Bioconjugation Strategies for Tobacco Mild Green Mosaic Virus

Abstract

Tobacco mild green mosaic virus (TMGMV) is a plant virus closely related to Tobacco mosaic virus (TMV), sharing many of its structural and chemical features. These rod-shaped viruses, comprised of 2130 identical coat protein subunits, have been utilized as nanotechnological platforms for a myriad of applications, ranging from drug delivery to precision agriculture. This versatility for functionalization is due to their chemically active external and internal surfaces. While both viruses are similar, they do exhibit some key differences in their surface chemistry, suggesting the reactive residue distribution on TMGMV should not overlap with TMV. In this work, we focused on the establishment and refinement of chemical bioconjugation strategies to load molecules into or onto TMGMV for targeted delivery. A combination of NHS, EDC, and diazo coupling reactions in combination with click chemistry were used to modify the N-terminus, glutamic/aspartic acid residues, and tyrosines in TMGMV. We report loading with over 600 moieties per TMGMV via diazo-coupling, which is a >3-fold increase compared to previous studies. We also report that cargo can be loaded to the solvent-exposed N-terminus and carboxylates on the exterior/interior surfaces. Mass spectrometry revealed the most reactive sites to be Y12 and Y72, both tyrosine side chains are located on the exterior surface. For the carboxylates, interior E106 (66.53 %) was the most reactive for EDC-propargylamine coupled reactions, with the exterior E145 accounting for >15 % reactivity, overturning previous assumptions that only interior glutamic acid residues are accessible. A deeper understanding of the chemical properties of TMGMV further enables its functionalization and use as a multifunctional nanocarrier platform for applications in medicine and precision farming.

Keywords: bioconjugation; drug delivery; nanocarriers; plant viruses; precision agriculture.

Fig. 1.

A structural model of TMGMV (PDB: 1VTM) showing different reactive residues (rendered in UCSF Chimera X). A) Exterior surface of TMGMV with areas of interest highlighted. B-C) exterior glutamic acid (red) and tyrosine (blue) residues. B) accessibility of E145 and Y12/Y72. C) accessibility of E131 and the tyrosine binding pocket (Y12/Y17/Y68/Y70). D-E) surface exposure of the N-terminus (yellow) and Y2. F) cross-section of the interior surface of TMGMV. G-H) accessibility of the interior glutamic acid residues G) E106 and H) E95. The colored boxes in (A) and (F) correspond to the areas with the matching insets in the remaining panels.

Fig. 2.

The structure of TMGMV. (A) The atomic model of TMGMV showing a helical structure assembled of 2130 CP subunits. N-terminus, tyrosine and glutamic acid residues are highlighted in yellow, blue and red, respectively. (B) Top view and (C) side view of a short segment of TMGMV displaying exposed N-terminus (yellow), tyrosine (blue) and glutamic acid (red) residues. (D) Reactive surface of TMGMV, N-terminus (yellow), tyrosine residues (blue) and glutamic acid residues (red), and a summary of the reactive moieties on the surface of TMGMV. (E) Model compounds for TMGMV bioconjugation (Sulfo-Cy5, ATTO488 and biotin), where * denotes the reactive position on the molecule. (F) Reactive groups required for bioconjugation using the residue in the same row, with NHS-amine coupling (upper) and with azide-alkyne cycloaddition via azo coupling of tyrosine and a diazonium salt (middle) and via glutamic acid activation by EDC (lower). (G) The reactive activated intermediate and the final compound structure for each type of bioconjugation. R denotes the target molecule (E).

Fig. 3.

Characterization of TMGMV labeled with Sulfo-Cy5 (TMGMV-Cy5) at reactive amines from the N-terminus (left) and reactive alkynes from tyrosine residues (middle) and glutamic acid residues (right). (A) Quantification of Cy5 per TMGMV as determined by UV/vis spectroscopy. Averaged values from three independent experiments are shown; error bars represent the standard deviation. (B) SDS-PAGE of TMGMV-Cy5 visualized under UV light allowing for dye detection and one under white light after Gel Code staining allowing for protein detection. M: SeeBlue Plus2 Protein Standards, (1): non-modified TMGMV control, (2)–(7): increasing ratios of Cy5:CP: (2) 1:1, (3) 2:1, (4) 5:1, (5) 10:1, (6) 20:1, (7) 50:1. (C) TEM images of Cy5:CP ratio = 10:1 (scale bar: 200 nm).

Fig. 4.

Characterization of biotin-labeled TMGMV at the N-terminus (top), tyrosine residues (middle) and or glutamic acid residues (lower). (A) SDS-PAGE (left) and (B) Western blot (right) of TMGMV-biotin. The gel was visualized after Gel Code Blue staining under white light and blots were probed with alkaline phosphatase-conjugated streptavidin. M: SeeBlue Plus2 Protein Standards, (1) non-modified TMGMV control, (2) non-modified TMGMV-alkyne (3)–(7): increasing ratios of Biotin:CP: (3) 1:1, (4) 2:1, (5) 5:1, (6) 10:1, (7) 20:1, (8) 50:1. (C) TEM of immunogold staining of TMGMV-biotin using gold-labeled anti-biotin antibodies. Yellow arrows show gold anti-biotin labeled nanoparticles.