title: Further studies on hepatitis C virus NS5A-SH3 domain interactions: identification of residues critical for binding and implications for viral RNA replication and modulation of cell signalling.
abstract: The NS5A protein of hepatitis C virus has been shown to interact with a subset of Src homology 3 (SH3) domain-containing proteins. The molecular mechanisms underlying these observations have not been fully characterized, therefore a previous analysis of NS5A-SH3 domain interactions was extended. By using a semi-quantitative ELISA assay, a hierarchy of binding between various SH3 domains for NS5A was demonstrated. Molecular modelling of a polyproline motif within NS5A (termed PP2.2) bound to the FynSH3 domain predicted that the specificity-determining RT-loop region within the SH3 domain did not interact directly with the PP2.2 motif. However, it was demonstrated that the RT loop did contribute to the specificity of binding, implicating the involvement of other intermolecular contacts between NS5A and SH3 domains. The modelling analysis also predicted a critical role for a conserved arginine located at the C terminus of the PP2.2 motif; this was confirmed experimentally. Finally, it was demonstrated that, in comparison with wild-type replicon cells, inhibition of the transcription factor AP-1, a function previously assigned to NS5A, was not observed in cells harbouring a subgenomic replicon containing a mutation within the PP2.2 motif. However, the ability of the mutated replicon to establish itself within Huh-7 cells was unaffected. The highly conserved nature of the PP2.2 motif within NS5A suggests that functions involving this motif are of importance, but are unlikely to play a role in replication of the viral RNA genome. It is more likely that they play a role in altering the cellular environment to favour viral persistence.
authors.affiliation: School of Biochemistry and Microbiology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Macdonald
authors.affiliation: School of Biochemistry and Microbiology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Sabine
authors.initials: S
authors.lastname: Mazaleyrat
authors.affiliation: School of Biochemistry and Microbiology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Christopher
authors.initials: C
authors.lastname: McCormick
authors.affiliation: School of Biochemistry and Microbiology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Street
authors.affiliation: School of Biochemistry and Microbiology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Burgoyne
authors.affiliation: School of Biochemistry and Microbiology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Richard M
authors.initials: RM
authors.lastname: Jackson
authors.affiliation: School of Biochemistry and Microbiology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Virginie
authors.initials: V
authors.lastname: Cazeaux
authors.affiliation: School of Biochemistry and Microbiology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Holly
authors.initials: H
authors.lastname: Shelton
authors.affiliation: Institute of Medical Technology, Tampere University Hospital, FIN-33014 Tampere, Finland.
authors.forename: Kalle
authors.initials: K
authors.lastname: Saksela
authors.affiliation: School of Biochemistry and Microbiology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Harris
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D005786:Gene Expression Regulation; D016174:Hepacivirus / Q000378:metabolism / Q000472:pathogenicity*; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D051076:Proto-Oncogene Proteins c-fyn; D012367:RNA, Viral / Q000378:metabolism; D015398:Signal Transduction; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D018909:src Homology Domains; D019061:src-Family Kinases / Q000737:chemistry*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D011518:Proto-Oncogene Proteins; D051076:Proto-Oncogene Proteins c-fyn; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; D019061:src-Family Kinases; C494957:FYN protein, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase