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Journal of Virology, February 2001, p. 1211-1219, Vol. 75, No. 3
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.3.1211-1219.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Proteinase-Polymerase Precursor as the Active Form of Feline Calicivirus RNA-Dependent RNA Polymerase

Lai Wei,1 Jason S. Huhn,1 Aaron Mory,1 Harsh B. Pathak,1 Stanislav V. Sosnovtsev,2 Kim Y. Green,2 and Craig E. Cameron1,*

Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania,1 and Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland2

Received 28 July 2000/Accepted 26 October 2000

The objective of this study was to identify the active form of the feline calicivirus (FCV) RNA-dependent RNA polymerase (RdRP). Multiple active forms of the FCV RdRP were identified. The most active enzyme was the full-length proteinase-polymerase (Pro-Pol) precursor protein, corresponding to amino acids 1072 to 1763 of the FCV polyprotein encoded by open reading frame 1 of the genome. Deletion of 163 amino acids from the amino terminus of Pro-Pol (the Val-1235 amino terminus) caused a threefold reduction in polymerase activity. Deletion of an additional one (the Thr-1236 amino terminus) or two (the Ala-1237 amino terminus) amino acids produced derivatives that were 7- and 175-fold, respectively, less active than Pro-Pol. FCV proteinase-dependent processing of Pro-Pol in the interdomain region preceding Val-1235 was not observed in the presence of a catalytically active proteinase; however, processing within the polymerase domain was observed. Inactivation of proteinase activity by changing the catalytic cysteine-1193 to glycine permitted the production and purification of intact Pro-Pol. Biochemical analysis of Pro-Pol showed that this enzyme has properties expected of a replicative polymerase, suggesting that Pro-Pol is an active form of the FCV RdRP.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Pennsylvania State University, 201 Althouse Laboratory, University Park, PA 16802. Phone: (814) 863-8705. Fax: (814) 863-7024. E-mail: cec9{at}psu.edu.

Journal of Virology, February 2001, p. 1211-1219, Vol. 75, No. 3
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.3.1211-1219.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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