Multiparametric assay to screen and dissect the mode of action of anti-human immunodeficiency virus envelope drugs

Abstract

A flow cytometry-based assay was used to simultaneously quantify X4 and R5 human immunodeficiency virus (HIV) envelope-mediated cell-to-cell viral transfer, cell death, and cell-to-cell fusion. In this assay, different anti-HIV envelope drugs showed characteristic inhibitory profiles for each measured parameter, allowing for the rapid identification of the mode of action of active compounds.

FIG. 1.

Quantifications of transfer, death, and fusion. Flow cytometry analysis of cocultures of CD4 T cells with uninfected (UNINF; left) or CI-1-SI infected (right) MOLT/CCR5 cells. Forward (FSC)-by-side scatter (SSC) plots (upper panels) allowed the identification of different cell populations. R1 and R2 reginos were defined as the living and dead target cell populations, respectively (detail is shown in the middle panels). Morphology of target cells was used as a marker of cell death. R3 was defined as the effector (uninfected or HIV-infected) cell population. Absolute cell counts were obtained by adding a constant number of fluorescent beads to cultures. Beads were identified by morphology (R4) and fluorescence intensity (not shown). P24 antigen staining allowed for the evaluation of HIV particles transferred to the living (R1) target cell population (low panels). Parameters were set up by using uninfected cocultures as a control. Lymph., lymphocytes.

FIG. 2.

Validation of the absolute cell count. (A) Correlation of the assessment of cell death by morphological or mitochondrial parameters. A total of 159 samples from CD4 T cells cultured in different conditions with infected MOLT cells were analyzed for mitochondrial membrane potential after DIOC staining (6) and cell morphology (as shown in Fig. 1). (B) The effect of fixation (FIX) and permeabilization (PERM) on absolute CD4 T-cell count was assessed for unfixed (left) and fixed/permeabilized CD4 T cells cocultured for 24 h with NL4-3-, BaL-, or CI-1-SI-infected MOLT/CCR5 cells. The percentage of dead cells in each case is shown. Data are means ± standard deviations of three experiments. UNINF, uninfected. (C) CD4 T cells were cultured in the absence or the presence of the indicated concentrations of puromycin for 24 h, and the absolute numbers of morphologically living and dead cells were assessed. The numbers of living cells (black columns) and dead cells (white columns) are shown. Data are means ± standard deviations of triplicate samples. Ctrol, control; FIX, fixated; PERM, permeabilized. (D) Correlation between calculated CD4 T-cell loss induced by NL4-3-infected MOLT/CCR5 cells and the surface of syncytia evaluated in micrographs of the same cocultures by using NIH Image 1.63 software. Correlations in panels A and D were examined with the Pearson correlation coefficient.

FIG. 3.

Evaluation of the inhibition of HIV envelope functions by several drugs. The effects of Leu3a (filled squares), AMD3100 (open squares), TAK779 (filled triangles), C34 (open triangles), and AZT (circles) on HIV transfer (left), cell death (middle), and fusion (right) were determined in cocultures of CD4 T cells with NL4-3 (upper graphs), CI-1-SI (middle graphs), or BaL (lower graphs) infected MOLT/CCR5 cells, in the presence of increasing concentration of each inhibitor. Dotted lines represent background levels found in uninfected control cocultures. Data are representative of one experiment of three performed. Dose-response inhibition curves were fitted by nonlinear regression using the GraphPad Prism 4 software.