Development of RNA-free particles of Cowpea mosaic virus for applications in Nanotechnology
A method for the efficient production of RNA-free particles of Cowpea mosaic virus (CPMV) has been developed. These are generated by co-expression of the precursor of the coat proteins (VP60) and the viral proteinase (24K) using the highly-efficient plant expression system, CPMV-HT, in the model plant Nicotiana benthamiana. Particles thus produced were shown to be identical to CPMV on the outside and devoid of RNA on the inside and were hence named CPMV empty virus-like particles (eVLPs). The availability of large quantities of purified eVLPs represents a significant milestone in the development of CPMV-based particle technologies and their potential applications in nanotechnology have been investigated. eVLPs were shown be genuinely empty unlike other VLPs which package random cellular RNAs from the host. The high specificity of CPMV in packaging led to the investigation of the requirements for efficient packaging in CPMV where the functional coupling of replication and encapsidation was identified. Methods have been presented to extend this approach for packaging heterologous nucleic acids in eVLPs for their application as delivery vehicles. To obtain a continuous supply of eVLPs, methods for its stable expression were developed for which the suppressor of silencing deployed in the CPMV-HT system, P19, was modified as the use of wt P19 inhibits regeneration of leaf tissue. A mutant form of P19, R43W, with reduced but still substantial suppressor activity was shown to permit the regeneration of transgenic plants. P19/R43W was used for the stable expression of a variety of heterologous proteins showing the broad applicability of this system. To reduce the possibility of homologous recombination, an alternative to the CPMV-HT system was developed by deploying the UTRs from CPMV RNA-1. Expression with RNA-1 UTRs was rapid as compared to CPMV-HT and hence, the expression system was named Rapid-Trans.
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