Description: Phosphorylated tyrosine 93 of hepatitis C virus nonstructural protein 5A is essential for interaction with host c-Src and efficient viral replication

This title appears in the Scientific Report : 2019

Phosphorylated tyrosine 93 of hepatitis C virus nonstructural protein 5A is essential for interaction with host c-Src and efficient viral replication

The 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...

Personal Name(s):	Klinker, Stefan
	Stindt, Sabine / Gremer, Lothar / Bode, Johannes G. / Gertzen, Christoph G. W. / Gohlke, Holger / Weiergräber, Oliver H. / Hoffmann, Silke / Willbold, Dieter (Corresponding author)
Contributing Institute:	Strukturbiochemie; ICS-6 John von Neumann - Institut für Computing; NIC Jülich Supercomputing Center; JSC
Published in:	The journal of biological chemistry, 294 (2019) S. 7388-7402
Imprint:	Bethesda, Md. Soc. 2019
DOI:	10.1074/jbc.RA119.007656
PubMed ID:	30862675
Document Type:	Journal Article
Research Program:	Physical Basis of Diseases Computational Science and Mathematical Methods
	Publikationsportal JuSER

Please use the identifier: http://dx.doi.org/10.1074/jbc.RA119.007656 in citations.

The 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.