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

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 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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Calcium-Dependent Calpain Proteases Are Implicated in Processing of the Hepatitis C Virus NS5A Protein

Journal of Virology ◽

10.1128/jvi.78.21.11865-11878.2004 ◽

2004 ◽

Vol 78 (21) ◽

pp. 11865-11878 ◽

Cited By ~ 33

Author(s):

M. Kalamvoki ◽

P. Mavromara

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Replication ◽

Intracellular Calcium ◽

Calcium Homeostasis ◽

Mammalian Cells ◽

Calcium Dependent ◽

Ns5a Protein ◽

Genotype 1A ◽

Immunofluorescence Studies

ABSTRACT The nonstructural 5A (NS5A) protein of the hepatitis C virus (HCV) is a multifunctional phosphoprotein that is implicated in viral replication and HCV-mediated pathogenesis. We report here that the NS5A protein from the HCV genotype 1a is processed into shorter distinct forms when expressed in mammalian cells (Vero, HepG2, HuH-7, and WRL68) infected with an NS5A-expressing HSV-1-based amplicon vector or when transiently transfected with NS5A-expressing plasmids in the absence of exogenous apoptotic stimuli. Inhibitor studies combined with cell-free cleavage assays suggest that calcium-dependent calpain proteases, in addition to caspase-like proteases, are involved in NS5A processing. Interestingly, His-tagging experiments indicated that all the detectable NS5A-cleaved products are N-terminal forms of the protein. Additionally, immunofluorescence studies showed that, despite proteolytic cleavage, the NS5A protein exhibits a cytoplasm-perinuclear localization similar to that of the full-length protein. Thus, our results are consistent with recent data that demonstrated that NS5A is capable of perturbing intracellular calcium homeostasis and suggest that NS5A is both an inducer and a substrate of the calcium-dependent calpain protease(s). This may imply that cleavage of NS5A by calpain(s) could play a role in the modulation of NS5A function.

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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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Branched‐chain amino acids reduce hepatic iron accumulation and oxidative stress in hepatitis C virus polyprotein‐expressing mice

Liver International ◽

10.1111/liv.12675 ◽

2014 ◽

Vol 35 (4) ◽

pp. 1303-1314 ◽

Cited By ~ 16

Author(s):

Masaaki Korenaga ◽

Sohji Nishina ◽

Keiko Korenaga ◽

Yasuyuki Tomiyama ◽

Naoko Yoshioka ◽

...

Keyword(s):

Oxidative Stress ◽

Amino Acids ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Branched Chain Amino Acids ◽

Iron Accumulation ◽

Hepatic Iron ◽

Branched Chain ◽

Virus Polyprotein ◽

And Oxidative Stress

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The hepatitis C virus NS5A protein and response to interferon α: mutational analyses in patients with chronic HCV genotype 3a infection from India

Medical Microbiology and Immunology ◽

10.1007/s00430-006-0024-z ◽

2006 ◽

Vol 196 (1) ◽

pp. 11-21 ◽

Cited By ~ 7

Author(s):

Ankur Goyal ◽

Wolf P. Hofmann ◽

Eva Hermann ◽

Stella Traver ◽

Syed S. Hissar ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Interferon Α ◽

Hcv Genotype ◽

Ns5a Protein ◽

Chronic Hcv ◽

Genotype 3A

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TRIM14 inhibits hepatitis C virus infection by SPRY domain-dependent targeted degradation of the viral NS5A protein

Scientific Reports ◽

10.1038/srep32336 ◽

2016 ◽

Vol 6 (1) ◽

Cited By ~ 29

Author(s):

Shanshan Wang ◽

Yongzhi Chen ◽

Chunfeng Li ◽

Yaoxing Wu ◽

Lei Guo ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Infection ◽

Hepatitis C Virus Infection ◽

Spry Domain ◽

Ns5a Protein

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Recombinant streptavidin fusion proteins as signal reporters in rapid test of human hepatitis C virus infection

Journal of Clinical Laboratory Analysis ◽

10.1002/jcla.22701 ◽

2018 ◽

Vol 33 (3) ◽

pp. e22701

Author(s):

Shengliang Zhou ◽

Shinian Cao ◽

Guoliang Ma ◽

Tielin Ding ◽

Jingjing Mu ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Infection ◽

Fusion Proteins ◽

Rapid Test ◽

Hepatitis C Virus Infection ◽

Human Hepatitis

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Mechanisms of Liver Injury. III. Oxidative stress in the pathogenesis of hepatitis C virus

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00522.2005 ◽

2006 ◽

Vol 290 (5) ◽

pp. G847-G851 ◽

Cited By ~ 204

Author(s):

Jinah Choi ◽

J.-H. James Ou

Keyword(s):

Oxidative Stress ◽

Hepatocellular Carcinoma ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Liver Injury ◽

Iron Overload ◽

Antioxidant Defense ◽

Hcv Infection ◽

Nitrogen Species ◽

Systemic Oxidative Stress

Hepatitis C virus (HCV) is a major cause of viral hepatitis that can progress to hepatic fibrosis, steatosis, hepatocellular carcinoma, and liver failure. HCV infection is characterized by a systemic oxidative stress that is most likely caused by a combination of chronic inflammation, iron overload, liver damage, and proteins encoded by HCV. The increased generation of reactive oxygen and nitrogen species, together with the decreased antioxidant defense, promotes the development and progression of hepatic and extrahepatic complications of HCV infection. This review discusses the possible mechanisms of HCV-induced oxidative stress and its role in HCV pathogenesis.

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