title: Reduction of hepatitis C virus NS5A phosphorylation through its interaction with amphiphysin II.
abstract: Hepatitis C virus non-structural protein 5A (NS5A) is a pleiotropic protein with key roles in viral RNA replication, modulation of cellular-signaling pathways and interferon (IFN) responses. To search for possible host factors involved in mediating these functions of NS5A, we adopted an affinity purification approach coupled with mass spectrometry to examine protein-protein interactions, and found that human amphiphysin II (also referred to as Bin1) specifically interacts with NS5A in mammalian cells. Pull-down assays showed that the Src homology 3 (SH3) domain of amphiphysin II is required for NS5A interaction and that c-Src also interacts with NS5A in cells. IFN-alpha treatment reduced the interaction of NS5A with c-Src, but not amphiphysin II, suggesting that the latter is independent of the IFN-signaling pathway. NS5A is a phosphoprotein and its phosphorylation status is considered to have an effect on viral RNA replication. In vitro kinase assays demonstrated that its interaction with amphiphysin II inhibits phosphorylation of NS5A. These results suggest that amphiphysin II participates in the HCV life cycle by modulating the phosphorylation of NS5A.
authors.affiliation: Department of Safety Research on Blood and Biological Products, National Institute of Infectious Diseases, Tokyo, Japan. amasumi@nih.go.jp
authors.forename: Atsuko
authors.initials: A
authors.lastname: Masumi
authors.forename: Hideki
authors.initials: H
authors.lastname: Aizaki
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Suzuki
authors.forename: James B
authors.initials: JB
authors.lastname: DuHadaway
authors.forename: George C
authors.initials: GC
authors.lastname: Prendergast
authors.forename: Katsutoshi
authors.initials: K
authors.lastname: Komuro
authors.forename: Hidesuke
authors.initials: H
authors.lastname: Fukazawa
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000737:chemistry* / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D025521:Tumor Suppressor Proteins / Q000737:chemistry* / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000737:chemistry* / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D009687:Nuclear Proteins; D025521:Tumor Suppressor Proteins; D017361:Viral Nonstructural Proteins; C101410:BIN1 protein, human; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase