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Browsing by Author "Basic, Marta"

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  • Basic, Marta (2015)
    Plant viruses are a prominent subject of scientific research due to their ability to annually decimate agricultural crop yields. Amongst all of the groups of plant viruses, the potyvirus group is one of the largest and therefore contributes to a large portion of all plant virus-induced economic and agricultural deficiencies. The potyvirus field has become a relevant area of research, ultimately aiming to provide a more comprehensive understanding of the infection process in the hopes of yielding virus-resistant crops, subsequently increasing food production and reducing the plant virus-related economic burden. Potato Virus A (PVA) is a positive-sense, single-stranded RNA virus of the genus Potyvirus. It has a single open reading frame that is translated into a polyprotein and latterly cleaved into 10 mature proteins: P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb, and CP. Helper component-proteinase (HC-Pro) is a multifunctional viral protein and the main subject of this study. It is involved in many stages of the virus life cycle; however, its role as a suppressor of RNA silencing, an anti-viral defense mechanism employed by the host organism, makes it a particularly interesting candidate for protein interaction studies. Because viruses are heavily dependent on the host's cellular machinery for survival, understanding the interactions between viral and host proteins is crucial for elucidating the molecular mechanisms underlying infection. The goal of this thesis was to identify potentially novel interacting partners of potyviral HC-Pro. This study utilized a Twin-Strep-tag affinity purification method for isolating HC-Pro associated binding partners from the total cell lysate of PVA-infected plant leaves. The purified macromolecular complexes were then analyzed using mass spectrometry and numerous individual proteins were identified. Gene Ontology (GO) functional annotations were generated for the obtained proteome using several R scripts and their distributions visualized as graphs. This study identified a total of 190 proteins purified from specific HC-Pro-associated complexes. Two identified host proteins were of particular interest due to their potential involvement in the RNA interference pathway and are currently being studied in more detail. Overall, this study successfully yielded numerous potentially novel protein interactions and presents an effective approach to pinpointing host binding partners of particular interest for future studies.