THE TOBACCO ETCH POTYVIRUS 6-KILODALTON PROTEIN IS MEMBRANE-ASSOCIATED AND INVOLVED IN VIRAL REPLICATION

The tobacco etch potyvirus (TEV) genome encodes a polyprotein that is processed by three virus-encoded proteinases. Although replication of TEV likely occurs in the cytoplasm, two replication-associated proteins, VPg-proteinase (nuclear inclusion protein a) (NIa) and RNA-dependent RNA polymerase (nuclear inclusion protein b) (NIb), accumulate in the nucleus of infected cells. The 6-kDa protein is located adjacent to the N terminus of NIa in the TEV polyprotein, and, in the context of a 6-kDa protein/NIa (6/NIa) polyprotein, impedes nuclear translocation of NIa (M. A. Restrepo-Hartwig and J. C Carrington, J. Virol. 66:5662-5666, 1992). The 6-kDa protein and three polyproteins containing the 6-kDa protein were identified by affinity chromatography of extracts from infected plants. Two of the polyproteins contained NIa or the N-terminal VPg domain of NIa linked to the 6-kDa protein. To investigate the role of the 6-kDa protein in vivo, insertion and substitution mutagenesis was targeted to sequences coding for the 6-kDa protein and its N- and C-terminal cleavage sites. These mutations were introduced into a TEV genome engineered to express the reporter protein P-glucuronidase (GUS), allowing quantitation of virus amplification by a fluorometric assay. Three-amino-acid insertions at each of three positions in the 6-kDa protein resulted in viruses that were nonviable in tobacco protoplasts. Disruption of the N-terminal cleavage site resulted in a virus that was approximately 10% as active as the parent, while disruption of the C-terminal processing site eliminated virus viability. The subcellular localization properties of the 6-kDa protein were investigated by fractionation and immunolocalization of 6-kDa protein/GUS (6/GUS) fusion proteins in transgenic plants. Nonfused GUS was associated with the cytosolic fraction (30,000 X g centrifugation supernatant), while 6/GUS and GUS/6 fusion proteins sedimented with the crude membrane fraction (30,000 x g centrifugation pellet). The GUS/6 fusion protein was localized to apparent membranous proliferations associated with the periphery of the nucleus. These data suggest that the 6-kDa protein is membrane associated and is necessary for virus replication.