Hepatitis C Virus NS5A Protein Triggers Oxidative Stress by Inducing NADPH Oxidases 1 and 4 and Cytochrome P450 2E1

Replication of hepatitis C virus (HCV) is associated with the induction of oxidative stress, which is thought to play a major role in various liver pathologies associated with chronic hepatitis C. NS5A protein of the virus is one of the two key viral proteins that are known to trigger production of reactive oxygen species (ROS). To date it has been considered that NS5A induces oxidative stress by altering calcium homeostasis. Herein we show that NS5A-induced oxidative stress was only moderately inhibited by the intracellular calcium chelator BAPTA-AM and not at all inhibited by the drug that blocks the Ca2+flux from ER to mitochondria. Furthermore, ROS production was not accompanied by induction of ER oxidoreductins (Ero1), H2O2-producing enzymes that are implicated in the regulation of calcium fluxes. Instead, we found that NS5A contributes to ROS production by activating expression of NADPH oxidases 1 and 4 as well as cytochrome P450 2E1. These effects were mediated by domain I of NS5A protein. NOX1 and NOX4 induction was mediated by enhanced production of transforming growth factorβ1 (TGFβ1). Thus, our data show that NS5A protein induces oxidative stress by several multistep mechanisms.

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P228 HEPATITIS C VIRUS NS5A PROTEIN TRIGGERS OXIDATIVE STRESS BY INDUCING NADPH OXIDASES 1 AND 4 AND CYTOCHROME P450 2E1

Journal of Hepatology ◽

10.1016/s0168-8278(14)60390-1 ◽

2014 ◽

Vol 60 (1) ◽

pp. S142

Author(s):

O.A. Smirnova ◽

O.N. Ivanova ◽

F. Mukhtarov ◽

B. Bartosch ◽

S.N. Kochetkov ◽

...

Keyword(s):

Oxidative Stress ◽

Cytochrome P450 ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Cytochrome P450 2E1 ◽

Nadph Oxidases ◽

Ns5a Protein

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Hepatitis C Virus (HCV) Proteins Induce NADPH Oxidase 4 Expression in a Transforming Growth Factor β-Dependent Manner: a New Contributor to HCV-Induced Oxidative Stress

Journal of Virology ◽

10.1128/jvi.01059-09 ◽

2009 ◽

Vol 83 (24) ◽

pp. 12934-12946 ◽

Cited By ~ 91

Author(s):

Howard E. Boudreau ◽

Suzanne U. Emerson ◽

Agnieszka Korzeniowska ◽

Meghan A. Jendrysik ◽

Thomas L. Leto

Keyword(s):

Oxidative Stress ◽

Hepatitis C Virus ◽

Liver Disease ◽

Hepatitis C ◽

Growth Factor ◽

Nadph Oxidase ◽

Transforming Growth Factor ◽

Transforming Growth Factor Β ◽

Dominant Negative ◽

Ros Production

ABSTRACT Viral hepatitis-induced oxidative stress accompanied by increased levels of transforming growth factor β (TGF-β) and hepatic fibrosis are hallmarks of hepatitis C virus (HCV) infection. The mechanisms of redox regulation in the pathogenesis of HCV-induced liver disease are not clearly understood. The results of our current studies suggest that reactive oxygen species (ROS) derived from Nox4, a member of the NADPH oxidase (Nox) family, could play a role in HCV-induced liver disease. We found that the expression of HCV (genotype 1a) cDNA constructs (full-length and subgenomic), core protein alone, viral RNA, or replicating HCV (JFH-AM2) induced Nox4 mRNA expression and ROS generation in human hepatocyte cell lines (Huh-7, Huh-7.5, HepG2, and CHL). Conversely, hepatocytes expressing Nox4 short hairpin RNA (shRNA) or an inactive dominant negative form of Nox4 showed decreased ROS production when cells were transfected with HCV. The promoters of both human and murine Nox4 were used to demonstrate transcriptional regulation of Nox4 mRNA by HCV, and a luciferase reporter tied to an ∼2-kb promoter region of Nox4 identified HCV-responsive regulatory regions modulating the expression of Nox4. Furthermore, the human Nox4 promoter was responsive to TGF-β1, and the HCV core-dependent induction of Nox4 was blocked by antibody against TGF-β or the expression of dominant negative TGF-β receptor type II. These findings identified HCV as a regulator of Nox4 gene expression and subsequent ROS production through an autocrine TGF-β-dependent mechanism. Collectively, these data provide evidence that HCV-induced Nox4 contributes to ROS production and may be related to HCV-induced liver disease.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.171311298 ◽

2001 ◽

Vol 98 (17) ◽

pp. 9599-9604 ◽

Cited By ~ 430

Author(s):

G. Gong ◽

G. Waris ◽

R. Tanveer ◽

A. Siddiqui

Keyword(s):

Oxidative Stress ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Intracellular Calcium ◽

Ns5a Protein ◽

Human Hepatitis

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Hepatitis C virus core protein, cytochrome P450 2E1, and alcohol produce combined mitochondrial injury and cytotoxicity in hepatoma cells

Gastroenterology ◽

10.1053/j.gastro.2004.10.045 ◽

2005 ◽

Vol 128 (1) ◽

pp. 96-107 ◽

Cited By ~ 102

Author(s):

Kazuhiro Otani ◽

Masaaki Korenaga ◽

Michael R. Beard ◽

Kui Li ◽

Ting Qian ◽

...

Keyword(s):

Cytochrome P450 ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Core Protein ◽

Hepatoma Cells ◽

Cytochrome P450 2E1 ◽

Mitochondrial Injury ◽

Virus Core

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Hepatitis C Virus Proteins Core and NS5A Are Highly Sensitive to Oxidative Stress-Induced Degradation after eIF2α/ATF4 Pathway Activation

Viruses ◽

10.3390/v12040425 ◽

2020 ◽

Vol 12 (4) ◽

pp. 425

Author(s):

W. Alfredo Ríos-Ocampo ◽

María-Cristina Navas ◽

Manon Buist-Homan ◽

Klaas Nico Faber ◽

Toos Daemen ◽

...

Keyword(s):

Oxidative Stress ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Replication ◽

Protein Degradation ◽

Cellular Stress ◽

Selective Degradation ◽

Ns5a Protein ◽

Adaptive Mechanisms ◽

Huh7 Cells

Hepatitis C virus (HCV) infection is accompanied by increased oxidative stress and endoplasmic reticulum stress as a consequence of viral replication, production of viral proteins, and pro-inflammatory signals. To overcome the cellular stress, hepatocytes have developed several adaptive mechanisms like anti-oxidant response, activation of Unfolded Protein Response and autophagy to achieve cell survival. These adaptive mechanisms could both improve or inhibit viral replication, however, little is known in this regard. In this study, we investigate the mechanisms by which hepatocyte-like (Huh7) cells adapt to cellular stress in the context of HCV protein overexpression and oxidative stress. Huh7 cells stably expressing individual HCV (Core, NS3/4A and NS5A) proteins were treated with the superoxide anion donor menadione to induce oxidative stress. Production of reactive oxygen species and activation of caspase 3 were quantified. The activation of the eIF2α/ATF4 pathway and changes in the steady state levels of the autophagy-related proteins LC3 and p62 were determined either by quantitative polymerase chain reaction (qPCR) or Western blotting. Huh7 cells expressing Core or NS5A demonstrated reduced oxidative stress and apoptosis. In addition, phosphorylation of eIF2α and increased ATF4 and CHOP expression was observed with subsequent HCV Core and NS5A protein degradation. In line with these results, in liver biopsies from patients with hepatitis C, the expression of ATF4 and CHOP was confirmed. HCV Core and NS5A protein degradation was reversed by antioxidant treatment or silencing of the autophagy adaptor protein p62. We demonstrated that hepatocyte-like cells expressing HCV proteins and additionally exposed to oxidative stress adapt to cellular stress through eIF2a/ATF4 activation and selective degradation of HCV pro-oxidant proteins Core and NS5A. This selective degradation is dependent on p62 and results in increased resistance to apoptotic cell death induced by oxidative stress. This mechanism may provide a new key for the study of HCV pathology and lead to novel clinically applicable therapeutic interventions.

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Nonstructural 5A Protein of Hepatitis C Virus Regulates Soluble Resistance-Related Calcium-Binding Protein Activity for Viral Propagation

Journal of Virology ◽

10.1128/jvi.02493-15 ◽

2015 ◽

Vol 90 (6) ◽

pp. 2794-2805 ◽

Cited By ~ 7

Author(s):

Giao V. Q. Tran ◽

Trang T. D. Luong ◽

Eun-Mee Park ◽

Jong-Wook Kim ◽

Jae-Woong Choi ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protein Interaction ◽

Calcium Binding ◽

Crucial Role ◽

Binding Protein ◽

Binding Activity ◽

Calcium Binding Protein ◽

Virus Propagation ◽

Ns5a Protein

ABSTRACTHepatitis C virus (HCV) is a major cause of chronic liver disease and is highly dependent on cellular proteins for virus propagation. To identify the cellular factors involved in HCV propagation, we recently performed protein microarray assays using the HCV nonstructural 5A (NS5A) protein as a probe. Of 90 cellular protein candidates, we selected the soluble resistance-related calcium-binding protein (sorcin) for further characterization. Sorcin is a calcium-binding protein and is highly expressed in certain cancer cells. We verified that NS5A interacted with sorcin through domain I of NS5A, and phosphorylation of the threonine residue 155 of sorcin played a crucial role in protein interaction. Small interfering RNA (siRNA)-mediated knockdown of sorcin impaired HCV propagation. Silencing of sorcin expression resulted in a decrease of HCV assembly without affecting HCV RNA and protein levels. We further demonstrated that polo-like kinase 1 (PLK1)-mediated phosphorylation of sorcin was increased by NS5A. We showed that both phosphorylation and calcium-binding activity of sorcin were required for HCV propagation. These data indicate that HCV modulates sorcin activity via NS5A protein for its own propagation.IMPORTANCESorcin is a calcium-binding protein and regulates intracellular calcium homeostasis. HCV NS5A interacts with sorcin, and phosphorylation of sorcin is required for protein interaction. Gene silencing of sorcin impaired HCV propagation at the assembly step of the HCV life cycle. Sorcin is phosphorylated by PLK1 via protein interaction. We showed that sorcin interacted with both NS5A and PLK1, and PLK1-mediated phosphorylation of sorcin was increased by NS5A. Moreover, calcium-binding activity of sorcin played a crucial role in HCV propagation. These data provide evidence that HCV regulates host calcium metabolism for virus propagation, and thus manipulation of sorcin activity may represent a novel therapeutic target for HCV.

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