title: Biochemical and genetic studies of the VPg uridylylation reaction catalyzed by the RNA polymerase of poliovirus.
abstract: The first step in poliovirus (PV) RNA synthesis is the covalent linkage of UMP to the terminal protein VPg. This reaction can be studied in vitro with two different assays. The simpler assay is based on a poly(A) template and requires synthetic VPg, purified RNA polymerase 3D(pol), UTP, and a divalent cation. The other assay uses specific viral sequences [cre(2C)] as a template for VPg uridylylation and requires the addition of proteinase 3CD(pro). Using one or both of these assays, we analyzed the VPg specificities and metal requirements of the uridylylation reactions. We determined the effects of single and double amino acid substitutions in VPg on the abilities of the peptides to serve as substrates for 3D(pol). Mutations in VPg, which interfered with uridylylation in vitro, were found to abolish viral growth. A chimeric PV containing the VPg of human rhinovirus 14 (HRV14) was viable, but substitutions of HRV2 and HRV89 VPgs for PV VPg were lethal. Of the three rhinoviral VPgs tested, only the HRV14 peptide was found to function as a substrate for PV1(M) 3D(pol) in vitro. We also examined the metal specificity of the VPg uridylylation reaction on a poly(A) template. Our results show a strong preference of the RNA polymerase for Mn(2+) as a cofactor compared to Mg(2+) or other divalent cations.
authors.affiliation: Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook, 11794, USA.
authors.forename: Aniko V
authors.initials: AV
authors.lastname: Paul
authors.forename: Julia
authors.initials: J
authors.lastname: Peters
authors.forename: JoAnn
authors.initials: J
authors.lastname: Mugavero
authors.forename: Jiang
authors.initials: J
authors.lastname: Yin
authors.forename: Jacques H
authors.initials: JH
authors.lastname: van Boom
authors.forename: E
authors.initials: E
authors.lastname: Wimmer
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D017955:Poliovirus / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D020798:Two-Hybrid System Techniques; D014758:Viral Core Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D014758:Viral Core Proteins; D012321:DNA-Directed RNA Polymerases