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Hepatitis C virus NS5A inhibits mixed lineage kinase 3 to block apoptosis.

Amako Y, Igloi Z, Mankouri J, Kazlauskas A, Saksela K, Dallas M, Peers C, Harris M - J. Biol. Chem. (2013)

Bottom Line: We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1.An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis.We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K(+) homeostasis.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.

ABSTRACT
Hepatitis C virus (HCV) infection results in the activation of numerous stress responses including oxidative stress, with the potential to induce an apoptotic state. Previously we have shown that HCV attenuates the stress-induced, p38MAPK-mediated up-regulation of the K(+) channel Kv2.1, to maintain the survival of infected cells in the face of cellular stress. We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1. In this study, we investigated the host cell proteins targeted by NS5A to mediate Kv2.1 inhibition. We screened a phage-display library expressing the entire complement of human SH3 domains for novel NS5A-host cell interactions. This analysis identified mixed lineage kinase 3 (MLK3) as a putative NS5A interacting partner. MLK3 is a serine/threonine protein kinase that is a member of the MAPK kinase kinase (MAP3K) family and activates p38MAPK. An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis. We further demonstrate a novel role of MLK3 in the modulation of Kv2.1 activity, whereby MLK3 overexpression leads to the up-regulation of channel activity. Accordingly, coexpression of NS5A suppressed this stimulation. Additionally we demonstrate that overexpression of MLK3 induced apoptosis, which was also counteracted by NS5A. We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K(+) homeostasis.

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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 800 of Kv2.1, which is a key phosphorylation event involved in the insertion of Kv2.1 into the plasma membrane. The upsurge in the efflux of K+ lowers the intracellular concentration of K+, causing an alteration of ionic homeostasis. At the same time MLK3 stimulates the induction of apoptosis. In this study, we show that NS5A inhibits MLK3 activation, thereby blocking both the phosphorylation and activation of Kv2.1, and MLK3-mediated apoptosis.
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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 800 of Kv2.1, which is a key phosphorylation event involved in the insertion of Kv2.1 into the plasma membrane. The upsurge in the efflux of K+ lowers the intracellular concentration of K+, causing an alteration of ionic homeostasis. At the same time MLK3 stimulates the induction of apoptosis. In this study, we show that NS5A inhibits MLK3 activation, thereby blocking both the phosphorylation and activation of Kv2.1, and MLK3-mediated apoptosis.

Mentions: 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 Fig. 8. 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).


Hepatitis C virus NS5A inhibits mixed lineage kinase 3 to block apoptosis.

Amako Y, Igloi Z, Mankouri J, Kazlauskas A, Saksela K, Dallas M, Peers C, Harris M - J. Biol. Chem. (2013)

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 800 of Kv2.1, which is a key phosphorylation event involved in the insertion of Kv2.1 into the plasma membrane. The upsurge in the efflux of K+ lowers the intracellular concentration of K+, causing an alteration of ionic homeostasis. At the same time MLK3 stimulates the induction of apoptosis. In this study, we show that NS5A inhibits MLK3 activation, thereby blocking both the phosphorylation and activation of Kv2.1, and MLK3-mediated apoptosis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750171&req=5

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 800 of Kv2.1, which is a key phosphorylation event involved in the insertion of Kv2.1 into the plasma membrane. The upsurge in the efflux of K+ lowers the intracellular concentration of K+, causing an alteration of ionic homeostasis. At the same time MLK3 stimulates the induction of apoptosis. In this study, we show that NS5A inhibits MLK3 activation, thereby blocking both the phosphorylation and activation of Kv2.1, and MLK3-mediated apoptosis.
Mentions: 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 Fig. 8. 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).

Bottom Line: We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1.An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis.We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K(+) homeostasis.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular and Cellular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, United Kingdom.

ABSTRACT
Hepatitis C virus (HCV) infection results in the activation of numerous stress responses including oxidative stress, with the potential to induce an apoptotic state. Previously we have shown that HCV attenuates the stress-induced, p38MAPK-mediated up-regulation of the K(+) channel Kv2.1, to maintain the survival of infected cells in the face of cellular stress. We demonstrated that this effect was mediated by HCV non-structural 5A (NS5A) protein, which impaired p38MAPK activity through a polyproline motif-dependent interaction, resulting in reduction of phosphorylation activation of Kv2.1. In this study, we investigated the host cell proteins targeted by NS5A to mediate Kv2.1 inhibition. We screened a phage-display library expressing the entire complement of human SH3 domains for novel NS5A-host cell interactions. This analysis identified mixed lineage kinase 3 (MLK3) as a putative NS5A interacting partner. MLK3 is a serine/threonine protein kinase that is a member of the MAPK kinase kinase (MAP3K) family and activates p38MAPK. An NS5A-MLK3 interaction was confirmed by co-immunoprecipitation and Western blot analysis. We further demonstrate a novel role of MLK3 in the modulation of Kv2.1 activity, whereby MLK3 overexpression leads to the up-regulation of channel activity. Accordingly, coexpression of NS5A suppressed this stimulation. Additionally we demonstrate that overexpression of MLK3 induced apoptosis, which was also counteracted by NS5A. We conclude that NS5A targets MLK3 with multiple downstream consequences for both apoptosis and K(+) homeostasis.

Show MeSH
Related in: MedlinePlus