Identification and characterization of amphiphysin II as a novel cellular interaction partner of the hepatitis C virus NS5A protein

Birgit Zech1, Alexander Kurtenbach1, Nicole Krieger2, Dennis Strand3, Stephanie Blencke1, Monika Morbitzer1, Kostas Salassidis1, Matt Cotten1, Josef Wissing4, Sabine Obert1, Ralf Bartenschlager2, Thomas Herget1 and Henrik Daub1

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 Stra{beta}e 63, 55131 Mainz, Germany
4 Department of Biochemistry, Technical University of Braunschweig, Mascheroder Weg 1, 38124 Braunschweig, Germany

Correspondence
Henrik Daub
daub{at}axxima.com


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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.


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Chronic infection with hepatitis C virus (HCV) bears a substantial risk of developing severe liver disease such as chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. 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-{alpha} (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 {beta}3 (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 interaction partners suggesting a role in membrane-associated replication (Tu et al., 1999). The physiological significance of these cellular NS5A-binding proteins for the HCV infectious cycle remains unclear.

Moreover, NS5A is phosphorylated by as yet unidentified host-cell protein kinases in cultured cells (Katze et al., 2000). To address the important issue of how NS5A might interact with cellular kinase signalling networks, we set out to isolate cellular phosphoproteins that could specifically associate with NS5A protein. For this purpose, we first inserted the complete coding region of NS5A (Lohmann et al., 1999) into the pGEX5x1 vector for generation of recombinant glutathione S-transferase (GST)–NS5A fusion protein in E. coli. GST–NS5A was subjected to in vitro association with total lysates from HuH-7 cells that had been metabolically labelled with 100 µCi [33P]orthophosphate ml-1 for 3 h. Lysates were prepared essentially as described by Daub et al. (2002) and added to GST–NS5A fusion protein immobilized on glutathione–Sepharose (Amersham). Bound proteins were analysed by two-dimensional (2-D) gel electrophoresis. For the first dimension, isoelectric focussing was performed using linear 24 cm immobilized pH 4–7 gradient drystrips on an IPGphor according to the manufacturer's instructions (Amersham). As shown in Fig. 1(a), several cellular 33P-labelled protein species were detected in the acidic region of the 2-D gel and these proteins specifically associated with GST–NS5A and precisely co-migrated with Coomassie-stainable protein spots. Corresponding spots were subjected to mass spectrometry (MS) analysis by nano-electrospray ionization on a Q-TOF mass spectrometer (Borchers et al., 2000). The spectra generated allowed sequencing of four different peptides, which all were identical to sequences found in human amphiphysin II. The human amphiphysin II (also referred to as Bin1) gene encodes various different isoforms, which result from alternative splicing (Wechsler-Reya et al., 1997; Tsutsui et al., 1997). As none of our peptides mapped to alternatively spliced regions of amphiphysin II, it was not possible to discriminate between different isoforms based on our MS analysis. The Src homology 3 (SH3) domain-containing protein amphiphysin II has been implicated in clathrin-mediated endocytosis (Wigge et al., 1997). Amphiphysin II was also found to interact with the myc oncogene and implicated in myc-induced apoptosis (Sakamuro et al., 1996; DuHadaway et al., 2001).



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Fig. 1. (a) Purification of NS5A-interacting phosphoproteins. After metabolic labelling of HuH-7 cells with [33P]orthophosphate, cell lysates were used for in vitro association with GST–NS5A. Bound proteins were resolved by 2-D gel electrophoresis and visualized by Coomassie staining (left) and autoradiography (right). Arrows indicate the positions of two NS5A-interacting phosphoproteins identified as amphiphysin II by mass spectrometry. (b) Specific in vitro association of endogenous amphiphysin II with GST–NS5A fusion proteins. Total cell lysates prepared from HuH-7 cells were subjected to in vitro association with either GST or different GST–NS5A fusion proteins containing full-length NS5A, NS5ApolyP- carrying three alanine substitutions for proline (P350A, P353A and P354A) or truncated NS5A{Delta}C terminating at aa 277. Bound proteins were resolved by 10 % SDS-PAGE followed by immunoblotting with anti-amphiphysin II antibody. Molecular mass is indicated on the left and amphiphysin II isoforms are marked by arrows. (c) Specific interactions of transiently expressed amphiphysin II splice variants and the amphiphysin II SH3 domain with GST–NS5A and GST–NS5ApolyP-. HuH-7 cells were transiently transfected with plasmids encoding FLAG-tagged amphiphysin II2, amphiphysin II2 short or the amphiphysin II SH3 domain (1·5 µg DNA of each per well). After 24 h, cells were lysed and extracts were subjected to in vitro association with GST, GST–NS5A or GST–NS5ApolyP-proteins. After 15 % SDS-PAGE, immunoblotting was performed with an anti-FLAG antibody. The positions of FLAG-tagged proteins are marked by arrows.

 
To confirm amphiphysin II as a novel NS5A-interacting protein, we performed an immunoblot analysis of GST–NS5A-associated proteins from HuH-7 cells employing a monoclonal antibody recognizing amphiphysin II (UBI). As shown in Fig. 1(b), two splice variants of amphiphysin II of approximately 66 and 73 kDa interacted with the GST–NS5A fusion protein, but not with GST alone. To characterize the domains of NS5A required for amphiphysin II binding, three out of five prolines were changed to alanines in a class II proline-rich region of NS5A, which has previously been shown to mediate SH3 domain-dependent binding of the adaptor protein Grb2 (Tan et al., 1999). Binding of endogenous amphiphysin II to the resulting GST–NS5ApolyP- mutant fusion protein (P350A, P353A and P354A substitutions in NS5A) was strongly reduced but not completely abrogated. No detectable amphiphysin II binding was observed when the C-terminal part of NS5A (aa 278–447) was deleted in the GST fusion protein (Fig. 1b).

We then PCR-amplified two amphiphysin II splice variants from human cDNA libraries and cloned them into the pPM7 plasmid to allow CMV promoter-driven ectopic protein expression (Daub et al., 2002). Sequencing revealed that one construct represented the amphiphysin II2 splice variant of 482 amino acids (GenBank accession no. AF001383.1), whereas the other clone was a shorter version of amphiphysin II2 harbouring a deletion of aa 304–346. We therefore referred to this construct as amphiphysin II2 short. Both amphiphysin II variants were fused to N-terminal FLAG epitopes. We also generated a pPM7-FLAG-amph II-SH3 expression plasmid to express the C-terminal SH3 domain of amphiphysin II (residues 393–482 of amphiphysin II2). All three plasmids were then used for transient transfection of HuH-7 cells as described (Daub et al., 2002). Cell lysates were subjected to in vitro association with GST, GST–NS5A or GST–NS5ApolyP- fusion proteins. Protein complexes were then analysed by immunoblotting using monoclonal anti-FLAG antibody (Sigma). Both amphiphysin II2 and amphiphysin II2 short interacted with GST–NS5A fusion protein but not with GST itself, confirming the specific NS5A–amphiphysin complex formation observed for endogenous amphiphysin II (Fig. 1c). Interaction of both amphiphysin II variants with the GST–NS5ApolyP- fusion protein was somewhat weaker, although the reduction in affinity was not as pronounced as observed with endogenous amphiphysin II, most likely due to the high amphiphysin II expression levels obtained in the transient expression experiments. Specific association with GST–NS5A was also observed when only the SH3 domain was expressed, but this interaction depended on the presence of the class II proline-rich region of NS5A (Fig. 1c). We concluded from these in vitro association results that, although a class II proline-rich region comprising aa 350–356 of NS5A significantly contributes to binding through interaction with the amphiphysin II SH3 domain, other determinants residing in the C-terminal part of NS5A play a role in NS5A–amphiphysin II complex formation.

To address the important issue of the physiological significance of the NS5A–amphiphysin II interaction, we employed the HuH-7-derived 5-15 replicon cell clone as a relevant model system. These cells carry a selectable self-replicating HCV RNA and functionally express NS5A protein in the context of an NS3 to 5B polyprotein fragment (Lohmann et al., 1999). Cell lysates were prepared from either the 5-15 replicon cell clone or control HuH-7 cells expressing only the neo-resistance gene and subjected to immunoprecipitation with monoclonal anti-NS5A antibody (Biogenesis). As shown in Fig. 2(a), two variants of amphiphysin II of identical molecular mass, as found in our in vitro association experiments, were specifically detected in anti-NS5A immunoprecipitates from 5-15 replicon cells. Immunoprecipitation of NS5A protein from the 5–15 replicon, but not from control HuH-7 cells, was confirmed by parallel immunoblotting with anti-NS5A antibody. Moreover, pretreatment of cells with 1000 units interferon-{alpha} ml-1 (Calbiochem) for 48 h prior to cell lysis not only strongly reduced NS5A expression but also abrogated amphiphysin II co-precipitation (Fig. 2a). As an additional control, we verified that amphiphysin II was expressed at similar levels in all lysates (data not shown). Thus, endogenous amphiphysin II specifically binds NS5A under physiologically relevant conditions and can be isolated from 5-15 cells in a stable complex with NS5A. Similar results were obtained with replicon cell clones 9-13 and 11-7 (data not shown).



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Fig. 2. (a) Co-precipitation of NS5A with endogenous amphiphysin II from HCV replicon cells. HuH-7-neo control cells or 5-15 replicon cells were pretreated with 1000 units IFN-{alpha} ml-1 prior to cell lysis where indicated. Lysates were subjected to immunoprecipitation with anti-NS5A antibody. After gel electrophoresis and transfer on to nitrocellulose membrane, immunoblotting was performed with either anti-amphiphysin II antibody (upper panel) or anti-NS5A antibody (lower panel). Amphiphysin II isoforms and NS5A are marked by arrows. (b) Co-localization of endogenous NS5A and amphiphysin II. NS5A was detected with a Cy3-labelled secondary antibody (left upper panel) and Amphiphysin II with an FITC-labelled secondary antibody (middle upper panel) in cell clone 9-13. Both proteins co-localized in distinct granules that probably correspond to the membranous web (right upper panel). Naive HuH-7 cells stained for amphiphysin II served as a reference and the same cells stained for NS5A were used as a negative control (lower panels).

 
To analyse the subcellular localization of endogenous NS5A–amphiphysin II complexes, we performed an immunofluorescence analysis by co-staining of paraformaldehyde-fixed 9-13 replicon cells with monoclonal anti-amphiphysin II antibody and rabbit polyclonal NS5A-specific antibody. Bound primary antibodies were detected with Cy3-conjugated goat anti-rabbit IgG (Dianova), or FITC-conjugated goat anti-mouse IgG (Sigma) at a dilution of 1 : 5000 or 1 : 500, respectively. The cell nucleus was counterstained using bis-benzimide (Hoechst 33342; Sigma) at 10 µg ml-1. Confocal microscopy of 9-13 replicon cells revealed that both NS5A and endogenous amphiphysin II co-localized to distinct granules (Fig. 2b). These structures probably correspond to the membranous web, where all HCV proteins including NS5A assemble into multiprotein complexes (Egger et al., 2002). Only weak, punctate amphiphysin II staining was detectable in the cytoplasm of parental HuH-7 cells. In both parental and 9-13 replicon cells, the majority of amphiphysin II immunoreactivity was found in the nuclei. Thus, our data suggest that NS5A recruits a certain fraction of cellular amphiphysin II to the sites of viral RNA replication, which is mediated through NS5A-containing HCV protein complexes at the membranous web (R. Gosert & D. Moradpour, personal communication).

We next investigated whether the NS5A–amphiphysin II interaction is essential for HCV RNA replication in cell culture. For this purpose, we generated a recombinant adenovirus for ectopic expression of the FLAG-tagged amphiphysin II SH3 domain according to previously described procedures (Daub et al., 2002) and infected 5-15 replicon cells at an m.o.i. of 3000. Expression of the dominant-interferring SH3 domain specifically disrupted endogenous NS5A–amphiphysin II complexes (Fig. 3a). As a readout for HCV replication, we analysed NS5A protein expression 2 and 4 days after adenovirus infection. However, we found no reduction in NS5A protein levels on amphiphysin II SH3 domain expression (Fig. 3b). Thus, it appeared that the NS5A–amphiphysin II interaction is dispensible for HCV RNA replication in 5-15 cells. Moreover, as measured by a previously described luciferase-based reporter gene assay (Krieger et al., 2001), transient HCV RNA replication was reduced by only about 50 % on introduction of the combined P350A, P353A and P354A substitutions into NS5A of a replicon construct. This modest reduction may result from changes in NS5A secondary structure due to substitution of three adjacent alanine residues for proline rather than from the reduced interaction of NS5A with SH3 domain-containing proteins. Taken together, our data argue against a major role for the NS5A–amphiphysin II interaction in HCV RNA replication in the replicon cell culture model. However, based on the intracellular interaction data presented above, we propose that amphiphysin II might be an essential host-cell factor during natural HCV infection, whose function cannot be studied in replicon cells due to the limitations of this experimental system. One potential clue comes from recent observations that splice variants of amphiphysin II can induce tubular membrane invaginations (Lee et al., 2002). Moreover, the N-terminal domain of the highly related amphiphysin I protein is sufficient to alter membrane topology by forcing vesicles into high-curvature tubular structures (Takei et al., 1999). Based on these findings, it is tempting to speculate that NS5A binds amphiphysin II at the sites of virus replication at the membranous web. Amphiphysin II could then confer topological changes to the local membrane environment, thereby facilitating HCV RNA replication under the physiological conditions of natural HCV infection. Alternatively, amphiphysin II might play a role during the HCV life cycle at steps distinct from viral RNA replication and therefore evade functional characterization in the HuH-7 replicon system. Further research may overcome the limitations of current model systems and might deliver novel tools to evaluate the role of the NS5A interaction partner amphiphysin II in more detail.



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Fig. 3. NS5A–amphiphysin II complex formation appears to be dispensible for subgenomic HCV RNA replication in cell culture. (a) HuH-7-neo or 5-15 replicon cells were infected at an m.o.i. of 3000 with either AdAmph II-SH3 or control adenovirus. On the second day after infection, cell lysates were prepared and subjected to immunoprecipitation with anti-NS5A antibody. Samples were resolved in parallel by 7·5 % SDS-PAGE for immunoblotting with anti-amphiphysin II antibody and by 15 % SDS-PAGE for immunoblotting with anti-NS5A and anti-FLAG antibodies. (b) Total lysates from replicon cell clone 5-15 were prepared 2 and 4 days after infection at an m.o.i. of 3000 with either AdAmph II-SH3 or control adenovirus, resolved by 15 % SDS-PAGE and subjected to immunoblotting with anti-NS5A and anti-FLAG antibodies. The positions of NS5A and the FLAG-tagged amphiphysin II SH3 domain are indicated on the right.

 


   ACKNOWLEDGEMENTS
 
We are grateful to Rainer Gosert and Darius Moradpour for communication of unpublished results. We thank Axel Ullrich for critical reading of the manuscript. Birgit Zech, Alexander Kurtenbach and Nicole Krieger contributed equally to this work.


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Received 29 August 2002; accepted 19 November 2002.