Hepatitis C virus NS5A and core proteins induce oxidative stress-mediated calcium signalling alterations in hepatocytes.
OBJECTIVE
The hepatitis C virus (HCV) structural core and non-structural NS5A proteins induce in liver cells a series of intracellular events, including elevation of reactive oxygen and nitrogen species (ROS/RNS). Since oxidative stress is associated to altered intracellular Ca(2+) homeostasis, we aimed to investigate the effect of these proteins on Ca(2+) mobilization in human hepatocyte-derived transfected cells, and the protective effect of quercetin treatment.


METHODS
Ca(2+) mobilization and actin reorganization were determined by spectrofluorimetry. Production of ROS/RNS was determined by flow cytometry.


RESULTS
Cells transfected with NS5A and core proteins showed enhanced ROS/RNS production and resting cytosolic Ca(2+) concentration, and reduced Ca(2+) concentration into the stores. Phenylephrine-evoked Ca(2+) release, Ca(2+) entry and extrusion by the plasma membrane Ca(2+)-ATPase were significantly reduced in transfected cells. Similar effects were observed in cytokine-activated cells. Phenylephrine-evoked actin reorganization was reduced in the presence of core and NS5A proteins. These effects were significantly prevented by quercetin. Altered Ca(2+) mobilization and increased calpain activation were observed in replicon-containing cells.


CONCLUSIONS
NS5A and core proteins induce oxidative stress-mediated Ca(2+) homeostasis alterations in human hepatocyte-derived cells, which might underlie the effects of both proteins in the pathogenesis of liver disorders associated to HCV infection.
Dionisio
Natalia
N
Department of Physiology (Cell Physiology Research Group), University of Extremadura, Cáceres, Spain.
Garcia-Mediavilla
Maria V
MV
Sanchez-Campos
Sonia
S
Majano
Pedro L
PL
Benedicto
Ignacio
I
Rosado
Juan A
JA
Salido
Gines M
GM
Gonzalez-Gallego
Javier
J
D000199:Actins / Q000378:metabolism; D000975:Antioxidants / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000502:physiology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D016207:Cytokines / Q000378:metabolism; D016174:Hepacivirus / Q000502:physiology; D006526:Hepatitis C / Q000188:drug therapy / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D006706:Homeostasis / Q000187:drug effects / Q000502:physiology; D006801:Humans; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D011794:Quercetin / Q000494:pharmacology; D026361:Reactive Nitrogen Species / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D014758:Viral Core Proteins / Q000502:physiology*; D017361:Viral Nonstructural Proteins / Q000502:physiology*; D014779:Virus Replication / Q000187:drug effects / Q000502:physiology; D012324:RNA-Dependent RNA Polymerase
D000199:Actins; D000975:Antioxidants; D002118:Calcium; D002135:Calcium-Binding Proteins; D015853:Cysteine Proteinase Inhibitors; D016207:Cytokines; D011794:Quercetin; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; C069130:nucleocapsid protein, Hepatitis C virus; C033668:calpastatin; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase