Development of RNA-free particles of Cowpea mosaic virus for applications in Nanotechnology

Saxena, Pooja (2012) Development of RNA-free particles of Cowpea mosaic virus for applications in Nanotechnology. Doctoral thesis, University of East Anglia.

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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
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.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Users 2259 not found.
Date Deposited: 02 May 2013 11:54
Last Modified: 02 May 2013 11:54


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