Identifying novel interaction partners for the hepatitis C virus NS5A protein by screening a human SH3 domain phage display library

Abstract

The hepatitis C virus is a worldwide cause of liver disease by infecting approximately 2 % of the population. One of its non-structural protein, NS5A, Is known to interact with many cellular proteins involved in cell signaling pathways. Doing so modifies the outcome of the signalling for the benefit of virus replication and survival. We have previously demonstrated that NSSA contains two Class II poly-proline motifs in the second low complexity sequence, with the consensus sequence Pro-X-X-Pro-X-Lys/ Arg, which mediate the interaction with SH3 domain containing proteins. Substitution of the prolines with alanines did not have an effect on RNA replication but disrupted interactions with SH3 domain containing proteins. In order to identify novel NSSA binding partners, genotype 2a (JFH1) NSSA was expressed with a biotin tag individually or in the context of the replicon as an experimental requirement to screen an all human SH3 domain displaying library. In total, sixteen SH3 domain containing binding partners were identified, from which Mlk3, RelA, Vinexin, PACSIN1 RBP2 and STAC1 were novel targets. The interaction of NS5A with Amph1, CMS, Nck1 and Ponsin were investigated in more details. Interactions of NS5A with Amph1 and CMS were confirmed using immunoprecipitation, immunofluorescence and biochemical assays. Interestingly these proteins have been implicated in clathrin-mediated endocytosis and epidermal growth factor receptor trafficking. Upon overexpression of wt but not the proline mutant NSSA the cellular localization of Amph1 was altered and interaction with dynamin2, a well characterized partner of Amph1, was disrupted. Furthermore, as it was reported before, NS5A diverts the epidermal growth factor receptor away from the late endosomes thereby modifying its degradation rate and signalling capability towards amongst other targets the Ras-Erk mitogen-activated protein kinase pathway, a signalling cascade known to be inhibited by NS5A in a PxxPxR dependent manner. NS5A-CMS and EGFR was found to co-localise to vesicles upon EGF stimulation and overexpression of CMS was found to reverse the effect of NS5A on EGFR.EThOS - Electronic Theses Online ServiceGBUnited Kingdo