FIGURE 10:

Arf6 regulates HIV-1 viral fusion with CD4⁺ lymphocytes, without affecting CCR5 and CXCR4 internalization. (A) Western blot analysis of endogenous Arf6 knockdown 24 h after siRNA nucleofection of CEM-CCR5 cells, quantified as the band intensity ratios to α-tubulin. Scrambled oligonucleotides represent the negative control for RNAi. A representative experiment of three is shown. (B) Specific silencing of endogenous Arf6 specifically affects the early steps of viral infection. Control (scrambled)– or siRNA-Arf6–treated CEM-CCR5 cells were incubated for 3 h with equivalent viral inputs (determined by standard p24-ELISA) of X4-tropic or R5-tropic pNL4-3.Luc.R-E- virions containing the BlaM-Vpr fusion protein. After adsorption for 3 h, cells were treated with CCF2-AM and analyzed by fluorescence spectrophotometry after 16 h. VSV-G virions containing the BlaM-Vpr fusion protein were used to control the specificity of Arf6-mediated effects on HIV-1 viral fusion. The percentages of HIV-1–fused cells were determined by measuring the ratio of blue (447 nm; cleaved CCF2) to green (520 nm; intact CCF2) fluorescence signals in target cells. Each assay was done in triplicate, and results are representative of three independent experiments. (C) Western blot analysis of endogenous Arf6 knockdown, 24 h after control- or siRNA-Arf6–pEGFP-N2-RNAi nucleofection of CEM-CCR5 cells, quantified as the band intensity ratios to α-tubulin. Control and siRNA oligonucleotides were transfected using the pEGFP-N2-RNAi plasmid; therefore treated cells expressed the EGFP protein, which serves as a control of cell treatment. A representative experiment of three is shown. (D) Effect of Arf6 knockdown on ligand-induced CXCR4 (SDF-1α) and CCR5 (RANTES) endocytosis in CEM-CCR5 cells. Control or siRNA-Arf6–pEGFP-N2-RNAi–treated cells were exposed to SDF-1α (200 nM) or RANTES (200 nM) for 1 h at 37°C. Then CXCR4 or CCR5 expression was analyzed by flow cytometry in control/EGFP⁺ and siRNA-Arf6/EGFP⁺ cells using PE-conjugated specific mAbs against cell-surface CXCR4 or CCR5. Data are mean ± SEM of three independent experiments carried out in triplicate and refer to CXCR4 or CCR5 expression in the absence of SDF-1α or RANTES, respectively, taken as 100%. Asterisk indicates p < 0.05, t test. (E) Western blot analysis of endogenous Arf6, WT Arf6–, Arf6-Q67L–, and Arf6-T44N–EGFP expression in CEM-CCR5 cells. α-Tubulin and pEGFP-N1 are the controls for total protein and intact Arf6-EGFP expression, respectively. (F). Effect of different Arf6-EGFP constructs on ligand-induced CXCR4 (SDF-1α) and CCR5 (RANTES) endocytosis in CEM-CCR5 cells. The experiments were carried out as indicated in (D) but in cells overexpressing each Arf6-EGFP construct. Data are mean ± SEM of three independent experiments carried out in triplicate and refer to cell-surface CXCR4 or CCR5 expression in WT Arf6–EGFP–transfected cells in the absence of SDF-1α or RANTES, respectively, taken as 100%. Asterisk indicates p < 0.05, t test.