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Dual Site-Selective Presentation of Functional Handles on Protein-Engineered Cowpea Chlorotic Mottle Virus-Like Particles.

ABSTRACT: Protein cages hold much promise as carrier systems in nanomedicine, due to their well-defined size, cargo-loading capacity, and inherent biodegradability. In order to make them suitable for drug delivery, they have to be stable under physiological conditions. In addition, often surface modifications are required, for example, to improve cell targeting or reduce the particle immunogenicity by PEGylation. For this purpose, we investigated the functionalization capacity of the capsid of cowpea chlorotic mottle virus (CCMV), modified at the interior with a stabilizing elastin-like polypeptide (ELP) tag, by employing a combination of protein engineering and bio-orthogonal chemistry. We first demonstrated the accessibility of the native cysteine residue in ELP-CCMV as a site-selective surface-exposed functional handle, which was not available in the native CCMV capsid. An additional bio-orthogonal functional handle was introduced by incorporation of the noncanonical amino acid, azido-phenylalanine (AzF), using the amber suppression mechanism. Dual site-selective presentation of both a cell-penetrating TAT peptide and a fluorophore to track the particles was demonstrated successfully in HeLa cell uptake studies.

SUBMITTER: Vervoort DFM

PROVIDER: S-EPMC8154214 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background & methodsWithin the last decade Virus-Like Particles (VLPs) have increasingly received attention from scientists for their use as a carrier of (peptide) molecules or as scaffold to present epitopes for use in subunit vaccines. To test the feasibility of Cowpea chlorotic mottle virus (CCMV) particles as a scaffold for epitope presentation and identify sites for epitope fusion or insertion that would not interfere with virus-like-particle formation, chimeric CCMV coat protein (CP) gene constructs were engineered, followed by expression in E. coli and assessment of VLP formation. Various constructs were made encoding a 6x-His-tag, or selected epitopes from Influenza A virus [IAV] (M2e, HA) or Foot and Mouth Disease Virus [FMDV] (VP1 and 2C). The epitopes were either inserted 1) in predicted exposed loop structures of the CCMV CP protein, 2) fused to the amino- (N) or carboxyl-terminal (C) ends, or 3) to a N-terminal 24 amino acid (aa) deletion mutant (N∆24-CP) of the CP protein.ResultsHigh levels of insoluble protein expression, relative to proteins from the entire cell lysate, were obtained for CCMV CP and all chimeric derivatives. A straightforward protocol was used that, without the use of purification columns, successfully enabled CCMV CP protein solubilization, reassembly and subsequent collection of CCMV CP VLPs. While insertions of His-tag or M2e (7-23 aa) into the predicted external loop structures did abolish VLP formation, high yields of VLPs were obtained with all fusions of His-tag or various epitopes (13- 27 aa) from IAV and FMDV at the N- or C-terminal ends of CCMV CP or N∆24-CP. VLPs derived from CCMV CP still encapsulated RNA, while those from CCMV CP-chimera containing a negatively charged N-terminal domain had lost this ability. The usefulness and rapid ease of exploitation of CCMV VLPs for the production of potential subunit vaccines was demonstrated with the synthesis of chimeric CCMV VLPs containing selected sequences from the GN and GC glycoproteins of the recently emerged Schmallenberg orthobunyavirus at both termini of the CP protein.ConclusionsCCMV VLPs can be successfully exploited as scaffold for epitope fusions up to 31 aa at the N- and C-terminus, and at a N-terminal 24 amino acid (aa) deletion mutant (N∆24-CP) of the CP protein.

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Dual Site-Selective Presentation of Functional Handles on Protein-Engineered Cowpea Chlorotic Mottle Virus-Like Particles.

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