Aleem Siddiqui

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

The nonstructural protein 5A (NS5A) encoded by the human hepatitis C virus RNA genome is shown here to induce the activation of NF-κB and STAT-3 transcription factors from its cytoplasmic residence via oxidative stress. NS5A causes the disturbance of intracellular calcium. Ca2+ signaling triggers the elevation of reactive oxygen species in mitochondria, leading to the translocation of NF-κB 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 Ca2+ chelators (EGTA-AM, TMB-8), NS5A-induced activation of NF-κB 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.

Mitochondrially associated hepatitis B virus X protein constitutively activates transcription factors STAT-3 and NF-kappa B via oxidative stress.

ABSTRACT The hepatitis B virus X protein (HBx) plays essential roles in viral replication and the generation of hepatocellular carcinoma. In spite of a large number of suggestive cellular targets and functions, a clear picture of its mechanism(s) of action has remained elusive. In this report, we continue to characterize its recently described mitochondrial association and further examine its impact on mitochondrial functions. HBx was previously shown to bind to a voltage-dependent anion channel (VDAC3) and alter the mitochondrial transmembrane potential (ΔΨm). Here we show that, as a consequence of association with mitochondria, HBx constitutively induces activation of transcription factors, which include STAT-3 and NF-κB. This induction of activation was sensitive to the antioxidantsN-acetyl l-cysteine and pyrrolidine dithiocarbamate, as well as to overexpression of Mn-superoxide dismutase. These results therefore implicate a potential role of reactive oxygen species (ROS) in a process that ultimately leads to the activation of STAT-3 and NF-κB. Evidence is also presented for the HBx-induced generation of ROS. The ability of HBx to induce the activation of STAT-3 and NF-κB was demonstrated by mobility shift and reporter gene expression assays with lysates from HBx-transfected HepG2 cells. A C-terminal HBx deletion mutant, HBxΔ99, failed to bind VDAC3 and activate STAT-3 and NF-κB. These studies shed new light on the physiological significance of HBxs mitochondrial association and its role in inducing oxidative stress which can contribute to the liver disease pathogenesis associated with the hepatitis B virus infection.

Hepatitis C virus hijacks host lipid metabolism.

Hepatitis C virus (HCV) enhances its replication by modulating host cell lipid metabolism. HCV circulates in the blood in association with lipoproteins. HCV infection is associated with enhanced lipogenesis, reduced secretion, and beta-oxidation of lipids. HCV-induced imbalance in lipid homeostasis leads to steatosis. Many lipids are crucial for the virus life cycle, and inhibitors of cholesterol/fatty acid biosynthetic pathways inhibit virus replication, maturation and secretion. HCV negatively modulates the synthesis and secretion of very low-density lipoproteins (VLDL). Components involved in VLDL assembly are also required for HCV morphogenesis/secretion, suggesting that HCV co-opts the VLDL secretory pathway for its own secretion. This review highlights HCV-altered lipid metabolic events that aid the virus life cycle and ultimately promote liver disease.

Hepatitis B Virus X Protein Colocalizes to Mitochondria with a Human Voltage-Dependent Anion Channel, HVDAC3, and Alters Its Transmembrane Potential

ABSTRACT Understanding the mechanism(s) of action of the hepatitis B virus (HBV)-encoded protein HBx is fundamental to elucidating the underlying mechanisms of chronic liver disease and hepatocellular carcinoma caused by HBV infection. In our continued attempts to identify cellular targets of HBx, we have previously reported the identification of a novel cellular protein with the aid of a yeast two-hybrid assay. This cellular gene was identified as a third member of the family of human genes that encode the voltage-dependent anion channel (HVDAC3). In the present study, physical interaction between HBx and HVDAC3 was established by standard in vitro and in vivo methods. Confocal laser microscopy of transfected cells with respective expression vectors colocalized HVDAC3 and HBx to mitochondria. This novel, heretofore unreported subcellular distribution of HBx in mitochondria implies a functional role of HBx in functions associated with mitochondria. Using a stable cationic fluorophore dye, CMXRos, we show that HBx expression in cultured human hepatoma cells leads to alteration of mitochondrial transmembrane potential. Such functional roles of HBx in affecting mitochondrial physiology have implications for HBV-induced liver injury and the development of hepatocellular carcinoma.

Hepatitis C virus induces proteolytic cleavage of sterol regulatory element binding proteins and stimulates their phosphorylation via oxidative stress.

ABSTRACT Hepatic steatosis is a common histological feature of chronic hepatitis C. Hepatitis C virus (HCV) gene expression has been shown to alter host cell cholesterol/lipid metabolism and thus induce hepatic steatosis. Since sterol regulatory element binding proteins (SREBPs) are major regulators of lipid metabolism, we sought to determine whether genotype 2a-based HCV infection induces the expression and posttranslational activation of SREBPs. HCV infection stimulates the expression of genes related to lipogenesis. HCV induces the proteolytic cleavage of SREBPs. HCV core and NS4b derived from genotype 3a are also individually capable of inducing the proteolytic processing of SREBPs. Further, we demonstrate that HCV stimulates the phosphorylation of SREBPs. Our studies show that HCV-induced oxidative stress and subsequent activation of the phosphatidylinositol 3-kinase (PI3-K)-Akt pathway and inactivation (phosphorylation) of PTEN (phosphatase and tensin homologue) mediate the transactivation of SREBPs. HCV-induced SREBP-1 and -2 activities were sensitive to antioxidant (pyrrolidine dithiocarbamate), Ca2+ chelator 1,2-bis(aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-tetra(acetoxymethyl) ester (BAPTA-AM), and PI3-K inhibitor (LY294002). Collectively, these studies provide insight into the mechanisms of hepatic steatosis associated with HCV infection.

Hepatitis C Virus Subgenomic Replicons Induce Endoplasmic Reticulum Stress Activating an Intracellular Signaling Pathway

ABSTRACT Hepatitis C virus (HCV) replicates from a ribonucleoprotein (RNP) complex that is associated with the endoplasmic reticulum (ER) membrane. The replication activities of the HCV subgenomic replicon are shown here to induce ER stress. In response to this stress, cells expressing HCV replicons induce the unfolded protein response (UPR), an ER-to-nucleus intracellular signaling pathway. The UPR is initiated by the proteolytic cleavage of a transmembrane protein, ATF6. The resulting cytoplasmic protein fragment of ATF6 functions as a transcription factor in the nucleus and activates selective genes required for an ER stress response. ATF6 activation leads to increased transcriptional levels of GRP78, an ER luminal chaperone protein. However, the overall level of GRP78 protein is decreased. While ER stress is also known to affect translational attenuation, cells expressing HCV replicons have lower levels of phosphorylation of the α subunit of eukaryotic initiation factor 2. Interestingly, cap-independent internal ribosome entry site-mediated translation directed by the 5′ noncoding region of HCV and GRP78 is activated in cells expressing HCV replicons. These studies provide insight into the effects of HCV replication on intracellular events and the mechanisms underlying liver pathogenesis.

Hepatitis C virus (HCV) constitutively activates STAT-3 via oxidative stress: role of STAT-3 in HCV replication.

ABSTRACT The hepatitis C virus (HCV) causes chronic hepatitis, which often results in liver cirrhosis and hepatocellular carcinoma. We have previously shown that HCV nonstructural proteins induce activation of STAT-3 via oxidative stress and Ca2+ signaling (G. Gong, G. Waris, R. Tanveer, and A. Siddiqui, Proc. Natl. Acad. Sci. USA 98:9599-9604, 2001). In this study, we focus on the signaling pathway leading to STAT-3 activation in response to oxidative stress induced by HCV translation and replication activities. Here, we demonstrate the constitutive activation of STAT-3 in HCV replicon-expressing cells. The HCV-induced STAT-3 activation was inhibited in the presence of antioxidant (pyrrolidine dithiocarbamate) and Ca2+ chelators (BAPTA-AM and TMB-8). Previous studies have shown that maximum STAT-3 transactivation requires Ser727 phosphorylation in addition to tyrosine phosphorylation. Using a series of inhibitors and dominant negative mutants, we show that HCV-induced activation of STAT-3 is mediated by oxidative stress and influenced by the activation of cellular kinases, including p38 mitogen-activated protein kinase, JNK, JAK-2, and Src. Our results also suggest a potential role of STAT-3 in HCV RNA replication. We also observed the constitutive activation of STAT-3 in the liver biopsy of an HCV-infected patient. These studies provide an insight into the mechanisms by which HCV induces intracellular events relevant to liver pathogenesis associated with the viral infection.

Hepatitis C Virus Stimulates the Expression of Cyclooxygenase-2 via Oxidative Stress: Role of Prostaglandin E2 in RNA Replication

ABSTRACT Hepatitis C virus (HCV) infection is a major cause of chronic liver disease, which can lead to the development of liver cirrhosis and hepatocellular carcinoma. Recently, the activation of cyclooxygenase-2 (Cox-2) has been implicated in the HCV-associated hepatocellular carcinoma. In this study, we focus on the signaling pathway leading to Cox-2 activation induced by HCV gene expression. Here, we demonstrate that the HCV-induced reactive oxygen species and subsequent activation of NF-κB mediate the activation of Cox-2. The HCV-induced Cox-2 was sensitive to antioxidant (pyrrolidine dithiocarbamate), Ca2+ chelator (BAPTA-AM), and calpain inhibitor (N-acetyl-Leu-Leu-Met-H). The levels of prostaglandin E2 (PGE2), the product of Cox-2 activity, are increased in HCV-expressing cells. Furthermore, HCV-expressing cells treated with the inhibitors of Cox-2 (celecoxib and NS-398) showed significant reduction in PGE2 levels. We also observed the enhanced phosphorylation of Akt and its downstream substrates glycogen synthase kinase-3β and proapoptotic Bad in the HCV replicon-expressing cells. These phosphorylation events were sensitive to inhibitors of Cox-2 (celecoxib and NS-398) and phosphatidylinositol 3-kinase (LY294002). Our results also suggest a potential role of Cox-2 and PGE2 in HCV RNA replication. These studies provide insight into the mechanisms by which HCV induces intracellular events relevant to liver pathogenesis associated with viral infection.

Hepatitis C Virus NS5A and Subgenomic Replicon Activate NF-κB via Tyrosine Phosphorylation of IκBα and Its Degradation by Calpain Protease

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-κB via oxidative stress (22). In this study, we investigate the molecular mechanism(s) of NF-κB activation in response to oxidative stress induced by NS5A protein. In contrast to the classic Ser32,36 phosphorylation of IκBα, we report here that tyrosine phosphorylation of IκBα at Tyr42 and Tyr305 residues is induced by the HCV NS5A and the subgenomic replicons in the NF-κB activation process. Use of IκBα-Tyr42,305 double mutant provided the evidence for their key role in the activation of NF-κB. Activation of NF-κB was blocked by a series of tyrosine kinase inhibitors but not by IκB kinase inhibitor BAY 11-7085. More specifically, a ZAP-70 knock-out cell line expressing NS5A and other nonstructural proteins respectively prevented the NF-κB 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 IκBα degradation. These studies collectively define an alternate pathway of NF-κB activation by NS5A alone or in the context of the HCV subgenomic replicon. Constitutive activation of NF-κB by HCV has implications in the chronic liver disease including hepatocellular carcinoma associated with HCV infection.

Endoplasmic reticulum (ER) stress: hepatitis C virus induces an ER-nucleus signal transduction pathway and activates NF-κB and STAT-3

Human hepatitis C virus (HCV) is the leading cause of chronic hepatitis, which often results in liver cirrhosis and hepatocellular carcinoma. The HCV RNA genome codes for at least ten proteins. The HCV non-structural protein 5A (NS5A) has generated considerable interest due to its effect on interferon sensitivity via binding and inactivating the cellular protein kinase, PKR. It has been shown that NS5A engages in the endoplasmic reticulum (ER)-nucleus signal transduction pathway. The expression of NS5A in the ER induces an ER stress ultimately leading to the activation of STAT-3 and NF-kappaB. This pathway is sensitive to inhibitors of Ca(2+) uptake in the mitochondria (ruthenium red), Ca(2+) chelators (TMB-8, EGTA-AM), and antioxidants (PDTC, NAC, Mn-SOD). The inhibitory effect of protein tyrosine kinase (PTK) inhibitors indicates the involvement of PTK in NF-kappaB activation by NS5A. This implicates an alternate pathway of NF-kappaB activation by NS5A. The actions of NS5A have also been studied in the context of an HCV subgenomic replicon inducing a similar intracellular event. Thus, activation of NF-kappaB leads to the induction of cellular genes, which are largely antiapoptotic in function. These studies suggest a potential function of NS5A in inducing chronic liver disease and hepatocellular carcinoma associated with HCV infection.