Isopeptide Bonding In Planta Allows Functionalization of Elongated Flexuous Proteinaceous Viral Nanoparticles, including Non-Viable Constructs by Other Means

Daniel A Truchado; Sara Rincón; Lucía Zurita; Flora Sánchez; Fernando Ponz

doi:10.3390/v15020375

Isopeptide Bonding In Planta Allows Functionalization of Elongated Flexuous Proteinaceous Viral Nanoparticles, including Non-Viable Constructs by Other Means

Viruses. 2023 Jan 28;15(2):375. doi: 10.3390/v15020375.

Authors

Daniel A Truchado¹, Sara Rincón¹, Lucía Zurita¹, Flora Sánchez¹, Fernando Ponz¹

Affiliation

¹ Centro de Biotecnología y Genómica de Plantas (CBGP) (UPM-INIA/CSIC), Autopista M40, km 38, Campus Montegancedo, Pozuelo de Alarcón, 28223 Madrid, Spain.

Abstract

Plant viral nanoparticles (VNPs) have become an attractive platform for the development of novel nanotools in the last years because of their safety, inexpensive production, and straightforward functionalization. Turnip mosaic virus (TuMV) is one example of a plant-based VNP used as a nanobiotechnological platform either as virions or as virus-like particles (VLPs). Their functionalization mainly consists of coating their surface with the molecules of interest via chemical conjugation or genetic fusion. However, because of their limitations, these two methods sometimes result in non-viable constructs. In this paper, we applied the SpyTag/SpyCatcher technology as an alternative for the functionalization of TuMV VLPs with peptides and proteins. We chose as molecules of interest the green fluorescent protein (GFP) because of its good traceability, as well as the vasoactive intestinal peptide (VIP), given the previous unsuccessful attempts to functionalize TuMV VNPs by other methods. The successful conjugation of VLPs to GFP and VIP using SpyTag/SpyCatcher was confirmed through Western blot and electron microscopy. Moreover, the isopeptide bond between SpyTag and SpyCatcher occurred in vivo in co-agroinfiltrated Nicotiana benthamiana plants. These results demonstrated that SpyTag/SpyCatcher improves TuMV functionalization compared with previous approaches, thus implying the expansion of the application of the technology to elongated flexuous VNPs.

Keywords: Spytag/SpyCatcher; VIP; VLPs; functionalization; potyvirus.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Blotting, Western
Green Fluorescent Proteins
Nanoparticles*
Potyvirus*

Substances

Green Fluorescent Proteins

Supplementary concepts

Turnip mosaic virus

Grants and funding

This work was conceived as transversal to efforts experimentally aimed to TuMV nanobiotechnological functionalizations. As a consequence, it has benefited from different grants obtained along the years. These are P2018/BAA-4574, COV20/00114, and PanGreen-CM from the Comunidad de Madrid; RTA2015-00017-00-00 from INIA; and ARIMNet-2 618127, an ERANet project. D.A.T. was supported by a Margarita Salas postdoctoral grant funded by the Spanish Ministry of Universities and UCM (CT31/21).