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Agriculture and Agri-Food Canada, 6660 NW Marine Drive, Vancouver, British Columbia V6T 1X2, Canada
We have cloned the region of tomato ringspot nepovirus (TomRSV) RNA-1 coding for the putative TomRSV 3C-related protease (amino acids 1213 to 1508) in a transcription vector and in a transient expression vector. Using cell-free transcription and translation systems and plant protoplasts, we have demonstrated that proteins produced from these clones possess a proteolytic activity in trans on the cleavage site between the TomRSV movement and coat proteins. By amino acid homology of the TomRSV 3C-related protease with other nepo- and comovirus proteases, His1283, Glu1331 (or Asp1354) and Cys1433 have been predicted to constitute the catalytic triad. Site-directed mutagenesis of His1283 to Asp abolished the TomRSV protease activity, in vitro and in vivo. The cleavage site between the TomRSV movement and coat proteins has been determined to be Q/G, by direct protein sequencing. Previously, His1451 located in the substrate binding pocket of the TomRSV 3C-related protease has been suggested to be involved in the cleavage site specificity. We show that an inactive TomRSV 3C-related protease is obtained after substitution of His1451 with Leu. These results are discussed in light of the possible relation of the TomRSV 3C-related protease to 3C-related proteases of nepo-, como- and potyviruses.
* Author for correspondence. Fax +1 604 666 4994. e-mail SANFACON@PARGVA.AGR.CA
Received 13 September 1994;
accepted 15 November 1994.
This article has been cited by other articles:
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V. Thole and R. Hull Characterization of a protein from Rice tungro spherical virus with serine proteinase-like activity J. Gen. Virol., December 1, 2002; 83(12): 3179 - 3186. [Abstract] [Full Text] [PDF]
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S. Leonard, J. Chisholm, J.-F. Laliberte, and H. Sanfacon Interaction in vitro between the proteinase of Tomato ringspot virus (genus Nepovirus) and the eukaryotic translation initiation factor iso4E from Arabidopsis thaliana J. Gen. Virol., August 1, 2002; 83(8): 2085 - 2089. [Abstract] [Full Text] [PDF]
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K. Carrier, Y. Xiang, and H. Sanfacon Genomic organization of RNA2 of Tomato ringspot virus: processing at a third cleavage site in the N-terminal region of the polyprotein in vitro J. Gen. Virol., July 1, 2001; 82(7): 1785 - 1790. [Abstract] [Full Text] [PDF]
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A. Wang and H. Sanfaçon Proteolytic processing at a novel cleavage site in the N-terminal region of the tomato ringspot nepovirus RNA-1-encoded polyprotein in vitro J. Gen. Virol., November 1, 2000; 81(11): 2771 - 2781. [Abstract] [Full Text]
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A Wang, K Carrier, J Chisholm, A Wieczorek, C Huguenot, and H Sanfacon Proteolytic processing of tomato ringspot nepovirus 3C-like protease precursors: definition of the domains for the VPg, protease and putative RNA-dependent RNA polymerase J. Gen. Virol., March 1, 1999; 80(3): 799 - 809. [Abstract]
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