Fig. 1

a Titration of combination reporter viruses bearing the CXCR4-tropic HIV Envs JOTO.TA1.2248, IIIB/LAI, or TYBE or the CCR5-tropic Envs REJO.D12.1972, JRCSF, or ADA. Flow cytometry was used to measure the percentage of fusion(+) CD4+ T cells by detection of CCF2-AM dye cleavage and EGFP(+) cells by the combination of EGFP expression and Nef-mediated CD4 downregulation. Closed and open symbols represent data from separate normal donors. b Equation for Poisson distribution to predict the percentage of cells (P(n)) infected with n viruses. m is the multiplicity of infection (MOI) and e is Euler’s number. The example shows the expected frequency of cells infected by 0, 1, 2, 3, 4, or 5 cells at a MOI of 1. c Representative experiment (1 of 3) showing the percentage of unstimulated CD4+ T cells undergoing fusion (left) or expressing EGFP (right) following infection with 1–2048 ng p24 equivalents of combination reporter viruses pseudotyped with the primary X4-tropic isolate JOTO.TA1.2247. These experimental data are compared with data predicted by Poisson distribution if a threshold (x) number of 1, 2, or 3 viruses is required for signal detection. Thresholds greater than x = 3 have increasingly steep slopes that do not match the experimental data. Multiplicity of infection (MOI) values were calculated by determining the MOI that best fit the experimental data using the Poisson distribution formula. MOI varied depending on the readout (fusion or EGFP signal) and whether the cells were spinoculated or not despite a constant number of cells and identical viral titrations. (Note: titrations <16 ng p24 equivalents were not included for EGFP in the absence of spinoculation because these concentrations did not result in signals above the background fluorescence of 0.02–0.03 % in the EGFP channel)