000861817 001__ 861817 000861817 005__ 20220930130208.0 000861817 0247_ $$2doi$$a10.1074/jbc.RA119.007656 000861817 0247_ $$2ISSN$$a0021-9258 000861817 0247_ $$2ISSN$$a1067-8816 000861817 0247_ $$2ISSN$$a1083-351X 000861817 0247_ $$2altmetric$$aaltmetric:60072151 000861817 0247_ $$2pmid$$apmid:30862675 000861817 0247_ $$2WOS$$aWOS:000468402900025 000861817 037__ $$aFZJ-2019-02245 000861817 082__ $$a540 000861817 1001_ $$0P:(DE-HGF)0$$aKlinker, Stefan$$b0 000861817 245__ $$aPhosphorylated tyrosine 93 of hepatitis C virus nonstructural protein 5A is essential for interaction with host c-Src and efficient viral replication 000861817 260__ $$aBethesda, Md.$$bSoc.$$c2019 000861817 3367_ $$2DRIVER$$aarticle 000861817 3367_ $$2DataCite$$aOutput Types/Journal article 000861817 3367_ $$0PUB:(DE-HGF)16$$2PUB:(DE-HGF)$$aJournal Article$$bjournal$$mjournal$$s1579181345_24206 000861817 3367_ $$2BibTeX$$aARTICLE 000861817 3367_ $$2ORCID$$aJOURNAL_ARTICLE 000861817 3367_ $$00$$2EndNote$$aJournal Article 000861817 520__ $$aThe hepatitis C virus (HCV) nonstructural protein 5A (NS5A) plays a key role in viral replication and virion assembly, and regulation of the assembly process critically depends on phosphorylation of both serine and threonine residues in NS5A. We previously identified SRC proto-oncogene, non-receptor tyrosine kinase (c-Src) as an essential host component of the HCV replication complex consisting of NS5A, the RNA-dependent RNA polymerase NS5B, and c-Src. Pull-down assays revealed an interaction between NS5A and the Src-homology 2 (SH2) domain of c-Src; however, the precise binding mode remains undefined. In this study, using a variety of biochemical and biophysical techniques, along with molecular dynamics simulations, we demonstrate that the interaction between NS5A and the c-Src SH2 domain strictly depends on an intact, phosphotyrosine binding-competent SH2 domain and on tyrosine phosphorylation within NS5A. Detailed analysis of c-Src SH2 domain binding to a panel of phosphorylation-deficient NS5A variants revealed that phosphorylation of Y93 located within domain 1 of NS5A, but not of any other tyrosine residue, is crucial for complex formation. In line with these findings, effective replication of subgenomic HCV replicons as well as production of infectious virus particles in mammalian cell culture models were clearly dependent on the presence of tyrosine at position 93 of NS5A. These findings indicate that phosphorylated Y93 in NS5A plays an important role during viral replication by facilitating NS5A’s interaction with the SH2 domain of c-Src. 000861817 536__ $$0G:(DE-HGF)POF3-511$$a511 - Computational Science and Mathematical Methods (POF3-511)$$cPOF3-511$$fPOF III$$x0 000861817 536__ $$0G:(DE-HGF)POF3-553$$a553 - Physical Basis of Diseases (POF3-553)$$cPOF3-553$$fPOF III$$x1 000861817 588__ $$aDataset connected to CrossRef 000861817 7001_ $$0P:(DE-HGF)0$$aStindt, Sabine$$b1 000861817 7001_ $$0P:(DE-Juel1)145165$$aGremer, Lothar$$b2 000861817 7001_ $$0P:(DE-HGF)0$$aBode, Johannes G.$$b3 000861817 7001_ $$0P:(DE-HGF)0$$aGertzen, Christoph G. 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