Changes Of Redox State In Host Cells During Dengue Virus Infection

Dengue virus (DV) is a member of mosquito-borne flaviviruses and possesses a positive-sense RNA genome consisting of one single open-reading frame (ORF). The genomic RNA encodes a single polyprotein precursor, NH2-C-prM-E-NS1-NS2A-NS2B- NS3-NS4A-NS4B-NS5-COOH, which is post- and co-translationally cleaved by both host and viral proteases to yield mature proteins. It's transmitted from human to human by the mosquito Aedes aegypti and Aedes albopictus. Infection with DV can result in classical dengue fever (DF) and/or dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). Epidemics of DHF/DSS have more frequent since the 1980s in the tropics and subtropics worldwide such as Southeast Asia and South PRC. Therefore, DV infection has become the severe problem of the public health.Viral multiplication occurs exclusively within the host cell and thus influences 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 viral-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 is a cysteine-containing tripeptide (y-glutamyl-cysteinyl-glycine), found in eukaryotic cells, which has a number of important functions in cell physiology. GSH provides cells with a large supply of reducing equivalents, thus acting as a major cellular antioxidant, via metabolic interconversion with its oxidized disulphide form (GSSG). It was reported that many viruses were associated with oxidative stress resulting in a decrease in the total concentration GSH. Whether cells infected DV had a decrease of antioxidants is not reported. In the present study, we have investigated the effect of HepG2 cell lines infected DV on endogenous GSH content and assayed GSH content of ECV304 cells expressing DV C, prM and prM/E protein and HepG2 cell expressing DV NS3.RESULTS1. Production of antibodies against dengue virus nonstructural protein 3(NS3)①The NS3 gene was successfully amplified from genomic cDNA of DV by PCR.②The PCR product of NS3 gene was inserted into pQE31 vector and transformed into E.coli JM109. The recombinant pQE-NS3/JM109 expressed the fusion protein, nearly all of which was in the inclusion-body.○3 The inclusion body of fusion protein was extract from cultures of stably transformed E.coli JM109, and then dissolved in 8mol/L urea. The fusion NS3 protein was captured by Ni-NTA column.④The purified fusion protein was renatured through dialysis.⑤A rabbit and five Blab/c mouses were immunized subcutaneously with NS3 protein. After three-time immunization, the antiserum was taken. Antiserum preparations were titrated by ELISA assay. The titration of rabbit antisera was more than 1:12 800 and the mouse antisera is 1:6400. The characteristic of the NS3 antibody was analysed by Western blot and Immunofluorescence. The productions of antisera were specific to NS3 protein of DV2 and could recognize native NS3 protein in ECV304 cells infected DV.2. Establishment of DV NS3 stably-expressed HepG2 cellThe NS3 gene was successfully amplified from pQE-NS3 vector by PCR. The PCR product of NS3 gene was inserted into pReceiver vector and transformed into E.coli JM109. The recombinant pRE-NS3/JM109 was obtained. The expression vector pRE-NS3 containing DV NS3 cDNA was transduced into HepG2 cell line by lipid-mediated gene transfection method. The NS3 transfected cells were obtained after being selected with G418. The transfected cells were cloned by a limited dilution method and detected by Western blot and Immunofluorescence. The HepG2 cell strain that expressed DV NS3 protein stably, was established successfully, naming pRE-NS3/HepG2. As control, The HepG2 cell strain containing pReceiver vector was established, naming pReceiver/HepG2.3. The assay of the intracellular GSH level of HepG2 cells infected with DV-2In order to study the effect of viral infection on the intracellular level of GSH, confluent monolayers of HepG2 cells were infected with DV2. The monolayers were carefully washed with PBS and assayed for intracellular GSH. One set of cells was mock-infected and used as control. A significant decrease in intracellular GSH levels during the 24 h after virus infection was detected in DV2 infected cells, reaching 32.8% of the control level. A significant decrease in the GSH content was observed compared with mock cells after 24 h of culture (P < 0.01 between infected and uninfected cells at the same time point). The results indicated that DV can affect the host cell pro-/antioxidant balance.4. The assay of the intracellular GSH level of HepG2 cells expressing DV NS3It was proved that the effect of DV infection on the intracellular level of GSH was involved. We presumed that DV proteins maybe also affect intracellular level of GSH. We studied the levels of GSH in HepG2 cells expressing DV NS3 protein and compared them with control cells pReceiver/HepG2. We found that GSH levels were significantly decreased in pRE-NS3/HepG2 cells reaching 20.9% compared with control cells pReceiver/HepG2. A significant decrease in the GSH content was observed compared with that of pReceiver/HepG2 cell (P < 0.01). We also studied the levels of GSH in ECV304 cells expressing DV C, prM and prM/E protein and compared them to control cells pReceiver/ECV304. GSH levels were significantly decreased 34.5%, 30.1%, 32.8%, respectively compared to control cells. A significant decrease in the GSH content was observed compared with that of pReceiver/ECV304 cell (P < 0.05). No significant difference in the GSH content of ECV304 cells expressing DV C, prM and prM/E proteins was observed (P﹥0.05). Our data indicated that DV NS3, C, prM and prM/E proteins were involved in decrease of intracellular level of GSH.Thus, based on this favourable profile, further research concerning the potential clinical efficacy of GSH against DV infection is warranted.