The cauliflower mosaic virus ORF II encoding the aphid transmission factor (ATF) was mutagenized to introduce a BamHl restriction site upstream from the initiation codon and then cloned into an eukaryotic viral expression vector (Autographa californica nuclear polyhedrosis virus). All recombinant viruses tested in Spodopterafrugiperda (SF21) cells expressed a protein of about 18 kD which comigrated in PAGE with ATF from infected plants. Western blotting using an oligopeptide antiserum to ATF confirmed the identity of the 18-kD protein from infected cells as the product of the ORF II sequences (PI8). Subcellular fractionation of cells infected with the recombinant AcMNPV demonstrated that the expressed PI8 accumulated intracellularly in an insoluble form. Antiserum was produced in rabbit against the partially purified PI 8 expressed in SF21 cells. When used to immunogold label ultrathin sections of cauliflower mosaic virus (CaMV)-infected turnip tissue, this antiserum was shown to be highly specific, labelling only the electron-lucent inclusion bodies (containing PI8) and not other plant cellular components.

Keywords:
CaMV, Aphid transmission factor, Baculovirus expression
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© 1992 S. Karger AG, Basel
1992
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