Abstract
Hepatitis C virus (HCV) non-structural 5A (NS5A) protein is associated with a wide variety of host signaling pathways by binding to C-terminal polyproline (PxxP) motifs of various proteins. In this study, we used yeast two-hybrid analysis and a GST pull-down assay to screen a novel NS5A interacting protein and elucidate the binding site and cellular signaling by focusing on recombinant human epidermal growth factor (rhEGF)-mediated ERK1/2 activation. Screening a liver cDNA library revealed that h-prune, a member of the DHH (Asp-His-His) protein superfamily, directly interacted with HCV NS5A C-terminus. In particular, a mutation of five proline amino acids to alanine in this region revealed that these two proteins produced strong interaction through this domain. It is known that h-prune possesses a highly conserved DHH motif, which has exopolyphosphatase activity that accelerates hydrolysis of inorganic polyphosphate. A time-chasing analysis after rhEGF treatment demonstrated that h-prune overexpression almost restored NS5A-mediated attenuation of ERK1/2 phosphorylation, but h-prune itself did not alter this signaling. Although the detailed mechanisms need to be clarified, this study demonstrates that h-prune interacts directly with the PxxP motif of the HCV NS5A C-terminus and that this binding alters the rhEGF-mediated ERK1/2 signaling cascade in liver cells.
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This work was supported by the Chungnam National University (2013-0631).
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Nam, M., Kim, CH., Kim, DU. et al. h-Prune as a novel binding protein of NS5A that regulates ERK1/2 activation. Appl Biol Chem 59, 543–551 (2016). https://doi.org/10.1007/s13765-016-0193-4
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DOI: https://doi.org/10.1007/s13765-016-0193-4