Differential contribution of hepatitis C virus NS5A and core proteins to the induction of oxidative and nitrosative stress in human hepatocyte-derived cells.
OBJECTIVE
We aimed to explore the effects of hepatitis C virus (HCV) core and NS5A proteins on reactive oxygen (ROS) and nitrogen species (RNS) formation and on gene expression profile of iNOS in human hepatocyte-derived cells.


METHODS
Production of ROS and RNS and nitrotyrosine residues accumulation were determined by flow cytometry and fluorescent microscopy as well as by Western blot, respectively, in NS5A- and core-transfected cells. Northern blot, Western blot, real-time PCR, and luciferase assays were used to assess iNOS gene expression in both transfectants.


RESULTS
Cytokine-activated NS5A- and core-transfected cells induced ROS and RNS production but an earlier and more marked increase was observed in NS5A-expressing cells. Superoxide production was also augmented, showing a similar temporal pattern of appearance in both NS5A- and core-transfected cells. Although both NS5A and core HCV proteins were able to up-regulate iNOS gene expression, accompanied by a nitrotyrosine-containing proteins accumulation, an earlier iNOS overexpression was observed in NS5A-expressing cells, suggesting a different time course of iNOS activation pattern for core and NS5A HCV proteins.


CONCLUSIONS
Our results indicate a differential contribution of both HCV proteins to oxidative and nitrosative stress generation.
García-Mediavilla
María Victoria
MV
Departamento de Fisiología, Universidad de León, Campus Universitario, 24071 León, Spain.
Sánchez-Campos
Sonia
S
González-Pérez
Pilar
P
Gómez-Gonzalo
Marta
M
Majano
Pedro Lorenzo
PL
López-Cabrera
Manuel
M
Clemente
Gerardo
G
García-Monzón
Carmelo
C
González-Gallego
Javier
J
D002460:Cell Line; D005434:Flow Cytometry; D017930:Genes, Reporter; D016174:Hepacivirus / Q000502:physiology*; D022781:Hepatocytes / Q000502:physiology* / Q000821:virology*; D006801:Humans; D007249:Inflammation; D008156:Luciferases / Q000032:analysis / Q000235:genetics; D008954:Models, Biological; D009566:Nitrates / Q000378:metabolism*; D017672:Nitrogen Compounds; D018384:Oxidative Stress; D016133:Polymerase Chain Reaction; D017382:Reactive Oxygen Species; D014162:Transfection; D014758:Viral Core Proteins / Q000502:physiology*
D009566:Nitrates; D017672:Nitrogen Compounds; D017382:Reactive Oxygen Species; D014758:Viral Core Proteins; C069130:nucleocapsid protein, Hepatitis C virus; D008156:Luciferases