The Generation of Turnip Crinkle Virus-Like Particles in Plants by the Transient Expression of Wild-Type and Modified Forms of Its Coat Protein.

Keith Saunders,George P Lomonossoff

doi:10.3389/fpls.2015.01138

Keith Saunders, George P Lomonossoff

Open Access

https://doi.org/10.3389/fpls.2015.01138

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Abstract

Turnip crinkle virus (TCV), a member of the genus carmovirus of the Tombusviridae family, has a genome consisting of a single positive-sense RNA molecule that is encapsidated in an icosahedral particle composed of 180 copies of a single type of coat protein. We have employed the CPMV-HT transient expression system to investigate the formation of TCV-like particles following the expression of the wild-type coat protein or modified forms of it that contain either deletions and/or additions. Transient expression of the coat protein in plants results in the formation of capsid structures that morphologically resemble TCV virions (T = 3 structure) but encapsidate heterogeneous cellular RNAs, rather than the specific TCV coat protein messenger RNA. Expression of an amino-terminal deleted form of the coat protein resulted in the formation of smaller T = 1 structures that are free of RNA. The possibility of utilizing TCV as a carrier for the presentation of foreign proteins on the particle surface was also explored by fusing the sequence of GFP to the C-terminus of the coat protein. The expression of coat protein-GFP hybrids permitted the formation of VLPs but the yield of particles is diminished compared to the yield obtained with unmodified coat protein. Our results confirm the importance of the N-terminus of the coat protein for the encapsidation of RNA and show that the coat protein's exterior P domain plays a key role in particle formation.

Highlights

Virus-like particles (VLPs) are increasingly being utilized as vaccines and are finding use in emerging roles in the rapidly and ever expanding bionanotechnology field
Denaturing polyacrylamide gel analysis and Instant Blue staining of extracts from Nicotiana benthamiana leaves infiltrated with pEAQ-HT-P38 containing the sequence of the full-length Turnip crinkle virus (TCV) coat protein (Figure 2), revealed the presence of a protein that had gel migration equivalent to that of TCV coat protein (P38) which had been extracted from purified TCV particles
To test whether the transiently expressed P38 can form TCV-like particles, extracts from infiltrated leaves were subjected to the TCV extraction and purification protocol and the resulting material analyzed on 10–50% (w/v) sucrose gradients

Summary

Introduction

Virus-like particles (VLPs) are increasingly being utilized as vaccines and are finding use in emerging roles in the rapidly and ever expanding bionanotechnology field. Denaturing polyacrylamide gel analysis and Instant Blue staining of extracts from Nicotiana benthamiana leaves infiltrated with pEAQ-HT-P38 containing the sequence of the full-length TCV coat protein (Figure 2), revealed the presence of a protein that had gel migration equivalent to that of TCV coat protein (P38) which had been extracted from purified TCV particles (data not shown).

Results

Conclusion