Env glycoproteins are dispensable for the incorporation of CD40L within HIV-1. NL4-3/CD40L and NL4-3 Env−/CD40L virus stocks were incubated with streptavidin-coated magnetic beads coated with anti-CD40L antibodies (i.e., 5c8) or isotype-matched irrelevant antibodies (i.e., IgG2a). The amounts of precipitated viruses were estimated by a p24 test. The data shown are the means ± standard deviations of triplicate samples and are representative of three independent transfections.

CD40L-bearing HIV-1 particles, but not viruses lacking host CD40L, induce Il-6 production. Purified B lymphocytes from human tonsils were treated with IL-4 and IL-10 and incubated for 72 h at 37°C with (A) either Mock/CD40L, the indicated virus preparations, or G28.5, (B) either medium alone, Mock/CD40L, the indicated virus stocks, or G28.5, or (C) either medium alone, the listed virus preparations, or G28.5. The amounts of IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12(p70), IL-13, TNF-α, IFN-γ, MCP-1, and RANTES were measured in cell-free supernatants with the Luminex technology (A). In some studies, the levels of IL-6 were determined with a commercial ELISA test (B and C). In panel A, the data shown represent the means ± standard deviations of triplicate samples for two independent experiments, whereas in panels B and C, the data shown represent the means ± standard deviations of triplicate samples and are representative of three different experiments. The asterisks indicate statistically significant differences (i.e., P < 0.05) between the following matched pairs: NL4-3/CD40L versus NL4-3/null and NL4-3balenv/CD40L versus NL4-3balenv/null (A), NL4-3/CD40L versus medium alone (B and C), and NL4-3 Env−/CD40L versus medium alone (C).

CD40L-bearing virions, but not viruses lacking host CD40L, induce a dose-dependent nuclear translocation of NF-κB. (A) B lymphocytes purified from human tonsils were either left untreated or treated for 30 min at 37°C with medium alone, the listed virus stocks (30 ng of p24 per 1 × 10⁵ B cells), Mock/CD40L, or phorbol myristate acetate-ionomycin (used as a positive control). The nuclear extracts were incubated with a labeled NF-κB probe, and the complexes were resolved on a native 4% polyacrylamide gel. Competitions were performed with a 100-fold molar excess of either specific (i.e., NF-κB) (S) or nonspecific (Oct 2A) (NS) oligonucleotides. (B) For the supershift assays, the reactions were also conducted in the absence or presence of antibodies specific for p50 and p65. The arrows on the right indicate the positions of the specific DNA-protein complexes. (C) In some experiments, B lymphocytes purified from human tonsils were either left untreated or treated for 60 min at 37°C with medium alone, the listed virus stocks (at 10 or 30 ng of p24 per 1 × 10⁵ B cells), Mock/CD40L, or phorbol myristate acetate-ionomycin. Competition assays were performed with a 100-fold molar excess of either specific (S) (i.e., NF-κB) or nonspecific (NS) (i.e., Oct 2A) oligonucleotides. The signal band intensities are shown at the bottom of the graph and were quantified by laser densitometry scanning. The data shown are representative of three independent experiments.

B-cell mediated transmission of HIV-1 to autologous CD4⁺ T lymphocytes is increased with CD40L-bearing virions. B lymphocytes purified from human tonsils were incubated for 1 h at 37°C with NL4-3/null (triangles), NL4-3/CD40L (squares), or NL4-3/CD40L used in combination with sCD40L (10 μg/ml) (circles). Thereafter, the virus-cell mixtures were extensively washed and cocultured with PHA/IL-2-treated autologous CD4⁺ T cells. The supernatants were harvested to estimate the p24 content by ELISA at 4, 6, and 8 days following the initiation of the coculture. The data shown represent the means ± standard deviations of triplicate samples and are representative of three independent experiments. The asterisks indicate statistically significant differences (i.e., P < 0.05) between the following matched pair: NL4-3/CD40L versus NL4-3/null.

CD40L-bearing virions, but not viruses lacking host CD40L, induce IgG production. B lymphocytes purified from human tonsils were treated with IL-4 and IL-10 and incubated at 37°C either in medium alone or with G28.5 (used as a positive control) or one of the following: (A) Mock/CD40L, NL4-3/null, or NL4-3/CD40L, (B) Mock/CD40L, NL4-3balenv/null, or NL4-3balenv/CD40L, or (C) NL4-3/CD40L or NL4-3 Env−/CD40L. After 5 (filled bars) and 12 (empty bars) days in culture, supernatants were harvested and the IgG content was measured using a commercial ELISA. In panels A and B, the data shown represent the means ± standard deviations of triplicate samples for three independent experiments, whereas in panel C, the data shown represent the means ± standard deviations of triplicate samples and are representative of three independent experiments. The asterisks indicate statistically significant differences (i.e., P < 0.05) between the following matched pairs: NL4-3/CD40L versus NL4-3/null (A), NL4-3balenv/CD40L versus NL4-3balenv/null (B), NL4-3/CD40L versus medium alone (C) and NL4-3 Env−/CD40L versus medium alone (C).

CD40L-bearing virions, but not viruses lacking host CD40L, mediate homotypic B-cell adhesion. B lymphocytes purified from human tonsils were treated with IL-4 and IL-10 and were incubated for 72 h at 37°C with either (A) medium alone, Mock/CD40L, NL4-3balenv/null, NL4-3balenv/CD40L, or G28.5, or (B) medium alone, NL4-3/CD40L, NL4-3 Env−/CD40L, or G28.5. The images were observed at ×100 magnification, and each is representative of three independent experiments.

HIV-1 attachment to B lymphocytes is increased by interactions between virus-anchored host CD40L and cell surface CD40. B lymphocytes purified from human tonsils were incubated for 1 h at 37°C with NL4-3/null or NL4-3/CD40L virions in either the absence or presence of sCD40L. Next, the virus-cell mixture was extensively washed, lysed, and tested for the p24 content. The data shown represent the means ± standard deviations of triplicate samples and are representative of three independent experiments. The asterisk indicates statistically significant differences (P < 0.05) between the following matched pairs: NL4-3/CD40L versus NL4-3/null and NL4-3/CD40L versus NL4-3/CD40L+sCD40L.