Abstract
Hepatitis C virus nonstructural protein 5A (NS5A) has been implicated in the HCV antiviral resistance, replication, and transactivation of cellular gene expression. We have recently shown that HCV NS5A activates NF-kappaB via oxidative stress (22). In this study, we investigate the molecular mechanism(s) of NF-kappaB activation in response to oxidative stress induced by NS5A protein. In contrast to the classic Ser32,36 phosphorylation of IkappaBalpha, we report here that tyrosine phosphorylation of IkappaBalpha at Tyr42 and Tyr305 residues is induced by the HCV NS5A and the subgenomic replicons in the NF-kappaB activation process. Use of IkappaBalpha-Tyr42,305 double mutant provided the evidence for their key role in the activation of NF-kappaB. Activation of NF-kappaB was blocked by a series of tyrosine kinase inhibitors but not by IkappaB kinase inhibitor BAY 11-7085. More specifically, a ZAP-70 knock-out cell line expressing NS5A and other nonstructural proteins respectively prevented the NF-kappaB activation, indicating the involvement of ZAP-70 as a probable tyrosine kinase in the activation process. Evidence is also presented for the possible role of calpain proteases in the NS5A-induced IkappaBalpha degradation. These studies collectively define an alternate pathway of NF-kappaB activation by NS5A alone or in the context of the HCV subgenomic replicon. Constitutive activation of NF-kappaB by HCV has implications in the chronic liver disease including hepatocellular carcinoma associated with HCV infection.
Highlights
We have recently shown that HCV nonstructural protein 5A (NS5A) activates NF-B break-through in the HCV field [9]
NS5A Induces Tyrosine Phosphorylation of IB␣—To initiate these studies, we sought to determine the ability of NS5A alone or in the context of all the HCV nonstructural proteins (NS) to induce the tyrosine phosphorylation of IB␣
The involvement of ROS and Ca2ϩ signaling in the IB␣ tyrosine phosphorylation induced by HCV NS5A was further evaluated by using antioxidant Pyrrolidine dithiocarbamate (PDTC) and calcium chelator BAPTA-AM
Summary
We have recently shown that HCV NS5A activates NF-B break-through in the HCV field [9]. HCV NS5A Induces NF-B Activation through IB␣ Degradation been observed during ischemia/reprefusion of the liver [33], suggesting its functional role in this pathway. We investigated the mechanism(s) of NF-B activation in human hepatoma cell line expressing NS5A protein and the HCV subgenomic replicons respectively. NF-B activation by NS5A involves Ca2ϩ signaling and the generation of ROS in the cells. These data collectively suggest a novel pathway of ER-nucleus signaling by the HCV NS5A protein either alone or in the context of the subgenomic replicon. The intracellular events triggered by the HCV translation and replication activities in the cells are relevant to the mechanism(s) of liver disease pathogenesis associated with the HCV infection
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