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.
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.


CONCLUSIONS
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.
Shelton
Holly
H
Institute of Molecular and Cellular Biology, Faculty of Biological Sciences and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK. h.a.shelton@reading.ac.uk
Harris
Mark
M
D019943:Amino Acid Substitution / Q000235:genetics; D016174:Hepacivirus / Q000502:physiology*; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D011485:Protein Binding; D025941:Protein Interaction Mapping; D051076:Proto-Oncogene Proteins c-fyn / Q000235:genetics / Q000378:metabolism*; D020349:Surface Plasmon Resonance; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D018909:src Homology Domains / Q000235:genetics*; D012324:RNA-Dependent RNA Polymerase
D051076:Proto-Oncogene Proteins c-fyn; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase