FIG 2.
Contribution of CD4 binding, coreceptor binding, and fusion to cell attachment and endocytotic uptake of HIV-1 particles in SupT1-R5 cells. (A) Cell attachment. SupT1-R5 cells were incubated for 3 h at 16°C with HIV-1MA.mCherry lacking Env [Env(−)] or carrying wild-type EnvNL4-3 or a CD4- or a coreceptor binding-deficient variant [EnvCD4(−) and EnvCoR(−), respectively]. Cells were imaged by wide-field microscopy, and the number of particles bound per cell was determined on the basis of maximum projections. Mean values and SDs for 25 individual view fields (corresponding to a total of ∼900 particles) from one representative experiment are shown. VLPs, virus-like particles. (B to E) Uptake by endocytosis. Cells were incubated for 3 h with HIV-1MA.mCherry particles pseudotyped with the indicated Env variants, fixed, and stained with phalloidin-Atto488. z-stacks covering whole cell volumes were recorded by SDCM, and particle uptake was quantified as described in the legend to Fig. 1. The proportion of cells scoring positive for one or more internalized particles is shown. More than 150 cells corresponding to 10 randomly selected view fields from at least two independent experiments were analyzed for each condition. Dotted lines, values obtained for particles lacking a viral Env protein. (B) Internalization of particles carrying or lacking EnvNL4-3 in SupT1-R5 or activated primary human CD4+ T cells. (C) Requirement for CD4. SupT1-R5 cells were incubated with HIV-1mCherry either carrying EnvNL4-3 preincubated in the presence or absence of sCD4 or carrying EnvCD4(−). (D) Requirement for a coreceptor. The internalization of HIV-1mCherry carrying X4-tropic (EnvNL4-3; black bars) or R5-tropic (EnvJR-FL; white bars) Env variants was assessed in the absence or presence of specific coreceptor antagonists (AMD-3100 [AMD] for CXCR4 or MVC for CCR5). EnvCoR(−), a variant with a mutation in the coreceptor binding V3 loop of EnvNL4-3. (E) Effect of inhibition of membrane fusion. Internalization of HIV-1MA.mCherry carrying EnvNL4-3 was quantitated in the presence or absence of the fusion inhibitor T-1249, and the results were compared to those for particles carrying a fusion-incompetent Env mutant [EnvFus(V2E)]. For all experiments, mean values and SDs are shown. P values were determined by a two-tailed Student's t test. ns, not significant; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001.