Nicotiana benthamiana as a Source of Cowpea Mosaic Virus-Derived Particles That Specifically Package Designer RNAs

The coupling of replication and encapsidation in the bipartite plant virus, cowpea mosaic virus (CPMV), has been used to specifically incorporate designer RNA molecules into viral particles via transient expression in Nicotiana benthamiana. This is achieved by placing the desired RNA sequence between the 5' and 3' UTRs of CPMV RNA-2 so that the cargo RNA can be recognised by the RNA-1-encoded replicase. Replication and encapsidation of cargo RNA are achieved by co-infiltrating with CPMV RNA-1 to provide replication functions and a non-replicating construct that encodes the precursor of the viral coat proteins, VP60. Encapsidation within CPMV particles stabilises the RNA such that it can be stored for prolonged periods at +4°C. Investigation of the length and sequence of the RNA that can be efficiently incorporated showed that a wide range of different RNA molecules are tolerated, provided that the length does not significantly exceed that of RNA-1 (6.0 kb). In contrast to previously published conclusions, we show here that a self-replicating version of RNA-1 is not required for the replication and packaging of cargo RNA constructs. Use of a non-replicating version of RNA-1 not only eliminates the packaging of RNA-1 but also increases the yield of particles containing the desired cargo RNA. Such particles have the potential to be used as delivery systems for bespoke RNAs to target cells.