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.
Abbreviations used: Ad-GFP, recombinant adenovirus encoding GFP; Ad-NS5A, recombinant adenovirus encoding NS5A; AP-1, activator protein-1; CMV, cytomegalovirus; DCF, 2´,7´-dichlorofluorescein; DCFH-DA, 2´,7´-dichlorofluorescein diacetate; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; ERK, extracellular-signal-regulated kinase; GFP, green fluorescent protein; GST, glutathione S-transferase; HCV, hepatitis C virus; JNK, c-Jun N-terminal kinase; MAPK, mitogen-activated protein kinase; NAC, N-acetylcysteine; NFκB, nuclear factor κB; NS5A, non-structural protein 5A; PKR, double-stranded-RNA-dependent protein kinase; SOD, superoxide dismutase; SRCAP, SNF2-related CBP activator protein [where CBP is CREB (cAMP response element binding protein) binding protein]; STAT5b, signal transduction and activators of transcription 5b; SV40, simian virus 40; TBP, TATA box binding protein; TNF, tumour necrosis factor; TRE, PMA (‘TPA’)-responsive element.