EXPRESSION OF ENZYMATICALLY ACTIVE-RABBIT HEMORRHAGIC-DISEASE VIRUS RNA-DEPENDENT RNA-POLYMERASE IN ESCHERICHIA-COLI

The rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) RNA-depen dent RNA-polymerase (3D(pol)) coding region was expressed in Escherich ia coli by using a glutathione S-transferase-based vector, which allow ed milligram purification of a homogeneous enzyme with an expected mol ecular mass of about 58 kDa, The recombinant polypeptide exhibited rif ampin-and actinomycin D-resistant, poly(A)-dependent poly(U) polymeras e, The enzyme also showed RNA polymerase activity in in vitro reaction s with synthetic RHDV subgenomic RNA in the presence or absence of an oligo(U) primer, Template-size products were synthesized in the oligo( U)-primed reactions, whereas in the absence of added primer, RNA produ cts up to mice the length of the template were made, The double-length RNA products were double stranded and hybridized to both positive- an d negative-sense probes.