Evidence that the potyvirus P1 proteinase functions in trans as an accessory factor for genome amplification.
The tobacco etch potyvirus (TEV) polyprotein is proteolytically processed by three viral proteinases (NIa, HC-Pro, and P1). While the NIa and HC-Pro proteinases each provide multiple functions essential for viral infectivity, the role of the P1 proteinase beyond its autoproteolytic activity is understood poorly. To determine if P1 is necessary for genome amplification and/or virus movement from cell to cell, a mutant lacking the entire P1 coding region (delta P1 mutant) was produced with a modified TEV strain (TEV-GUS) expressing beta-glucuronidase (GUS) as a reporter, and its replication and movement phenotypes were assayed in tobacco protoplasts and plants. The delta P1 mutant accumulated in protoplasts to approximately 2 to 3% the level of parental TEV-GUS, indicating that the P1 protein may contribute to but is not strictly required for viral RNA amplification. The delta P1 mutant was capable of cell-to-cell and systemic (leaf-to-leaf) movement in plants but at reduced rates compared with parental virus. This is in contrast to the S256A mutant, which encodes a processing-defective P1 proteinase and which was nonviable in plants. Both delta P1 and S256A mutants were complemented by P1 proteinase expressed in a transgenic host. In transgenic protoplasts, genome amplification of the delta P1 mutant relative to parental virus was stimulated five- to sixfold. In transgenic plants, the level of accumulation of the delta P1 mutant was stimulated, although the rate of cell-to-cell movement was the same as in nontransgenic plants. Also, the S256A mutant was capable of replication and systemic infection in P1-expressing transgenic plants. These data suggest that, in addition to providing essential processing activity, the P1 proteinase functions in trans to stimulate genome amplification.
Verchot
J
J
Department of Biology, Texas A&M University, College Station 77843-3258, USA.
Carrington
J C
JC
D010450:Endopeptidases / Q000378:metabolism*; D005784:Gene Amplification; D005816:Genetic Complementation Test; D016679:Genome, Viral; D009154:Mutation; D030821:Plants, Genetically Modified; D017800:Potyvirus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D014176:Protein Biosynthesis; D011523:Protoplasts / Q000821:virology; D012333:RNA, Messenger / Q000235:genetics; D014764:Viral Proteins / Q000378:metabolism*
C071896:35-kDa protein, Potyvirus; D012333:RNA, Messenger; D014764:Viral Proteins; D010450:Endopeptidases