Specific Packaging of Custom RNA Molecules into Cowpea Mosaic Virus-like Particles

Recent discoveries in the dynamics of genome replication and packaging in the plant virus Cowpea mosaic virus (CPMV) has led to the development of a novel method for specifically packaging an RNA molecule of choice into virus-like particles (VLPs) of CPMV. Thanks to modern gene synthesis and molecular cloning methods, the DNA sequence corresponding to an RNA sequence of interest can be cloned into a suitable expression plasmid for transient expression in plants. We describe here a method for ensuring that this RNA sequence will be packaged within VLPs of CPMV in plant cells by replication-dependent RNA packaging. This requires co-expression of the CPMV replication machinery alongside the CPMV coat protein precursor. These components are co-expressed in the leaves of the Nicotiana benthamiana plant and this co-expression results in the production of large quantities of VLPs that contain the RNA sequence of choice. These VLPs are easy to extract and purify from the plant tissue, and are stable for months in refrigerated conditions. These VLPs can then be used for a variety of different applications, such as RNA delivery or control reagents in RT-qPCR.