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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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NS5A suppresses Kv2. 1 channel activity. HEK293 cells expressing Kv2.1 were transfected with bicistronic vectors encoding NS5A and GFP, or GFP alone (shown in Fig. 1B). At 24 h after transfection, Kv2.1 expression was induced by tetracycline treatment for 12 h, prior to electrophysiological analysis either without (A), or following (B) of DTDP stimulation (n = 6). Insets show representative traces of outward K+ currents in patch-clamp recordings. Top; control trace in GFP-expressing cells, below: trace from cells expressing NS5A and GFP. C, comparison of current density measurements from A and B at +40 mV. *, p < 0.002, **, p < 0.02, unpaired t test.
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Figure 5: NS5A suppresses Kv2. 1 channel activity. HEK293 cells expressing Kv2.1 were transfected with bicistronic vectors encoding NS5A and GFP, or GFP alone (shown in Fig. 1B). At 24 h after transfection, Kv2.1 expression was induced by tetracycline treatment for 12 h, prior to electrophysiological analysis either without (A), or following (B) of DTDP stimulation (n = 6). Insets show representative traces of outward K+ currents in patch-clamp recordings. Top; control trace in GFP-expressing cells, below: trace from cells expressing NS5A and GFP. C, comparison of current density measurements from A and B at +40 mV. *, p < 0.002, **, p < 0.02, unpaired t test.

Mentions: We previously demonstrated that HCV genotype 1b NS5A fused to GFP was able to inhibit basal Kv2.1 activity in Huh7 cells (14). To confirm that this phenotype was also a property of the JFH-1 NS5A, and to test the requirement for S800 phosphorylation, we transfected the tetracycline inducible HEK293 Kv2.1 cell lines with a bicistronic vector encoding NS5A and GFP, (Fig. 1B). Following the addition of tetracycline, whole cell recordings were performed from GFP-positive cells. JFH-1 NS5A also effectively suppressed channel activity by up to 50% either without or with oxidative stimuli (Fig. 5, A and B, respectively). As shown in Fig. 5C, the current densities measured at +40 mV were reduced by ∼50% in NS5A-expressing cells in both cases.


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)

NS5A suppresses Kv2. 1 channel activity. HEK293 cells expressing Kv2.1 were transfected with bicistronic vectors encoding NS5A and GFP, or GFP alone (shown in Fig. 1B). At 24 h after transfection, Kv2.1 expression was induced by tetracycline treatment for 12 h, prior to electrophysiological analysis either without (A), or following (B) of DTDP stimulation (n = 6). Insets show representative traces of outward K+ currents in patch-clamp recordings. Top; control trace in GFP-expressing cells, below: trace from cells expressing NS5A and GFP. C, comparison of current density measurements from A and B at +40 mV. *, p < 0.002, **, p < 0.02, unpaired t test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: NS5A suppresses Kv2. 1 channel activity. HEK293 cells expressing Kv2.1 were transfected with bicistronic vectors encoding NS5A and GFP, or GFP alone (shown in Fig. 1B). At 24 h after transfection, Kv2.1 expression was induced by tetracycline treatment for 12 h, prior to electrophysiological analysis either without (A), or following (B) of DTDP stimulation (n = 6). Insets show representative traces of outward K+ currents in patch-clamp recordings. Top; control trace in GFP-expressing cells, below: trace from cells expressing NS5A and GFP. C, comparison of current density measurements from A and B at +40 mV. *, p < 0.002, **, p < 0.02, unpaired t test.
Mentions: We previously demonstrated that HCV genotype 1b NS5A fused to GFP was able to inhibit basal Kv2.1 activity in Huh7 cells (14). To confirm that this phenotype was also a property of the JFH-1 NS5A, and to test the requirement for S800 phosphorylation, we transfected the tetracycline inducible HEK293 Kv2.1 cell lines with a bicistronic vector encoding NS5A and GFP, (Fig. 1B). Following the addition of tetracycline, whole cell recordings were performed from GFP-positive cells. JFH-1 NS5A also effectively suppressed channel activity by up to 50% either without or with oxidative stimuli (Fig. 5, A and B, respectively). As shown in Fig. 5C, the current densities measured at +40 mV were reduced by ∼50% in NS5A-expressing cells in both cases.

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