Abstract
Hepatitis C virus (HCV) is a blood-borne pathogen causing acute and chronic hepatitis. A significant number of people chronically infected with HCV develop cirrhosis and/or liver cancer. The pathophysiologic mechanisms of hepatocyte damage associated with chronic HCV infection are not fully understood yet, mainly due to the lack of an in vitro system able to recapitulate the stages of infection in vivo. Several studies underline that HCV virus replication depends on redox-sensitive cellular pathways; in addition, it is known that virus itself induces alterations of the cellular redox state. However, the exact interplay between HCV replication and oxidative stress has not been elucidated. In particular, the role of reduced glutathione (GSH) in HCV replication and infection is still not clear. We set up an in vitro system, based on low m.o.i. of Huh7.5 cell line with a HCV infectious clone (J6/JFH1), that reproduced the acute and persistent phases of HCV infection up to 76 days of culture. We demonstrated that the acute phase of HCV infection is characterized by the elevated levels of reactive oxygen species (ROS) associated in part with an increase of NADPH-oxidase transcripts and activity and a depletion of GSH accompanied by high rates of viral replication and apoptotic cell death. Conversely, the chronic phase is characterized by a reestablishment of reduced environment due to a decreased ROS production and increased GSH content in infected cells that might concur to the establishment of viral persistence. Treatment with the prooxidant auranofin of the persistently infected cultures induced the increase of viral RNA titer, suggesting that a prooxidant state could favor the reactivation of HCV viral replication that in turn caused cell damage and death. Our results suggest that targeting the redox-sensitive host-cells pathways essential for viral replication and/or persistence may represent a promising option for contrasting HCV infection.
Highlights
Hepatitis C virus (HCV), an RNA virus belonging to the Flaviviridae family, represents a major worldwide concern causing about 400,000 deaths worldwide every year [1]
Cell culture viability was monitored at different time points, and viral replication was evaluated by both real-time room temperature (RT)-PCR and immunofluorescence assay for HCV core protein
During the first two passages (8 days postinfection), about 80% of the cells were stained for HCV core protein, indicating a high rate of HCV replication corresponding to the “acute” phase of infection (Figure 1(a) left side)
Summary
Hepatitis C virus (HCV), an RNA virus belonging to the Flaviviridae family, represents a major worldwide concern causing about 400,000 deaths worldwide every year [1]. An imbalance between the reactive oxygen species (ROS) production and their clearance by scavenging molecules, has Oxidative Medicine and Cellular Longevity been recognized as a leading factor in inducing hepatocyte death, inflammation, and fibrogenesis, which are responsible for induction and perpetuation of liver damage [5]. Other studies report an increase in the antioxidant defenses, such as superoxide dismutase (SOD), peroxiredoxin (PRDX), glutathione S-transferase (GST) enzyme activity, and GSH levels [14, 20,21,22,23]. Hepatocytes overexpressing HCV core protein have reduced GSH levels and increased the oxidation of thioredoxin (Trx), while the overexpression of viral NS5A protein (known for its ability to cause oxidative stress) [16] increases antioxidant enzymes (MnSOD and catalase), heme oxygenase-1 (HO-1), and GSH content. In patients chronically infected with genotype 1a/b, a sharp decrease of reduced GSH level has been observed with respect to the other genotypes, suggesting the more serious disease associated with this genotype [29]
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