UBC Theses and Dissertations
In vitro translation of cucumber necrosis virus RNA Johnston, Julie Catherine
The in vitro translation products directed by cucumber necrosis virus (CNV) RNA were analyzed in both rabbit reticulocyte lysate and wheat germ extract cell-free translation systems. In rabbit reticulocyte lysates, one major protein of ca. 33 Mr was produced. In wheat germ extracts, four proteins of ca. 41, 33, 21 and 20 Mr were produced. Hybrid-arrested translation (HART) studies using synthetic CNV antisense RNA corresponding to the entire CNV genome demonstrated that the four major proteins synthesized from CNV virion RNA in wheat germ extracts are virus-specific translation products. The genomic locations of the CNV in vitro translation products were determined using a number of experimental approaches including: (1) HART using antisense RNA corresponding to selected regions of the CNV genome; (2) in vitro translation of synthetic messenger-sense CNV transcripts; (3) immunoprecipitation of in vitro translation products with CNV polyclonal antisera and (4) in vitro translation of size-fractionated CNV virion RNA. Together, these experiments demonstrated that the ca. 33 Mr protein is derived from the 5' proximal coding region, the ca. 41 Mr protein is derived from an internal coding region, and that at least one but probably both of the ca. 20 and 21 Mr proteins are derived from the 3' terminal coding region(s) of the CNV genome. In addition, immunoprecipitation experiments provided further evidence that the ca. 41 Mr protein is the viral coat protein. The size, number, and genomic locations of the CNV in vitro translation products reported here are in agreement with those predicted from nucleotide sequence data (Rochon & Tremaine, 1989). The natural template for the expression of downstream cistrons in the CNV genome was investigated by in vitro translation of sucrose fractionated CNV virion RNA as well as in vitro translation of messenger-sense synthetic transcripts. These studies indicate that in vitro, both subgenomic and genomic-length CNV RNA molecules may act as templates for the synthesis of the ca. 41,21 and 20 Mr proteins as well as the ca. 33 Mr protein.
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