Production of Hybrid Chimeric PVX Particles Using a Combination of TMV and PVX-Based Expression Vectors.

Christina Dickmeis,Rainer Fischer,Ulrich Commandeur,Mareike Michaela Antonia Honickel

doi:10.3389/fbioe.2015.00189

Abstract

We have generated hybrid chimeric potato virus X (PVX) particles by coexpression of different PVX coat protein fusions utilizing tobacco mosaic virus (TMV) and PVX-based expression vectors. Coinfection was achieved with a modified PVX overcoat vector displaying a fluorescent protein and a TMV vector expressing another PVX fluorescent overcoat fusion protein. Coexpression of the PVX-CP fusions in the same cells was confirmed by epifluorescence microscopy. Labeling with specific antibodies and transmission electron microscopy revealed chimeric particles displaying green fluorescent protein and mCherry on the surface. These data were corroborated by bimolecular fluorescence complementation. We used split-mCherry fragments as PVX coat fusions and confirmed an interaction between the split-mCherry fragments in coinfected cells. The presence of assembled split-mCherry on the surface confirmed the hybrid character of the chimeric particles.

Highlights

Plant virus particles offer an excellent tool for pharmaceutical and nanotechnological applications
Coinfections with two vectors based on the same virus (e.g., potato virus X (PVX)-green fluorescent protein (GFP)-2A-coat protein (CP) and PVX-mCherry-2A-CP) led to the spatial separation of the viruses as they spread through the host plant (Figures 3C,D), and in most cases, one vector became dominant
In coinfections with PVX and tobacco mosaic virus (TMV) vectors expressing different CPPVX fusions, we observed the coexpression of the different fluorescent proteins in the same parts of the plant (Figures 3A,B)

Summary

Introduction

Plant virus particles offer an excellent tool for pharmaceutical and nanotechnological applications. They can consist of multiple identical copies of one or two coat protein (CP) subunits and can be modified by genetic engineering or chemical conjugation (Meunier et al, 2004; Chatterji et al, 2005; Steinmetz, 2010; Rego et al, 2013; Lee et al, 2014). There is a growing demand for inexpensive vaccines that can be distributed and stored without maintaining the cold chain, in developing countries which lack an appropriate infrastructure (Ma et al, 2005b) This exacerbates even simple infectious diseases and leads to a high child mortality rate (Hefferon, 2013).

Methods

Results

Conclusion