The roles of turnip yellow mosaic virus genes in virus replication Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/ht24wn57g

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  • Turnip yellow mosaic virus is a monopartite, plus sense RNA virus infecting the Cruciferae, and is a model system for the study of RNA virus replication. A cDNA clone (pTYMC) representing an infectious RNA genome of the European isolate of TYMV was constructed and used to assess the importance of virus genes in virus infectivity. Derivatives of pTYMC with alterations in open reading frame 69 (ORF- 69) were made. The mutations disrupted the expression of ORF-69 in vitro as predicted. Although the ORF-69 mutants were competent for replication in protoplasts, none of the mutants detectably infected turnip or Chinese cabbage plants, except where reversion mutations led to the restoration of an uninterrupted ORF-69. The data suggest a role for ORF-69 expression in the cell-to-cell movement of the virus. Mutant RNAs with a deletion or frameshift in the coat protein ORF infected protoplasts and plant leaves. No systemic infection symptoms were generated by these mutants, and no viral products were detected in young, expanding tissue of infected plants. When the coat protein deletion mutant and an ORF-69 mutant were co-inoculated onto plants, only a virus producing a coat protein of wild type size was detected in symptomatic, systemic tissue in these inoculations, emphasizing a requirement for the expression of native size coat protein for the systemic translocation of TYMV infection. The role of ORF-206 expression in TYMV replication was examined. Three classes of mutants were made in ORF-206: those affecting the synthesis of the 150 kDa protein, those affecting the synthesis of the 70 kDa protein, and those affecting the synthesis of both the 150 and the 70 kDa proteins. All ORF- 206 mutations eliminated RNA infectivity. Protoplast inoculations using mixtures of individual ORF-206 mutant RNAs and a helper genome demonstrated that co-replication of defective genomes could occur. Moreover, inoculations in which individual 150 kDa and 70 kDa protein mutant RNAs were combined showed that complementation between these two classes of mutants was possible. The data indicate that RNAs expressing wild type 150 kDa protein are favored replication substrates in mixed infections, and suggest that the 150 kDa protein functions preferentially in cis.
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