Cistron mapping of tobacco vein mottling virus

Abstract

The location and order of cistrons in the RNA of the potyvirus tobacco vein mottling virus (TVMV) were investigated. Hybrid-arrested translation, using cloned single-stranded DNA probes complementary to various regions of the viral RNA, was performed and the resulting translation products were analyzed by electrophoresis. The pattern of polypeptides produced with each probe was different from that of control reactions containing RNA alone. Immunoprecipitation of reaction products with antisera to five potyviral proteins revealed that, in some cases, portions of cistrons were masked by the DNA probe resulting in the precipitation of altered polypeptides. In other cases, the entire cistron was affected, resulting in the total loss of immunoreactive material. By correlating the location of each DNA probe with the resulting pattern of translation products, it was possible to construct a map of known cistrons and potential coding regions for additional cistrons in the genomic RNA. DNA probes representing several regions of the viral RNA were expressed in E. coli. Immunoprecipitation of cell proteins revealed the presence of TVMV-related polypeptides; the results were consistent with the cistron order determined by hybrid-arrested translation experiments. Our proposed cistron order and the sizes in kilodaltons of the corresponding polypeptides are: 5′-25 kDa unidentified protein-53 kDa helper component protein-50 kDa unidentified protein-70 kDa cylindrical inclusion protein-52 kDa nuclear inclusion protein-56 kDa nuclear inclusion protein-32 kDa coat protein-3′.