Geminivirus replication-association protein (Rep) as a target for the development of small molecule inhibitors by

Kgomokaboya, Maite Codlinne
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Replication-association protein (Rep) is an indispensable protein for the replication of Geminivirus single-stranded DNA in its life cycle. Geminivirus, in particular African Cassava Mosaic Virus (ACMV), affects agricultural crops such as Cassava (Manihot esculenta Crantz) that have the potential to bridge food insecurity in sub-Saharan Africa where it is mostly produced. For this virus to replicate, it encode for a Rep protein to initiate the rolling cycle replication (RCR) therefore making the protein indispensable for viral life cycle. Understanding the mechanism of viral replication proteins is essential because of the involvement with replication and spread of the disease. There is an urgent need to develop novel strategies to control the virus. Therefore, the main aim of this study was to investigate ACMV Rep protein functional activity as a target for the development of small molecule inhibitors. To achieve this aim, overexpression of full length ACMV Rep recombinant protein in Escherichia coli (E.coli) BL21 (DE3) pLysS bacterial cells and purification on nickel affinity chromatography was successfully done. Biochemical and biophysical characterisation is a crucial step to follow in order to understand the role of proteins in the viral life cycle. he structural determination of ACMV Rep protein was first predicted using online bioinformatics tool ExPASy and further determined with Fourier-transform infrared (FTIR)and intrinsic fluorescence spectroscopy. The secondary structure prediction from ExPASy resulted in the prediction of helices, sheets and coils. From FTIR spectroscopy analyses, the Amide I region was detected which was reported to be common in proteins representing the secondary structure. The overlapping peaks of the Amide I region were resolved through deconvolution on OriginPro 8 and this resulted inα-helices, β-sheets, β-turns and coils which correlates with the ExPASy results. Intrinsic fluorescence spectra showed an emission maximum wavelength of 355 nm at both 280 nm and 290 nm excitation wavelengths. The emission maxima shows that the tryptophan is exposed to the solvent. Assessing the activity of proteins is another step to determine proper folding of the protein. This was determined with binding and cleavage assays using Electromobility Shift Assay (EMSA). Results showed that purified ACMV Rep was functional in both the binding and cleavage activity at increasing concentrations (≥0.4 μM). Enzyme-linked Immunosorbent Assay (ELISA), a microtiter plate assay for protein functionality, was also developed for the interaction of ACMV Rep and ACMV DNA. This assay is amenable to high throughput screening of potential inhibitors. ELISA results also confirmed the binding activity of the purified Rep protein to ACMV DNA, this assay can be adapted to screen potential small molecule inhibitors of ACMV Rep interaction with ACMV DNA. The identification of small molecules that inhibit the activity of ACMV Rep protein to viral DNA on EMSA was done. Natural phenolic compounds such as Epigallocatechin gallate (EGCG) and Chicoric acid (CA) have inhibition activity on the binding of ACMV Rep to DNA at a concentration of ≥100 μM. In addition, EGCG inhibits the cleavage activity of DNA by ACMV Rep protein with a concentration of ≥ 100 μM while CA showed no inhibition towards cleavage activity of the ACMV Rep on the DNA. From this study, more can be done to understand the crystal structure of the protein and its mechanism of interaction
A dissertation submitted in fulfilment of the requirements for the degree Master of Science in Molecular and Cell Biology in the Faculty of Science, University of the Witwatersrand, Johannesburg, 2020