Chemical addressability of potato virus X for its applications in bio/nanotechnology

Potato virus X (PVX), a type member of the plant virus potexvirus group, offers a unique nanotechnology platform based on its high aspect ratio and flexible filamentous shape. The PVX platform has already been engineered and studied for its uses in imaging, drug delivery, and immunotherapies. While genetic engineering procedures are well established for PVX, there is limited information about chemical conjugation strategies for functionalizing PVX, partly due to the lack of structural information of PVX at high resolution. To overcome these challenges, we built a structural model of the PVX particle based on the available structures from pepino mosaic virus (PepMV), a close cousin of PVX. Using the model and a series of chemical conjugation experiments, we identified and probed the addressability of cysteine side chains. Chemical reactivity of cysteines was confirmed using Michael-addition and thiol-selective probes, including fluorescent dyes and biotin tags. LC/MS/MS was used to map Cys 121 as having the highest selectivity for modification. Finally, building on the availability of two reactive groups, the newly identified Cys and previously established Lys side chains, we prepared multifunctional PVX nanoparticles by conjugating Gd-DOTA for magnetic resonance imaging (MRI) to lysines and fluorescent dyes for optical imaging to cysteines. The resulting functionalized nanofilament could have applications in dual-modal optical-MRI imaging applications. These results further extend the understanding of the chemical properties of PVX and enable development of novel multifunctional platforms in bio/nanotechnology.