Human hepatitis C virus NS5A protein alters intracellular calcium levels, induces oxidative stress, and activates STAT-3 and NF-κB

Abstract

The nonstructural protein 5A (NS5A) encoded by the human hepatitis C virus RNA genome is shown here to induce the activation of NF-B and STAT-3 transcription factors from its cytoplasmic residence via oxidative stress. NS5A causes the disturbance of intracellular calcium. Ca 2؉ signaling triggers the elevation of reactive oxygen species in mitochondria, leading to the translocation of NF-B and STAT-3 into the nucleus. Evidence is presented for the constitutive activation of STAT-3 by NS5A. In the presence of antioxidants [pyrrolidine dithiocarbamate (PDTC), N-acetyl L-cysteine (NAC)] or Ca 2؉ chelators (EGTA-AM, TMB-8), NS5A-induced activation of NF-B and STAT-3 was eliminated. These results provide an insight into the mechanism by which NS5A can alter intracellular events relevant to liver pathogenesis associated with the viral infection.H epatitis C virus (HCV) causes acute͞chronic hepatitis with a significant risk of end-stage cirrhosis and hepatocellular carcinoma (1). The single-stranded RNA genome of the human HCV is a 9.6-kb-long positive-sense molecule, which encodes a polyprotein of about 3,000 aa (2, 3). The polyprotein is posttranslationally cleaved by both viral͞cellular proteases to produce about 10 polypeptides that include structural (core and E1 and E2) and nonstructural (NS2, NS3-NS5A͞B) proteins (2, 3). The single long ORF is preceded by 332 or 342 nt of the 5Ј untranslated region, which harbors an internal ribosome entry site capable of initiating translation at an internal site (4-6). The 3Ј end of the RNA genome contains a unique sequence of the untranslated region that is necessary for initiating RNA replication. Although the infectious cDNA clones that have been generated could infect chimpanzees, they failed to replicate in vitro in cultured cell lines (7). Lohmann et al. (8) described the generation of efficient replicating HCV RNA subgenomic replicons coexpressing a neomycin-resistance marker. A high level of replication of subgenomic replicons was characterized, resulting from adaptive mutations, which were scattered throughout the HCV genes of the replicon (9, 10).The HCV NS5A has generated a significant level of interest as several cellular targets have been identified. NS5A is a serine phosphoprotein, which exists as a polypeptide of p56 or p58 with varying degrees of phosphorylation (11,12). The identity of cellular kinase(s) responsible for NSS5A phosphorylation has not been firmly established. NS4A, an integral membrane protein, has been shown to modulate NS5A phosphorylation at least for some HCV subtypes (13). NS5A is localized to the cytoplasm in the perinuclear reticular network characteristic of the endoplasmic reticulum (ER) membrane (3,14). NS5A came into prominence because of its suggested role in IFN resistance. It was shown that NS5A directly interacted with double-stranded RNA-dependent kinase (PKR) and inactivated its function, thus modulating the IFN-stimulated antiviral response (15). Neither the sites of hyperphosphorylation nor the region designated ISDR (IFN-sen...

Role of free radicals in liver diseases

“…Host antioxidant defenses, such as GSH, catalase, MnSOD, and heme oxygenase-1, are augmented, suggesting adaptation to ROS/RNS stress [92,104,105].…”

Redox modulation of the hepatitis C virus replication complex is calcium dependent

“…HCV infection is associated with increases in various markers of oxidative stress in patients [10][11][12][13]. In addition to chronic inflammation, iron overload and some of HCV proteins may help increase the oxidative burden [10,[14][15][16][17][18][19][20][21].…”

“…Therefore, in this study, we tested whether ROS rapidly suppressed the activity HCV replication complex through redox signaling, to understand the biological consequence of increased oxidative stress in hepatitis C. The effect of H 2 O 2 on HCV RNA replication is also compared with that of interferon gamma (IFNγ). Interestingly, various HCV proteins have been suggested to modulate calcium metabolism [17,23,24]. HCV p7 protein may act as a calcium channel [24].…”

Exploiting hepatitis C virus activation of NFκB to deliver HCV-responsive expression of interferons α and γ

“…The responsiveness of IFN to HCV was expected to reflect the ability of HCV NS5a to activate NFkB, 7 which in turn would upregulate HIVLTR as a promoter. 8 We did not directly address the mechanism of HCV activation of NFkB, but we found that mutating NFkB binding sites in the HIVLTR eliminated HCV responsiveness in IFN production and secretion.…”

“…For gene delivery to the liver, rSV40 vectors have been shown to deliver enduring transgene expression to very high percentages of unselected hepatocytes, whether resting or dividing, in vitro or in vivo. 5,6 The goal of rendering transgene expression responsive to the presence of the offending HCV virus was approached by exploiting the reported ability of the HCV gene product NS5a to activate the cellular transcription factor NFkB, 7 as well as the known responsiveness of the promoter activity of the HIV-1 long terminal repeat (LTR) to NFkB. 8 Accordingly, we engineered rSV40 vectors to carry the HIVLTR as a promoter, to drive expression of murine and human IFNa and IFNg as transgenes.…”