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Journal of Virology, February 2002, p. 1707-1717, Vol. 76, No. 4
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.4.1707-1717.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Comparison of Turnip Crinkle Virus RNA-Dependent RNA Polymerase Preparations Expressed in Escherichia coli or Derived from Infected Plants{dagger}

K. S. Rajendran, J. Pogany, and P. D Nagy*

Department of Plant Pathology, University of Kentucky, Lexington, Kentucky 40546

Received 24 July 2001/ Accepted 6 November 2001

Turnip crinkle virus (TCV) is a small, plus-sense, single-stranded RNA virus of plants. A virus-coded protein, p88, which is required for replication has been expressed and purified from Escherichia coli. In vitro assays revealed that the recombinant p88 has an RNA-dependent RNA polymerase (RdRp) activity and can also bind to RNA. Deletion of the N-terminal region in p88 resulted in a more active RdRp, while further deletions abolished RdRp activity. Comparison of the E. coli-expressed p88, the N-terminal deletion mutant of p88, and a TCV RdRp preparation obtained from infected plants revealed that these preparations show remarkable similarities in RNA template recognition and usage. Both the recombinant and the plant TCV RdRp preparations are capable of de novo initiation on both plus- and minus-strand satC and satD templates, which are small parasitic RNAs associated with TCV infections. In addition, these RdRp preparations can efficiently recognize the related Tomato bushy stunt virus promoter sequences, including the minus- and plus-strand initiation promoters. Heterologous viral and artificial promoters are recognized poorly by the recombinant and the plant TCV RdRps. Further comparison of the single-component recombinant TCV RdRp and the multicomponent plant TCV RdRp will help dissect the functions of various components of the TCV replicase.

* Corresponding author. Mailing address: Department of Plant Pathology, University of Kentucky, S-305, Ag. Sci. Bldg.-N, Lexington, KY 40546. Phone: (859) 257-2889. Fax: (859) 323-1961. E-mail: pdnagy2{at}pop.uky.edu.

{dagger} Publication no. 01-12-115 of the Kentucky Agricultural Experiment Station

Journal of Virology, February 2002, p. 1707-1717, Vol. 76, No. 4
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.4.1707-1717.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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