Figure 2.

Effect of the different conformations of the loop peptide on HIV-1 ENV-mediated fusion by utilizing 3 fusion systems: virus cell infection, cell-cell lipid mixing, and cell-cell content mixing. A–C) Increasing concentrations of a loop peptide: reduced L27 (A), oxidized L27 (B), and mutant L27 (CC/AA) (C) were tested in the 3 fusion systems. Virus infectivity assay (♦): fully contagious HXBc2 HIV-1 viruses were allowed to infect TZM-bl cells, and the infection rate was determined by luciferase activity. Fitting curve is denoted by a continuous gray line. Cell-cell lipid-mixing assay (▴): Jurkat HXBc2 effector cells and Jurkat E6-1 target cells were labeled with the lipid dyes DID and DiI, respectively. Cells were coincubated, and dye transfer was monitored by flow cytometry. Fitting curve is denoted by a continuous black line. Cell-cell content-mixing assay (■): Jurkat HXBc2 effector cells and Jurkat E6-1 target cells were labeled with the cytoplasmatic dyes calcein AM and CMTMR, respectively. Cells were coincubated, and dye transfer was monitored by flow cytometry. Fitting curve is denoted by a dotted black line. Percentage of fusion enhancement or inhibition was determined by calculating the difference between the fusion rate in the presence or absence of a peptide. Error bars = se; n = 3. D) Effect of each loop peptide (at a concentration of 4 μM) was tested in cell-cell fusion assay mediated by influenza HA. Human erythrocytes were labeled with the lipid dye PKH26 and coincubated with HA-expressing cells. Dye transfer was monitored during the fusion event. Lipid-mixing values in the presence of peptides were normalized to that in the absence of peptides to calculate percentage of change from control (100% lipid mixing). Results are presented as averages ± se; n = 5.