Abstract
Plant viruses can cause devastating losses to agriculture and are therefore a major threat to food security. The rapid identification of virally-infected crops allowing containment is essential to limit such threats, but plant viral diseases can be extremely challenging to diagnose. An ideal method for plant virus diagnosis would be a device which can be implemented easily in the field. Such devices require a binding reagent that is specific for the virus of interest. We chose to investigate the use of Affimer reagents, artificial binding proteins and a model plant virus Cowpea Mosaic virus (CPMV) empty virus like particles (eVLPs). CPMV-eVLP mimic the morphology of wild-type (WT) CPMV but lack any infectious genomic material and so do not have biocontainment issues. We have produced and purified an Affimer reagent selected for its ability to bind to CPMV-eVLP and have shown that the selected Affimer also specifically binds to WT CPMV. We have produced a 3.4 Å structure of WT CPMV bound to the Affimer using cryo-electron microscopy. Finally, we have shown that this Affimer is capable of reliably detecting the virus in crude extracts of CPMV-infected leaves and can therefore form the basis for the future development of diagnostic tests.
Highlights
Food security is a global challenge; over 6% of global food production is lost due to pathogens including viruses[1]
We demonstrate that the selected Affimer can detect the presence of Cowpea Mosaic Virus (CPMV) directly within crude extracts of infected leaves, showing that the empty virus-like particles (eVLPs)/Affimer combination is a potential route to the development of new in-field diagnostics
The protein components of CPMV-B, CPMV-M, CPMV-T and a recombinantly-expressed empty Virus-like particles (VLPs) are almost identical structurally (Fig. 1a)[13,14,15,16] with the only significant difference being in the degree of cleavage of a 24 amino acid extension to the C-terminus of the S subunit, which occurs during particle purification and storage[13,14]
Summary
Food security is a global challenge; over 6% of global food production is lost due to pathogens including viruses[1]. Affimer proteins are small (11 kDa, 3 nm in diameter), stable (70 °C < Tm < over 100 °C), monomeric proteins that lack disulphide bonds[3] Their structure consists of a single α-helix and four β-strands, with the molecular recognition sites being located within two variable loops, each up to nine amino acids in length[3]. We sought to investigate whether VLPs could be used to generate an Affimer reagent that would be cross-reactive to a WT virus For this analysis we used Cowpea Mosaic Virus (CPMV), a plant virus from the order Picornavirales that has been used extensively in biotechnology and as a model for single-stranded RNA viruses more generally. We demonstrate that the selected Affimer can detect the presence of CPMV directly within crude extracts of infected leaves, showing that the eVLP/Affimer combination is a potential route to the development of new in-field diagnostics
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