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 Strae 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
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() |
---|
![]() |
MAIN TEXT |
---|
![]() ![]() ![]() ![]() |
---|
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
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. GSTNS5A 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 GSTNS5A fusion protein immobilized on glutathioneSepharose (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 47 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 GSTNS5A 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
).
|
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 304346. 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 393482 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, GSTNS5A or GSTNS5ApolyP- fusion proteins. Protein complexes were then analysed by immunoblotting using monoclonal anti-FLAG antibody (Sigma). Both amphiphysin II2 and amphiphysin II2 short interacted with GSTNS5A fusion protein but not with GST itself, confirming the specific NS5Aamphiphysin complex formation observed for endogenous amphiphysin II (Fig. 1c
). Interaction of both amphiphysin II variants with the GSTNS5ApolyP- 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 GSTNS5A 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 350356 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 NS5Aamphiphysin II complex formation.
To address the important issue of the physiological significance of the NS5Aamphiphysin 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 515 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-
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).
|
We next investigated whether the NS5Aamphiphysin 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 NS5Aamphiphysin II complexes (Fig. 3
a). 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 NS5Aamphiphysin 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 NS5Aamphiphysin 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.
|
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() |
---|
Blight, K. J., Kolykhalov, A. A. & Rice, C. M. (2000). Efficient initiation of HCV RNA replication in cell culture. Science 290, 19721974.
Borchers, C., Peter, J. F., Hall, M. C., Kunkel, T. A. & Tomer, K. B. (2000). Identification of in-gel digested proteins by complementary peptide mass fingerprinting and tandem mass spectrometry data obtained on an electrospray ionization quadrupole time-of-flight mass spectrometer. Anal Chem 72, 11631168.[CrossRef][Medline]
Brass, V., Bieck, E., Montserret, R., Wolk, B., Hellings, J. A., Blum, H. E., Penin, F. & Moradpour, D. (2002). An amino-terminal amphipathic alpha-helix mediates membrane association of the hepatitis C virus nonstructural protein 5A. J Biol Chem 277, 81308139.
Chung, K. M., Lee, J., Kim, J. E., Song, O. K., Cho, S., Lim, J., Seedorf, M., Hahm, B. & Jang, S. K. (2000). Nonstructural protein 5A of hepatitis C virus inhibits the function of karyopherin beta3. J Virol 74, 52335241.
Daub, H., Blencke, S., Habenberger, P., Kurtenbach, A., Dennenmoser, J., Wissing, J., Ullrich, A. & Cotten, M. (2002). Identification of SRPK1 and SRPK2 as the major cellular kinases phosphorylating hepatitis B virus core protein. J Virol 76, 81248137.
DuHadaway, J. B., Sakamuro, D., Ewert, D. L. & Prendergast, G. C. (2001). Bin1 mediates apoptosis by c-Myc in transformed primary cells. Cancer Res 61, 31513156.
Egger, D., Wolk, B., Gosert, R., Bianchi, L., Blum, H. E., Moradpour, D. & Bienz, K. (2002). Expression of hepatitis C virus proteins induces distinct membrane alterations including a candidate viral replication complex. J Virol 76, 59745984.
Enomoto, N., Sakuma, I., Asahina, Y., Kurosaki, M., Murakami, T., Yamamoto, C., Izumi, N., Marumo, F. & Sato, C. (1995). Comparison of full-length sequences of interferon-sensitive and resistant hepatitis C virus 1b. Sensitivity to interferon is conferred by amino acid substitutions in the NS5A region. J Clin Invest 96, 224230.[Medline]
Enomoto, N., Sakuma, I., Asahina Y. & 7 other authors (1996). Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection. N Engl J Med 334, 7781.
Gale, M. J., Jr, Korth, M. J., Tang, N. M., Tan, S. L., Hopkins, D. A., Dever, T. E., Polyak, S. J., Gretch, D. R. & Katze, M. G. (1997). Evidence that hepatitis C virus resistance to interferon is mediated through repression of the PKR protein kinase by the nonstructural 5A protein. Virology 230, 217227.[CrossRef][Medline]
Gale, M., Jr, Blakely, C. M., Kwieciszewski, B. & 7 other authors (1998). Control of PKR protein kinase by hepatitis C virus nonstructural 5A protein: molecular mechanisms of kinase regulation. Mol Cell Biol 18, 52085218.
Ghosh, A. K., Majumder, M., Steele, R., Yaciuk, P., Chrivia, J., Ray, R. & Ray, R. B. (2000). Hepatitis C virus NS5A protein modulates transcription through a novel cellular transcription factor SRCAP. J Biol Chem 275, 71847188.
Gong, G., Waris, G., Tanveer, R. & Siddiqui, A. (2001). Human hepatitis C virus NS5A protein alters intracellular calcium levels, induces oxidative stress, and activates STAT-3 and NF-kappa B. Proc Natl Acad Sci U S A 98, 95999604.
Hijikata, M., Mizushima, H., Tanji, Y., Komoda, Y., Hirowatari, Y., Akagi, T., Kato, N., Kimura, K. & Shimotohno, K. (1993). Proteolytic processing and membrane association of putative nonstructural proteins of hepatitis C virus. Proc Natl Acad Sci U S A 90, 1077310777.[Abstract]
Kato, N., Lan, K. H., Ono-Nita, S. K., Shiratori, Y. & Omata, M. (1997). Hepatitis C virus nonstructural region 5A protein is a potent transcriptional activator. J Virol 71, 88568859.[Abstract]
Katze, M. G., Kwieciszewski, B., Goodlett, D. R., Blakely, C. M., Neddermann, P., Tan, S.-L. & Aebersold, R. (2000). Ser2194 is a highly conserved major phosphorylation site of the hepatitis C virus nonstructural protein NS5A. Virology 278, 501513.[CrossRef][Medline]
Krieger, N., Lohmann, V. & Bartenschlager, R. (2001). Enhancement of hepatitis C virus RNA replication by cell culture-adaptive mutations. J Virol 75, 46144624.
Lee, E., Marcucci, M., Daniell, L., Pypaert, M., Weisz, O. A., Ochoa, G.-C., Farsad, K., Wenk, M. R. & De Camilli, P. (2002). Amphiphysin 2 (Bin1) and T-tubule biogenesis in muscle. Science 297, 11931196.
Lohmann, V., Korner, F., Koch, J., Herian, U., Theilmann, L. & Bartenschlager, R. (1999). Replication of subgenomic hepatitis C virus RNAs in a hepatoma cell line. Science 285, 110113.
Lohmann, V., Korner, F., Dobierzewska, A. & Bartenschlager, R. (2001). Mutations in hepatitis C virus RNAs conferring cell culture adaptation. J Virol 75, 14371449.
Majumder, M., Ghosh, A. K., Steele, R., Ray, R. & Ray, R. B. (2001). Hepatitis C virus NS5A physically associates with p53 and regulates p21/waf1 gene expression in a p53-dependent manner. J Virol 75, 14011407.
Park, K. J., Choi, S. H., Lee, S. Y., Hwang, S. B. & Lai, M. M. (2002). Nonstructural 5A protein of hepatitis C virus modulates tumor necrosis factor alpha-stimulated nuclear factor kappa B activation. J Biol Chem 277, 1312213128.
Polyak, S. J., Khabar, K. S., Paschal, D. M., Ezelle, H. J., Duverlie, G., Barber, G. N., Levy, D. E., Mukaida, N. & Gretch, D. R. (2001). Hepatitis C virus nonstructural 5A protein induces interleukin-8, leading to partial inhibition of the interferon-induced antiviral response. J Virol 75, 60956106.
Sakamuro, D., Elliott, K. J., Wechsler-Reya, R. & Prendergast, G. C. (1996). BIN1 is a novel MYC-interacting protein with features of a tumor suppressor. Nat Genet 14, 6976.[Medline]
Shirota, Y., Luo, H., Qin, W., Kaneko, S., Yamashita, T., Kobayashi, K. & Murakami, S. (2002). Hepatitis C virus (HCV) NS5A binds RNA-dependent RNA polymerase (RdRP) NS5B and modulates RNA-dependent RNA polymerase activity. J Biol Chem 277, 1114911155.
Takei, K., Slepnev, V. I., Haucke, V. & De Camilli, P. (1999). Functional partnership between amphiphysin and dynamin in clathrin-mediated endocytosis. Nat Cell Biol 1, 3339.[CrossRef][Medline]
Tan, S. L., Nakao, H., He, Y., Vijaysri, S., Neddermann, P., Jacobs, B. L., Mayer, B. J. & Katze, M. G. (1999). NS5A, a nonstructural protein of hepatitis C virus, binds growth factor receptor-bound protein 2 adaptor protein in a Src homology 3 domain/ligand-dependent manner and perturbs mitogenic signaling. Proc Natl Acad Sci U S A 96, 55335538.
Tanimoto, A., Ide, Y., Arima, N., Sasaguri, Y. & Padmanabhan, R. (1997). The amino terminal deletion mutants of hepatitis C virus nonstructural protein NS5A function as transcriptional activators in yeast. Biochem Biophys Res Commun 236, 360364.[CrossRef][Medline]
Tsutsui, K., Maeda, Y., Tsutsui, K., Seki, S. & Tokunaga, A. (1997). cDNA cloning of a novel amphiphysin isoform and tissue-specific expression of its multiple splice variants. Biochem Biophys Res Commun 236, 178183.[CrossRef][Medline]
Tu, H., Gao, L., Shi, S. T. & 7 other authors (1999). Hepatitis C virus RNA polymerase and NS5A complex with a SNARE-like protein. Virology 263, 3041.[CrossRef][Medline]
Wechsler-Reya, R., Sakamuro, D., Zhang, J., Duhadaway, J. & Prendergast, G. C. (1997). Structural analysis of the human BIN1 gene. J Biol Chem 272, 3145331458.
Wigge, P., Köhler, K., Vallis, Y., Doyle, C. A., Owen, D., Hunt, S. P. & McMahon, H. T. (1997). Amphiphysin heterodimers: potential role in clathrin-mediated endocytosis. Mol Biol Cell 8, 20032015.
Received 29 August 2002;
accepted 19 November 2002.