South African cassava mosaic virus movement and nuclear shuttle proteins: uncovering their structures

Abstract

South African cassava mosaic virus (SACMV) is a plant-infecting circular ssDNA bipartite begomovirus, whose genome comprises of DNA-A (encodes six genes) and DNA-B [encodes BC1 cell-to-cell movement (MP) and BV1 nuclear shuttle protein (NSP)]. Expression of these viral proteins in vitro and their purification have not been achieved to date and this study aimed to achieve this for a truncated region of the MP (N-ter) and NSP (C-ter), respectively. Furthermore, SACMV movement and nuclear shuttle proteins were truncated due to the difficulties in working with the full-length proteins; and the secondary structures of the truncated proteins were then investigated. Computational characterization and homology modelling were also undertaken. The truncated MP (amino acid 1-258) and NSP (amino acid 171-258) were cloned into a pCOLDI expression vector and transformed into BL21 (DE3) pLYSs and BL21 (DE3) E. coli respectively. Optimal conditions for the induced expression of the MP were found to be 0.50 mM IPTG at an OD600 of 0.45 expressed for 24 h. In contrast, the optimal conditions of the expression of the truncated NSP were found to be 0.25 mM IPTG at an OD600 of 0.40 expressed for 24 h. 8-Anilinonaphthalene-1- sulfonic acid (ANS) has been used as a folding/unfolding monitoring tool. This study showed that both proteins bind ANS with greater affinity in the denatured form, indicating that there are more hydrophobic regions for the polar fluorescent dye to bind. Both truncated proteins do not have the ability to bind ATP, and this was determined by using the MANT-ATP intrinsic fluorescence assay. In order to confirm that both proteins were refolded and did not bind ATP, intrinsic tryptophan fluorescence was assessed. This assay confirmed that both proteins were refolded due to the observed difference in spectra profiles. The assay also confirmed that ATP caused no conformational changes in both proteins in the native or denatured forms. Secondary structure analysis employing Circular Dichroism confirmed that the truncated NSP and MP contained predominantly β-sheet secondary structures. The estimated molecular weights by SDS-PAGE, were determined to be 23 kDa and 13kDa for MP and NSP, respectively. The employment of Mass Spectrometry in future studies could assist in confirming the exact molecular weight of the protein as Size Exclusion High Performance Liquid Chromatography did not yield desirable results as 2 the proteins eluted at the incorrect retention times possibly due to the proteins being expressed in the insoluble form.