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J Virol, April 1998, p. 2999-3004, Vol. 72, No. 4
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Expression of Enzymatically Active Rabbit Hemorrhagic Disease Virus RNA-Dependent RNA Polymerase in Escherichia coli

Ana López Vázquez, José M. Martín Alonso, Rosa Casais, José A. Boga, and Francisco Parra*

Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006 Oviedo, Spain

Received 18 June 1997/Accepted 6 January 1998

The rabbit hemorrhagic disease virus (RHDV) (isolate AST/89) RNA-dependent RNA-polymerase (3Dpol) coding region was expressed in Escherichia coli by using a glutathione S-transferase-based vector, which allowed milligram purification of a homogeneous enzyme with an expected molecular mass of about 58 kDa. The recombinant polypeptide exhibited rifampin- and actinomycin D-resistant, poly(A)-dependent poly(U) polymerase. The enzyme also showed RNA polymerase activity in in vitro reactions 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 products up to twice the length of the template were made. The double-length RNA products were double stranded and hybridized to both positive- and negative-sense probes.

* Corresponding author. Mailing address: Departamento de Bioquímica y Biología Molecular, Instituto Universitario de Biotecnología de Asturias, Universidad de Oviedo, 33006 Oviedo, Spain. Phone: 34-8-5103563. Fax: 34-8-5103157. E-mail: parra{at}biosun.quimica.uniovi.es.




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