Lolium latent virus (Alphaflexiviridae) coat proteins: expression and functions in infected plant tissue.

The genome of Lolium latent virus (LoLV; genus Lolavirus, family Alphaflexiviridae) is encapsidated by two carboxy-coterminal coat protein (CP) variants (about 28 and 33 kDa), in equimolar proportions. The CP ORF contains two 5'-proximal AUGs encoding Met 1 and Met 49, respectively promoting translation of the 33 and 28 kDa CP variants. The 33 kDa CP N-terminal domain includes a 42 aa sequence encoding a putative chloroplast transit peptide, leading to protein cleavage and alternative derivation of the approximately 28 kDa CP. Mutational analysis of the two in-frame start codons and of the putative proteolytic-cleavage site showed that the N-terminal sequence is crucial for efficient cell-to-cell movement, functional systemic movement, homologous CP interactions and particle formation, but is not required for virus replication. Blocking production of the 28 kDa CP by internal initiation shows no major outcome, whereas additional mutation to prevent proteolytic cleavage at the chloroplast membrane has a dramatic effect on virus infection.

Improvement of PVX/CMV CP expression tool for display of short foreign antigens.

We have previously reported that Potato virus X-expressed coat protein of Cucumber mosaic virus (CMV) formed virus-like particles (VLPs), which served as carriers for display of different neutralizing epitopes of Newcastle disease virus (NDV). In this work, we further modified the purification protocol of recombinant VLPs carrying short neutralizing epitopes of the NDV proteins and demonstrated that self-contained capsid protein subunits of CMV transiently expressed from heterologous virus packaged into individual virions morphologically resembling and/or indistinguishable from wild type CMV particles. Homogeneity of the final preparation represents an advance over our previous study, where VLPs were found to be of variable size. Chickens immunized with purified VLPs developed antigen-specific response.

Mutations in the alpha-helical region of the amino terminus of the Maize rayado fino virus capsid protein and CP:RNA ratios affect virus-like particle encapsidation of RNAs.

Viral-based nanoplatforms rely on balancing the delicate array of virus properties to optimally achieve encapsidation of foreign materials with various potential objectives. We investigated the use of Maize rayado fino virus (MRFV)-virus-like particles (VLPs) as a multifunctional nanoplatform and their potential application as protein cages. MRFV-VLPs are composed of two serologically related, carboxy co-terminal coat proteins (CP1 and CP2) which are capable of self-assembling in Nicotiana benthamiana plants into 30nm particles with T=3 symmetry. The N-terminus of CP1 was targeted for genetic modification to exploit the driving forces for VLP assembly, packaging and retention of RNA in vivo and in vitro. The N-terminus of MRFV-CP1 contains a peptide sequence of 37 amino acids which has been predicted to have an alpha-helical structure, is rich in hydrophobic amino acids, facilitates CP-RNA interactions, and is not required for self-assembly. Amino acid substitutions were introduced in the 37 amino acid N-terminus by site-directed mutagenesis and the mutant VLPs produced in plants by a Potato virus X (PVX)-based vector were tested for particle stability and RNA encapsidation. All mutant CPs resulted in production of VLPs which encapsidated non-viral RNAs, including PVX genomic and subgenomic (sg) RNAs, 18S rRNA and cellular and viral mRNAs. In addition, MRFV-VLPs encapsidated GFP mRNA when was expressed in plant cells from the pGD vector. These results suggest that RNA packaging in MRFV-VLPs is predominantly driven by electrostatic interactions between the N-terminal 37 amino acid extension of CP1 and RNA, and that the overall species concentration of RNA in the cellular pool may determine the abundance and species of the RNAs packaged into the VLPs. Furthermore, RNA encapsidation is not required for VLPs stability, VLPs formed from MRFV-CP1 were stable at temperatures up to 70°C, and can be disassembled into CP monomers, which can then reassemble in vitro into complete VLPs either in the absence or presence of RNAs.

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.

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, vaccines, electronic materials, chemical tools, and molecular electronic containers. 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. 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. VLPs produced in tobacco plants were examined as a scaffold on which a variety of peptides can be covalently displayed. 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, Carnation mottle virus, Cowpea chlorotic mottle virus, Tobacco mosaic virus, Turnip yellow mosaic virus, and MRFV.

Maize rayado fino virus virus-like particles expressed in tobacco plants: A new platform for cysteine selective bioconjugation peptide display.

Maize rayado fino virus (MRFV) virus-like-particles (VLPs) produced in tobacco plants were examined for their ability to serve as a novel platform to which a variety of peptides can be covalently displayed when expressed through a Potato virus X (PVX)-based vector. To provide an anchor for chemical modifications, three Cys-MRFV-VLPs mutants were created by substituting several of the amino acids present on the shell of the wild-type MRFV-VLPs with cysteine residues. The mutant designated Cys 2-VLPs exhibited, under native conditions, cysteine thiol reactivity in bioconjugation reactions with a fluorescent dye. In addition, this Cys 2-VLPs was cross-linked by NHS-PEG4-Maleimide to 17 (F) and 8 (HN) amino acid long peptides, corresponding to neutralizing epitopes of Newcastle disease virus (NDV). The resulting Cys 2-VLPs-F and Cys 2-VLPs-HN were recognized in Western blots by antibodies to MRFV as well as to F and HN. The results demonstrated that plant-produced MRFV-VLPs have the ability to function as a novel platform for the multivalent display of surface ligands.

Transient expression of the ectodomain of matrix protein 2 (M2e) of avian influenza A virus in plants.

We have previously reported an expression system based on the capsid protein gene (CP) of cucumber mosaic virus (CMV) placed under transcriptional control of a potato virus X (PVX)-based vector. PVX-expressed CMV CP formed virus-like particles, which served as carriers for heterologous antigens of the Newcastle disease virus (NDV). In this work, we applied our expression tool toward the development of plant-derived vaccine candidate against avian influenza A virus. Twenty-three amino acid-long extracellular domain of the viral M2 protein (M2e) was engineered into the internal motif 5 of CMV CP and the recombinant gene then was transiently expressed in plants through a PVX vector. Chimeric CMV capsids reacted with specific antibodies produced to synthetic M2e epitope of the H5N1 strain of the virus. In addition, CMV CP-M2e protein was expressed to high levels in Escherichia coli bacterial cells and was recognized by antibodies to both CMV and M2e. This initial study demonstrates the feasibility of using plant virus-based vectors for expression of antigenic epitopes of H5N1 avian influenza in plants.