Analysis of the VPg-Proteinase (NIa) Encoded by Tobacco Etch Potyvirus: Effects of Mutations on Subcellular Transport, Proteolytic Processing, and Genome Amplification Public Deposited

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  • A mutational analysis was conducted to investigate the functions of the tobacco etch potyvirus VPgproteinase (NIa) protein in vivo. The NIa N-terminal domain contains the VPg attachment site, whereas the C-terminal domain contains a picornavirus 3C-like proteinase. Cleavage at an internal site separating the two domains occurs in a subset of NIa molecules. The majority of NIa molecules in TEV-infected cells accumulate within the nucleus. By using a reporter fusion strategy, the NIa nuclear localization signal was mapped to a sequence within amino acid residues 40 to 49 in the VPg domain. Mutations resulting in debilitation of NIa nuclear translocation also debilitated genome amplification, suggesting that the NLS overlaps a region critical for RNA replication. The internal cleavage site was shown to be a poor substrate for NIa proteolysis because of a suboptimal sequence context around the scissile bond. Mutants that encoded NIa variants with accelerated internal proteolysis exhibited genome amplification defects, supporting the hypothesis that slow internal processing provides a regulatory function. Mutations affecting the VPg attachment site and proteinase activesite residues resulted in amplification-defective viruses. A transgenic complementation assay was used to test whether NIa supplied in trans could rescue amplification-defective viral genomes encoding altered NIa proteins. Neither cells expressing NIa alone nor cells expressing a series of NIa-containing polyproteins supported increased levels of amplification of the mutants. The lack of complementation of NIa-defective mutants is in contrast to previous results obtained with RNA polymerase (NIb)-defective mutants, which were relatively efficiently rescued in the transgenic complementation assay. It is suggested that, unlike NIb polymerase, NIa provides replicative functions that are cis preferential
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  • Schadd, M. C., Haldeman-Cahill, R., Cronin, S., & Carrington, J. C. (1996, October). Analysis of the VPg-Proteinase (NIa) Encoded by Tobacco Etch Potyvirus: Effects of Mutations on Subcellular Transport, Proteolytic Processing, and Genome Amplification. Journal of Virology, 70(10), 7039-7048
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  • description.provenance : Approved for entry into archive by Sue Kunda(sue.kunda@oregonstate.edu) on 2011-05-26T00:37:11Z (GMT) No. of bitstreams: 1 CarringtonJamesC.BotanyPlantPathology.AnalysisVPgProteinase (NIa).pdf: 492673 bytes, checksum: 6b10475810e03dca1e457b3cc98ea2c2 (MD5)
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