Functional differences between precursor and mature forms of the RNA-dependent RNA polymerase from rabbit hemorrhagic disease virus.
The genome region encoding the RNA-dependent RNA polymerase 3CD-like precursor from rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) was cloned and expressed in Escherichia coli using polyhistidine fusion-based vectors. The full-length recombinant 3CD-like precursor polypeptide could not be purified as a consequence of its autoproteolytic processing. A Cys-->Gly substitution of the 3C-like catalytic cysteine (C1212) impeded the cleavage and allowed the purification of the precursor at high yields using a polyhistidine fusion expression vector. Equimolar amounts of purified recombinant precursor (C1212G mutant) and mature 3D-like polymerase showed significant activity differences in genome-linked protein (VPg) uridylylation and RNA polymerization using in vitro assays. The data indicated that the precursor was more active than the mature polymerase in catalysing RHDV VPg uridylylation, whereas the latter enzyme form had higher activity than its precursor in RNA polymerization in vitro assays using a heteropolymeric RNA template.
Machín
Angeles
A
Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006 Oviedo, Spain.
Martín Alonso
José M
JM
Dalton
Kevin P
KP
Parra
Francisco
F
D017929:Hemorrhagic Disease Virus, Rabbit / Q000201:enzymology* / Q000235:genetics; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D011498:Protein Precursors; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase