Exploitation Of The Microtubule Cytoskeleton During Dengue Virus Infection

Dengue virus (DV) is a member of mosquito-borne flaviviruses and possesses a positive-sense RNA genome. 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. The life cycle of DV can be divided into three essential steps: entry into a host cell, replication, and egress to ultimately infect another cell. Previous experiments showed that many viruses take advantage of microtubule for their long range retrograde and anterograde transport. Therefore, we considered that microtubule may play a role during DV infection. Two kinds of drugs are commonly used to perturb the microtubule treadmilling cycle: one binds to the tubulin and prevents its addition to the plus ends inhibiting the polymerization of microtubule and disrupting it, such as demecolcine, nocodazole; the other stabilizes microtubule by binding to a polymer, such as paclitaxel and taxol. In the present study, we used demecolcine ,nocodazole and paclitaxel separately to investigate whether mocrotubule is employed in the dengue virus serotype 2 (DV2) infection of ECV304 and HepG2 cell lines. RESULTES 1. Growth course of DV in ECV304 cells In our experiments, ECV304 cells were infected with DV(MOI=1) and virus titer was detected at different time point after infection. It increased gradually, reached peak value at 72h post-infection with titer of 106PFU/ml and than decreased. We also detected virus titer at early stage of the infection. Our data showed that virus titer were 1.10×10~3, 1.20×10~3, 4.25×103, 1.05×104 PFU/ml at 0min, 2h, 5h, 8h post-infection respectively. The results indicated that DV might begin to release during 5-8h post-infection. 2. DV-2 infection induced reorganization microtubule network, DV-2 antigen and microtubule showed co-location in ECV304 cell line. In course of our experiments, indirect immunofluorescence staining showed that the normal morphology of the microtubule cytoskeleton was replaced by morphologically aberrant microtubule forms at 24 h post-infection. These aberrant forms could be broadly classified into three types: (1) cells with a disorganized microtubule network where microtubule seem randomly oriented; (2) cells in which microtubule form rings around the nucleus and throughout the cytoplasm; (3) cells with long projections consisting of microtubule bundles. The similar reorganization of the microtubule cytoskeleton were also observed by 48 h post-infection. Indirect double immuno-staining also showed that the distribution of viral antigen were concentrated in the area coinciding with the center of the microtubule or the peri-nuclear. With the process of infection, the number of DV antigen-positive cells and load of DV antigens in the cytoplasm had dominant increasing. DV antigens accumulation often co-localized with microtubule bundles. Moreover, co-localization of DV-2 antigen and microtubule were often observed in ECV304 cell line using laser confocal scanning microscope. Our data indicated that microtubule may play an important role in DV infection. 3. Demecolcine and paclitaxel did not impair ECV304 cells endocytosis In our experiments, we confirmed that demecolcine or paclitaxel did not impair the cell internalization mechanism by observing the internalization of FITC-dextran,which is a routine test to evaluate the integrity of the cellular endocytic pathways. ECV304 cells were treated with demecolcine or paclitaxel. After 2 h treatment, culture media were replaced by fresh media containing FITC-dextran. Cells were incubated for other five hours and then were washed to remove extracellular FITC-dextran. At this point they were fixed with standard techniques. Samples were observed with fluorescence microscope. All the samples showed the same pattern of the distribution of internalized FITC-dextran as cytoplasm fluorescent dots, no matter with or without the drug treatment on the cells. This result indicated that the cell endocytic pathways remained functional in the presence of demecolcine or paclitaxel.4. Demecolcine and paclitaxel have less effect on DV entry In our experiments, cells were pretreated with demecolcine or paclitaxel for 1 h at 37°C, then culture media were replaced by media containing DV and drugs for 1 h at 37°C. At 5h post-infection, titers of extracellular and intracellular virus have no significant difference between the groups treated with drugs and with infection alone. The results showed that treatment with demecolcine or paclitaxel did not inhibit of DV internalization. 5. Demecolcine induces an enhancement of the DV release, whereas paclitaxel has less effect on DV release. From our results of primary experiments, we suggest that the DV began to release during 5~8 h post-infection. In our experiments, demecolcine and paclitaxel were added at 5h post-infection respectively. Infected cells were treated with drugs for 3h at 37°C prior to measuring virus release. Our data showed that in group treated with demecolcine, levels of extracellular virus were higher than that in group with infection alone. At the same time, the intracellular virus titer was lower than untreated group. In contrast, when cells were treated with paclitaxel, there was no effect on DV titer. This indicated that demecolcine induced disruption of microtubule may enhance DV release. For further demonstration role of the microtubule in DV infection, ECV304 and HepG2 cells were pretreated and infected in the presence of demecolcine or nocodazole. Cells were left in the presence of these drugs until 8h or 24h postin-fection. In group treated with demecolcine or nocodazole, levels of extracellular virus were higher than that in group with infection alone. Compared with untreated group, levels of intracellular virus have not significant difference. These results suggest that disruption of the microtubule may enhance DV infection in ECV304 and HepG2 cells.