Substrate recognition by the NIa proteinase of two potyviruses involves multiple domains: characterization using genetically engineered hybrid proteinase molecules.
The proteolytic activity associated with the small nuclear inclusion protein (NIa proteinase) of tobacco etch virus (TEV), a potyvirus, catalyzes several cleavages at sites within the polyprotein derived from the TEV RNA genome. The homologous proteinase of tobacco vein mottling virus (TVMV), a closely related potyvirus, cleaves at similar, yet distinct, recognition sites. We examined these proteinases, in a cell-free cleavage system, in an attempt to define the biochemical basis of substrate specificity. Each proteinase was specific for its own cleavage site sequence in cell-free trans processing reactions, and no processing of the heterologous cleavage site was evident. Domains of the proteinase which were important in determining this substrate specificity were identified by generating hybrid proteinase genes containing both TEV and TVMV NIa proteinase coding sequences. Using site-directed mutagenesis and standard recombinant DNA techniques, plasmids were constructed which contained coding sequences for hybrid TEV-TVMV proteinases. These plasmids were expressed and tested in a cell-free environment for their ability to cleave both TEV and TVMV substrates. The data suggest that the carboxy-terminal 150 amino acids of the NIa protein contain the necessary information to specifically recognize a particular cleavage site sequence, and that specificity determinants are contained in at least three interactive subdomains within this region.
Parks
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Department of Microbiology, Oregon State University, Corvallis 97331-3804.
Dougherty
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D000595:Amino Acid Sequence; D010450:Endopeptidases; D008969:Molecular Sequence Data; D010942:Plant Viruses / Q000201:enzymology* / Q000235:genetics; D011506:Proteins / Q000378:metabolism; D012328:RNA Viruses / Q000201:enzymology* / Q000235:genetics; D011993:Recombinant Fusion Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*
D011506:Proteins; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D010450:Endopeptidases; C059992:nuclear inclusion protein a, mosaic viruses