Potyviruses constitute a large genus of plant RNA viruses that cause significant economic losses worldwide in agriculture by infecting a wide range of hosts. Due to their small genomes, they encode a set of multifunctional proteins, among which the HCPro protein plays a central role. Through complex interactions with viral and host proteins, HCPro facilitates viral replication and spread while simultaneously suppressing the plant's defense system. Interactions between HCPro from various potyviruses and selected plant molecules, identified through diverse methodological approaches, have already been documented in the literature. The aim of this master's thesis was to qualitatively and quantitatively analyse the interactions of the potato virus A (PVA) HCPro protein with five tobacco (Nicotiana tabacum) proteins, eIF(iso)4E, CF1β, MinD, WD40 and HIP2, under controlled in vitro conditions. Recombinant proteins were expressed in the bacterial system Escherichia coli. Due to inadequate expression or solubility of MinD, WD40 and HIP2, further experimental work was limited to the proteins eIF(iso)4E and CF1β. By introducing an additional polyhistidine tag, we successfully optimized the isolation and purification protocols for both proteins. Their purity was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while structural integrity and stability were assessed using native polyacrylamide gel electrophoresis, dynamic light scattering, circular dichroism and differential scanning fluorimetry. Using the pull-down assay, we confirmed the direct interaction of both proteins with HCPro, and their binding affinity was determined via microscale thermophoresis. Dissociation constant values in the low micromolar range indicate the specificity and transient nature of these interactions. These findings provide an important foundation for further structural and functional research into the interactions between viral and plant (host) molecules.
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