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Hepatitis C virus NS5A protein binds the SH3 domain of the Fyn tyrosine kinase with high affinity: mutagenic analysis of residues within the SH3 domain that contribute to the interaction

Holly Shelton12 and Mark Harris1*

Author Affiliations

1 Institute of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK

2 Department of Microbiology, School of Biological Sciences, The University of Reading, Whiteknights, RG6 6AJ, Reading, UK

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Virology Journal 2008, 5:24  doi:10.1186/1743-422X-5-24

Published: 11 February 2008

Abstract

Background

The hepatitis C virus (HCV) non-structural 5A protein (NS5A) contains a highly conserved C-terminal polyproline motif with the consensus sequence Pro-X-X-Pro-X-Arg that is able to interact with the Src-homology 3 (SH3) domains of a variety of cellular proteins.

Results

To understand this interaction in more detail we have expressed two N-terminally truncated forms of NS5A in E. coli and examined their interactions with the SH3 domain of the Src-family tyrosine kinase, Fyn. Surface plasmon resonance analysis revealed that NS5A binds to the Fyn SH3 domain with what can be considered a high affinity SH3 domain-ligand interaction (629 nM), and this binding did not require the presence of domain I of NS5A (amino acid residues 32–250). Mutagenic analysis of the Fyn SH3 domain demonstrated the requirement for an acidic cluster at the C-terminus of the RT-Src loop of the SH3 domain, as well as several highly conserved residues previously shown to participate in SH3 domain peptide binding.

Conclusion

We conclude that the NS5A:Fyn SH3 domain interaction occurs via a canonical SH3 domain binding site and the high affinity of the interaction suggests that NS5A would be able to compete with cognate Fyn ligands within the infected cell.