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

Summary

A method to analyze the solvent accessibility of the thiol group of cysteine residues of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) followed by a peptide cross-linking reaction is described. The method takes advantage of the availability of several chemical groups on the surface of the VLPs that can be targets for specific reactions.

Abstract

Mimicking and exploiting virus properties and physicochemical and physical characteristics holds promise to provide solutions to some of the world's most pressing challenges. The sheer range and types of viruses coupled with their intriguing properties potentially give endless opportunities for applications in virus-based technologies. Viruses have the ability to self- assemble into particles with discrete shape and size, specificity of symmetry, polyvalence, and stable properties under a wide range of temperature and pH conditions. Not surprisingly, with such a remarkable range of properties, viruses are proposed for use in biomaterials ⁹, vaccines ^{14, 15}, electronic materials, chemical tools, and molecular electronic containers^{4, 5, 10, 11, 16, 18, 12}.

In order to utilize viruses in nanotechnology, they must be modified from their natural forms to impart new functions. This challenging process can be performed through several mechanisms including genetic modification of the viral genome and chemically attaching foreign or desired molecules to the virus particle reactive groups ⁸. The ability to modify a virus primarily depends upon the physiochemical and physical properties of the virus. In addition, the genetic or physiochemical modifications need to be performed without adversely affecting the virus native structure and virus function. Maize rayado fino virus (MRFV) coat proteins self-assemble in Escherichia coli producing stable and empty VLPs that are stabilized by protein-protein interactions and that can be used in virus-based technologies applications ⁸. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed ¹³. Here, we describe the steps to 1) determine which of the solvent-accessible cysteines in a virus capsid are available for modification, and 2) bioconjugate peptides to the modified capsids. By using native or mutationally-inserted amino acid residues and standard coupling technologies, a wide variety of materials have been displayed on the surface of plant viruses such as, Brome mosaic virus ³, Carnation mottle virus ¹², Cowpea chlorotic mottle virus ⁶, Tobacco mosaic virus ¹⁷, Turnip yellow mosaic virus ¹, and MRFV ¹³.

Protocol

1. Virus Inoculation and VLPs Purification from Nicotiana benthamiana Plants

1. Produce capped T7-RNA transcripts from Potato virus X (PVX)-based vector plasmids carrying MRFV wild-type (wt) and Cys-mutated coat protein (CP) genes ¹², using Ambion's T7-mMessage mMachine Kit.
2. For each T7 transcript reaction, inoculate two fully expanded leaves of N. benthamiana with 10 μl reactions and incubate the plants for 10 days in greenhouse, at 60% humidity for 16 hr with light (25,000-30,000 lux) at 25 °C and 8 hr dark at 20 °C.
3. Harvest N. benthamiana virus-infected leaves 10 days post inocul....

Results

Transient expression of mutant MRFV coat protein (CP) genes in N. benthamiana plants in a PVX-based vector producing VLPs is described in Figure 1. The modified MRFV coat protein gene is amplified by PCR and then placed under the transcriptional control of the duplicated subgenomic CP promoter in a PVX-based vector, pP2C2S ², (a gift of D. Baulcombe, Sainsbury Laboratories, Norwich, England). In vitro RNA transcription produces RNA transcripts that are then used to ino.......

Discussion

The method presented here enables a very sensitive and rapid analysis of reactive cysteines present on the surface of plant-produced VLPs as well as on other protein complexes. Maleimides are thiol-specific reagents, which react with free sulfhydryl-containing molecules to form stable thioether bonds. This method draws on the specificity of the maleimides to react with sulfhydryl groups not involved in interactions with other amino acids. Preserving the native structure of the VLPs is very important through the en.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Thinwall, Ultra-Clear Tubes	Beckman	344059
mMESSAGE mMACHINE T7 Kit	Life Tecnologies	AM1344M
Fluorescein-5-Maleimide	Thermo Scientific Life Technologies	46130 F150	46130 is out of order substitute with F150
Pierce Biotin Quantitation Kit	Thermo Scientific	28005
EZ-Link Maleimide-PEG2-Biotin, No-Weigh Format	Thermo Scientific	21901
SM(PEG)n Crosslinkers	Thermo Scientific	22107
10-20 % Tris-Glycine gel	Invitrogen	EC61352
Laemmli Buffer	Bio-Rad	1610737
Tris Glycine SDS Running Buffer	Invitrogen	LC2675
Tris Glycine Transfer Buffer	Invitrogen	LC3675
Nitrocellulose Membrane Filter Paper Sandwich	Invitrogen	LC2001
Phosphatase Labeled Affinity Purified Antibody to Rabbit IgG	Kirkegaard and Perry Laboratories	0751516
NBT/BCIP Phosphatase Substrate	Kirkegaard and Perry Laboratories	508107