Hepatitis C virus NS5A protein interacts with beta-catenin and stimulates its transcriptional activity in a phosphoinositide-3 kinase-dependent fashion.

Hepatitis C virus (HCV) infection is increasingly associated with the development of hepatocellular carcinoma (HCC). HCV is not thought to be directly oncogenic but, by modulating a range of cellular functions, may predispose patients to the development of liver tumours. However, the molecular mechanisms by which HCV infection might contribute to HCC remain to be characterized. In this regard, we showed previously that the HCV NS5A protein bound to the p85 regulatory subunit of phosphoinositide-3 kinase (PI3K), thereby stimulating the activity of the p110 catalytic subunit of the enzyme. One of the downstream consequences of this was the stabilization of the proto-oncogene, beta-catenin, with a concomitant stimulation of its transcriptional activity. Here, we further analyse the mechanism by which NS5A mediates activation of beta-catenin. Although our previous data were consistent with a role for the PI3K downstream effector kinases, Akt and glycogen synthase kinase-3beta, in NS5A-mediated activation of beta-catenin, we demonstrate here that it is in fact independent of both of these kinases. Truncation analysis revealed that both the N and C termini of NS5A are required for full activation of beta-catenin. Furthermore, we demonstrate that NS5A, either alone or in complex with p85, is able to bind directly to beta-catenin; again both N and C termini contribute to this interaction. We propose that NS5A activates beta-catenin via a novel mechanism that involves a direct interaction between the two proteins and is augmented by PI3K activity. This may contribute to the association between chronic HCV infection and the development of HCC.

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

GB virus B (GBV-B) is the closest relative to hepatitis C virus (HCV) with which it shares a common genome organization, however, unlike HCV in humans, it generally causes an acute resolving hepatitis in New World monkeys. It is important to understand the factors regulating the different disease profiles of the two viruses and in this regard, as well as playing a key role in viral RNA replication, the HCV NS5A non-structural protein modulates a variety of host-cell signalling pathways. We have shown previously that HCV NS5A, expressed either alone, or in the context of the complete polyprotein, inhibits the Ras-extracellular-signal-regulated kinase (Erk) pathway and activates the phosphoinositide 3-kinase (PI3K) pathway. In this report, we investigate whether these functions are shared by GBV-B NS5A. Immunofluorescence analysis revealed that a C-terminally FLAG-tagged GBV-B NS5A exhibited a punctate cytoplasmic distribution. However, unlike HCV NS5A, the GBV-B protein did not partially co-localize with early endosomes. Utilizing a transient luciferase reporter system, we observed that GBV-B NS5A failed to inhibit Ras-Erk signalling, however GBV-B NS5A expression did result in the elevation of beta-catenin-dependent transcription via activation of the PI3K pathway. These effects of GBV-B and HCV NS5A on the PI3K and Ras-Erk pathways were confirmed in cells harbouring subgenomic replicons derived from the two viruses. Based on these data we speculate that the differential effects of the two NS5A proteins on cellular signalling pathways may contribute to the differences in the natural history of the two viruses.

Sodium orthovanadate induced tyrosine phosphorylation of platelet nitric oxide synthase negatively regulates enzyme activity.

The post-translational regulation of platelet nitric oxide synthase (NOS) activity is poorly understood. In the present study we examined how tyrosine phosphorylation of NOS, induced by the tyrosine phosphatase inhibitor sodium orthovanadate (VO4), influenced enzyme activity. Platelet NOS was basally tyrosine phosphorylated, but incubation with VO4 (100-1000 microM) led to a concentration-dependent increase in tyrosine phosphorylation of the enzyme with maximal effects observed at 500 microM. Importantly, we observed no change in serine(1179) or threonine(497) phosphorylation. The increased tyrosine phosphorylation was associated with reduced NOS activity and NO bioavailability, as evidenced by measurement of [(3)H]-L-citrulline and cGMP respectively. The signalling events underlying the effects of VO4 were studied using specific inhibitors to kinases that are known to influence NOS activity. Preincubation of platelets with the Src kinase inhibitor PP2 (20 microM) blocked VO4-induced tyrosine phosphorylation of NOS and abolished the effects of VO4 on cGMP formation. The PKC inhibitor Ro-31-8220 (10 microM) had no effect on VO4-induced tyrosine phosphorylation, but did have a modest but significant effect on cGMP formation. In contrast, the PI-3-kinase inhibitor wortmannin (100 nM) had no effect on either tyrosine phosphorylation or cGMP formation. Our data indicate that tyrosine phosphorylation may act to repress NOS activity. Furthermore, VO4 induces a Src-dependent, and to a lesser degree PKC-dependent, inhibition of platelet NOS.