Adoption of the 2A Ribosomal Skip Principle to Tobacco Mosaic Virus for Peptide Display

Abstract

Plant viruses are suitable as building blocks for nanomaterials and nanoparticles because they are easy to modify and can be expressed and purified using plants or heterologous expression systems. Plant virus nanoparticles have been utilized for epitope presentation in vaccines, for drug delivery, as nanospheres and nanowires, and for biomedical imaging applications. Fluorescent protein fusions have been instrumental for the tagging of plant virus particles. The monomeric non-oxygen-dependent fluorescent protein iLOV can be used as an alternative to green fluorescent protein. In this study, the iLOV sequence was genetically fused either directly or via a glycine-serine linker to the C-terminus of the Tobacco mosaic virus (TMV) coat protein (CP) and also carried an N-terminal Foot-and-mouth disease virus (FMDV) 2A sequence. Nicotiana benthamiana plants were inoculated with recombinant viral vectors and a systemic infection was achieved. The presence of iLOV fusion proteins and hybrid particles was confirmed by western blot analysis and transmission electron microscopy. Our data suggest that TMV-based vectors are suitable for the production of proteins at least as large as iLOV when combined with the FMDV 2A sequence. This approach allowed the simultaneous production of foreign proteins fused to the CP as well as free CP subunits.

Optimization of aPlasmodium falciparumcircumsporozoite protein repeat vaccine using the tobacco mosaic virus platform

“…The N and C termini of the TMV capsid point outwards from an assembled TMV disk ( Fig. 1A) and majority of previous vaccine design efforts have used these N and C termini to display epitopes (41,(49)(50)(51)(52). Dedeo et al (53) reported a "circular permutant" of TMV where the N and C termini were re-engineered to the inner pore and a short loop was placed to close the sequence gap.…”

Systemic Infection of Nicotiana benthamiana with Potato virus X Nanoparticles Presenting a Fluorescent iLOV Polypeptide Fused Directly to the Coat Protein

“…The PVX-based vectors we used to produce recombinant particles displaying the iLOV polypeptide are shown in Figure 1(a) . The iLOV sequence was amplified from source vector pSC1001a (Dr. S. Chapman, The James Hutton Institute, Dundee, Scotland) using the primers listed in Table 1 and was transferred to pCR2.1-TOPO as previously described [ 57 ]. An iLOV fragment was isolated from this intermediate vector using NheI and BspEI and was transferred to vector pTCXIIc [ 37 ] either as a direct fusion (replacing the mCherry and FMDV 2A genes) or as fusion via the FMDV 2A sequence (replacing only the mCherry gene).…”

“…The integrity of the resulting vectors pPVX-iLOV-CP, pPVX-iLOV-2A-CP, and pPVX-iLOV-G 4 S-CP was tested by PCR and sequencing. The vectors were propagated in Escherichia coli strain DH5 α ready for the inoculation of Nicotiana benthamiana plants as previously described [ 57 ].…”

“…Proteins were extracted from systemically infected N. benthamiana leaves and were analyzed by SDS-PAGE [ 58 ] and western blot, with specific antibodies against iLOV and the PVX CP as previously described [ 57 ].…”

“…Pioloform-coated nickel grids (Plano, Wetzlar, Germany) were incubated on a drop of monoclonal antibody MAC58 against PVX (kindly provided by Professor L. Torrance, The James Hutton Institute) for 20 min at room temperature to capture the recombinant PVX particles. Immunogold decoration of captured or adsorbed particles with primary anti-PVX antibody or the anti-iLOV antiserum and secondary goat anti-rabbit (GAR) 15 nm gold conjugate (British BioCell, Cardiff, UK) and imaging was carried out as previously described [ 57 ].…”