FIG 7.
Contribution of internalized HIV-1 to productive infection of primary human CD4+ T cells. Data for primary CD4+ T cells from five different donors are shown. (A, B) Donor 1; (C, D) donor 2; (E) donor 3; (F to I, top) donor 4; (F to I, bottom) donor 5. (A, C). Cytoplasmic entry by membrane fusion of wild-type HIV-1NL4-3. Cells were incubated for either 5 h (A) or 16 h (C) with BlaM reporter virions at the indicated temperature in the presence or absence of T-20 and analyzed using the BlaM assay. Bars indicate the proportion of entry-positive cells from one representative experiment performed in triplicate. (B, D) Contribution of endocytotic uptake to HIV-1NL4-3 infectivity in primary CD4+ T cells. Cells were incubated with virus at a multiplicity of infection of 1 for 15 h at 22°C prior to addition of T-20. Cells were incubated for one more hour at 22°C prior to a shift to 37°C. Samples in which T-20 was present throughout the experiment (T-20; time zero) were included as controls. Infection was scored by immunostaining for intracellular CA and flow cytometry. (E) Effect of ammonium chloride on HIV-1 infection in primary CD4+ T cells. Cells were incubated with HIV-1NL4-3 in the presence or absence of 10 mM NH4Cl at a multiplicity of infection of 0.2 for 15 h at 22°C prior to addition of T-20. Cells were further incubated at 22°C for one more hour prior to a shift to 37°C. Samples in which T-20 was present throughout the experiment (T-20, time zero) were included as controls. Infection was scored by immunostaining for intracellular CA and flow cytometry. (F, H) Cytoplasmic entry by membrane fusion of HIV-1 particles pseudotyped with envelope glycoproteins directly derived from infected patients. Cells were incubated for either 5 h (F) or 16 h (H) with BlaM reporter virions pseudotyped with either the X4 T-cell-tropic Env5002 or the R5-tropic Env4051 at the indicated temperature in the presence or absence of T-20 and analyzed using the BlaM fusion assay. (Top and bottom) Infection of cells from two different donors, respectively, that were analyzed in parallel. Bars indicate the proportion of entry-positive cells from one representative experiment performed in triplicate. (G, I) Contribution of endocytotic uptake to infectivity of HIV-1 pseudotyped with envelope glycoproteins directly derived from HIV-infected patients in primary CD4+ T cells. Cells were incubated at 22°C with NL4-3Env5002 or NL4-3Env4051 at a multiplicity of infection of 1 for either 4 h (G) or 15 h (I) prior to addition of T-20. Cells were incubated for one more hour at 22°C prior to a shift to 37°C. Samples in which T-20 was present throughout the experiment (T-20, time zero) were included as controls. Infection was scored by immunostaining for intracellular CA and flow cytometry. For all experiments, mean values and SDs are shown. P values were determined by a two-tailed Student's t test. ***, P ≤ 0.001.