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We have used immunoblotting, immunocytochemical, and gene fusion methods to examine the differential subcellular partitioning of tobacco etch potyvirus proteins that are potentially associated with RNA replication. From the earliest timepoints at which viral proteins could be detected, proteins Nla (49-kilodalton proteinase) and Nlb (58-kilodalton polymerase) were localized primarily in the nucleus, whereas the 71-kilodalton cylindrical inclusion protein was identified in the cytoplasm. The Nla and Nlb coding regions were fused to the beta-glucuronidase (GUS) sequence in a plant expression vector, resulting in synthesis of chimeric proteins in transfected protoplasts and in transgenic plants. In situ localization of GUS activity revealed nuclear localization of the GUS-Nla and GUS-Nlb fusion proteins and cytoplasmic localization of nonfused GUS. These results indicate that both Nla and Nlb contain nuclear targeting signals, and that they may serve as useful models for studies of plant cell nuclear transport. A discussion of the general utility of the nuclear transport system described here, as well as the role of nuclear translocation of potyviral proteins, is presented.

The virus-encoded proteins of tobacco etch virus (TEV), a plant potyvirus, arise by proteolytic processing of a large polyprotein precursor. The TEV genome codes for two proteinases, a 49-kilodalton proteinase and helper component proteinase (HC-Pro), which cleave the polyprotein at specific sites. The only known cleavage event catalyzed by HC-Pro occurs at the HC-Pro carboxyl terminus. The proteolytic activity of HC-Pro was analyzed by expression of the enzyme in bacterial and cell-free systems. The carboxyl-terminal domain of HC-Pro exhibited proteolytic activity in Escherichia coli with a processing half-time of approximately 100 s. The processing kinetics of HC-Pro expressed in vitro by cell-free transcription and translation was variable, depending on the presence or absence of TEV polypeptide sequences at the amino terminus of the proteolytic domain. Cleavage of the HC-Pro carboxyl terminus appeared to proceed exclusively by an autocatalytic mechanism; the proteinase synthesized in vitro exhibited little or no proteolytic activity when reacted with the HC-Pro cleavage site in trans or biomolecular reactions.