Identification of the amino acid residue involved in rabbit hemorrhagic disease virus VPg uridylylation.
The virus genome-linked protein (VPg) coding region from rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) was expressed in Escherichia coli by using a glutathione S-transferase-based vector. The recombinant polypeptide could be purified in good yields and was uridylylated in vitro from [alpha-32P]UTP in a reaction catalyzed by the recombinant RNA-dependent RNA polymerase from RHDV in the absence of added template RNA. The use of deletion and point mutants allowed the identification of Tyr-21 as the residue involved in uridylylation and consequently in the linkage between VPg and the viral genome. These data constitute the first report on the identity of the amino acid residue involved in VPg uridylylation in a member of the Caliciviridae family.
Machín
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Departamento de Bioquimica y Biologia Molecular, Instituto Universitario de Biotecnologia de Asturias (Consejo Superior de Investigaciones Cientificas), Universidad de Oviedo, 33006 Oviedo, Spain.
Martín Alonso
J M
JM
Parra
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F
D000595:Amino Acid Sequence; D002412:Cations; D018076:DNA, Complementary / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D017929:Hemorrhagic Disease Virus, Rabbit / Q000737:chemistry* / Q000235:genetics; D007477:Ions; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014443:Tyrosine / Q000378:metabolism; D014542:Uridine Monophosphate / Q000378:metabolism*; D014758:Viral Core Proteins / Q000737:chemistry* / Q000235:genetics
D002412:Cations; D018076:DNA, Complementary; D007477:Ions; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins; D014443:Tyrosine; D014542:Uridine Monophosphate; D005982:Glutathione Transferase; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases