Induced oxidative stress and activated expression of manganese superoxide dismutase during hepatitis C virus replication: role of JNK, p38 MAPK and AP-1

Abstract

Activation of cellular kinases and transcription factors mediates the early phase of the cellular response to chemically or biologically induced stress. In the present study we investigated the oxidant/antioxidant balance in Huh-7 cells expressing the HCV (hepatitis C virus) subgenomic replicon, and observed a 5-fold increase in oxidative stress during HCV replication. We used MnSOD (manganese-superoxide dismutase) as an indicator of the cellular antioxidant response, and found that its activity, protein levels and promoter activity were significantly increased, whereas Cu/ZnSOD was not affected. The oxidative stress-induced protein kinases p38 MAPK (mitogen-activated protein kinase) and JNK (c-Jun N-terminal kinase) were activated in the HCV repliconcontaining cells and in Huh-7 cells transduced with Ad-NS5A [a recombinant adenovirus encoding NS5A (non-structural protein 5A)], coupled with a 4-5-fold increase in AP-1 (activator protein-1) DNA binding. Ava.1 cells, which encode a replication-defective HCV replicon, showed no significant changes in MnSOD, p38 MAPK or JNK activity. The AP-1 inhibitors dithiothreitol and N -acetylcysteine, as well as a dominant negative AP-1 mutant, significantly reduced AP-1 activation, demonstrating that this activation is oxidative stress-related. Exogenous NS5A had no effect on AP-1 activation in vitro, suggesting that NS5A acts at the upstream targets of AP-1 involving p38 MAPK and JNK signalling cascades. AP-1-dependent gene expression was increased in HCV subgenomic replicon-expressing Huh-7 cells. MnSOD activation was blocked by inhibitors of JNK (JNKI1) and p38 MAPK (SB203580), but not by an ERK (extracellular-signal-regulated kinase) inhibitor (U0126), in HCV-replicating and Ad-NS5A-transduced cells. Our results demonstrate that cellular responses to oxidative stress in HCV subgenomic replicon-expressing and Ad-NS5A-transduced cells are regulated by two distinct signalling pathways involving p38 MAPK and JNK via AP-1 that is linked to increased oxidative stress and therefore to an increased antioxidant MnSOD response.

Expression of Gastrointestinal Glutathione Peroxidase Is Inversely Correlated to the Presence of Hepatitis C Virus Subgenomic RNA in Human Liver Cells

“…Transfection of NS5a itself seems to be sufficient to trigger the elevation of ROS leading to the activation of several cellular transcription factors like NF-B and STAT-3 (44). Several studies demonstrated that HCV-infected liver cells in patients (45) and in vitro (46,47) are characterized by a high level of oxidative stress.…”

Oxidative stress modulation in hepatitis C virus infected cells

“…As expected there is a compensatory induction of anti-oxidant defenses in cells expressing viral proteins [31] . On the other hand, expression of non-structural protein 5A (NS5A) upregulates mitochondrial ROS levels because it induces the release of calcium from ER, binds to PI3K and triggers NF-kB signaling [19,[32][33][34] . This event is followed by a translocation of NF-kB to the nucleus, where it binds to DNA and activates several gene promoters.…”

Inhibition of hepatitis C virus replication by peroxidation of arachidonate and restoration by vitamin E

“…ROS generation has been reported in HCV infected patients (6-8), Huh7 cells infected by an infectious clone of HCV (9), and Huh7 cells that contain HCV replicons (10,11). ROS that are located in membranes are eradicated by lipid-soluble antioxidants, such as vitamin E (12).…”

“…This decline in lipid peroxidation allows inhibition of HCV replication without generation of cellular toxicity. (10,11), which have the potential to trigger lipid peroxidation. To monitor the production of reactive carbonyls generated by lipid peroxidation, we measured the amount of MDA produced by Huh7-K2040 cells, a line of Huh7 cells that harbor an HCV replicon (15).…”