Encapsidation of DNA..............

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J Gen Virol January 2012 vir.0.040170-0

Encapsidation of DNA, a protein and a fluorophore into virus-like particles by the capsid protein of cucumber mosaic virus

Xiaoyun Lu, Jeremy R. Thompson and Keith L. Perry

An important property of some spherical plant viruses is their ability to reassemble in vitro from native capsid protein (CP) and RNA into infectious, virus-like particles (VLPs). Virions of Cucumber mosaic virus (CMV) are stabilized by protein-RNA interactions and the nucleic acid is essential for assembly. In this study, we demonstrate that VLPs will form in the presence of both single stranded (ss) and double stranded (ds) DNA oligonucleotides, and with a lower size limit of 20nts. Based on urea disruption assays, assembled VLPs from CMV CP and RNA (ReCMV) exhibited a level of stability similar to that of virions purified from plants, while VLPs from CMV CP and a 20mer exhibited comparable or greater stability. Fluorescent labeling of VLPs was achieved by the encapsidation of an Alexa Fluor® 488-labeled oligonucleotide 45mer (ReCMV-Alexa488-45) and confirmed by transmission electron and confocal microscopy. Using ssDNA as a nucleating factor, encapsidation of fluorescently labeled streptavidin (53kDa) conjugated to a biotinylated oligonucleotide was observed. The biological activity and stability of ReCMV and ReCMV-Alexa488-45 was confirmed in infectivity assays and insect vector feeding assays. This work demonstrates the utility of the CMV CP as a protein cage for use in the growing repertoire of nanotechnological applications.

URL

http://vir.sgmjournals.org/conte ... 0.040170-0.abstract