Identification and characterization of amphiphysin II as a novel cellular interaction partner of the hepatitis C virus NS5A protein.
The hepatitis C virus (HCV) NS5A protein is highly phosphorylated by cellular protein kinases. To study how NS5A might be integrated in cellular kinase signalling, we isolated phosphoproteins from HuH-7 hepatoma cells that specifically interacted with recombinant NS5A protein. Subsequent mass spectrometry identified the adaptor protein amphiphysin II as a novel interaction partner of NS5A. Mutational analysis revealed that complex formation is primarily mediated by a proline-rich region in the C-terminal part of NS5A, which interacts with the amphiphysin II Src homology 3 domain. Importantly, we could further demonstrate specific co-precipitation and cellular co-localization of endogenous amphiphysin II with NS5A in HuH-7 cells carrying a persistently replicating subgenomic HCV replicon. Although the NS5A-amphiphysin II interaction appeared to be dispensable for replication of these HCV RNAs in cell culture, our results indicate that NS5A-amphiphysin II complex formation might be of physiological relevance for the HCV life cycle.
Zech
Birgit
B
Axxima Pharmaceuticals AG, Am Klopferspitz 19, 82152 Martinsried, Germany.
Kurtenbach
Alexander
A
Axxima Pharmaceuticals AG, Am Klopferspitz 19, 82152 Martinsried, Germany.
Krieger
Nicole
N
Department of Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 350, 69120 Heidelberg, Germany.
Strand
Dennis
D
Department of Internal Medicine, Johannes Gutenberg University Mainz, Obere Zahlbacher Straβe 63, 55131 Mainz, Germany.
Blencke
Stephanie
S
Axxima Pharmaceuticals AG, Am Klopferspitz 19, 82152 Martinsried, Germany.
Morbitzer
Monika
M
Axxima Pharmaceuticals AG, Am Klopferspitz 19, 82152 Martinsried, Germany.
Salassidis
Kostas
K
Axxima Pharmaceuticals AG, Am Klopferspitz 19, 82152 Martinsried, Germany.
Cotten
Matt
M
Axxima Pharmaceuticals AG, Am Klopferspitz 19, 82152 Martinsried, Germany.
Wissing
Josef
J
Department of Biochemistry, Technical University of Braunschweig, Mascheroder Weg 1, 38124 Braunschweig, Germany.
Obert
Sabine
S
Axxima Pharmaceuticals AG, Am Klopferspitz 19, 82152 Martinsried, Germany.
Bartenschlager
Ralf
R
Department of Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 350, 69120 Heidelberg, Germany.
Herget
Thomas
T
Axxima Pharmaceuticals AG, Am Klopferspitz 19, 82152 Martinsried, Germany.
Daub
Henrik
H
Axxima Pharmaceuticals AG, Am Klopferspitz 19, 82152 Martinsried, Germany.
D003593:Cytoplasm / Q000737:chemistry; D019084:Fluorescent Antibody Technique, Indirect; D016174:Hepacivirus / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006801:Humans; D015151:Immunoblotting; D016350:Leucine Zippers; D020930:MAP Kinase Kinase Kinases; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D011392:Proline; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012093:Replicon; D014407:Tumor Cells, Cultured; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11
D020930:MAP Kinase Kinase Kinases; D009419:Nerve Tissue Proteins; D011392:Proline; D017346:Protein Serine-Threonine Kinases; D012324:RNA-Dependent RNA Polymerase; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D000097767:Mitogen-Activated Protein Kinase Kinase Kinase 11; C075637:amphiphysin; C087036:NS-5 protein, hepatitis C virus