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

Abstract

Although hepatitis C virus (HCV) infection is an emerging global epidemic causing severe liver disorders, the molecular mechanisms of HCV pathogenesis remain elusive. The NS5A nonstructural protein of HCV contains several proline-rich sequences consistent with Src homology (SH) 3-binding sites found in cellular signaling molecules. Here, we demonstrate that NS5A specifically bound to growth factor receptor-bound protein 2 (Grb2) adaptor protein. Immunoblot analysis of anti-Grb2 immune complexes derived from HeLa S3 cells infected with a recombinant vaccinia virus (VV) expressing NS5A revealed an interaction between NS5A and Grb2 in vivo. An inactivating point mutation in the N-terminal SH3 domain, but not in the C-terminal SH3 domain, of Grb2 displayed significant diminished binding to NS5A. However, the same mutation in both SH3 regions completely abrogated Grb2 binding to NS5A, implying that the two SH3 domains bind in cooperative fashion to NS5A. Further, mutational analysis of NS5A assigned the SH3-binding region to a proline-rich motif that is highly conserved among HCV genotypes. Importantly, phosphorylation of extracellular signalregulated kinases 1 and 2 (ERK1͞2) was inhibited in HeLa S3 cells infected with NS5A-expressing recombinant VV but not recombinant VV control. Additionally, HeLa cells stably expressing NS5A were refractory to ERK1͞2 phosphorylation induced by exogenous epidermal growth factor. Moreover, the coupling of NS5A to Grb2 in these cells was induced by epidermal growth factor stimulation. Therefore, NS5A may function to perturb Grb2-mediated signaling pathways by selectively targeting the adaptor. These findings highlight a viral interceptor of cellular signaling with potential implications for HCV pathogenesis.

Perturbation of epidermal growth factor receptor complex formation and Ras signalling in cells harbouring the hepatitis C virus subgenomic replicon

“…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;, By doing so, it is possible that NS5A might inhibit the formation of the Grb2-Sos 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 Grb2-Sos complex (Tan et al, 1999), although these data have not been presented.…”

“…The mechanism of this inhibition is unknown, although it has been shown that NS5A is able to bind Grb2 (Tan et al, 1999;, By doing so, it is possible that NS5A might inhibit the formation of the Grb2-Sos 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 Grb2-Sos 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 Ras-ERK signalling by analysing EGFR-proximal signalling events in Huh-7 cells harbouring an HCV subgenomic replicon (expressing the nonstructural proteins NS3-NS5B) at very early time points following EGF stimulation.…”

“…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 Ras-ERK 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.…”

A comparative cell biological analysis reveals only limited functional homology between the NS5A proteins of hepatitis C virus and GB virus B

“…In this regard NS5A is implicated in the establishment of chronic HCV infection, due to its effects on IFN signalling, modulation of cell growth and the inhibition of apoptosis (for review see . Since it has been shown previously that HCV NS5A inhibits the Ras-Erk pathway (Tan et al, 1999;Macdonald et al, 2003;Georgopoulou et al, 2003) and simultaneously activates PI3K (Street et al, 2005), the current study investigated if this ability was shared by the GBV-B NS5A protein. We demonstrated that the GBV-B NS5A protein differs both in its ability to inhibit Ras-Erk signalling (Figs 3 and 4) and its cellular distribution when compared with HCV NS5A (Figs 1 and 2).…”

“…In addition NS5A has been shown to bind to and modulate a range of cellular proteins, influencing activation of signalling pathways . Of particular interest is the observation that NS5A inhibits the Ras-Erk mitogen-activated protein kinase (MAPK) pathway acting between the epidermal growth factor receptor (EGFR) and the activation of Ras (Georgopoulou et al, 2003;Macdonald et al, 2003Macdonald et al, , 2005aTan et al, 1999). Furthermore, NS5A has also been shown to bind to, and activate, phosphoinositide 3-kinase (PI3K), resulting in activation of the downstream effector serine/ threonine kinase Akt/protein kinase B (He et al, 2002;Street et al, 2004) and elevating b-catenin-dependent transcription (Street et al, 2005).…”