School of Biochemistry and Microbiology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
Correspondence
Mark Harris
m.harris{at}leeds.ac.uk
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ABSTRACT |
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Present address: MRC Protein Phosphorylation Unit, School of Life Sciences, University of Dundee, Dundee DD1 5EH, UK.
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INTRODUCTION |
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The NS5A protein is one of six non-structural proteins that are believed to form a membrane-bound RNA-replication complex (Macdonald & Harris, 2004). Further to this role, NS5A has been shown to modulate a range of cellular signalling pathways, including the mitogen-activated protein kinase (MAPK) pathways. In particular, we and others have shown that NS5A expression is able to inhibit the RasERK MAPK pathway (Tan et al., 1999
; Georgopoulou et al., 2003
; Macdonald et al., 2003
). This pathway is activated by growth factors binding to their cognate receptors; this induces autophosphorylation of the receptors on tyrosine residues, which then act as ligands for the Src homology 2 (SH2) domains of adaptor proteins, such as ShcA and Grb2. Grb2 associates with a guanine nucleotide-exchange factor, Sos, which induces GDP/GTP exchange on the GTP-binding protein Ras (Tari & Lopez-Berestein, 2001
). Ras then binds to and activates a serine kinase, Raf, which triggers a kinase cascade ultimately leading to c-Fos expression and concomitant activation of the AP-1 transcription factor (of which c-Fos is a component). We previously demonstrated that transfection of a dominant-active mutant of Ras was able to override the NS5A-mediated block to this pathway and we therefore concluded that NS5A was acting between the epidermal growth factor receptor (EGFR) and the activation of Ras (Macdonald et al., 2003
). The mechanism of this inhibition is unknown, although it has been shown that NS5A is able to bind Grb2 (Tan et al., 1999
; Macdonald et al., 2004
), By doing so, it is possible that NS5A might inhibit the formation of the Grb2Sos complex, thereby breaking the link between the activated EGFR and Ras. However, it has previously been stated that, in HeLa cells expressing NS5A, there was no effect on the levels of the Grb2Sos complex (Tan et al., 1999
), although these data have not been presented. We therefore sought to investigate further the mechanisms by which NS5A is able to disable EGF-mediated RasERK signalling by analysing EGFR-proximal signalling events in Huh-7 cells harbouring an HCV subgenomic replicon (expressing the non-structural proteins NS3NS5B) at very early time points following EGF stimulation. By using a combination of immunoprecipitation and immunoblotting, together with an assay for Ras activation, we have demonstrated that multiple EGFR-proximal events are perturbed in replicon cells, compared with naïve Huh-7 cells.
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METHODS |
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Immunoprecipitation and Western blot analysis.
To analyse the EGFR complex, cells were lysed in EGFR lysis buffer [50 mM Tris/HCl (pH 8·0), 50 mM NaCl, 1 % NP-40, 1 mM Na3VO4, 100 mM NaF and protease inhibitors (Boehringer Mannheim)] at 4 °C for 30 min. Lysates (500 µg total protein) were precipitated with antibodies directed against EGFR (Upstate), ShcA (Upstate), Grb2 (Santa Cruz) or Sos (Santa Cruz) for 2 h at 4 °C, followed by incubation with either protein G- or protein A-conjugated agarose beads (Sigma) for a further 2 h at 4 °C under rotation. Immune complexes were washed in lysis buffer and bound proteins were analysed by SDS-PAGE and Western blot. PVDF membranes were probed by using an in-house polyclonal antiserum to NS5A or antibodies to EGFR (Upstate), Grb2 (Santa Cruz), Sos (Santa Cruz), phosphotyrosine (4G10; Upstate) or ShcA (Upstate). Cell lysates (50 µg total protein) were also probed with antibodies for the phosphorylated forms of ERK, ShcA (Tyr-317), pan-ERK and Ras (all from Cell Signaling Technology) following the manufacturer's recommendations. Western blots were visualized by using the ECL system (Amersham Biosciences).
Protein expression and purification.
The bacterial-expression vector pGST-RBD encoding aa 51131 of Raf-1 fused to glutathione S-transferase (GST) (de Rooij & Bos, 1997) was obtained from Dr Walter Kolch (Beatson Institute for Cancer Research, Glasgow, UK). The GSTRBD fusion protein was purified by using standard techniques (Smith & Johnson, 1988
; Macdonald et al., 2004
).
Affinity precipitation of RasGTP.
Cells were seeded into 90 mm dishes and allowed to reach 7080 % confluence, then serum-starved overnight and stimulated with 100 ng EGF ml1. After treatment, cells were washed in ice-cold PBS, and 200 µl magnesium-containing lysis buffer [MLB: 25 mM HEPES (pH 7·5), 150 mM NaCl, 1 % (v/v) NP-40, 0·25 % sodium deoxycholate, 10 % (v/v) glycerol, 10 mM MgCl2, 1 mM EDTA and protease inhibitors (Boehringer Mannheim)] was added. Samples were lysed on ice for 20 min and centrifuged (13 000 g) for 10 min to pellet the debris. The protein concentration of each sample was determined by using the BCA assay (Pierce). Active Ras was precipitated from the cleared lysate (200 µg) by adding 20 µl glutathioneagarose beads pre-bound with GSTRBD and the samples were incubated under rotation at 4 °C for 30 min. The beads were washed three times in 500 µl MLB and resuspended in 30 µl 4x SDS loading buffer prior to analysis by SDS-PAGE. Levels of RasGTP were detected by Western blot analysis with a monoclonal anti-Ras antibody (Upstate) and compared with a blot of total cellular Ras, taken from the lysate.
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RESULTS |
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EGF signalling promotes an association of NS5A with the Grb2Sos complex
A C-terminal PxxP motif in NS5A has been shown to interact with the SH3 domains of Grb2; however, these domains also interact with PxxP motifs within Sos. Although the Grb2Sos interaction has been reported to be constitutive and unaffected by EGF signalling (Okada & Pessin, 1996), it is conceivable that the interaction between NS5A and Grb2 would disrupt the Grb2Sos interaction and thereby block RasERK signalling. To test this, we immunoprecipitated either Grb2 or Sos from EGF-stimulated parental Huh-7 or replicon cells and analysed the immunoprecipitates by Western blotting. Fig. 3[a(i)]
shows that, prior to EGF treatment, no NS5A was associated with Grb2; however, at 2 min post-treatment, there was a dramatic increase in the level of NS5A associated with Grb2. This interaction was sustained until 20 min after treatment, with only a slight decline. Likewise, when lysates were immunoprecipitated with an anti-Sos antibody, NS5A could be detected in the precipitates only after 2 min EGF treatment [Fig. 3b(i)
]. In both parental and replicon cells, the level of Grb2 associated with Sos showed a marginal decline over the 20 min time course [Fig. 3b(ii)
]. Although the decline appeared to be more pronounced in replicon cells, this was unlikely to be due to displacement by NS5A, as the interaction of NS5A with Grb2Sos preceded the decline in Sos-associated Grb2 by several minutes. Judging by the intensity of the NS5A signal in the Grb2 and Sos immunoprecipitates, it seemed likely that NS5A was binding directly to Grb2, rather than to Sos [compare Fig. 3a(i) and b(i)
].
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DISCUSSION |
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Our results are consistent with the hypothesis that NS5A, by interacting with the Grb2Sos complex, is able to perturb Sos-mediated Ras activation without disrupting the Grb2Sos interaction. The EGF-dependent association of NS5A with the Grb2Sos complex is highly reminiscent of the Sprouty (Spry1/2) protein family (Hanafusa et al., 2002). Spry1/2 has been shown to bind to Grb2 in a growth factor-dependent fashion, preventing recruitment of Grb2 to the receptor, but not disrupting the Grb2Sos complex. The mechanism underpinning this has been shown to involve growth factor-stimulated translocation of Spry to the membrane and subsequent tyrosine phosphorylation of Spry, facilitating association with the SH2 domain of Grb2. In contrast, NS5A interacts with the SH3 domains of Grb2; however, it is conceivable that this interaction might block the association of the Grb2 SH2 domain with either Shc or the EGFR, possibly by steric hindrance. This might explain why no Grb2 was observed complexed to EGFR in replicon cells, in comparison with naïve cells [Fig. 2a(iii)
]. Interestingly, the activation of Ras is only partially abrogated in replicon cells although, downstream of Ras, the block in ERK phosphorylation is more pronounced. This suggests that there may be further, as yet unidentified, ways in which NS5A and/or other non-structural proteins are able to modulate the RasERK pathway. In this regard, the hepatitis E virus ORF3 protein was recently shown to activate ERK by inactivating an ERK-specific phosphatase (Kar-Roy et al., 2004
). It is intriguing to speculate that, as in the case of the EGFR phosphatases discussed above, HCV might activate an ERK-specific phosphatase.
The physiological significance of ERK inhibition for virus replication remains to be determined. However, it is pertinent to note that ERK signalling is involved in many processes, such as control of the cell cycle: growth-factor signalling through ERK is required for hepatocytes to transit a block in late G1 (Talarmin et al., 1999) and many viruses, such as murine coronavirus (Chen et al., 2004
), human parvovirus B19 (Morita et al., 2003
) and Kaposi's sarcoma-associated herpesvirus (Izumiya et al., 2003
), have been shown to effect a G1 block to favour virus replication. Interestingly, a recent study observed elevated levels of G1-phase hepatocytes in biopsy material taken from patients with HCV compared with alcoholic liver disease, providing indirect evidence for a G1 block in HCV infection (Marshall et al., 2005
). We propose, therefore, that the inhibition of RasERK signalling by NS5A may play a role in cell-cycle perturbation during HCV infection. We have also shown that NS5A, either alone or in the context of the subgenomic replicon, can activate phosphoinositide 3-kinase, resulting in increased resistance to the induction of apoptosis (Street et al., 2004
). Given that a G1 block can result in the induction of apoptosis (Chang et al., 2004
), the differential effects of NS5A on these two signalling cascades may represent a coordinated strategy to modulate the cellular environment to favour virus replication and persistence. This is an attractive hypothesis that is currently under investigation in our laboratory. It also suggests that the interactions between NS5A and cellular SH3 domain proteins, such as Grb2, are valid targets for antiviral-drug development.
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ACKNOWLEDGEMENTS |
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Received 26 October 2004;
accepted 7 January 2005.
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