Moreover, 2-HP em /em CD exhibited a doseCresponse effect on infection (data not shown). As shown in Figure 1A, 2-HP em /em CD did not significantly affect VSV-G pseudotyped particle infection in HOS cells. HTSU-Fc or SUA-Fc Necrostatin-1 at 20C for 25 min. The lysates were removed, the cells were washed with PBS, a 1:25 mixture of FITC-labeled anti-rabbit IgG was added and incubated at 4C for 30 min. The cells were washed with PBS, fixed with 4% paraformaldehyde, and the coverslips were adhered to microscope slides with 8 em /em l of 75% glycerol. To visualize the HTLV-I receptor and ganglioside marker 1 (GM1), HOS cells were incubated with biotin-labeled cholera toxin (CtB, stock concentration 10 em /em g/ml, Sigma) at a ratio of 1 1:150 CtB to HTSU-Fc or SUA-Fc lysate, for 25 min at 20C. The cells were washed with PBS, treated with 1:100 Cy3-labeled anti-biotin antibody (stock concentration 10 em /em g/ml, Sigma) and 1:25 FITC-labeled antirabbit IgG for 25 min at 4C, and processed as described above. To visualize the HTLV-I and transferrin receptors (TfR), HOS cells were incubated with 200 em /em g/ml HTSU-Fc lysate for 25 min at 20C, washed with PBS, then incubated with 1:30 biotin-labeled anti-rabbit IgG antibody (stock concentration 80 em /em g/ml, Sigma, to bind HTSU-Fc) and 1:15 FITC-labeled anti-TfR antibody (stock concentration 10 em /em g/ml, Santa Cruz, for 25 min at room temperature. The cells were washed with PBS, treated with 1:100 Cy3-labeled anti-biotin antibody to label HTSU-Fc with Cy3 for 25 min at 4C, and processed as described above. RESULTS Cholesterol is important for HTLV-I envelope-mediated, pseudotyped particle infection We have previously shown that HTLV-I requires intact lipid raft membranes for viral budding from the host plasma membrane.13 In addition, Niyogi and Hildreth found that treatment of target cells with 2-HP em /em CD, a drug that GMFG binds cholesterol and disrupts lipid rafts, inhibits HTLV-I envelope-mediated syncytium formation.8 Similar findings have been described for HIV as well.14C16 These findings suggested that cell-free HTLV-I infection may also depend on the integrity of cholesterol-rich lipid rafts. To examine this issue, HOS cells were cultured with or without cyclodextrin, followed by infection with HIV particles encoding the firefly luciferase gene, pseudotyped with either HTLV-I, A-MLV, or vesicular stomatitis virus glycoproteins (VSV-G). Several different em /em -cyclodextrins, including em /em -cyclodextrin, methyl- em /em -cyclodextrin, and 2-hydroxypropyl- em /em -cyclodextrin, (2-HP em /em CD) were examined. Each reagent resulted in similar effects on pseudotyped particle infection, but the solubility and cell toxicity varied; 2-HP em /em CD was the most soluble and least toxic Necrostatin-1 agent under the assay conditions. Moreover, 2-HP em /em CD exhibited a doseCresponse effect on infection (data not shown). As shown in Figure 1A, 2-HP em /em CD did not significantly affect VSV-G pseudotyped particle infection in HOS cells. However, both HTLV-I and A-MLV envelope pseudotyped particles were sensitive to 2-HP em /em CD treatment, and exhibited a 85C90% reduction in luciferase activity. Similar assays were also performed with MAGI-5 cells, a HeLa cell line that expresses CD4, CXCR4, and CCR5, with or without prior 2-HP em /em CD treatment. In this case, HIV particles pseudotyped with an R5 HIV-1 envelope were used as an additional control. As shown in Figure 1B, the effect of 2-HP em /em CD on VSV-G pseudo-typed particle infection of MAGI-5 cells was not diminished. In contrast, entry of HTLV-I, A-MLV, and HIV-1 pseudotyped particles was sensitive to 2-HP em /em CD treatment, and luciferase activity decreased 80%, 60%, and 25%, respectively. These results demonstrate that HTLV-I pseudotyped particle infection depends on cholesterol, and further shows that 2-HP em /em CD does not negatively affect cell viability since VSV-G particle entry was not adversely affected by cholesterol extraction. Open in a separate window FIG. 1 2HP em /em CD inhibits infection of (A) HOS cells and (B) MAGI-5 cells by a lentivirus particles carrying a luciferase gene pseudotyped with HTLV-I Env. Cells were treated with (+) or without (?) 2HP em /em CD prior Necrostatin-1 to infection with HTLV-I, VSV, A-MLV, or HIV-I glycoprotein pseudotyped particles. After 2 days, luciferase activity was measured. Cholesterol is important for wild-type HTLV-I particle entry into B5 cells The cholesterol dependence of infection of wild-type HTLV-I particles was also examined. B5 cells, a monkey lung fibroblast cell line, is the most suitable cell line for efficient replication of free HTLV-I particles. These cells were treated with 2-HP em /em CD, and.