Subcellular partitioning and regulation of nuclear transport of tobacco etch potyvirus proteins

Abstract

The subcellular localization of several tobacco etch potyvirus (TEV) proteins in infected cells was analyzed. From the earliest time points at which the proteins were detected, the helper component-proteinase (HC-Pro), and cylindrical inclusion protein (Cl) were localized in the cytoplasm. The VPg/proteinase (NIa) and the catalytic subunit of the polymerase (Nib) accumulated in the nucleus. The sequence for the reporter protein β-glucuronidase (GUS) was fused to the sequences for NIa or NIb in a plant expression vector, resulting in synthesis of chimeric proteins in transfected protoplasts and transgenic plants. Both proteins mediated nuclear translocation of GUS, indicating that they have independent nuclear localization signals. To understand the factors that regulate the nuclear transport of NIa, transgenic plants that express genes encoding GUS fused to NIa or the 6/NIa polyprotein were generated. When located in a 6-kDa protein/NIa polyprotein, the 6-kDa protein impeded the nuclear translocation of NIa. The presence of free 6-kDa protein had no effect on NIa-mediated translocation. The 6-kDa protein and three polyproteins containing the 6- kDa protein were identified by affinity chromatography in extracts from infected plants. Two of these polyproteins contained NIa or the N-terminal domain of NIa (VPg). The subcellular localization properties of the 6-kDa protein were investigated by fractionation and immunolocalization of tissues from transgenic plants expressing 6-kDa protein/GUS fusion proteins. The GUS activity was detected in the cytosolic fraction (S30) of extracts derived from transgenic plants expressing non-fused GUS, while GUS activity was detected in the crude membrane fraction (P30) of extracts derived from transgenic plants expressing 6/GUS or GUS/6 fusion proteins. Transgenic plants expressing a GUS/6 fusion protein developed mitochondrial aggregates, and cytoplasmic membranous proliferations around and extending from the nucleus. The 6-kDa protein accumulated in these novel structures. The data presented in this dissertation suggest that differential processing and subcellular accumulation of proteins may be the mechanism by which potyviruses regulate the effective amounts of active replication proteins.