Characterisation of the RNA-dependent RNA polymerase from Rabbit hemorrhagic disease virus produced in Escherichia coli.
All positive-strand RNA viruses encode a RNA-dependent RNA polymerase which in most cases has been only identified on the basis of its sequence conservation. Catalytic activity has been experimentally demonstrated in only a handful of these viral proteins, including that from Rabbit hemorrhagic disease virus. Studies from our laboratory have reported that RHDV RNA polymerase produced in Escherichia coli was enzymatically active showing poly(A)-dependent poly(U) polymerase as well as RNA polymerase activity on heteropolymeric substrates. In this work, we have investigated the in vitro activity of the recombinant 3Dpol from RHDV, including ion requirements, resistance to inhibitors, substrate specificity as well as data on the initiation mechanism of the template-linked products derived from heteropolymeric RNA substrates. Our study demonstrates that in an in vitro reaction recombinant RHDV RNA polymerase generated the minus strand of the heteropolymeric RNA substrates by a "copy-back" mechanism that initiated at the template 3'-terminal OH.
Departamento de Bioquîmica y Biología Molecular, Instituto Universitario de Biotecnología de Asturias (CSIC), Universidad de Oviedo, Spain.
A L
AL
López Vázquez
J M
JM
Martín Alonso
F
F
Parra
D000818:Animals; D004926:Escherichia coli / Q000235:genetics; D017929:Hemorrhagic Disease Virus, Rabbit / Q000201:enzymology*; D012313:RNA / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013698:Templates, Genetic
D011994:Recombinant Proteins; D012313:RNA; D012324:RNA-Dependent RNA Polymerase