Development and evaluation of a complementation-dependent gene delivery system based on cucumber mosaic virus

To engineer cucumber mosaic virus (CMV-Ix) into a gene vector, genome compo nent RNA 3 of the virus,was modified and split into two subcomponents, RNA 3A and RNA 3B. In RNA 3A, the open reading frame of the movement protein (M P) was replaced by a reporter gene encoding the green fluorescent protein ( GFP), to monitor virus replication and movement. In RNA 3B, the coat protei n (CP) gene was eliminated and a multiple cloning site (MCS) was created fo r foreign gene insertion. Each sub-component alone is defective and relies on its companion sub-component to restore full RNA 3 function. The vector s ystem was evaluated for its ability to deliver and express the bacterial be ta -glucuronidase (GUS) gene and a modified bean yellow mosaic virus coat p rotein (BYMV-CP) gene in Nicotiana benthamiana plants. Results showed that the engineered virus was able to move from cell to cell in the inoculated l eaf and enter the minor veins of the inoculated leaf. Foreign gene expressi on was detected in the inoculated leaves. However, intermolecular recombina tion between RNA 3A and 3B occurred frequently, preventing efficient system ic expression of the foreign gene(s). Modifications and further evaluations are being undertaken to improve the gene delivery system.