A novel strategy for the expression of foreign genes from plant virus vectors

Potato virus X (PVX)-based vector constructs were generated to investigate the use of an internal ribosome entry site (IRES) sequence to direct transl ation of a viral gene. The 148-nucleotide IRESep sequence from a crucifer-i nfecting strain of tobacco mosaic virus was used to direct expression of th e PVX coat protein (CF), The IRES,vas inserted downstream of the gene encod ing green fluorescent protein (GFP) and upstream of the PVX CP, in either s ense or antisense orientation, such that CP expression depended on ribosome recruitment to the IRES. Stem-loop structures were inserted at either the 3 '- or 5 ' -end of the IRES sequence to investigate its mode of action. In vitro RNA transcripts were inoculated to Nicotiana benthamiana plants and protoplasts: levels of GFP and CP expression were analysed by enzyme-linked immunosorbent assay and the rate of virus cell-to-cell movement was determ ined by confocal laser scanning microscope imaging of GFP expression, PVX C P was expressed, allowing cell-to-cell movement of virus, from constructs c ontaining the IRES sequence in either orientation, and from the construct c ontaining a stem-loop structure at the 5 ' -end of the IRES sequence, No CP was expressed from a construct containing a stem-loop at the 3 ' -end of t he IRES sequence. Our results suggest that the IRES sequence is acting in v ivo to direct expression of the 3 ' -proximal open reading frame in a bicis tronic mRNA thereby demonstrating the potential of employing IRES sequences for the expression of foreign proteins from plant virus-based vectors. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier S cience B.V. All rights reserved.