In Planta Production of Fluorescent Filamentous Plant Virus-Based Nanoparticles

Viral nanoparticles are attractive platforms for biomedical applications and are frequently employed for optical imaging in tissue culture and preclinical animal models as fluorescent probes. Chemical modification with organic dyes remains the most common strategy to develop such fluorescent probes. Here we report a genetic engineering approach to incorporate fluorescent proteins in viral nanoparticles, which can be propagated in their plant host. The fluorescent viral nanoparticles so obtained obviate post-harvest modifications and thereby maximize yields. Our engineering approach transforms filamentous potato virus X (PVX) to display green fluorescent protein (GFP) or mCherry as N-terminal coat protein (CP) fusions at a 1:3 fusion protein to CP ratio through integration of the foot-and-mouth disease 2A sequence. The in planta propagation of recombinant GFP-PVX or mCherry-PVX thus produced in Nicotiana benthamiana can be easily documented using fluorescence imaging. Molecular farming protocols can be accordingly optimized by monitoring chimera stability over the course of the infection cycle. Moreover, we also demonstrate the utility of recombinant mCherry-PVX in optical imaging of human cancer cells and tumor tissue in preclinical mice model. Together, these features make genetically engineered fluorescent PVX particles ideally suited for molecular imaging applications.