Downloaded from www.microbiologyresearch.org by IP: 54.163.97.99 On: Sun, 02 Apr 2017 04:18:00 Short Communication Identification and characterization of amphiphysin II as a novel cellular interaction partner of the hepatitis C virus NS5A protein Birgit Zech, 1 Alexander Kurtenbach, 1 Nicole Krieger, 2 Dennis Strand, 3 Stephanie Blencke, 1 Monika Morbitzer, 1 Kostas Salassidis, 1 Matt Cotten, 1 Josef Wissing, 4 Sabine Obert, 1 Ralf Bartenschlager, 2 Thomas Herget 1 and Henrik Daub 1 Correspondence Henrik Daub daub@axxima.com 1 Axxima Pharmaceuticals AG, Am Klopferspitz 19, 82152 Martinsried, Germany 2 Department of Molecular Virology, University of Heidelberg, Im Neuenheimer Feld 350, 69120 Heidelberg, Germany 3 Department of Internal Medicine, Johannes Gutenberg University Mainz, Obere Zahlbacher Strabe 63, 55131 Mainz, Germany 4 Department of Biochemistry, Technical University of Braunschweig, Mascheroder Weg 1, 38124 Braunschweig, Germany Received 29 August 2002 Accepted 19 November 2002 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. Chronic infection with hepatitis C virus (HCV) bears a substantial risk of developing severe liver disease such as chronic hepatitis, liver cirrhosis and hepatocellular carci- noma. The interactions between cellular proteins and HCV gene products may provide clues for novel approaches to interfere with virus propagation and pathogenesis. The HCV non-structural protein 5A (NS5A) became the focus of studies concerning cellular binding partners when it was reported to be involved in HCV resistance to IFN-a (Enomoto et al., 1995, 1996). NS5A is presumed to be a component of the membrane-associated complex of HCV proteins that replicates the plus-strand RNA genome via a minus-strand RNA intermediate (Brass et al., 2002; Hijikata et al., 1993; Shirota et al., 2002). Cell culture-adaptive mutations in the NS5A sequence significantly enhance the replication efficiency of HCV replicons, supporting its role in RNA replication (Blight et al., 2000; Krieger et al., 2001; Lohmann et al., 2001). NS5A proteins of some HCV isolates associate with IFN- induced double-stranded RNA-activated protein kinase (PKR) and inhibit PKR activity (Gale et al., 1997, 1998). In addition, mechanisms for PKR-independent repression of IFN action by NS5A, such as the induction of IL-8 expression by transcriptional stimulation, have been reported (Polyak et al., 2001). Transcriptional activation mediated by NS5A is most pronounced for N-terminally truncated NS5A, which is transported into the nucleus, in contrast to the perinuclear, cytoplasmic full-length protein (Enomoto et al., 1996; Kato et al., 1997; Tanimoto et al., 1997). A cellular transcription factor (Ghosh et al., 2000), as well as a putative nucleoplasmic transporter, karyopherin b3 (Chung et al., 2000), were found as NS5A interaction partners in yeast two-hybrid screens. Ectopically expressed NS5A protein has been described to interact with Grb2 (Tan et al., 1999), p53 (Majumder et al., 2001), Cdk1 (Arima et al., 2001) and TRAF-2 (Park et al., 2002) and to cause changes in cell growth or cellular signalling (Gong et al., 2001; Park et al., 2002). A SNARE-like protein, h-VAP-33, found associated with the endoplasmic reticulum, Golgi and prelysosomal membranes, extends the list of NS5A inter- action partners suggesting a role in membrane-associated 0001-8801 G 2003 SGM Printed in Great Britain 555 Journal of General Virology (2003), 84, 555–560 DOI 10.1099/vir.0.18801-0