Because of the lack of an HCV infection system, we utilized four different surrogate systems to gain insights into the interaction between NS5A and cell signaling pathways: stable or transiently transfected Tet-Off cell lines expressing HCV NS5A, a recombinant, E3L-deficient VV expressing NS5A, and the recently developed HCV replicon cells expressing HCV NS proteins (2
). The Tet-Off cell line system allows the characterization of NS5A function in a tightly controlled, inducible manner. The recombinant VV system provides the context of viral infection, at least activation of the dsRNA-dependent antiviral pathway (25
), which is likely important for studying the function of a viral protein. Finally, the HCV replicon system, consisting of human liver cells replicating an HCV subgenomic RNA construct and expressing several HCV NS proteins (NS3, NS4A, NS4B, NS5A, and NS5B), is considered the “gold standard” of HCV model systems and enables the examination of NS5A behavior in the presence of viral genome replication and other HCV proteins.
A mutant form of NS5A, NS5A-Pro3, is unable to interact with Grb2 and inhibit EGF-induced ERK MAPK activation.
Grb2 is an adaptor protein that mediates signaling by nucleating the formation of signal transduction complexes in response to growth factor stimulation (14
). We previously showed that NS5A binds Grb2 in an Src homology 3 (SH3) domain/ligand-dependent manner and inhibits ERK1/2 MAPK activation by EGF (53
). As an extension of these observations, we determined whether mutations within the C-terminal proline-rich SH3 domain-binding motif affects the ability of NS5A to inhibit EGF-dependent ERK1/2 activation. Consistent with previous findings (53
), transient expression of NS5A in Tet-Off HeLa cells and in vivo coimmunoprecipitation analysis revealed that NS5A and Grb2 interaction was dependent on EGF stimulation of the cells (Fig. ). NS5A-Pro3, which carries three proline→alanine point mutations within the proline-rich motif, did not interact with Grb2, indicating that the proline-rich motif of NS5A is essential for its interaction with Grb2 (Fig. ). This finding was further supported by GST pulldown experiments with a GST-Grb2 fusion protein and lysates from HeLa cells transfected with either wild-type NS5A or NS5A-Pro3 constructs (data not shown). Importantly, the ability of NS5A-Pro3 to inhibit EGF-dependent ERK activation was severely abrogated while wild-type NS5A inhibited Erk1/2 phosphorylation in a transient manner, as shown in our previous studies (25
) (Fig. ). These results support the notion that NS5A binds Grb2 via an SH3 domain/ligand-dependent manner and demonstrate the requirement and specificity of the interaction for mediating downstream effects on cellular signaling. NS5A-Pro3 also provides an effective approach by which to test whether any effect of NS5A is dependent on Grb2 interaction.
In order to examine the physiological relevance of these results, we compared the level of ERK1/2 MAPK activation in Huh7 human liver cells with its level of activation in HCV replicon cells, which are Huh7-derived human liver cells that carry an HCV subgenomic construct and express several HCV NS proteins, including NS5A (2
). In this analysis, we found that activation of the Erk1/2 MAPKs was inhibited by approximately twofold in actively proliferating HCV replicon cells, compared with that in the parental Huh7 cells (Fig. ). This finding suggests that NS5A is able to perturb the MAPK pathway in the context of other HCV NS proteins and in the presence of viral genome replication. However, these factors may also exert their own effects on cell signaling pathways and may regulate or counteract the actions of NS5A. This may partially explain the fact that inhibition of the MAPK pathway in the replicon cells was not as dramatic as that observed in Tet-Off cells expressing NS5A only. In addition, the Tet-Off and replicon cells are of different types and may express different levels of NS5A protein.
NS5A indirectly associates with the Gab1 signaling complex in a Grb2-independent manner.
Members of the insulin receptor substrate family of proteins, of which Gab1 is one, play pivotal roles in cell signaling by associating with different growth factor, cytokine, and antigen receptors and by providing docking sites for components of multiple downstream pathways (26
). Since Gab1 also associates with the EGF receptor, either via the adaptor protein Grb2 or directly, and since it contributes to activation of the MAPK pathway by providing binding sites for the SH2 domain of Grb2 (32
), we tested whether NS5A could associate with the Gab1 signaling complex. Coimmunoprecipitation experiments were performed with anti-Gab1 antibody on lysates from untreated or EGF-treated, wild-type NS5A-expressing HeLa cells. NS5A was detected in the same immunocomplex as Gab1 only after EGF treatment, indicating that this association is regulated by cell signaling events (Fig. ), similar to the association between NS5A and Grb2 (53
). An NS5A-Gab1 complex was not detectable when a parallel coimmunoprecipitation was performed with a normal-serum control (Fig. ). To characterize the function of NS5A in the setting of a virus infection, we examined the association of NS5A with the Gab1 signaling complex in HeLa S3 cells infected with recombinant VV expressing NS5A protein (Fig. ). The VV system offers an additional advantage in that VV encodes an EGF homologue that can mimic EGF-induced tyrosine protein phosphorylation (4
). Coimmunoprecipitation analyses were carried out with lysates from HeLa S3 cells infected with a recombinant VV expressing NS5A (vpNS5A) or a recombinant VV control (vp1080) (Fig. ). Infection of HeLa S3 cells with vpNS5A, but not infection with vp1080, produced an NS5A-Gab1 complex, as revealed by coprecipitation of NS5A by anti-Gab1 antibody. When a parallel experiment was performed with a normal-serum control, neither NS5A nor Gab1 was detectable. Collectively, these results suggest that NS5A associates with the Gab1 multiprotein signaling complex in the context of both viral infection and EGF-induced cellular signaling.
FIG. 2. NS5A associates with the Gab1 signaling complex. (A) Lysates from wild-type NS5A-expressing Tet-Off HeLa cells treated with EGF (lanes 2 and 3) or left untreated (lane 1) were immunoprecipitated with either anti-Gab1 antibody (lanes 1 and 2) or a normal (more ...)
To test whether NS5A associates with Gab1 directly or through a bridging molecule, such as Grb2, we used recombinant GST-Gab1 as an affinity matrix to examine whether NS5A and Gab1 could form a complex in cell lysates prepared from wild-type NS5A-expressing Tet-Off HeLa cells. Indeed, we were able to demonstrate the retention of NS5A on glutathione-agarose beads with GST-Gab1, but not with GST or buffer alone (Fig. ). Also, the interaction of NS5A with GST-Gab1 was dependent on EGF treatment, further indicating that the interaction is regulated by cellular signaling events. We next examined whether purified NS5A could bind to purified GST-Gab1 in the absence of other proteins (Fig. ). While NS5A was efficiently coprecipitated with GST-Grb2, no direct interaction between NS5A and GST-Gab1 was detected. However, when cell lysates prepared from actively proliferating HeLa cells were added to the reaction mixture, NS5A was coprecipitated with GST-Gab1. These results suggest that the association between Gab1 and NS5A is mediated by an additional cellular factor(s) and is thus indirect.
FIG. 3. The NS5A-Gab1 association is indirect and independent of NS5A-Grb2 interaction. (A) Tet-Off HeLa cells stably expressing wild-type NS5A (lanes 1, 2, 4, and 5) or not expressing NS5A (lane 3) were either treated with EGF (lanes 1, 3, and 5) or left untreated (more ...)
We specifically tested if Grb2 could mediate the Gab1-NS5A association by using the in vitro GST pulldown system (Fig. ). As described above, GST-Gab1 was unable to interact with purified NS5A directly whereas GST-Grb2 efficiently interacted with NS5A in the same assay. Even when recombinant Grb2, purified from the cleavage product of GST-Grb2 fusion protein, was added to the system, no GST-Gab1-NS5A association was detected. The purity and amount of the recombinant Grb2 protein were confirmed by immunoblot analysis (data not shown). This indicates that Grb2 is unable to mediate Gab1-NS5A association, at least under the conditions used in this system. In order to further address the question of whether NS5A-Gab1 association is bridged by the Grb2 adaptor molecule, we tested the ability of wild-type NS5A and NS5A-Pro3, the mutant that does not interact with Grb2, to associate with the Gab1 signaling complex. The wild-type and mutant NS5A proteins were expressed in Tet-Off HeLa cells by transient transfection. Coimmunoprecipitation experiments were performed with lysates from HeLa cells expressing either wild-type NS5A or NS5A-Pro3 with a Gab1-specific antibody. Both the wild-type and mutant NS5A proteins coimmunoprecipitated with Gab1 in an EGF treatment-dependent manner (Fig. ). The same cell lysates were also used for GST pulldown assays with GST-Gab1 or the GST control, and similar results were obtained (data not shown). Combined, these results suggest that NS5A-Gab1 association is not dependent on direct interaction of NS5A with Grb2. Rather, the association between NS5A and Gab1 is likely bridged by additional signaling molecules in the Gab1 signaling complex, such as PI3K and PLC-γ, which possess SH3 domains (Fig. ) that can potentially interact with the other proline-rich motifs of NS5A (53
FIG. 4. NS5A interacts with the p85 subunit of PI3K. (A) Organization of the functional domains of p85 PI3K and PLC-γ. (B) Recombinant NS5A protein purified from insect cells was used for GST pulldown assays with GST-Grb2 (lane 2), GST-Gab1 (lane 3), (more ...) NS5A directly interacts with the p85 subunit of PI3K, which, in turn, mediates the NS5A-Gab1 association.
Primed by the observation that the NS5A-Gab1 association is not mediated by the Grb2 adaptor molecule, we tested whether NS5A is able to interact with other signaling molecules involved in the Gab1 complex, specifically, the SH3 domain-containing p85 subunit of PI3K and PLC-γ. A GST pulldown analysis was performed with purified NS5A protein and a panel of different purified GST fusion proteins (Fig. ). GST-Grb2 did, and GST-Gab1 did not, precipitate NS5A as previously shown. Significantly, the full-length GST-p85 protein, but not the two different SH2 domain fragments of p85 (p85 C-SH2 and p85 N-SH2) (Fig. ), interacted with NS5A. In addition, GST-PLC-γ, another candidate NS5A-interacting protein, did not interact with NS5A in the same experiments. These results demonstrated that NS5A is able to directly and specifically interact with the p85 subunit of PI3K. This interaction did not seem to be mediated by the SH2 domains of p85 and thus likely involved other regions of p85, especially the SH3 domain.
In order to test the in vivo interaction of NS5A with p85 PI3K, lysates from untreated or EGF-treated wild-type NS5A-expressing Tet-Off HeLa cells were used for coimmunoprecipitation assays with an antibody specific for the p85 subunit of PI3K, followed by immunoblot assays to detect the presence of NS5A and p85 in the immunocomplex. NS5A associated with p85 PI3K in EGF-treated cells but not in untreated cells (Fig. ). The association of NS5A with p85 PI3K was also demonstrated in the recombinant VV infection system by coimmunoprecipitation assays. NS5A was found to associate with p85 PI3K in vpNS5A-infected HeLa cells but not in cells infected with the control virus (vp1080) (Fig. ). These results showed that NS5A could interact with the p85 subunit of PI3K in vivo during both EGF-induced cell signaling and viral infection, alluding to a mechanism by which NS5A associates with the Gab1 signaling complex. In addition, coimmunoprecipitation analysis showed that both wild-type NS5A and NS5A-Pro3 interacted with p85 PI3K in HeLa cells treated with EGF (data not shown), suggesting that NS5A interacts with p85 PI3K in a Grb2 interaction-independent manner. Thus, NS5A interacts with p85 PI3K and Grb2 through different mechanisms.
Next, we tested the NS5A-p85 PI3K interaction in the HCV replicon system in order to better determine the physiological relevance of this observation. Lysates of untreated or EGF-treated HCV replicon cells were used for a coimmunoprecipitation analysis with antibody specific to the p85 subunit of PI3K (Fig. ). Significantly, the NS5A-p85 interaction was also demonstrated in the HCV replicon cells, in an EGF treatment-inducible manner. These results strongly support the physiological relevance of the NS5A-p85 interaction. Under the same conditions, p85 was not found to associate with NS4A, another HCV NS protein present in the replicon cells (Fig. ), further demonstrating the specificity of the NS5A-p85 PI3K interaction.
In order to determine the NS5A regions involved in the interaction with p85 PI3K, the p85 PI3K-interacting ability of wild-type NS5A and different N- or C-terminal deletion mutant forms of NS5A was examined next (Fig. ). A genotype 1b, full-length NS5A isolate, wild-type NS5A-NR, and NS5A-NR-derived deletion mutants lacking either the N-terminal 110 amino acids (ΔN110) or the C-terminal 117 amino acids (ΔC117) (39
) were separately expressed in Tet-Off HeLa cells by transient transfection. After EGF treatment, lysates of these cells expressing the different NS5A proteins were subjected to coimmunoprecipitation analysis with anti-p85 PI3K antibody as described above. Interestingly, both full-length NS5A and the C-terminal deletion mutant form of NS5A were able to interact with p85 PI3K. On the other hand, the mutant NS5A lacking the N-terminal region was unable to associate with p85. The expression of these different forms of NS5A was confirmed by immunoblot analysis, and similar expression levels were observed (data not shown). These results suggest that the NS5A-PI3K interaction involves the N-terminal, but not the C-terminal, region of NS5A. It is likely that the NS5A-PI3K interaction is mediated by the other highly conserved, proline-rich, SH3-binding motif within the N-terminal region of NS5A (53
). In addition, the association of these wild-type and mutant forms of NS5A with Gab1 was also tested by performing coimmunoprecipitation analysis with anti-Gab1 antibody as described above (Fig. ). Consistent with the above results and our hypothesis that NS5A associates with the Gab1 complex through direct NS5A-p85 PI3K interaction, both the full-length and the C-terminal deletion forms, but not the N-terminal deletion form, of NS5A associated with Gab1 in EGF-treated cells. These results strongly suggest that the NS5A-Gab1 association requires direct NS5A-p85 PI3K interaction, although the possibility that other, unknown, proteins mediate the NS5A-Gab1 association cannot be completely excluded.
FIG. 5. The NS5A-Gab1 association requires NS5A-p85 PI3K interaction. Tet-Off HeLa cells were transiently transfected with pTRE-NS5A-NR (full length) (lane 1), pTRE-NS5A-ΔN110 (lane 2), or pTRE-NS5A-ΔC117 (lane 3), and at 24 h posttransfection, (more ...) NS5A enhances activation of the PI3K-Akt pathway and regulates the downstream apoptosis pathway.
Upon stimulation with growth factors such as EGF, the multisite docking protein Gab1 is phosphorylated at multiple tyrosine residues, which could serve as docking sites for SH2 domain-containing signaling molecules, including previously identified Gab1-binding proteins p85 PI3K, PLC-γ, Shc, and Shp2 (26
). Knowing that NS5A directly interacts with p85 PI3K and associates with the Gab1 complex, we next investigated whether NS5A expression affects the activation of any Gab1-mediated signaling pathway, in particular, the PI3K pathway, which plays a pivotal role in regulating cell growth and survival (5
) and is a popular target of viral modulation of host cell signaling (9
). Lysates from the stable wild-type NS5A-expressing Tet-Off HeLa cell line were subjected to coimmunoprecipitation analysis by using anti-p85 PI3K antibody and immunoblot analysis with antiphosphotyrosine antibody. Following EGF stimulation, cells expressing NS5A displayed enhanced tyrosine phosphorylation of p85 PI3K (Fig. ) but not PLC-γ (data not shown), indicating sustained activation of PI3K by NS5A. On average, NS5A expression caused an approximately twofold increase in PI3K tyrosine phosphorylation at early time points after EGF treatment (Fig. ). No significant effect of NS5A on Gab1 tyrosine phosphorylation was observed (data not shown), consistent with the model in which NS5A interacts directly with p85 PI3K and associates with the Gab1 complex indirectly.
The AKT serine/threonine protein kinase is activated by phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, which are lipid products of PI3K, and AKT acts as an important signal mediator downstream of PI3K, regulating cell survival and proliferation (6
). We therefore tested the effect of NS5A expression on AKT pathway activation. For these studies, Tet-Off HeLa cells expressing NS5A were treated with EGF and immunoblot assays with antibody specific to the phosphorylated/activated forms of AKT kinase (Fig. ) were performed as described previously (30
). Consistent with the observation that NS5A enhances p85 PI3K phosphorylation, NS5A expression also enhanced AKT phosphorylation by about threefold in HeLa cells treated with EGF for 30 min. In addition, NS5A expression enhanced AKT phosphorylation/activation in HeLa S3 cells infected with recombinant VV expressing NS5A, compared with cells infected with the control VV (Fig. ). On average, in recombinant VV-infected cells, NS5A expression resulted in an approximately 2.5-fold increase in the AKT phosphorylation level. More importantly, NS5A expression did not cause a significant change in the level of p85 PI3K or AKT protein. Collectively, these results show that NS5A expression can enhance the PI3K-AKT pathway in both cellular and viral systems.
In order to test whether the effect of NS5A on the PI3K-AKT pathway requires NS5A-p85 PI3K interaction, AKT phosphorylation levels were compared in two stable Tet-Off HeLa cell lines expressing either full-length, wild-type NS5A-NR or an N-terminal deletion mutant form of NS5A (ΔN222) that does not interact with p85 PI3K. We found that expression of full-length NS5A-NR, which is capable of interacting with p85 PI3K, also enhanced AKT phosphorylation, showing that the effect of NS5A on the PI3K-AKT pathway is not limited to a particular NS5A isolate (Fig. , lanes 3 and 4). On the other hand, in cells expressing the N-terminal deletion form of NS5A, which lost its ability to interact with p85 PI3K, the ability to enhance the PI3K-AKT pathway was also lost (Fig. , lanes 7 and 8). These results suggest that the enhancement of the PI3K-AKT pathway by NS5A requires its interaction with p85 PI3K. In addition, by comparing HeLa cells infected with recombinant VV expressing either wild-type NS5A or NS5A-Pro3, we found that both wild-type NS5A and NS5A-Pro3 enhanced AKT phosphorylation to similar levels (data not shown). These results further support the notion that the ability of NS5A to associate with p85 PI3K and enhance the PI3K-AKT pathway is independent of Grb2 interaction.
The PI3K-AKT pathway plays a pivotal role in the regulation of cell survival and apoptosis. For example, AKT is known to cause serine phosphorylation of BAD, a proapoptotic Bcl-2 family member, and to inhibit BAD function, thus contributing to a blockade of apoptosis (8
). In order to examine the effect of NS5A upregulation of the PI3K-AKT pathway on the downstream apoptosis pathway, serine phosphorylation levels of BAD were compared in Tet-Off cells expressing wild-type NS5A either in the presence or in the absence of EGF treatment (Fig. ). As revealed by immunoblot analysis with antibody specific to serine-phosphorylated BAD, NS5A expression also enhanced AKT-mediated serine 136 phosphorylation of BAD, consistent with the effect of NS5A on the PI3K-AKT pathway. Similarly, NS5A expression also enhanced the serine phosphorylation levels of BAD in recombinant VV-infected cells (Fig. ), demonstrating the ability of NS5A to modulate the apoptosis machinery through the PI3K-AKT pathway during both cellular signaling and viral infection. Upregulation of the PI3K-AKT-BAD pathway may contribute to the antiapoptotic and oncogenic abilities of NS5A (19