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A DNA clone encoding the full-length infectious genome of odontoglossum ringspot tobamovirus and mutagenesis of its coat protein gene

Yu, H. H., Wong, S. M.
Archives of virology 1998 v.143 no.1 pp. 163-171
DNA, DNA-directed RNA polymerase, Nicotiana benthamiana, Northern blotting, Odontoglossum ringspot virus, Oncidium, RNA, Western blotting, electron microscopy, enzyme-linked immunosorbent assay, genes, mutagenesis, mutants, pathogenicity, phenotype, seedlings, start codon
A full-length DNA clone encoding the genome of odontoglossum ringspot tobamovirus (ORSV) was synthesized and placed adjacent to a bacteriophage T7 RNA polymerase promoter. Capped-RNA transcripts produced in vitro were highly infectious when mechanically inoculated onto seedlings of Nicotiana benthamiana and Oncidium Gower Ramsey. A representative clone, designated pOT2, caused a disease phenotype identical to that produced by parental viral RNA. ELISA, Western blot analysis, Northern blot hybridization and electron microscopy verified the infectivity of pOT2. A coat protein deficient mutant of the clone, pOΔCP1, was produced with the initiation codon of the coat protein cistron of ORSV abolished. Transcripts from pOΔCP1 were infective, able to move in N. benthamiana but produced no coat protein. This demonstrates that the coat protein was dispensable for RNA replication and for movement. This is believed to be the first report of an ORSV infectious clone driven by a T7 RNA polymerase promoter.