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Journal of Virology, December 1999, p. 10129-10136, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Mutational Analysis of Bovine Viral Diarrhea Virus RNA-Dependent RNA Polymerase

Vicky C. H. Lai,1 C. Cheng Kao,2 Eric Ferrari, Justin Park,2 Annette S. Uss,1 Jacquelyn Wright-Minogue,1 Zhi Hong,1 and Johnson Y. N. Lau1,*

Department of Antiviral Therapy, Schering-Plough Research Institute, Kenilworth, New Jersey,1 and Department of Biology, Indiana University, Bloomington, Indiana2

Received 21 April 1999/Accepted 27 August 1999

Recombinant bovine viral diarrhea virus (BVDV) nonstructural protein 5B (NS5B) produced in insect cells has been shown to possess an RNA-dependent RNA polymerase (RdRp) activity. Our initial attempt to produce the full-length BVDV NS5B with a C-terminal hexahistidine tag in Escherichia coli failed due to the expression of insoluble products. Prompted by a recent report that removal of the C-terminal hydrophobic domain significantly improved the solubility of hepatitis C virus (HCV) NS5B, we constructed a similar deletion of 24 amino acids at the C terminus of BVDV NS5B. The resulting fusion protein, NS5BDelta CT24-His, was purified to homogeneity and demonstrated to direct RNA replication via both primer-dependent (elongative) and primer-independent (de novo) mechanisms. Furthermore, BVDV RdRp was found to utilize a circular single-stranded DNA as a template for RNA synthesis, suggesting that synthesis does not require ends in the template. In addition to the previously described polymerase motifs A, B, C, and D, alignments with other flavivirus sequences revealed two additional motifs, one N-terminal to motif A and one C-terminal to motif D. Extensive alanine substitutions showed that while most mutations had similar effects on both elongative and de novo RNA syntheses, some had selective effects. Finally, deletions of up to 90 amino acids from the N terminus did not significantly affect RdRp activities, whereas deletions of more than 24 amino acids at the C terminus resulted in either insoluble products or soluble proteins (Delta CT179 and Delta CT218) that lacked RdRp activities.

* Corresponding author. Mailing address: Department of Antiviral Therapy, K-15-4650, Schering-Plough Research Institute, 2015 Galloping Hill Rd., Kenilworth, NJ 07033-0539. Phone: (908) 740-3451. Fax: (908) 740-3918. E-mail: johnson.lau{at}spcorp.com.

Journal of Virology, December 1999, p. 10129-10136, Vol. 73, No. 12
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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Copyright © 1999 by the American Society for Microbiology. All rights reserved.