Production of Plant\nVirus-Derived Hybrid Nanoparticles\nDecorated with Different Nanobodies

Viral nanoparticles (VNPs) are self-assembled nanometric\ncomplexes\nwhose size and shape are similar to those of the virus from which\nthey are derived. VNPs are arousing great attention due to potential\nbiotechnological applications in fields like nanomedicine and nanotechnology\nbecause they allow the presentation of polypeptides of choice linked\nto the virus structural proteins. Starting from tobacco etch virus\n(TEV), a plant plus-strand RNA virus that belongs to the genus <i>Potyvirus</i> (family <i>Potyviridae</i>), here we\ndescribe the development of recombinant hybrid VNPs in <i>Nicotiana\nbenthamiana</i> plants able of exposing simultaneously different\nproteins on their surface. This system is based on the synergic coinfection\nof TEV and potato virus X (PVX; <i>Potexvirus</i>), in which\nPVX provides a second TEV CP <i>in trans</i> allowing a\nmixed assembly. We first generated genetically modified hybrid VNPs\nsimultaneously displaying green and red fluorescent proteins on their\nsurface. A population of decorated and nondecorated CPs resulting\nfrom the insertion of the picornavirus F2A ribosomal escape peptide\nwas required for viral particle assembly. Correct assembly of the\nrecombinant mosaic VNPs presenting the exogenous peptides was successfully\nobserved by immunoelectron microscopy. We next achieved the production\nof hybrid VNPs expressing a nanobody against SARS-CoV-2 and a fluorescent\nreporter protein, whose functionality was demonstrated by ELISA and\n dot-blot assay. Finally, we engineered the production of hybrid multivalent\nVNPs carrying two different nanobodies against distinct epitopes of\nthe same SARS-CoV-2 antigenic protein, emulating a nanobody cocktail.\nThese plant-produced recombinant mosaic VNPs, which are filamentous\nand flexuous in shape, presenting two different fused proteins on\nthe surface, represent a molecular tool with several potential applications\nin biotechnology.