Analysis of Engineered Tobacco Mosaic Virus and Potato Virus X Nanoparticles as Carriers for Biocatalysts

Abstract

Plant virus nanoparticles are promising candidates for the development of novel materials, including nanocomposites and scaffolds/carriers for functional molecules such as enzymes. Their advantages for enzyme immobilization include a modular organization, a robust and programmable structure, and a simple, cost-effective production. However, the activity of many enzymes relies on posttranslational modification and most plant viruses replicate in the cytoplasm, so functional enzymes cannot be displayed on the virus surface by direct coat protein fusions. An alternative display system to present the Trichoderma reesei endoglucanase Cel12A on potato virus X (PVX) using SpyTag/SpyCatcher (ST/SC) technology was recently developed by the authors, which allows the carrier and enzyme to be produced separately before isopeptide conjugation. Although kinetic analysis clearly indicated efficient biocatalyst activity, the PVX carrier interfered with substrate binding. To overcome this, the suitability of tobacco mosaic virus (TMV) was tested, which can also accommodate a larger number of ST peptides. We produced TMV particles displaying ST as a new platform for the immobilization of enzymes such as Cel12A, and compared its performance to the established PVX-ST platform in terms of catalytic efficiency. Although more enzyme molecules were immobilized on the TMV-ST particles, we found that the rigid scaffold and helical spacing significantly affected enzyme activity.

Facile Purification and Use of Tobamoviral Nanocarriers for Antibody-Mediated Display of a Two-Enzyme System

“…It also allows their assembly into dense networks on solid substrates and in solution, as well as retention in hydrogels, to serve as 2D- and 3D-immobilization platforms for various molecule species or cultivated cells [ 15 , 16 , 17 , 18 ]. Key players in this context are tobamoviruses such as tobacco mosaic virus (TMV), potex-, and potyviruses including potato virus X (PVX), and turnip mosaic virus (TuMV) [ 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 ].…”

“…Its easy production, physical and chemical stability, and options for generating TMV-derived designer particles of altered shapes offer versatile prospects for multi-functionalization with, e.g., peptides, enzymes, drugs, and/or synthetic and inorganic compounds, and corresponding uses [ 31 ]. Increasing numbers of studies have employed TMV-based adapters for the display of enzymes, and for installing enzymatic and other types of bioreceptors in different types of sensors [ 20 , 25 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 ]. TMV, however, does not tolerate extensive genetic modifications.…”

“…Genetically encoded displays of the 125 aa fluorescent reporter iLOV [ 52 ] have only been achieved via a sophisticated construct containing the foot-and-mouth disease virus (FMDV) 2A ‘ribosome skip’ sequence, limiting the portion of extended CPs co-translationally [ 53 ]. Enzymatic (self-)ligation of peptide- or coenzyme-A-equipped CPs to compatibly tagged proteins of interest is a promising alternative for installing functions but has been evaluated only exemplarily so far for TMV, with variable success [ 25 , 51 ].…”

Surface Functionalization of Rod-Shaped Viral Particles for Biomedical Applications

“…SpyTagging rod VLPs at their C-termini allows robust, site-selective functionalization with a variety of SpyCatcher fusions (Figure d). This approach was recently applied to TMV and the PVX for conjugation with a variety of catalytic enzymes. , A similar scheme was used for coupling proteins to SpyCatcher-conjugated gold, upconverting nanoparticles, and quantum dots . The SpyTagged VLP platform thus shows great promise, although its medical applications have not yet been investigated.…”

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

“…Through the gene fusion of SpyTag and SpyCatcher to either the N-terminus or C-terminus of two proteins of interest, these two proteins will be finally joined covalently to each other to a high yield [ 18 , 19 ]. Recently, SpyTag/SpyCatcher technology has been applied for virus nanoparticle functionalization in different plant viruses [ 20 , 21 ], with foreign antigen display being the most common use in general [ 22 , 23 , 24 , 25 ]. However, to the best of our knowledge, potyvirus VNPs have never been functionalized through this method.…”