In this study we set out to investigate the molecular mechanism by which the HCV NS5A protein inhibits the activity of the Kv2.1 potassium channel. This investigation led us to the observation that NS5A interacts with, and inhibits, the MAP3K MLK3. This has a number of potential functional consequences, two of which we have investigated further. A schematic model of the effects of NS5A on MLK3 and the downstream consequences is presented in . First, inhibition of MLK3 abrogates the well-characterized pro-apoptotic role of this kinase. MLK3 was first shown to be pro-apoptotic nearly a decade ago (17
), and more recently has been shown to be involved in apoptosis in both pancreatic β-cells (29
) and hepatocytes (18
) where it was shown to contribute to acetaminophen-induced oxidative stress and hepatotoxicity. Intriguingly, MLK3 was recently shown to be activated by the hepatitis B virus X protein (HBx) leading to increased apoptosis in cultured HepG2 cells (30
). In relation to HCV infection, inhibition of MLK3 is therefore potentially important to allow the survival of infected cells and the establishment of a persistent infection. In this regard it is noteworthy that HCV causes elevated intracellular ROS production mediated by iron overload (31
), deregulation of ER Ca2+
storage (ER stress) (32
), and direct mitochondrial ROS production (33
). Elevated ROS production can in turn suppress HCV replication (34
) and cause a range of pathological features including apoptosis, steatosis, DNA damage, and tumorigenesis (35
FIGURE 8. A model of NS5A interference with MLK3/p38/Kv2.1 signaling. HCV infection and RNA replication triggers the elevated production of reactive oxygen species (ROS), which activates MLK3, leading to activation of p38MAPK. In turn p38MAPK phosphorylates serine (more ...)
It is important to note that as well as inhibition of MLK3, NS5A targets other cellular pathways to that would potentially block apoptosis and contribute to tumorigenesis. In this regard both we (36
), and others (38
), have demonstrated that NS5A activates the proto-oncogene β-catenin, leading to its stabilization and stimulating β-catenin-dependent transcription. Intriguingly, MLK3 has been shown to inhibit β-catenin-dependent transcription while paradoxically stabilizing β-catenin (40
). It is therefore likely that NS5A could utilize multiple mechanisms to stimulate β-catenin, both by directly binding to it and indirectly by inhibiting MLK3. Clearly, HCV must benefit from the stimulation of β-catenin activity as it has evolved multiple mechanisms to ensure that this occurs during infection.
The second consequence of MLK3 inhibition is that there is a concomitant inhibition of p38MAPK-mediated phosphorylation of the K+
channel; Kv2.1. Kv2.1 channel activity has been associated with the induction of apoptosis in neurons (10
), and indeed recent observations suggest that NS5A is able to block Kv2.1-mediated apoptosis in cultured rat neurons (42
). However, in the present study, we were not able to observe elevated levels of apoptosis in either HEK293 or Huh7 cells following overexpression of Kv2.1 (data not shown). We conclude therefore, that the induction of apoptosis we observed previously in Huh7 cells treated with DTDP (14
) was coincident with stimulation of Kv2.1 activity, and not dependent upon the latter. Both the induction of apoptosis and the stimulation of Kv2.1 activity could be inhibited by NS5A in a P2 motif-dependent manner. In the present study we show that both of these effects can be explained by interactions between NS5A and MLK3. Although alanine substitution of the conserved prolines in the P2 motif does not affect viral fitness or replication (23
), the same mutation caused a greater sensitivity to oxidative stress-induced apoptosis (14
). Additionally, the P2 motif was shown to be important for the establishment of infection in the chimpanzee model (43
). Coupled with our previous observation that primary hepatocytes were much more sensitive to oxidative stress than Huh7 (14
), we propose that the P2 motif may play a role in virus persistence by blocking the induction of apoptosis in infected hepatocytes. This could explain the absolute conservation of this motif in all HCV isolates.
As mentioned above, a recent study revealed that NS5A of a different genotype (1b) effectively blocked Kv2.1 mediated currents without interfering with S800 phosphorylation but by suppressing Y124 phosphorylation, which is mediated by c-Src kinase (42
). The data discrepancy may be due to different experimental conditions (e.g.
cell type), although it is still noteworthy to mention that NS5A targets c-Src kinase (44
). A recent report (45
) demonstrated that S440 and S537 of Kv2.1 are substrates of AMP-activated protein kinase (AMPK). Since we have shown that AMPK activation was significantly impaired by HCV infection (46
), it will be of great interest to see how Kv2.1 phosphorylation by AMPK is affected during either HCV infection or NS5A expression. Furthermore, a body of etiological evidence shows that HCV infection causes a range of metabolic disorders in patients, for instance fatty liver and insulin resistance. Future studies will hopefully establish how viral suppression or deregulation of Kv2.1 could contribute to such metabolic alterations caused by HCV.
Overall this study demonstrates that the SH3-binding P2 motif of NS5A can bind MLK3 to both suppress oxidative stress induced outward K+ currents, and inhibit host cell apoptosis. This may facilitate the maintenance of long term viral infection and in turn may enable HCV to establish a persistent infection. Understanding the mechanisms of NS5A modulation of MLK3 and Kv2.1 provides a target for the development of novel antivirals that may trigger apoptosis in HCV infected hepatocytes.