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Translation of cucumber necrosis virus RNA in vitro

Julie C. Johnston^1,

¹ Department of Plant Science, University of British Columbia, Vancouver, B.C., Canada V6T 2A2
and² Agriculture Canada, Vancouver Research Station, 6660 N.W. Marine Drive, Vancouver, B.C., Canada V6T 1X2

The in vitro translation products directed by cucumber necrosis virus (CNV) RNA were analysed in both rabbit reticulocyte lysate and wheatgerm extract cell-free translation systems. In rabbit reticulocyte lysates, one major protein of approximate M_r 34.6K was produced. In wheatgerm extracts, four proteins of approximate M_r values 41.6K, 34.6K, 24K and 20K were produced. The genomic locations of the CNV in vitro translation products were determined using several experimental approaches including, first, hybrid-arrested translation using negative-sense RNA corresponding to selected regions of the CNV genome, second, in vitro translation of synthetic positive-sense CNV transcripts and third, in vitro translation of CNV virion RNA fractionated according to size. Together these experiments demonstrated that the protein of M_r 34.6K is derived from the 5'-proximal coding region, the 41.6K protein is derived from an internal coding region, and that at least one but probably both the 24K and 20K proteins are derived from the 3'-terminal coding region. In addition, immunoprecipitation of in vitro translation products using anti-CNV polyclonal serum demonstrated that the 41.6K protein is the coat protein. The templates for the expression of CNV cistrons were investigated by in vitro translation of sucrose gradient-fractionated CNV virion RNA as well as in vitro translation of positive-sense synthetic transcripts.

Present address: Department of Microbiology, University of British Columbia, Vancouver, B.C., Canada V6T 2A2.

Received 17 April 1990; accepted 18 June 1990.

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