Effects Of Glutathione On Dengue Virus Production In HepG2 Cell

Dengue virus (DV), belonging to the family of Flaviviridae, is one of the most widespread mosquito-borne human pathogens worldwide. There are four serotypes (DV1–4) and their genomes contain a single open-reading frame of approximately 11 Kb encoding a polyprotein precursor that is proteolitically cleaved into three structural proteins [capsid (C), premembrane (prM), and envelope (E)] and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). DV causes classical dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). These diseases have emerged as significant threats to human health in affected areas. Nevertheless, the specific viral mechanisms involved in dengue infection remain unclear and no specific anti-viral drug is available as yet.Viral replication occurs exclusively within the host cell and thus depends on numerous factors that control cell machinery and metabolism. Several findings have demonstrated the involvement of the intracellular redox balance in the establishment of viral infection and the progression of virus-induced diseases. Reducing conditions are normally maintained within the cell by molecules such as glutathione (GSH), superoxide dismutase, thioredoxin, and catalase, which constitute the system developed by cells to counteract oxidation. GSH, a cysteine-containing tripeptide, is the most important and ubiquitous antioxidant molecule of eukaryotic cells. The resultant decrease in GSH may contribute to viral pathogenesis, regulation of viral replication, host defense, and modulation of cellular responses.Previous studies have demonstrated that cultured cells infected with herpes simplex virus type 1, Sendai virus, human immunodeficiency virus (HIV), influenza virus, and hepatitis C virus have decreased intracellular GSH levels, increased generation of reactive oxygen species (ROS), and enhanced oxidation of the cellular GSH pool. Evidence has accumulated that suggests that redox mechanisms play a fundamental role in cellular events. The most striking example is the effect of oxidative stress on redox-responsive transcription factors, such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1), which activate gene transcription in response to peroxide. The NF-κB pathway is a ubiquitous protein system that regulates the expression of many genes, including numerous cellular and viral genes. NF-κB-activating stimuli generally seem to use the oxidative stress pathway as a common signal transduction pathway to elicit their responses. Intracellular thiols play a key role in regulating NF-κB activation; low thiol levels are required for NF-κB activation and high levels inhibit NF-κB activation. However, the relationship between GSH levels and NF-κB activity remains unknown during DV infection.Recently, a mouse model further confirmed that the liver might be an important target organ for DV, and human hepatoma cell line, HepG2, could support DV2 replication as well as oxidative damage could be observed in dengue fever patients. Therefore, in the present study, GSH levels and NF-κB activation were investigated to explore the role of cellular redox in DV2 production in the infected HepG2. The main results were as follows:1. Alterations of intracellular GSH levels during DV2 infectionIn order to investigate the effect of DV2 infection on the intracellular level of GSH, HepG2 cells were infected with DV2 and levels of intracellular GSH were assayed at different time points post-infection. It was shown that DV2 infection caused a time-dependent alteration in the intracellular GSH content. At early stages of the infection, the decrease of GSH levels occurred at the beginning of DV2 adsorption and the lowest GSH value, about 73% as compared with mock infection (p < 0.01), was seen at 30 min after adsorption. At the end of adsorption (1 h), the GSH levels tended to recover, but the values were always significantly lower than those observed in mock-infected cells. At late stages of infection, a significant decrease in intracellular GSH levels was detected in DV2-infected cells at different time points, and the values at 2, 6, 12, and 24 h after infection were as low as 83%, 91%, 89%, and 67%, respectively, compared with mock-infected cells (p < 0.01). GSH levels tended to recover and reached normal levels at 48 h after infection. Meanwhile, large amounts of GSH were detected in supernatants of infected cells at 30 min, but not at 24 h, as compared with that of controls and mock-infected cells. These results indicated that DV2 infection could affect the host cells'intracellular levels of GSH.2. Expression of DV-E or DV-NS3 proteins decrease intracellular GSH levels in transfected HepG2 cellsFirst, HepG2 cell lines stably expressing prM/E or NS3 proteins were established. Immunostaining of both pRe-NS3/HepG2 and pRe-E/HepG2 cells showed diffuse fluorescence were noted in the perinuclear region or cytoplasm. With immunoblot analyses, polypeptide bands of ~70 kDa and ~55 kDa were detected in pRe-NS3/HepG2 or pRe-E/HepG2 cells, and the two bands corresponded to the theoretical masses of NS3 and prM/E proteins, respectively, suggesting that DV2 NS3 and prM/E proteins were expressed in the HepG2 cells. As control, the pCI-GFP/HepG2 expressing green fluorescent protein and pRe/HepG2 cells were established.We proved that DV2 infection caused decreases in the intracellular level of GSH. It is presumed that DV proteins may be involved in this process. For this purpose, we investigated the effect of the DV NS3 and E proteins as well as GFP expression on GSH levels using pRe-NS3/HepG2, pRe-E/HepG2, and pCI-GFP/HepG2 cell lines and compared them with the pRe/HepG2 control cell line. Intracellular GSH levels were significantly decreased by 79% and 77% in pRe-NS3/HepG2 and pRe-E/HepG2 cells, respectively, as compared with pRe/HepG2 control cells (p<0.01). In addition, GSH levels in the supernatants of pRe-NS3/HepG2 and pRe-E/HepG2 cells were significantly decreased, by 65% and 64%, respectively, as compared to that of control cells (p<0.05). In contrast, GFP expression showed little effect on both intracellular and extracellular GSH levels as compared with pRe/HepG2 controls. Our data indicated that DV NS3 and E proteins were closely associated with a decrease in intracellular GSH levels.3. Effect of exogenous GSH on DV2 infection in HepG2 cellsTo investigate the effects of GSH on DV2 infection in vitro, the cytotoxicity of these drugs to HepG2 cells was determined by monitoring their morphology and their ability to exclude the trypan blue stain. HepG2 cells were infected with DV2 and treated with GSH at the concentration of 10 mM and 20 mM respectively. Subsequently, the cells were cultured for 24 h in the presence of GSH and then intracellular viral titers were assessed. GSH significantly inhibited viral production in a dose-dependent manner. At 10 mM and 20 mM GSH, DV2 titers in supernatants decreased significantly, to 62% and 40% , respectively, as a percentage of infection alone (p<0.05) , indicating that treatment of cells with exogenous GSH inhibited virus production, but did not alter intracellular GSH levels (p>0.05). Therefore, we conclude that GSH conferred substantial protection against DV2 infection in HepG2 cells.4. Effect of treatment with BSO on DV2 infection in HepG2 cells To further confirm the relationship between intracellular GSH levels and DV2 infection, HepG2 cells were treated with BSO, which is a well-known inhibitor of GSH synthesis, and then infected with DV2. Treatment of cells with 0.2 mM or 1 mM BSO caused a decrease in intracellular levels of GSH of about 20% compared with that in HepG2 cells infected alone (p<0.05). In contrast, DV2 titers were two-fold higher than those of untreated infected cells, reaching 211% and 215% as a percentage of untreated infected cells (p< 0.05).5. DV2 and E NS3 protein induce NF-κB activation in DV2-infected cells To investigate the effect of alteration of intracellular GSH levels induced by DV2 infection on the redox-responsive transcription factor, NF-κB, HepG2 cells transfected with vectors containing NF-κB promoter regions were infected with DV2 and transcription activity was assayed. NF-κB activity was recorded as a percentage of mock infection. The results are that decreasion of inhibitor of NF-κB (IκB) was found at 24h in DV-infected HepG2 cells. NF-κB activity in DV2-infected HepG2 cells and GSH-treated infected cells was 184% and 124%, respectively, as compared to mock-infected HepG2 cells and that of GSH-treated mock-infected cells was no difference (p>0.05). Remarkably, NF-κB activity was as high as 271% in BSO-treated infected HepG2 cells (p<0.05). Meanwhile, the effects of expression of DV2 NS3 and E proteins on NF-kB activity were also assayed; we observed 309% and 243% NF-κB activity in pRe-NS3/HepG2 and pRe-E/HepG2 cells, respectively, as compared to pRe/HepG2 controls (p<0.05). As target gene of NF-kB, the production of IL-6 was increased at 48 h after infection compared with mock infection (p<0.01). However, there are no obvious changes in levels of TNF-αand IL-8 during the observed period. These results indicated that NF-κB activation could be induced by DV2 infection, or DV2 E or NS3 protein expression, and was closely associated with GSH levels in host cells.In summary, this study demonstrated that infection with DV2, as well as expression of DV E or NS3 proteins influenced the host's intracellular GSH concentration. Decreased GSH led to activation of NF-κB and a subsequent increase in DV2 production. Supplemental GSH significantly inhibited activation of NF-κB, resulting in decreased production of DV2 in HepG2 cells. Furthermore, treatment of HepG2 cells with BSO caused high activity of NF-κB and increased production of DV2. Our results thus suggest that GSH may inhibit DV2 production through modulations of NF-κB activity and may therefore be useful in the prevention of DV2 infection.