Critical role of Casein kinase 2 in hepatitis C NS5A-mediated inhibition of Kv2.1 K + channel function

Abstract

Inhibiting injury-induced increases in outward K +currents is sufficient to block cell death in cortical neuronal injury models. It is now known that apoptosis is facilitated in hepatocytes by the same K+ channel as in cortical neurons, namely the delayed rectifier K+ channel Kv2.1. The hepatitis C virus (HCV) protein NS5A prevents the apoptosis-enabling loss of intracellular potassium by inhibiting Kv2.1 function and thus blocking hepatocyte cell death. Critically, neurons expressing NS5A1b (from HCV genotype 1b), but not NS5A1a, can be protected from lethal injurious stimuli via a block of Kv2.1-mediated potassium currents. Here, we identify a key component unique to NS5A1b, which is necessary for restricting Kv2.1 currents and establishing neuroprotection. By comparing the sequence differences between NS5A1b and 1a we identify putative casein kinase 2 (CK2) phosphorylation regions unique to the 1b genotype. We show that selective inhibition of CK2 in cortical neurons results in loss of NS5A1b’s ability to depress outward potassium currents, and, surprisingly, potentiates currents in non-NS5A-expressingcells. As such, our results suggest that NS5A1b-mediated inhibition of Kv2.1 function is critically dependent on its phosphorylation status at genotypic-specific CK2-directed residues. Importantly, inhibiting NS5A viral replicative function with the novel HCV drug Ledipasvir does not impair the ability of this protein to block Kv2.1 function. This suggests that the modulation of NS5A function by CK2 may be a component of HCV unique to the regulation of apoptosis.

Molecular Neuroprotection Induced by Zinc-Dependent Expression of Hepatitis C–Derived Protein NS5A Targeting Kv2.1 Potassium Channels

“…Finally, we observed very similar channel voltage activation profiles in all of the experimental groups ( Fig. 2D), suggesting that 1) the observed changes in Kv2.1 function following TBOA exposure are distinct from other forms of channel modulation not directly related to cell death (Mohapatra et al, 2007); and 2) NS5A expression, as reported previously (Norris et al, 2012;Clemens et al, 2015), does not substantially affect normal channel function. The half-maximal activation voltages for all experimental groups were as follows: vector/vehicle, 21.09 6 2.79 mV (n 5 12); vector/T-BOA, 14.58 6 1.26 mV (n 5 12); NS5A/vehicle, 20.14 6 2.57 mV (n 5 12); and NS5A/TBOA 18.78 6 2.80 mV (n 5 10).…”

“…It is worth noting at this point that NS5A is also the site of action of ledipasvir, a principal component of a highly successful HCV antiviral drug (Pawlotsky, 2013). We found, however, that the presence of ledipasvir does not influence the activity of NS5A on the Kv2.1 channel, suggesting that other components of the viral protein are critical for K 1 channel inhibition (Clemens et al, 2015).…”

NMR and circular dichroism data for domain 2 of the HCV NS5A protein phosphorylated by the Casein Kinase II

“…Subsequently, the NS5A-D2 was in vitro phosphorylated by CKII ( Fig. 1 ) [8] , [9] , [10] , [11] . The resulting NS5A-D2_CKII sample was then analyzed by circular dichroism spectroscopy ( Fig.…”

“…So far, no precise molecular function has been identified for this viral protein (Ross-Thriepland and Harris, 2015) [6] which is required for viral replication (Tellinghuisen et al, 2008) [7] . In this article, we present datasets of NMR and circular dichroism analyses of the domain 2 of the HCV NS5A protein (NS5A-D2) phosphorylated in vitro by the Casein Kinase II (CKII) (Dal Pero et al, 2007; Clemens et al, 2015; Masak et al, 2014; Kim et al, 2014) [8] , [9] , [10] , [11] . We describe the in vitro phosphorylation of the serine 288 (pS288) of NS5A-D2 by CKII and report the circular dichroism spectrum of the phosphorylated domain (NS5-D2_CKII).…”