FIGURE 4.
Interference of Sms2 in ZS/Sms2 cells decreased HIV-1 Env-mediated cell-cell fusion susceptibility. StealthTM control siRNA, siSms2-1, or siSms2-2 were transfected into ZS/Sms2 cells by electroporation. After 2 days, the expression vectors of CD4/CCR5 or CD4/CXCR4 were transfected into these cells. These cells were harvested 24 h post-transfection and examined for Sms2 levels by Western blotting, cell-surface SM levels, and cell-cell fusion assay. A, Sms2 knockdown was assessed by Western blot analysis with the anti-V5 antibody in total cell lysates (5 μg of protein/lane). The same blots were also probed with an anti-GAPDH antibody for the loading control. One representative experiment is shown, and similar results were obtained in three independent experiments. IB, immunoblot. B, cell-surface SM levels of Sms2 knockdown cells were determined by flow cytometry after EGFP-lysenin staining. Ten thousand cells were measured in each sample. C and D, cell-surface expression levels of CD4/CCR5 or CD4/CXCR4 in Sms2 knockdown cells were analyzed by flow cytometry using each specific antibody (filled histograms). Open histograms indicate the isotype control. Ten thousand cells were measured in each sample. E, Sms2 knockdown cells that expressed the CD4/CCR5/reporter gene or CD4/CXCR4/reporter gene were cultured for 4 h with HEK293 cells that expressed HIV-1 Tat and EnvJRFL- or EnvNL4-3. Fusion susceptibility was determined by the amount of luciferase activity using the reporter gene activation assay. The values were expressed as the percentage of Env-induced fusion using control siRNA-transfected cells as the reference value. Values represent the mean ± S.D. from three independent experiments. *, p < 0.01.