| Summary: | 碩士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 106 === The hepatitis C virus (HCV) non-structural 5A protein (NS5A) is a phosphoprotein critical to virus propagation. It has two phosphorylation states: hypo- and hyper- phosphorylated NS5A which are believed to recruit different host interacting proteins to execute distinct functions. Our previous studies showed that serine 235 (S235) phosphorylation is a primary event accounting for hyper-phosphorylated NS5A. S235 is phosphorylated by casein kinase I isoform alpha that requires S232 to be phosphorylated/primed first. However, two issues remain to be solved: 1) what proteins hypo- and hyper-phosphorylated NS5A recruit to facilitate their functions in HCV- infected cells, 2) which kinase is responsible for S232 phosphorylation, thus leading to NS5A hyper-phosphorylation. Here, we establish an affinity purification-mass spectrometry (AP-MS) approach combining on-bead two step digestion and stable isotope dimethyl labeling and use hypo- and hyper-phosphorylated NS5A-specific antibodies to identify the interactors of hypo- and hyper-phosphorylated NS5A. We identified the polymerase II-associated factor I complex (Paf1 complex) proteins as potential interactors of hyper-phosphorylated NS5A. Moreover, by combining the AP- MS result and a kinase ranking based on Bayes’ theorem, we identified DNA-dependent protein kinase catalytic subunit (PRKDC) as an interactor of hypo-phosphorylated NS5A. In line with this, inhibition and knockdown of PRKDC reduce S232 phosphorylation. These results suggested a novel role of the Paf1 complex in HCV infection and a new function of PRKDC in phosphorylate NS5A at S232.
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