Hepatitis C virus (HCV) causes human diseases such as chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. HCV non-structural protein 5A (NS5A) is indispensable for viral replication and particle assembly. We performed biophysical and structural studies of NS5A alone, and in complex with SH3 domains from cellular proteins. We showed that the C-terminal part of NS5A is intrinsically disordered and mediates transient interactions also via non-canonical SH3-binding motifs. We furthermore reported that - in HCV infected cells - c-Src, which is important for regulation of cell growth and differentiation, is recruited into the viral replication machinery by HCV proteins NS5A and NS5B. Our results can help to understand the perturbations of cellular signaling pathways mediated by NS5A, which serves as a diverse molecular interaction platform.
This project was funded by the SFB 575. Major parts of the results have been collected at the IBS (B. Brutscher lab, Grenoble) or in cooperation with the the UKD (J. Bode, Düsseldorf).
Interactions of HCV proteins NS5A and NS5B with host cell protein c-Src
To understand the structural basis of the interactions between the tyrosine kinase c-Src and the hepatitis C virus proteins NS5A and NS5B we will pursue three-dimensional structures of complexes formed by c-Src, NS5A and NS5B. An important step to successful reconstitution of the c-Src /NS5A / NS5B complex in vitro is the optimization of the recombinant production and purification of the three components in E. coli. We will then map the relevant binding sites using a combined approach including mutagenesis, pull-downs, phosphotyrosine mapping and NMR spectroscopy. Binding affinities will be analyzed by e.g. SPR, ITC or MST. Since NS5A, NS5B and c-Src are membrane-attached proteins, studies with model membranes like nanodiscs will be performed to characterize the influence of lipid membranes e.g. on complex stability. Our final goal is the structure determination of the complexes, using NMR spectroscopy and/or x-ray crystallography. To develop specific inhibitors of these interactions lead compounds for inhibitor development are also planned to be identified. Consequently, we will use cell culture assays to confirm binding sites and to verify identified inhibitors (collaboration: J. Bode).
This project is funded by the SFB974.