Conventionally, many researches into plant RNA virus vectors have been performed by using the infectious clones of the tobacco mosaic virus (TMV) and bromo mosaic virus (BMV), which have extremely strong proliferation potency in infected plants. Several systems for expressing foreign genes have also been developed in this field based on differences in the expression patterns of viral genes and the like. In the case of TMV and Potyviruses, for example, vectors have been developed, which express proteins by means of a viral CP gene fused to a foreign gene (Sugiyama, Y., Hamamoto, H., Takemoto, S., Watanabe, Y. and Okada, Y., “Systemic production of foreign peptides on the particle surface of Tobacco mosaic virus,” FEBS Letters 359, 247-250 (1995); Fernandez-Fernandez, M. R., Martinez-Torrecuadrada, J. L., Casal, J. I. and Garcia, J. A., “Development of an antigen presentation system based on plum pox potyvirus,” FEBS Letters 427, 229-235 (1998)). A vector expressing a foreign gene via the subgenome by introducing a subgenomic promoter of Odontoglossum ringspot virus, a close relative of TMV has also been developed (Donson et al., “Systemic expression of a bacterial gene by a tobacco mosaic virus-based vector,” Proc. Natl. Acad. Sci. USA 88: 7204-7208 (1991)).
However, these viruses were developed using rod-shaped and string-shaped viruses, typically TMV and Potyviruses. The advantage of rod-shaped and string-shaped viruses is that they present fewer physical limitations than spherical viruses on the length of the foreign gene to be inserted. On the other hand, as an expression vector system using the spherical virus BMV, a method of inoculating tobacco expressing 1a protein and 2a protein replicase genes with RNA3 having a foreign gene has been reported (Mori, M., Kaido, M., Okuno, T. and Furusawa, I., “mRNA amplification system by viral replicase in transgenic plants,” FEBS Lett. 336, 171-174 (1993).
Several studies have also been done on CMV viral vectors. One of these involves the development of a 4-segment CMV vector by means of complementation (see Zhao, Y., Hammond, J., Tousignant, M. E. and Hammond, R. W., “Development and evaluation of a complementation-dependent gene delivery system based on Cucumber mosaic virus,” Archives of Virology 145, 2285-2295 (2000)). MP and CP are coded for the RNA3 of CMV, and both MP and CP are essential for intercellular movement of viruses (see Zhao et al., above). These inventors have already altered the RNA3 molecule to construct RNA3A (in which the MP region is replaced by the GFP gene) and RNA3B (which lacks the CP gene), and have developed a virus vector that moves between cells by means of complementation. When this was used as CMV having a 4-segment genome (RNA1, RNA2, RNA3A and RNA3B) to inoculate benthamiana, GFP fluorescence was observed in a minor vein of the inoculated leaf. Moreover, when a multicloning site was inserted into the missing CP region of RNA3B, and the GUS (bacterial-β-glucuronidase) gene and BYMV (Bean yellow mosaic virus) CP gene were introduced therein, GUS activity and BYMV-CP were detected in a minor vein of the infected leaf. However, expression of the introduced genes was not observed in the upper leaf because RNA3A and RNA3B underwent homologous RNA recombination with each other.
Thus, although there have been various studies of CMV virus vectors, up till now in developing CMV vectors, the methods for introducing foreign genes into RNA3 have been adopted, with the result that proliferation has not been observed in the upper leaf in any case, and there is a strong need for improvement. In light of the prior art, the inventors attempted to introduce the foreign gene GFP into the RNA2 molecule of CMV with the aim of developing a novel virus vector, and perfected the present invention when they succeeded in developing a novel CMV vector in which the RNA2 molecule of CMV is altered.
USPTO Backgrounds
USPTO Backgrounds