Mode of virus transmission differentially steers susceptibility to entry inhibition.

<p><b>(A) DEAE-Dextran is not required for effective cell-cell transmission of HIV-1_JR-FL to TZM-bl cells.</b> Serial dilutions of JR-FL infected PBMC were incubated with TZM-bl cells in presence (black circles) or absence (red circles) of 10 µg/ml DEAE-Dextran. Infectivity was measured by enzymatic activity of the luciferase reporter (relative light units (RLU)). Each infected cell input was probed in triplicate. Error bars represent SD (standard deviation). One of four independent experiments is shown. <b>(B) Omission of DEAE-Dextran as media supplement abolishes cell-free JR-FL infection of TZM-bl cells.</b> Serial dilutions of cell-free JR-FL virus were used to infect the luciferase reporter cell line TZM-bl in presence (black squares) or absence (red squares) of 10 µg/ml DEAE-Dextran. Infectivity was measured by induction of the luciferase reporter (relative light units (RLU)). Each virus dilution was probed in quadruplicates. Bars represent SD . One of four independent experiments is shown. <b>(C) Cell-cell transmission but not enforced contact between virus and target cell overcomes entry restriction.</b> The infectivity of cell-free virus without enforced attachment to TZM-bl target cells (gravity sedimentation), or upon spinoculation, magnetic bead virus adsorption and during cell-cell transmission was assessed in presence (solid lines) or absence (dotted lines) of 10 µg/ml DEAE-Dextran. Infection was determined by measuring luciferase production after 48 h (recorded as RLU). Each virus dilution was probed in duplicates. Bars represent SD. One of three independent experiments is shown. <b>(D) Inhibitory profiles of CD4-IgG2 and T-20 during cell-cell and cell-free transmission.</b> TZM-bl target cells were either cocultivated with JR-FL infected PBMC (red circles, no DEAE) or cell-free virus (black squares, with 10 µg/ml DEAE) in the presence of increasing doses of CD4-IgG2 (left panel) or T-20 (right panel). Infection was determined by measuring luciferase production after 48 h and recorded as RLU. Red and black values denote IC50 (nM) of during cell-cell and cell-free transmission, respectively. Data points represent means of duplicates from three independent inhibition experiments. Bars represent SEM. Lines depict fitted dose response curves. <b>(E) Decreased CD4-IgG2 sensitivity during cell-cell transmission is due to an inherent feature of cell-cell transmission.</b> TZM-bl target cells were mixed with replication competent infected JR-FL^rc PBMC in the presence of CD4-IgG2 or T-20 (red bars) in medium lacking DEAE Dextran. Cell-free JR-FL^rc was either spinoculated (hatched bars), adsorbed by magnetic beads (checkered bars) or added without enforced adsorption (grey bars) onto TZM-bl target cells in medium containing DEAE Dextran in the presence of the inhibitor. Fold increases in IC50 of cell-cell compared to cell-free infection are indicated on top of the respective bars. Bars depict means of three independent experiments in duplicates. Lines denote SD. Inhibition of cell-cell transmission by CD4-IgG2 and T-20 (red bars) was significantly less efficient than blocking of cell-free virus (grey bars) infection (Student t-test, p<0.0001 in both cases). <b>(F) Single round infection is highly resistant to CD4-IgG2 inhibition during cell-cell transmission.</b> TZM-bl target cells (no DEAE) were co-cultivated with JR-FL pseudovirus transfected 293-T cells in the presence of CD4-IgG2 or T-20. Cell-free JR-FL^pp-lucAM was added to the TZM-bl (with 10 µg/ml DEAE) in the presence of both inhibitors. Fold increases in IC50 of cell-cell compared to cell-free infection are indicated on top of the respective bars. Bars depict means of three independent experiments performed in duplicates. Lines denote SD. Inhibition of cell-cell transmission by CD4-IgG2 and T-20 (red bars) was significantly less efficient than blocking of cell-free virus (grey bars) infection (Student t-test, p<0.0001 in both cases).