, but functional studies have been limited by the lack of genetically tractable in vivo systems that focus exclusively on the RNA-processing activities of the viral proteins. Here we have developed such a system by engineering a viral capping enzyme-vaccinia virus D1(1-545)p, an RNA triphosphatase and RNA guanylyltransferase-to function in the budding yeast Saccharomyces cerevisiae in lieu of the endogenous fungal triphosphatase (Cet1p) and guanylyltransferase (Ceg1p). This was accomplished by fusion of D1(1-545)p to the C-terminal guanylyltransferase domain of mammalian capping enzyme, Mce1(211-597)p, which serves as a vehicle to target the viral capping enzyme to the RNA polymerase II elongation complex. An inactivating mutation (K294A) of the mammalian guanylyltransferase active site in the fusion protein had no impact on genetic complementation of cet1Deltaceg1Delta cells, thus proving that (i) the viral guanylyltransferase was active in vivo and (ii) the mammalian domain can serve purely as a chaperone to direct other proteins to the transcription complex. Alanine scanning had identified five amino acids of vaccinia virus capping enzyme-Glu37, Glu39, Arg77, Glu192, and Glu194-that are essential for gamma phosphate cleavage in vitro. Here we show that the introduction of mutation E37A, R77A, or E192A into the fusion protein abrogates RNA triphosphatase function in vivo. The essential residues are located within three motifs that define a family of viral and fungal metal-dependent phosphohydrolases with a distinctive capacity to hydrolyze nucleoside triphosphates to nucleoside diphosphates in the presence of manganese or cobalt. The acidic residues Glu37, Glu39, and Glu192 likely comprise the metal-binding site of vaccinia virus triphosphatase, insofar as their replacement by glutamine abolishes the RNA triphosphatase and ATPase activities.
authors.affiliation: Molecular Biology Program, Sloan-Kettering Institute, New York, New York 10021, USA.
authors.forename: C K
authors.initials: CK
authors.lastname: Ho
authors.forename: A
authors.initials: A
authors.lastname: Martins
authors.forename: S
authors.initials: S
authors.lastname: Shuman
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D020134:Catalytic Domain; D005800:Genes, Fungal / Q000235:genetics; D005814:Genes, Viral / Q000235:genetics; D005816:Genetic Complementation Test; D005818:Genetic Engineering; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D008670:Metals / Q000378:metabolism / Q000494:pharmacology; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D009097:Multienzyme Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012319:RNA Polymerase II / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics*; D014764:Viral Proteins; D000098556:mRNA Guanylyltransferases
chemical_list: D008670:Metals; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D008780:Methyltransferases; D009713:Nucleotidyltransferases; D012319:RNA Polymerase II; D000098556:mRNA Guanylyltransferases; D010744:Phosphoric Monoester Hydrolases; C027731:capping enzyme, vaccinia virus; D017766:Acid Anhydride Hydrolases; D000251:Adenosine Triphosphatases; C023090:RNA triphosphatase