Figure 3

Slit2N blocks HIV-1 entry, but does not affect HIV-1 receptor expression, envelope binding or HIV-1 virus binding to T-cells. (A) To analyze the effect of Slit2N on HIV-1 receptor expression, MT4 cells or PBMCs were treated with Slit2N for various time periods. MT4 cells were analyzed for CD4 and CXCR4 expression while PBMCs were analyzed for CCR5 expression, respectively, by flow cytometry. (B) MT4-cells were treated with Slit2N and analyzed for Gp120 binding by flow cytometry using FITC-tagged gp120. (C) For virus binding, untreated and Slit2N (3 nM and 6 nM) -treated MT4 cells were infected with HIV-1 IIIB (40 ng/10⁶ cells) and incubated for one hour at 4°C, and the cell lysates were analyzed for HIV-1 p24 levels. (D) MT4 cells infected with HIV-1 IIIB were incubated for 3 hours at 37 ° C in the presence or absence of Slit2N (3nM and 6nM). The cells were treated with trypsin, washed, lysed and analyzed for intracellular p24 antigen levels by ELISA. *P < 0.05 versus the control preparation. (E) Untreated and Slit2N-treated MT4 cells were incubated with BlaM-Vpr-containing virions (500 ng of p24), after which the cells were washed and incubated with CCF2-AM loading mix (GeneBLAzer detection kit; Invitrogen). The excess dye was washed off and cells were incubated for 16 h at room temperature before fixation with 4% paraformaldehyde. The entry of Blam-Vpr containing virions was measured as the ratio of the maximum fluorescence intensity between cleaved and intact CCF2. *P < 0.05 versus the control infected cells. Results are representative of 3 separate experiments.