Characterisation of the RNA-dependent RNA polymerase from Rabbit hemorrhagic disease virus produced in Escherichia coli

• Published in: Archives of virology Vol. 146; no. 1; pp. 59 - 69
• Main Authors: LOPEZ VAZQUEZ, A, MARTIN ALONSO, J. M, PARRA, F
• Format: Journal Article
• Language: English
• Published: Wien Springer 01.01.2001; New York, NY Springer Nature B.V
• Subjects: Animal viral diseases; Animals; Bacteriology; Biological and medical sciences; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Hemorrhagic Disease Virus, Rabbit - enzymology; Infectious diseases; Medical sciences; Microbiology; Pathogenicity, virulence, toxins, bacteriocins, pyrogens, host-bacteria relations, miscellaneous strains; Rabbit hemorrhagic disease virus; Rabbits; Recombinant Proteins - metabolism; RNA - metabolism; RNA Replicase - genetics; RNA Replicase - metabolism; Substrate Specificity; Templates, Genetic; Viral diseases; Disease; RNA; Escherichia coli; Rabbit; Rabbit hemorrhagic disease virus; DNA-directed RNA polymerase; Enzymatic activity; Caliciviridae; Bacteria; Enterobacteriaceae; Laboratory study; Enzyme; Transferases; Lagomorpha; In vitro; Protein; RNA-directed RNA polymerase; Virus; Resistance; Substrate; Calicivirus; Nucleotidyltransferases; Vertebrata; Mammalia; Inhibitor; Environmental protection
• ISSN: 0304-8608; 1432-8798
• DOI: 10.1007/s007050170191

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.