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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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Exogenous MLK3 expression induces apoptosis.A, Huh7 cells were seeded on coverslips and transfected with the indicated expression vectors. 24 h after transfection, cells were fixed and stained with DAPI. Fluorescence microscopy was performed to visualize transfected cells (GFP fluorescence) and the nucleus (DAPI). B, multiple fields were counted (n = 4). GFP-positive cells, more than 60 cells for each sample point, were counted as transfected cells and the percentage of GFP-positive cells showing fragmented nuclei are presented. C, Huh7 cells were transfected with the indicated plasmid vectors. Lysates were subjected to Western blotting to detect caspase-cleaved PARP (as a marker of apoptosis), as well as NS5A-GFP and FLAG-MLK3.
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Figure 3: Exogenous MLK3 expression induces apoptosis.A, Huh7 cells were seeded on coverslips and transfected with the indicated expression vectors. 24 h after transfection, cells were fixed and stained with DAPI. Fluorescence microscopy was performed to visualize transfected cells (GFP fluorescence) and the nucleus (DAPI). B, multiple fields were counted (n = 4). GFP-positive cells, more than 60 cells for each sample point, were counted as transfected cells and the percentage of GFP-positive cells showing fragmented nuclei are presented. C, Huh7 cells were transfected with the indicated plasmid vectors. Lysates were subjected to Western blotting to detect caspase-cleaved PARP (as a marker of apoptosis), as well as NS5A-GFP and FLAG-MLK3.

Mentions: We previously demonstrated that the inhibition of Kv2.1 activity by NS5A was accompanied by a concomitant protection of infected cells from oxidative stress-induced apoptosis (14). However, it was not clear whether this protection from apoptosis was due to the block in K+ efflux, or some other consequence of the inhibition of p38MAPK signaling. To address this question we next sought to determine the downstream effects of the NS5A-MLK3 interaction. In particular we wished to assess whether MLK3 could mediate the induction of apoptosis and, if so, whether NS5A could abrogate this effect. It had been previously shown that when ectopically overexpressed wild type MLK3 can function as a dominant active kinase via homo-dimerization and subsequent auto-phosphorylation activation, whereas the kinase-inactive K144E mutant functioned as a dominant negative (28). Huh7 cells were therefore transfected with bicistronic constructs expressing MLK3 (either wild type or K144E) and either GFP or a NS5A (wild type or PA2) GFP fusion (Fig. 1B). The induction of apoptosis was assessed by the presence of fragmented nuclei at 24 h after transfection. Fig. 3A shows that, whereas either GFP or NS5A-GFP expression did not induce apoptosis, as anticipated ectopic MLK3 expression resulted in significant induction of apoptosis. Interestingly, expression of K144E MLK3 did not induce apoptosis suggesting that MLK3-mediated cell death is mediated by the kinase activity of MLK3. When MLK3 was expressed together with wild type NS5A-GFP, MLK3-mediated cell death was inhibited. Importantly, this inhibition was not observed upon co-expression of PA2 NS5A (Fig. 3A-VI). These results were quantified and expressed as an apoptotic cells in the GFP positive population (Fig. 3B). To confirm the induction of apoptosis by MLK3, we utilized an alternative assay for the caspase-3 mediated cleavage of poly[ADP-ribose]polymerase (PARP), a well characterized apoptosis effector molecule. Wild type MLK3 induced strong activation of PARP (Fig. 3C, lane 2) whereas this effect was abrogated by the K144E mutation of MLK3 (Fig. 3C, lane 3). Cotransfection of wild type NS5A, but not the PA2 mutant, blocked the appearance of cleaved PARP (lanes 4 and 5). Taken together, these observations suggest that NS5A interacts with MLK3 and inhibits its kinase activity and/or accessibility to substrate proteins, thus blocking MLK3-induced apoptosis.


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)

Exogenous MLK3 expression induces apoptosis.A, Huh7 cells were seeded on coverslips and transfected with the indicated expression vectors. 24 h after transfection, cells were fixed and stained with DAPI. Fluorescence microscopy was performed to visualize transfected cells (GFP fluorescence) and the nucleus (DAPI). B, multiple fields were counted (n = 4). GFP-positive cells, more than 60 cells for each sample point, were counted as transfected cells and the percentage of GFP-positive cells showing fragmented nuclei are presented. C, Huh7 cells were transfected with the indicated plasmid vectors. Lysates were subjected to Western blotting to detect caspase-cleaved PARP (as a marker of apoptosis), as well as NS5A-GFP and FLAG-MLK3.
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Figure 3: Exogenous MLK3 expression induces apoptosis.A, Huh7 cells were seeded on coverslips and transfected with the indicated expression vectors. 24 h after transfection, cells were fixed and stained with DAPI. Fluorescence microscopy was performed to visualize transfected cells (GFP fluorescence) and the nucleus (DAPI). B, multiple fields were counted (n = 4). GFP-positive cells, more than 60 cells for each sample point, were counted as transfected cells and the percentage of GFP-positive cells showing fragmented nuclei are presented. C, Huh7 cells were transfected with the indicated plasmid vectors. Lysates were subjected to Western blotting to detect caspase-cleaved PARP (as a marker of apoptosis), as well as NS5A-GFP and FLAG-MLK3.
Mentions: We previously demonstrated that the inhibition of Kv2.1 activity by NS5A was accompanied by a concomitant protection of infected cells from oxidative stress-induced apoptosis (14). However, it was not clear whether this protection from apoptosis was due to the block in K+ efflux, or some other consequence of the inhibition of p38MAPK signaling. To address this question we next sought to determine the downstream effects of the NS5A-MLK3 interaction. In particular we wished to assess whether MLK3 could mediate the induction of apoptosis and, if so, whether NS5A could abrogate this effect. It had been previously shown that when ectopically overexpressed wild type MLK3 can function as a dominant active kinase via homo-dimerization and subsequent auto-phosphorylation activation, whereas the kinase-inactive K144E mutant functioned as a dominant negative (28). Huh7 cells were therefore transfected with bicistronic constructs expressing MLK3 (either wild type or K144E) and either GFP or a NS5A (wild type or PA2) GFP fusion (Fig. 1B). The induction of apoptosis was assessed by the presence of fragmented nuclei at 24 h after transfection. Fig. 3A shows that, whereas either GFP or NS5A-GFP expression did not induce apoptosis, as anticipated ectopic MLK3 expression resulted in significant induction of apoptosis. Interestingly, expression of K144E MLK3 did not induce apoptosis suggesting that MLK3-mediated cell death is mediated by the kinase activity of MLK3. When MLK3 was expressed together with wild type NS5A-GFP, MLK3-mediated cell death was inhibited. Importantly, this inhibition was not observed upon co-expression of PA2 NS5A (Fig. 3A-VI). These results were quantified and expressed as an apoptotic cells in the GFP positive population (Fig. 3B). To confirm the induction of apoptosis by MLK3, we utilized an alternative assay for the caspase-3 mediated cleavage of poly[ADP-ribose]polymerase (PARP), a well characterized apoptosis effector molecule. Wild type MLK3 induced strong activation of PARP (Fig. 3C, lane 2) whereas this effect was abrogated by the K144E mutation of MLK3 (Fig. 3C, lane 3). Cotransfection of wild type NS5A, but not the PA2 mutant, blocked the appearance of cleaved PARP (lanes 4 and 5). Taken together, these observations suggest that NS5A interacts with MLK3 and inhibits its kinase activity and/or accessibility to substrate proteins, thus blocking MLK3-induced apoptosis.

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