title: Membrane-bound tomato mosaic virus replication proteins participate in RNA synthesis and are associated with host proteins in a pattern distinct from those that are not membrane bound.
abstract: Extracts of vacuole-depleted, tomato mosaic virus (ToMV)-infected plant protoplasts contained an RNA-dependent RNA polymerase (RdRp) that utilized an endogenous template to synthesize ToMV-related positive-strand RNAs in a pattern similar to that observed in vivo. Despite the fact that only minor fractions of the ToMV 130- and 180-kDa replication proteins were associated with membranes, the RdRp activity was exclusively associated with membranes. A genome-sized, negative-strand RNA template was associated with membranes and was resistant to micrococcal nuclease unless treated with detergents. Non-membrane-bound replication proteins did not exhibit RdRp activity, even in the presence of ToMV RNA. While the non-membrane-bound replication proteins remained soluble after treatment with Triton X-100, the same treatment made the membrane-bound replication proteins in a form that precipitated upon low-speed centrifugation. On the other hand, the detergent lysophosphatidylcholine (LPC) efficiently solubilized the membrane-bound replication proteins. Upon LPC treatment, the endogenous template-dependent RdRp activity was reduced and exogenous ToMV RNA template-dependent RdRp activity appeared instead. This activity, as well as the viral 130-kDa protein and the host proteins Hsp70, eukaryotic translation elongation factor 1A (eEF1A), TOM1, and TOM2A copurified with FLAG-tagged viral 180-kDa protein from LPC-solubilized membranes. In contrast, Hsp70 and only small amounts of the 130-kDa protein and eEF1A copurified with FLAG-tagged non-membrane-bound 180-kDa protein. These results suggest that the viral replication proteins are associated with the intracellular membranes harboring TOM1 and TOM2A and that this association is important for RdRp activity. Self-association of the viral replication proteins and their association with other host proteins may also be important for RdRp activity.
authors.affiliation: Plant-Microbe Interactions Research Unit, Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba, Japan.
authors.forename: Masaki
authors.initials: M
authors.lastname: Nishikiori
authors.forename: Koji
authors.initials: K
authors.lastname: Dohi
authors.forename: Masashi
authors.initials: M
authors.lastname: Mori
authors.forename: Tetsuo
authors.initials: T
authors.lastname: Meshi
authors.forename: Satoshi
authors.initials: S
authors.lastname: Naito
authors.forename: Masayuki
authors.initials: M
authors.lastname: Ishikawa
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D018551:Solanum lycopersicum / Q000821:virology; D008565:Membrane Proteins / Q000378:metabolism*; D030821:Plants, Genetically Modified; D011523:Protoplasts / Q000821:virology; D012367:RNA, Viral / Q000096:biosynthesis*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism*; D014026:Nicotiana / Q000821:virology; D017898:Tobamovirus / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: D008565:Membrane Proteins; D012367:RNA, Viral; D012324:RNA-Dependent RNA Polymerase