Analysis of the Solvent Accessibility of Cysteine Residues on Maize rayado fino virus Virus-like Particles Produced in Nicotiana benthamiana Plants and Cross-linking of Peptides to VLPs

The overall goal of the following experiment is to perform a solvent accessibility analysis of Cysteine residues on the surface of virus-like particles or VLPs, followed by cross-linking of peptides to Cysteine on the virus-like particles. This is achieved by first cloning the Maize rayado fino virus or MRFV coat protein in a potato virus-X based virus vector. Next N. benthamiana plants are inoculated with the modified virus and MRFV VLPs formed from the coat protein are purified. Finally, VLPs and peptides are cross-linked using ammonia-PEG4-maleimide and detected by western blot. Results are obtained that identify the production of VLP peptide complexes through western blot analysis. The technique extends the knowledge base on how to chemically modify surface of viruses and virus-like particle to produce new material for nanotechnology.

After producing Capped T7-RNA transcripts from potato virus-X based vector plasmids carrying MRFV wild type and cys-mutated coat protein genes, use two aliquots of 10 microliters each to inoculate two N. benthamiana leaves. Incubate the plants for 10 days in a greenhouse at 60% humidity and 16 hours of light at 25 degrees Celsius and eight hours of dark at 20 degrees Celsius. At the end of the incubation harvest the virus infected leaves, weigh the plant tissue and place it on ice. After purifying the virus-like particles according to the text protocol, prepare a one millimolar solution of Fluorescein-5-maleimide in 50 millimolar sodium phosphate buffer, pH 7.0, 1 millimolar EDTA. Vortex to completely dissolve and use aluminum foil to protect the solution from light. Mix approximately 30 microliters of one microgram per microliter of VLPs and 60 microliters of the Fluorescein-5-maleimide and incubate for two hours at room temperature or overnight at four degrees Celsius. Terminate the reaction by adding DTT at a final concentration of 50 millimolar. Using a desalting spin column remove the non-reactive fluorescein. To visualize the VLPs with available cysteine residues for cross-linking, perform SDS page analysis by adding 10 microliters of 2x sample buffer to 10 microliters of each reaction. Store the labeled protein protected from light at four degrees Celsius for up to one month or in single use aliquots at negative 20 degrees Celsius up to three months.

Prepare a Fresh crosslinking stock solution by dissolving 10 milligrams of NHS-PEG4-maleimide crosslinker in 680 microliters of dimethyl sulfoxide. Dissolve a 17 (F)-amino acid long peptide in 50 microliters of conjugation buffer at a concentration of 0.1 millimolar. Add four microliters of crosslinker to 50 microliters of the dissolved peptide for a one millimolar final concentration. Incubate the reaction for two hours at four degrees Celsius. Then use a desalting column to purify the reaction. Using VLPs with available cysteine residues for cross-linking and the cross-linked peptide, set up a reaction mix at a molar ratio determined by the number of thiols and activated amines involved in the reaction and the difference in molecular weight between VLPs and peptide. After incubating the reaction for two hours at room temperature, use it to carry out SDS page and western blotting according to the text protocol.

An example of reactive amino acids of the MRFV coat protein is shown here. Each amino acid selected can be used to attach moieties if it is surface exposed. The chemical techniques include traditional bio-conjugation strategies such as the acylation of the amino group of lysine, alkylation of the sulfhydryl group of Cysteine, and activation of carboxylic acid residues and coupling with added amines.

In addition, the aromatic groups of tyrosine and tryptophan can be attractive targets for bio conjugation through diazotization and alkylation. Illustrated here is the solvent accessibility of Cys-residues of VLPs to thiol-specific reagents. Purified wild type VLPs and cys-VLP mutants carrying Cysteine residues in position 107 to 111 and 192 to 194 of the CP gene are unreactive in native conditions with a thiol specific reagent. Fluorescein-5-maleimide imparts orange fluorescence only for the Cys-VLP mutant carrying Cysteine residues in position 125 to 129 of the CP gene. The result shows that VLPs in lane two have a geometric arrangement of Cysteine residues resulting in solvent exposed free thiol groups, whereas the Cysteine residues in the samples in lanes one and three are perhaps involved in di-sulfide bridges in the folding of the coat protein. Labeling reactions with maleimide-PEG2-biotin followed by a biotinylating assay represent another example of analysis of solvent accessibility of cys-residues. Because biotin is a relatively small molecule, it can be conjugated in several copies on the VLPs, each of which combined one molecule of avidin as shown here. The level of biotinylation, 84.74 mole of biotin per mole of protein, indicates the number of the biotin molecules attached to the VLPs, and consequently the number of ligands that can be displayed on the exterior surface. Because of the availability of several thiol groups on the VLPs peptides can be attached by crosslinking reactions with NHS-PEG4-maleimide.

This reagent is a hetero-bifunctional crosslinker, which contains reactive ends such as N-hydroxy succinimide or NHS-Ester and maleimide groups allowing covalent conjugation of amine and sulfhydryl containing molecules. In this example, the NHS Ester reacts with primary amines forming amide bonds, whereas maleimide reacts with sulfhydryl groups forming stable thioester bonds. As seen here, cross-linking reactions produce VLP-peptide complexes that are immunoreactive with the specific antibodies in western blot.

This development demonstrates to researchers in the field of virology how to explore the use of virus particles as a scaffold for the attachment of a large variety of ligands in a regular geometric array for numerous applications.