1 School of Biochemistry and Microbiology and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK
2 Institute of Medical Technology, Tampere University Hospital, FIN-33014 Tampere, Finland
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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One key group of SH3 domain-containing proteins is the Src family of tyrosine kinases (Tatosyan & Mizenina, 2000). These signalling proteins are common targets for viral interference, particularly in the case of viruses that establish a chronic infection. In this regard, one of the best-studied SH3 binding viral proteins is the human immunodeficiency virus type 1 (HIV-1) Nef protein, which has been shown to bind the SH3 domains of the Src family kinases Hck, Lyn, Fyn and Src (Saksela et al., 1995
). Indeed, the interaction between Nef and the HckSH3 domain, as determined by surface plasmon resonance (SPR), is amongst the strongest SH3PxxP interactions reported to date (Lee et al., 1995
). Our group and others have reported that NS5A interacts with a variety of SH3 domain-containing proteins, including Grb2, amphiphysin II, PI3K p85 subunit and members of the Src family (Macdonald et al., 2004
; Street et al., 2004
; Tan et al., 1999
; Zech et al., 2003
). Interestingly, as is the case for Nef, NS5A shows remarkable selective characteristics: although able to bind a subset of Src kinase SH3 domains, namely Hck, Lck, Lyn and Fyn, it does not bind that of Src itself; nor does it bind to a range of other SH3 domains, including those of the Vav guanine nucleotide-exchange factor, the adaptor proteins Crk and Nck and the Abl tyrosine kinase (Macdonald et al., 2004
; Tan et al., 1999
). As with other viruses, these interactions with SH3 domain-containing proteins are likely to aid in either virus replication or immune evasion, but, at this stage, their exact functions remain obscure. Molecular characterization of the interaction between NS5A and SH3 domains could therefore not only elucidate the structural basis of SH3 binding specificity, but may also be helpful for development of novel therapeutic strategies aimed at inhibition of NS5A function.
In this study, we have used a semi-quantitative assay to examine the differences in binding of various SH3 domains to NS5A and show that NS5A exhibits a hierarchy of binding affinities to SH3 domains. By using a molecular-modelling approach, we predicted the interaction surface between the PP2.2 motif of NS5A and the FynSH3 domain. Coupled with experimental data, this analysis revealed that residues in the variable region of the RT loop of the SH3 domain do not contact the PP2.2 motif, but play a role in determining the specificity of NS5A-mediated SH3 domain interactions, consistent with additional intermolecular contacts between NS5A and SH3 domains. We also demonstrate a requirement for salt-bridge formation for binding of NS5A to SH3 domains. Finally, we utilize a subgenomic replicon to analyse the potential role of NS5ASH3 domain interactions in HCV RNA replication and effects on signalling within replicon-harbouring cells.
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METHODS |
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Cell-culture procedures.
Cos-7 cells were propagated in Dulbecco's modified Eagle's medium supplemented with 10 % fetal calf serum, 2 mM L-glutamine, 100 IU penicillin ml1 and 100 µg streptomycin ml1 at 37 °C in a humidified 5 % CO2 incubator. Huh-7 cells were cultured in minimal essential medium supplemented as for Cos-7 cells, with the addition of 1 % non-essential amino acids. For transient transfection of NS5A-expression vectors, 1x106 cells were seeded in 90 mm dishes and incubated for 24 h at 37 °C, prior to transfection with pSG5 constructs expressing NS5A and mutants thereof by using Lipofectin (Invitrogen). Cells were incubated for 6 h, after which they were cultured in growth media for a further 24 h prior to lysis in Glasgow lysis buffer (GLB; Bentham et al., 2003) supplemented with protease inhibitors (Boehringer Mannheim). For the generation of Huh-7 cells harbouring subgenomic replicons, RNA transcripts were generated from pFK5.1, pFK5.1neo(PA2.2) and pBACrepGNDneoT7/NotI, and transfected as described previously (McCormick et al., 2004
).
Expression and purification of recombinant proteins.
Generation and use of the pGEX vectors for bacterial expression of the glutathione S-transferase (GST)SH3 domain fusion proteins was as described previously (Hiipakka et al., 1999; Macdonald et al., 2004
). GSTGrb2 was obtained from John Ladbury (University College London, UK). N-terminally His-tagged NS5A (genotype 1b) was expressed by a recombinant baculovirus in Sf9 cells and purified by Ni2+-NTA chromatography. The concentration and integrity of purified proteins were determined by Bradford assay (Bio-Rad), SDS-PAGE and Coomassie staining.
In vitro binding assays.
Binding assays were performed as described previously (Macdonald et al., 2004). Briefly, GSTSH3 domains were bound to glutathioneagarose beads overnight at 4 °C. Equal quantities of lysates from cells transiently transfected with the appropriate pSG5 vectors were added to the beads. After 3 h incubation, beads were washed extensively in lysis buffer and bound protein was analysed by SDS-PAGE and immunoblotting with a sheep polyclonal anti-NS5A antiserum. GST alone was used as a negative control.
ELISA protein-interaction assay.
Purified GSTSH3 domains (1 µg per well) were coated onto 96-well plates (PS Microplate; Greiner Bio-One) by incubation in 50 µl PBS/0·1 % Tween (PBT) per well overnight at 4 °C. Plates were washed in PBT and blocked in PBT containing 5 % non-fat dried milk for 2 h at room temperature. Plates were washed in PBT and purified HisNS5A or lysate from Cos-7 cells expressing NS5A and mutants thereof, diluted in GLB supplemented with protease inhibitors (50 µl per well), was added for 2 h at 4 °C. After three PBT washes, NS5A was detected by using a sheep polyclonal antiserum for 1 h followed by horseradish peroxidase-conjugated anti-sheep antibody (Sigma) for 1 h. Bound antibody was visualized by using o-phenylenediamine (Dako) and quantified at 490 nm with a reference filter at 630 nm, using an MRX microplate reader (Dynex).
Luciferase reporter assays.
Cells were transfected with plasmids expressing the appropriate luciferase reporter (0·5 µg) by using Lipofectin (Invitrogen). A Renilla luciferase reporter construct (pRLTK) was used as an internal control for transfection efficiency. Total DNA was kept constant by adjusting the amount of vector DNA. Cells were grown in reduced serum-containing media (0·5 %) for 24 h and lysed in passive lysis buffer (Promega) prior to analysis. Assays were performed in triplicate and analysed with dual luciferase reagent (Promega) and a luminometer as described previously (Macdonald et al., 2003).
Molecular modelling of the NS5ASH3 interaction.
Modelling of the NS5A PP2.2FynSH3 domain interaction was carried out by taking the peptide 71Thr-Pro-Gln-Val-Pro-Leu-Arg77, corresponding to the PxxP motif from HIV-1 Nef in complex with FynSH3 (1AVZ; Arold et al., 1997), and mutating it to the equivalent motif of NS5A. Individual mutations were created by using MutantDock, an online computational mutagenesis server (S. J. Campbell & R. M. Jackson, unpublished data), which uses SCWRL to predict the conformation of mutant-residue side-chain positions (Bower et al., 1997
) before refining the proteinpeptide interaction with MultiDock (Jackson et al., 1998
). The mutations required to convert the PP2.2 peptide were done sequentially in all possible orders; this did not, however, significantly change either the final predicted conformation or interaction energies. Results are shown for the model with the most favourable predicted interaction energy. The donor scaffold forms a polyproline helix, therefore mutating the peptide to/from proline did not affect the backbone conformation of the modelled peptide.
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RESULTS |
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We have previously shown that NS5A, by blocking RasERK pathway signalling, was able to inhibit both the basal and epidermal growth factor (EGF)-stimulated activity of the transcription factor AP-1 (Macdonald et al., 2003). We further showed that this was dependent on the PP2.2 motif: wild-type NS5A inhibited the expression of luciferase from an AP-1-responsive reporter construct by
50 %, whereas the PA2.2 mutant had no effect. These data implied that NS5ASH3 domain interactions were required for the inhibition of AP-1 activation and we predicted that abrogation of salt-bridge formation would therefore also block this function of NS5A. To test this, we used the luciferase reporter assay to screen all four mutants for the ability to inhibit AP-1-responsive transcription. Consistent with previous data, expression of NS5A(wt) resulted in a 40 % reduction in AP-1-responsive luciferase levels within transfected Cos-7 cells (Fig. 4c
); however, as expected, all of the mutations abolished this effect. These data confirm the validity of the molecular modelling and support the hypothesis that salt-bridge formation is critical for binding of NS5A to SH3 domains.
Mutations within the PP2.2 motif of a subgenomic replicon abolish NS5A signalling functions, but replicate to wild-type levels
To examine the role of NS5ASH3 domain interactions in HCV RNA replication, the PA2.2 mutation was introduced into the FK5.1 culture-adapted HCV subgenomic replicon (Krieger et al., 2001), generating FK5.1(PA2.2). Initially, the ability of FK5.1(PA2.2) to establish itself in cell culture was assessed by transfection into Huh-7 cells, in comparison with both the original 5.1 replicon and a polymerase knock-out (GND) control replicon transcript. Whilst no G418-resistant colonies were seen in cells transfected with the GND replicon, transfection of transcripts derived from the FK5.1(PA2.2) clone led to the formation of G418-resistant colonies, demonstrating that this latter replicon is still viable (Fig. 5a
). Furthermore, the number of colonies formed after transfection with FK5.1(PA2.2) was similar to that seen after transfection of Huh-7 cells with the original FK5.1 replicon (Fig. 5b
), demonstrating that the PP2.2 PxxP motif is dispensable for replicon establishment. We confirmed that the PA2.2 mutation had not reverted during G418 selection by extracting RNA from the FK5.1(PA2.2) replicon cells, amplifying the NS5A coding region by RT-PCR and sequencing five independent clones of the PCR products. No mutations were observed (data not shown); however, we cannot at this stage exclude the possibility of second-site compensatory mutations elsewhere in the replicon.
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DISCUSSION |
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By using a semi-quantitative ELISA binding-assay format (Bentham et al., 2003), we compared the binding abilities of various SH3 domains for NS5A. Our data highlight significant differences between the various SH3 domains, with a clear hierarchy of binding affinities over a 10-fold range, from LynSH3 down to LckSH3. The ELISA assay was also used to confirm the previous observation that mutation of the N-terminal class I polyproline motif (PP1) had no effect on binding of NS5A to SH3 domains (Macdonald et al., 2004
; Street et al., 2004
; Tan et al., 1999
). This is consistent with the observation that the PP1 motif resides within the N-terminal amphipathic helix of NS5A, which can act as an endoplasmic reticulum membrane anchor (Brass et al., 2002
) and, as such, is unlikely to be available for binding to cytoplasmic proteins. Our data also highlight the differential effects of mutating the PP2.2 motif within NS5A. Previous data have shown that mutating this motif abrogated the interactions between NS5A and the SH3 domains of Hck, Lck, Fyn and Grb2 (Macdonald et al., 2004
; Tan et al., 1999
), but had no effect on binding to LynSH3. The quantitative nature of the ELISA assay, compared with the GST-binding assay, demonstrated that this mutation reduced binding to LynSH3 twofold (Fig. 2c
), whereas the double (PA2.1/2.2) mutant abrogated binding to LynSH3 completely. This is consistent with our previous data demonstrating the ability of LynSH3 to bind either the PP2.1 or PP2.2 motifs (Macdonald et al., 2004
).
Molecular modelling of the PP2.2FynSH3 domain interaction suggested that the central variable region of the RT loop did not interact directly with the polyproline helix. This was consistent with data from other systems (e.g. HIV-1 Nef), showing that this region of the RT loop plays a key role in determining the specificity of binding by interacting with other parts of the ligand thus, a 12-mer peptide spanning the Nef PxxP motif bound equally (and rather poorly) to Fyn and HckSH3, whereas full-length Nef bound tightly to HckSH3, but not to FynSH3 (Saksela et al., 1995). It was shown previously that replacing the Hck RT loop (EAIHHE) with that of Fyn (EARTED) reduced binding to Nef considerably (Lee et al., 1995
). The opposite was true for NS5A we saw an increase in binding of NS5A to HckSH3 when the RT loop was replaced with that of Fyn, and a corresponding decrease in binding to FynSH3 with an Hck-like RT-loop sequence. These data imply that, as well as the PP2.2 motif, other sequences within NS5A contribute to the interaction. The identity of these sequences remains to be elucidated; however, we predict that these sequences will lie N-terminal to PP2.2, as our data show that a C-terminal deletion from residue 363 was able to bind SH3 domains as well as full-length NS5A (data not shown). Furthermore, we have shown that the N-terminal 270 residues of NS5A are dispensable for SH3 domain binding. A recent study provided evidence for a three-domain structure of NS5A (Tellinghuisen et al., 2004
), with the second domain consisting of residues 250342 and the polyproline region corresponding to a flexible linker between domains 2 and 3. We are thus focusing our attention on domain 2 to identify additional intermolecular contacts between NS5A and SH3 domains.
Molecular modelling also predicted a key role for Arg356 in the stabilization of the PP2.2SH3 interaction by the formation of a salt bridge with an acidic residue (Asp100 in FynSH3). This prediction was confirmed by experimental analysis. Interestingly, the binding of NS5A to LynSH3 was only abolished when the mutation Arg356Ala was introduced in the context of PA2.2. The data presented here suggest that, although the proline residues within PP2.2 are dispensable for binding to LynSH3 (as LynSH3 can bind PP2.1), Arg356 is not. It is not clear why binding of LynSH3 to the PP2.1 motif is dependent on Arg356; however, this may be the result of structural constraints. In this regard, secondary-structure predictions suggest that the two class II motifs are highly hydrophilic and are likely to be surface-exposed; furthermore, mutation of the arginines to alanines is predicted to reduce both the hydrophilicity and surface probability of these motifs (data not shown). Further mutagenesis, combined with sensitive quantitative methods such as SPR, will be required to fully characterize the interactions between NS5A and SH3 domains; such work is currently under way in our laboratory.
The highly conserved nature of the PP2.2 motif suggested that it was likely to play an important role at some stage during the virus replication cycle. The absence of a robust in vitro system that recapitulates the complete infectious cycle of HCV makes this a difficult question to address; however, as a first stage in this process, we used the replicon system to ask whether the PP2.2 motif plays a role in viral genomic RNA replication. Our data show that this motif is dispensable for replicon function: in colony-forming assays, the FK5.1(PA2.2) replicon was quantitatively and kinetically indistinguishable from the parental FK5.1 replicon. Although we demonstrated that the PA2.2 mutation had not reverted during selection, we cannot rule out the possibility of second-site compensatory mutations within the NS35B coding sequence that would allow the PA2.2 mutant to replicate. This seems unlikely, however, particularly in light of the observation (Zech et al., 2003) that transient replication of a luciferase reporter-based PA2.2 mutant replicon was only reduced twofold. A detailed analysis of viral RNA replication and protein expression in FK5.1(PA2.2) replicon cells is required to clarify this situation such studies are under way. It is also possible that the PP2.2 motif might be dispensable for viral RNA replication in Huh-7 cells, a transformed tumour-cell line, but could play a role in the natural site of virus replication (primary human hepatocytes).
In this study, we show that activation of the transcription factor AP-1 is reduced in Huh-7 cells harbouring the FK5.1 replicon, but not the FK5.1(PA2.2) mutant. Given that we have previously shown that ERK activation is reduced in replicon cells (Macdonald et al., 2003), the reduction in AP-1 activation is most likely to be due to the NS5A-mediated inhibition of the Ras-ERK pathway (Georgopoulou et al., 2003
; Macdonald et al., 2003
). Interestingly, interleukin 1 (IL1) has been shown to reduce replicon replication approximately fivefold via the activation of ERK (Zhu & Liu, 2003
); we therefore predict that, by reducing ERK activation, NS5A is able to effect a corresponding resistance to IL1 treatment and, furthermore, that the PA2.2 mutant replicon would exhibit an increased sensitivity to IL1 treatment. Again, these ideas remain to be tested.
In conclusion, we have shown that NS5ASH3 interactions are not absolutely required for viral RNA replication in Huh-7 cells, but do modulate host-cell signalling pathways; it is likely, therefore, that these interactions may play more subtle roles in either the replication or pathogenesis of HCV infection.
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ACKNOWLEDGEMENTS |
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Received 28 October 2004;
accepted 13 December 2004.