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

FEBS Letters

Santa Cruz, Simon¹  Chapman, Sean¹  Carr, Fiona¹  Toth, Rachel L¹ 

[1] Mylnefield Research Services Ltd and Division of Biochemistry and Cell Biology, Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK

关键词: Potato virus X;    Virus vector;    Internal ribosome entry site;    Internal initiation;   

DOI  :  10.1016/S0014-5793(01)02091-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

Potato virus X (PVX)-based vector constructs were generated to investigate the use of an internal ribosome entry site (IRES) sequence to direct translation of a viral gene. The 148-nucleotide IREScp sequence from a crucifer-infecting strain of tobacco mosaic virus was used to direct expression of the PVX coat protein (CP). The IRES was inserted downstream of the gene encoding green fluorescent protein (GFP) and upstream of the PVX CP, in either sense 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 determined by confocal laser scanning microscope imaging of GFP expression. PVX CP was expressed, allowing cell-to-cell movement of virus, from constructs containing the IRES sequence in either orientation, and from the construct containing 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 the IRES sequence. Our results suggest that the IRES sequence is acting in vivo to direct expression of the 3′-proximal open reading frame in a bicistronic mRNA thereby demonstrating the potential of employing IRES sequences for the expression of foreign proteins from plant virus-based vectors.

【 授权许可】

Unknown