Exploring the hepatitis C virus encoded protease NS3/4A in relation to Autophagy
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Autophagy is an evolutionarily conserved intracellular bulk degradation system essential for the cell homeostasis. In addition, autophagy is emerging as a central component in viral host defense. Many pathogens have evolved mechanisms to evade, subvert, or exploit autophagy. Recent studies have suggested that hepatitis C virus (HCV) utilize autophagic structures and /or proteins to promote viral replication. However, the underlying molecular mechanisms remain to be elucidated. The multifunctional HCV - encoded protein NS3/4A, a serine protease, is known to be essential for both viral lifecycle and evasion of host immune defence. In this thesis, we wanted to explore NS3/4A in relation to autophagy. Therefore, we studied the effect of NS3/4A on autophagy and searched for novel NS3/4A protease targets and interacting proteins among proteins known to be involved in autophagy. In addition, we explored potential interactions between virus- induced RIG-I signaling and autophagy of mitochondria (mitophagy). In that aspect we also studied the effect of NS3/4Amediated cleavage of the RIG-I signaling adapter protein MAVS. In this thesis, HEK293 cells were used for transfection experiments. Bioinformatics were applied to predict potential interacting proteins. Various methods were used for studying the effect of HCV NS3/4A expression, including western blotting, (co-) immunoprecipitation, luciferase reporter assays and confocal microscopy. We found that NS3/4A did not affect the levels of the autophagy marker protein LC3. By using the bioinformatic web tool, ScanProsite, fourteen proteins known to be involved in autophagy were predicted to contain NS3/4A substrate cleavage site. Three of them, p62, beclin-1 and mTOR were studied in experiments. However, we failed to verify these proteins as real targets of NS3/4A. NS3/4A was also predicted to be a LC3- interacting protein, this interaction was neither verified experimentally. Initial experiments indicated that MAVS may associates with the mitophagic adapter protein NIX in response to Sendai virus infection, and that NS3/4A may interrupt this association. However, these results have to be verified and further explored.