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Team:Wageningen UR/VLPs
From 2012.igem.org
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Virus-Like Particles (overview)
A Virus-Like Particle (VLP) is a shell of viral Coat Proteins (CPs) that spontaneously assemble with the right conditions. Although a VLP resembles the original virus in shape and size, it lacks both the external sites that are usually required for the infection of cells and the internal machinery needed for viral replication. Moreover, they also lack the viral genetics to be transcribed and replicated.
We want to create a standardized virus-like particle with a Plug ‘n Apply (PnA) System on both the inside and the outside. As a proof of concept for different kinds of attachment, we selected three distinct viruses that each have their specific qualities.
Cowpea Chlorotic Mottle Virus (CCMV)
Our iGEM team is located at Wageningen URs Microbiology faculty, where at the start of our project, neither we nor the staff had much expertise on viruses. However, on the other side of town stands the Virology faculty. Dr. Kormelink of this faculty was able to provide us with a plasmid harbouring the coding region for CCMV's Coat Protein. Since a validated protocol for isolating the VLPs from Escherichia coli was also provided, we decided to use CCMV as a starting point for VLP formation.
We produced CCMV VLPs and analysed their stability in various circumstances ([http://partsregistry.org/Part:BBa_K883001 BBa_K883001]). We also made various mutants of the Coat Protein to explore different possibilities regarding VLP inside modification.
Hepatitis B Virus (HBV)
For all its qualities, CCMV still lacks some aspects that we want our end product to have. To start with, the Hepatitis B VLP has not yet been modified on the outside. Furthermore, since some of our major applications are in the medical area, we need our VLP to be stable under blood-like conditions. For these reasons, we selected the Hepatitis B VLP as another platform to work with.
We produced Hepatitis B VLPs in E. coli. Moreover, we attempt to produce modified versions, presenting a Plug and Apply System either on the inside for targeted packaging of small peptides or exposed on the surface for standardized binding of either ligands or epitopes.
Polerovirus (PLRV & TuYV)
Modification of any protein will alter the way it folds in vivo. In the case of viral coat proteins, an altered tertiary structure is likely to result in loss of capsid formation (quaternary structure). To prevent this, we try use existing knowledge about examples of successful VLP modifications in our project. However, any individual modification may have a distinctly different effect on protein folding than another. Moreover, most existing examples of VLP modification include mixing of modified and wildtype subunits. This increases the chance of VLP formation by decreasing the induced stress: To ensure that additions (i.e. a Plug and Apply System) will experience no steric hindrance from each other, a capsid will consist of mostly wildtype subunits and incorporate only several modified versions.
As is often the case, a solution can be found by looking in nature itself. In the Wageningen area, one of the major agricultural crops is the potato. A naturally occurring virus species here is the Polerovirus, another well-studied plant virus. This virus offers a rather elegant solution to our problems. It has rather large ‘spikes’ sticking out of its capsid structure, that are each covalently bound to one of the coat proteins. Like in most modified VLPs, this is the result of mixing of both ‘normal’ coat proteins and ‘modified’ versions, which harbour the spike. Unlike most manmade constructs though, the Polerovirus does not need two separate genes to achieve this. Instead, it utilises a clever ‘stop codon readthrough’ system to fuse its outer spike to only some of the coat proteins. We try to use this system in our project.
