Generation of virus-like particles in plants expressing the capsid protein of Cymbidium Ringspot Virus

Agroinfiltration of Nicotiana benthamiana leaves with a construct expressing the coat protein of Cymbidium ringspot virus (CyRSV) resulted in the production of virus-like particles (VLPs) which showed a preferential localization within mitochondria with an apparently intact bounding membrane. VLPs were either non penetrated or penetrated by the negative stain. The former had the same outward aspect and size of wild type CymRSV and were assumed to encapsidate the CP messanger RNA, which was recovered from virus preparations purified from agroinfiltrated tissues. In addition, VLPs were shown to be able to encapsidate tombusviral satellite RNAs. Immunoblot analysis of dissociated VLPs showed that they were made up of a protein indistinguishable from the native CymRSV CP. A mitochondrial targeting signal was identified in the N-terminal region of CymRSV CP at amino acid (aa) position 10-27. The fusion protein CPMyc, containing the 10-aa Myc epitope fused to the CymRSV CP C-terminus, formed VLPs that were decorated by a Myc-specific antiserum.