Trans-infection but not infection from within endosomal compartments after cell-to-cell HIV-1 transfer to CD4+ T cells

Abstract

Cellular contacts between HIV-1-infected donor cells and uninfected primary CD4(+) T lymphocytes lead to virus transfer into endosomes. Recent evidence suggests that HIV particles may fuse with endosomal membranes to initiate a productive infection. To explore the role of endocytosis in the entry and replication of HIV, we evaluated the infectivity of transferred HIV particles in a cell-to-cell culture model of virus transmission. Endocytosed virus led to productive infection of cells, except when cells were cultured in the presence of the anti-gp120 mAb IgGb12, an agent that blocks virus attachment to CD4, suggesting that endocytosed virus was recycled to the outer cell surface. Confocal microscopy confirmed the colocalization of internalized virus antigen and the endosomal marker dynamin. Additionally, virus transfer, fusion, or productive infection was not blocked by dynasore, dynamin-dependent endosome-scission inhibitor, at subtoxic concentrations, suggesting that the early capture of virus into intracellular compartments did not depend on endosomal maturation. Our results suggest that endocytosis is not a mechanism of infection of primary CD4 T cells, but may serve as a reservoir capable of inducing trans-infection of cells after the release of HIV particles to the extracellular environment.

FIGURE 1.

CD4-dependent transfer of HIV antigen after cell-to-cell contacts. A, experimental procedure was overnight cocultures of MOLT_NL4-3 cells with primary CD4⁺ T lymphocytes. B, cocultures were performed in the presence of 80 nm anti-gp120 mAb IgGb12; 4 μm RT inhibitor AZT; 12.5 μm CXCR4 antagonist AMD3100; 80 μm dynamin inhibitor dynasore (DYN); 100 nm BFLA1, and 20 nm CON A. Results are represented as the percentage of intracellular CAp24⁺ target cells, using the coculture between CD4⁺ T cells and uninfected MOLT cells as a negative control. Results are the mean ± S.D. (error bars) of three independent experiments (**, p < 0.005; *, p < 0.05).

FIGURE 2.

IgGb12 and AMD3100 but not dynasore blocked virus-cell fusion after cell to cell transfer of virus. A, experimental procedure: measurement of viral fusion in cocultures of HEK293-T cells transfected with pNL4-3 and Vpr-BlaM plasmids and primary CD4⁺ T cells. B, dot plots of CCF2-loaded cells (FITC-labeled) versus CCF2-cleaved cells (Pacific blue-labeled). A representative experiment is shown. C, relative increase of CCF2-cleaved target cells compared with untreated condition. Data are the mean ± S.D. (error bars) of three independent experiments (**, p < 0.005; *, p < 0.05) (DYN, dynasore).

FIGURE 3.

Infection of CD4⁺ T cells by HIV particles captured into trypsin-resistant compartments was inhibited by mAb IgGb12. A, experimental procedure: isolation and culture of CAp24-loaded CD4⁺ T cells. After 5 days of culture, HIV infection was assessed by supernatant CAp24 antigen production, expressed in pg/ml (B) and quantification of total viral DNA as the copy number of total DNA/RNaseP, expressed relative to the untreated condition (cells untreated during the coculture and culture phase) (C). Results represent the mean ± S.D. (error bars) of three independent experiments (**, p < 0.005; *, p < 0.05) (DYN, dynasore).

FIGURE 4.

Trans-infection by released HIV viruses from antigen-loaded cells. A, in this experimental procedure, supernatants from cocultures were collected and used to infect MT4 T cells. B, after 12 h of culture, p24-antigen content was evaluated in the supernatant (white bars) and in the purified cells (black bars) by CAp24 ELISA and intracellular CAp24 antigen staining, respectively. C, total viral DNA was also quantified in purified cells by PCR using infected CD4⁺ T cells as a positive control. Results represent the total viral DNA copy number relative to the cellular control gene RNaseP. D, infection of MT4 T cells by collected supernatants was evaluated at day 5 by supernatant CAp24-antigen content. Data are the mean ± S.D. (error bars) of three independent experiments.

FIGURE 5.

Dynasore prevents PMA-induced down-regulation of CD4 receptor. Primary CD4⁺ T lymphocytes were pretreated for 30 min with or without 160, 80, 16, 4, and 1 μm of dynasore and then cultured in the absence (white bars) or the presence of PMA at 1 μg/ml (black bars) for an additional 30 min. Then, cells were fixed with 2% of formaldehyde, and surface CD4 expression (mean fluorescence intensity, MFI) was evaluated with an anti-CD4 mAb. Cells were analyzed by flow cytometry and identified by morphology. Dynasore inhibited PMA-induced CD4 down-regulation in a dose-dependent manner. Data are the mean ± S.D. (error bars) of three independent experiments. *, p < 0.05.

FIGURE 6.

Uptake of HIV particles into intracellular CD4⁺ compartments in primary T lymphocytes was not blocked by dynasore. Primary CD4⁺ T lymphocytes were cocultured overnight with HIV-infected MOLT_NL4-3 cells in the presence or the absence of dynasore (80 μm). Recovered cells were trypsinized to remove membrane-bound viruses and immunostained with antibodies against HIV CAp24 antigen, CD4 receptor, and dynamin. Sections of single target CD4⁺ T cells were viewed and analyzed by confocal microscopy. Colocalization between (A) HIVp24 antigen (green) and dynamin protein (red) or between (B) HIVp24 antigen (green) and CD4 receptor (red) was performed for uninfected (upper panels), untreated (middle panels), and dynasore-treated (lower panels) cocultures. The images show the phase-contrast (left column), the single stainings, the overlay (yellow), and the colocalized pixels (white). A CD4⁺ T lymphocyte representative of each coculture is shown from at least two independent experiments.