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13:04
A Protocol for Analyzing Hepatitis C Virus Replication
Published on: June 26, 2014
Redox modulation of the hepatitis C virus replication complex is calcium dependent.
Jinah Choi1, Henry Jay Forman, Jing-hsiung Ou
1School of Natural Sciences, University of California at Merced, CA 95344, USA. jchoi@ucmerced.edu
Free Radical Biology & Medicine
|October 7, 2006
Summary
Hydrogen peroxide (H2O2) disrupts hepatitis C virus (HCV) replication by increasing intracellular calcium. This calcium elevation, originating from the endoplasmic reticulum, is key to suppressing HCV RNA replication, suggesting a role for redox and calcium signaling in viral infections.
Area of Science:
- Molecular Virology
- Cellular Redox Biology
- Hepatitis C Virus (HCV) Pathogenesis
Background:
- Redox balance perturbation is linked to viral disease pathogenesis, including hepatitis C.
- Previous studies indicated hydrogen peroxide (H2O2) disrupts the HCV replication complex (RC) without harming host cells, suggesting a signaling role.
Purpose of the Study:
- To investigate the mechanism by which H2O2 suppresses HCV RNA replication.
- To determine the role of intracellular calcium in H2O2-mediated inhibition of the HCV RC.
Main Methods:
- HCV subgenomic and genomic RNA replication assays in Huh7 cells.
- Measurement of intracellular calcium concentration ([Ca2+]i) using fluorescent indicators.
- Experiments involving extracellular and intracellular calcium manipulation.
Main Results:
- H2O2 and interferon-gamma exhibited comparable suppression of HCV RNA replication.
- H2O2 induced a gradual increase in [Ca2+]i, which was essential for both rapid and sustained suppression of HCV RNA replication.
- The H2O2-induced [Ca2+]i elevation and subsequent HCV RC suppression were independent of extracellular calcium but required intracellular calcium stores, likely from the endoplasmic reticulum.
Conclusions:
- Oxidants, such as H2O2, may modulate the HCV replication complex through calcium signaling.
- Elevated intracellular calcium appears sufficient to suppress HCV RNA replication.
- These findings highlight potential regulatory roles for redox and calcium signaling in viral infections.
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