title: The tobacco etch potyvirus 6-kilodalton protein is membrane associated and involved in viral replication.
abstract: 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 beta-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.
authors.affiliation: Department of Biology, Texas A&M University, College Station 77843.
authors.forename: M A
authors.initials: MA
authors.lastname: Restrepo-Hartwig
authors.forename: J C
authors.initials: JC
authors.lastname: Carrington
mesh_terms: D000595:Amino Acid Sequence; D002451:Cell Compartmentation; D002458:Cell Fractionation; D005784:Gene Amplification; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D010947:Plants, Toxic; D017800:Potyvirus / Q000235:genetics / Q000254:growth & development* / Q000648:ultrastructure; D012367:RNA, Viral / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana / Q000382:microbiology* / Q000648:ultrastructure; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins