Obtain an overall assessment of DENV spread and infection in these cell lines. In the foci count, free virus transmission is limited by 0.5 Title Loaded From File methocellulose in the medium. Among these three cell lines at high MOI infection, no obvious difference was observed in the intracellular DENV 4G2 protein and viral RNA levels (Fig. 4). This observation implied that BST2 and its variant did not inhibit DENV viral entry, viral replication, and 18325633 protein translation. While DENV was freely transmitted in the infection system without restriction of 0.5 methocellulose, intracellular 4G2 protein markedly increased in all three cell lines at high MOI infection, whereas BST2 still moderately inhibited viral replication in Huh7-BST2 cells (Fig. 5). These results suggest that multiple rounds of infection from progeny virions occurred in this free transmission system that was partially inhibited by BST2.BST2 inhibits virion release and cell-to-cell transmissionThe low MOI infection plot, as shown in Fig. 4A, shows the representative DENV-infected cell foci from the cultures of the three cell lines. The quantitative analysis showed that theTetherin Inhibits DENV SecretionFigure 2. Immunofluorescent staining for DENV infection in Huh7-BST2 and Huh7-BST2CV5 cells. Cells were infected with DENV at indicated MOI and harvested on day 2. Cells were double-stained for DENV envelope protein 4G2 (top panel, green) and BST2 (middle panel, red). Cell nuclei were stained with DAPI (bottom panel, blue). doi:10.1371/journal.pone.0051033.ginfectious foci per well were decreased to about 30 by BST2 (Fig. 4B). However, BST2CV5 did not exert any effect. The average DENV-positive cell number per foci is 18297096 238 in Huh7 cells, whereas the cell numbers in the Huh7-BST2 and Huh7BST2CV5 cell foci were 78 and 175, respectively. Altogether, the expression of BST2 but not BSTCV5 inhibited DENV release and cell-to-cell transmission in Huh7 cells.DiscussionBST2 is a transmembrane protein that contains a short Nterminal cytoplasmic domain, a membrane-spanning alpha-helix, a coiled-coil ectodomain, and a C-terminal GPI anchor [31]. This antiviral protein localizes at the plasma membrane as well as themembranes of multiple intracellular vesicles, including endosomes and the trans-Golgi network [32,33]. At the plasma membrane, BST2 is found within cholesterol-enriched lipid rafts, presumably due to its C-terminal GPI modification. This optimally positions BST2 to interfere directly with virion release, since several lipidenveloped viruses, including HIV-1 and Ebola, bud selectively from raft domains [34?8]. Consistent with these reports, our results showed that BST2 localizes to both the cell membrane and cytoplasm. The addition of the V5 tag at the C-terminus of BST2 demonstrated an altered intracellular distribution (Fig. 1). Furthermore, similar as previous report [25,39], we found that three bands of BST2 distributed in the range from 30 to 36kd by western blot. We supposed that the different level of modification of BST2 likely cause the different size of BST2. However, forFigure 3. Viral The assays. Thus, patients in the MC and NE groups had infectivity detection of supernatant DENV in Huh7-BST2 and Huh7-BST2CV5 cells. The viral infectivity of supernatant DENV was determined by TCID50 method. The cells were infected with DENV at indicated MOI for 1 h; the media were replaced with complete media and cultured for 2 days. Dengue E protein was assayed by A cell-based flavivirus immunodetection assay. The values represent average from 3 independen.Obtain an overall assessment of DENV spread and infection in these cell lines. In the foci count, free virus transmission is limited by 0.5 methocellulose in the medium. Among these three cell lines at high MOI infection, no obvious difference was observed in the intracellular DENV 4G2 protein and viral RNA levels (Fig. 4). This observation implied that BST2 and its variant did not inhibit DENV viral entry, viral replication, and 18325633 protein translation. While DENV was freely transmitted in the infection system without restriction of 0.5 methocellulose, intracellular 4G2 protein markedly increased in all three cell lines at high MOI infection, whereas BST2 still moderately inhibited viral replication in Huh7-BST2 cells (Fig. 5). These results suggest that multiple rounds of infection from progeny virions occurred in this free transmission system that was partially inhibited by BST2.BST2 inhibits virion release and cell-to-cell transmissionThe low MOI infection plot, as shown in Fig. 4A, shows the representative DENV-infected cell foci from the cultures of the three cell lines. The quantitative analysis showed that theTetherin Inhibits DENV SecretionFigure 2. Immunofluorescent staining for DENV infection in Huh7-BST2 and Huh7-BST2CV5 cells. Cells were infected with DENV at indicated MOI and harvested on day 2. Cells were double-stained for DENV envelope protein 4G2 (top panel, green) and BST2 (middle panel, red). Cell nuclei were stained with DAPI (bottom panel, blue). doi:10.1371/journal.pone.0051033.ginfectious foci per well were decreased to about 30 by BST2 (Fig. 4B). However, BST2CV5 did not exert any effect. The average DENV-positive cell number per foci is 18297096 238 in Huh7 cells, whereas the cell numbers in the Huh7-BST2 and Huh7BST2CV5 cell foci were 78 and 175, respectively. Altogether, the expression of BST2 but not BSTCV5 inhibited DENV release and cell-to-cell transmission in Huh7 cells.DiscussionBST2 is a transmembrane protein that contains a short Nterminal cytoplasmic domain, a membrane-spanning alpha-helix, a coiled-coil ectodomain, and a C-terminal GPI anchor [31]. This antiviral protein localizes at the plasma membrane as well as themembranes of multiple intracellular vesicles, including endosomes and the trans-Golgi network [32,33]. At the plasma membrane, BST2 is found within cholesterol-enriched lipid rafts, presumably due to its C-terminal GPI modification. This optimally positions BST2 to interfere directly with virion release, since several lipidenveloped viruses, including HIV-1 and Ebola, bud selectively from raft domains [34?8]. Consistent with these reports, our results showed that BST2 localizes to both the cell membrane and cytoplasm. The addition of the V5 tag at the C-terminus of BST2 demonstrated an altered intracellular distribution (Fig. 1). Furthermore, similar as previous report [25,39], we found that three bands of BST2 distributed in the range from 30 to 36kd by western blot. We supposed that the different level of modification of BST2 likely cause the different size of BST2. However, forFigure 3. Viral infectivity detection of supernatant DENV in Huh7-BST2 and Huh7-BST2CV5 cells. The viral infectivity of supernatant DENV was determined by TCID50 method. The cells were infected with DENV at indicated MOI for 1 h; the media were replaced with complete media and cultured for 2 days. Dengue E protein was assayed by A cell-based flavivirus immunodetection assay. The values represent average from 3 independen.
