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Title: DEVELOPMENT AND EVALUATION OF A COMPLEMENTATION-DEPENDENT GENE DELIVERY SYSTEM BASED ON CUCUMBER MOSAIC VIRUS

Author
item Zhao, Yan
item Hammond, John
item TOUSIGNANT, MARIE - RETIRED SUPPORT SCIENTIST
item Hammond, Rosemarie

Submitted to: Archives of Virology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/18/2000
Publication Date: N/A
Citation: N/A

Interpretive Summary: Plant virus-based vectors as transient expression systems provide attractive alternatives to conventional breeding and genetic transformation technology. They have great potential for the expression of pharmaceutical compounds, including vaccines and therapeutic drugs, in plants. In this report, we describe the development of a novel strategy to express foreign genes in plants using cucumber mosaic virus, a virus that infects over 600 plant species. The strategy is based on the ability of two defective components of the virus to complement one another so that function of the virus is maintained. Although the engineered virus could move only a short distance in inoculated leaf tissue, it could not move throughout the plant, indicating that further modifications are needed. The information will be of use to scientists who are developing new technologies based on viral vectors that will benefit consumers by providing safe and affordable pharmaceutical products and to growers by providing alternative value-added products in plants.

Technical Abstract: To engineer cucumber mosaic virus (CMV-Ix) into a gene vector, genome component RNA 3 of the virus was modified and split into two sub-components, RNA 3A and RNA 3B. In RNA 3A, the open reading frame of the movement protein (MP) was replaced by a reporter gene, the green fluorescent protein (GFP), to monitor virus replication and movement. In RNA 3B, the coat protein (CP) gene was eliminated and a multiple cloning site (MCS) was created for foreign gene insertion. Each sub-component alone is defective and relies on its companion sub-component to restore full RNA 3 function. The vector system was evaluated for its ability to deliver and express the bacterial B-glucuronidase (GUS) gene and a modified bean yellow mosaic virus coat protein (BYMV-CP) gene in Nicotiana benthamiana plants. Results showed that the engineered virus was able to move from cell to cell in the inoculated leaf and enter the minor veins of the inoculated leaf. Foreign gene expression was detected in the inoculated leaves as well as some of the systemically infected leaves. However, intermolecular recombination between RNA 3A and 3B occurred frequently, preventing efficient systemic expression of the foreign gene(s). Modifications and further evaluations are being undertaken to improve the gene delivery system.