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HIV-1 cell to cell transfer across an Env-induced, actin-dependent synapse.

Jolly C, Kashefi K, Hollinshead M, Sattentau QJ - J. Exp. Med. (2004)

Bottom Line: Receptor recognition by HIV-1 occurs via interactions between the viral surface envelope glycoprotein (Env), gp120, and CD4 and a chemokine receptor, CCR5 or CXCR4.Recruitment of these membrane molecules into polarized clusters was dependent on Env engagement of CD4 and CXCR4 and required remodelling of the actin cytoskeleton.We propose that receptor engagement by Env directs the rapid, actin-dependent recruitment of HIV receptors and adhesion molecules to the interface, resulting in a stable adhesive junction across which HIV infects the target cell.

View Article: PubMed Central - PubMed

Affiliation: The Sir William Dunn School of Pathology, University of Oxford, UK.

ABSTRACT
Direct cell-cell transfer is an efficient mechanism of viral dissemination within an infected host, and human immunodeficiency virus 1 (HIV-1) can exploit this mode of spread. Receptor recognition by HIV-1 occurs via interactions between the viral surface envelope glycoprotein (Env), gp120, and CD4 and a chemokine receptor, CCR5 or CXCR4. Here, we demonstrate that the binding of CXCR4-using HIV-1-infected effector T cells to primary CD4(+)/CXCR4(+) target T cells results in rapid recruitment to the interface of CD4, CXCR4, talin, and lymphocyte function-associated antigen 1 on the target cell, and of Env and Gag on the effector cell. Recruitment of these membrane molecules into polarized clusters was dependent on Env engagement of CD4 and CXCR4 and required remodelling of the actin cytoskeleton. Transfer of Gag from effector to target cell was observed by 1 h after conjugate formation, was independent of cell-cell fusion, and was probably mediated by directed virion fusion with the target cell. We propose that receptor engagement by Env directs the rapid, actin-dependent recruitment of HIV receptors and adhesion molecules to the interface, resulting in a stable adhesive junction across which HIV infects the target cell.

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Kinetics of Env-mediated cell–cell fusion. Effector (red) and target (green) cells were labeled with cytoplasmic dyes, and conjugates were formed at 37°C in the presence or absence of DP178 for various times. The cells were fixed, and the conjugates were analyzed for fusion and mixing of cytoplasmic dyes (yellow). After 3 h of conjugate formation (left), no transfer of dye is detected; by 24 h, transfer of dye is apparent (middle) and this is blocked in conjugates formed for 24 h in the presence of DP178 (right).
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fig6: Kinetics of Env-mediated cell–cell fusion. Effector (red) and target (green) cells were labeled with cytoplasmic dyes, and conjugates were formed at 37°C in the presence or absence of DP178 for various times. The cells were fixed, and the conjugates were analyzed for fusion and mixing of cytoplasmic dyes (yellow). After 3 h of conjugate formation (left), no transfer of dye is detected; by 24 h, transfer of dye is apparent (middle) and this is blocked in conjugates formed for 24 h in the presence of DP178 (right).

Mentions: One outcome of contact between HIV-1–infected and receptor-expressing target cells is cell–cell fusion. To establish whether cell–cell fusion was taking place under our experimental conditions, we labeled the target and effector cells with, respectively, green and red cytoplasmic dyes. These dyes do not leach out of the cells over time, so the detection of dye transfer in conjugates is good evidence of cytoplasmic exchange via fusion pores. Effector–target cell conjugates were formed and incubated at 37°C for up to 24 h. By 24 h after initiation of conjugate formation, dye transfer from effector to target cells was observed in ∼80% of conjugates, although there was no evidence of syncytium formation, suggesting that cell–cell fusion in our system is inefficient. In contrast, dye transfer was not observed in any conjugates at 1, 3, or 6 h (Fig. 6 and not depicted). To ensure that the transfer was via gp41-mediated fusion, we incubated conjugates for 24 h in the presence of the gp41-derived fusion inhibitor DP178. Under these conditions, dye transfer was eliminated. The dramatic difference in the kinetics of Gag transfer and cell–cell fusion exclude the possibility that HIV movement from effector to target cells is via direct cell–cell fusion.


HIV-1 cell to cell transfer across an Env-induced, actin-dependent synapse.

Jolly C, Kashefi K, Hollinshead M, Sattentau QJ - J. Exp. Med. (2004)

Kinetics of Env-mediated cell–cell fusion. Effector (red) and target (green) cells were labeled with cytoplasmic dyes, and conjugates were formed at 37°C in the presence or absence of DP178 for various times. The cells were fixed, and the conjugates were analyzed for fusion and mixing of cytoplasmic dyes (yellow). After 3 h of conjugate formation (left), no transfer of dye is detected; by 24 h, transfer of dye is apparent (middle) and this is blocked in conjugates formed for 24 h in the presence of DP178 (right).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211771&req=5

fig6: Kinetics of Env-mediated cell–cell fusion. Effector (red) and target (green) cells were labeled with cytoplasmic dyes, and conjugates were formed at 37°C in the presence or absence of DP178 for various times. The cells were fixed, and the conjugates were analyzed for fusion and mixing of cytoplasmic dyes (yellow). After 3 h of conjugate formation (left), no transfer of dye is detected; by 24 h, transfer of dye is apparent (middle) and this is blocked in conjugates formed for 24 h in the presence of DP178 (right).
Mentions: One outcome of contact between HIV-1–infected and receptor-expressing target cells is cell–cell fusion. To establish whether cell–cell fusion was taking place under our experimental conditions, we labeled the target and effector cells with, respectively, green and red cytoplasmic dyes. These dyes do not leach out of the cells over time, so the detection of dye transfer in conjugates is good evidence of cytoplasmic exchange via fusion pores. Effector–target cell conjugates were formed and incubated at 37°C for up to 24 h. By 24 h after initiation of conjugate formation, dye transfer from effector to target cells was observed in ∼80% of conjugates, although there was no evidence of syncytium formation, suggesting that cell–cell fusion in our system is inefficient. In contrast, dye transfer was not observed in any conjugates at 1, 3, or 6 h (Fig. 6 and not depicted). To ensure that the transfer was via gp41-mediated fusion, we incubated conjugates for 24 h in the presence of the gp41-derived fusion inhibitor DP178. Under these conditions, dye transfer was eliminated. The dramatic difference in the kinetics of Gag transfer and cell–cell fusion exclude the possibility that HIV movement from effector to target cells is via direct cell–cell fusion.

Bottom Line: Receptor recognition by HIV-1 occurs via interactions between the viral surface envelope glycoprotein (Env), gp120, and CD4 and a chemokine receptor, CCR5 or CXCR4.Recruitment of these membrane molecules into polarized clusters was dependent on Env engagement of CD4 and CXCR4 and required remodelling of the actin cytoskeleton.We propose that receptor engagement by Env directs the rapid, actin-dependent recruitment of HIV receptors and adhesion molecules to the interface, resulting in a stable adhesive junction across which HIV infects the target cell.

View Article: PubMed Central - PubMed

Affiliation: The Sir William Dunn School of Pathology, University of Oxford, UK.

ABSTRACT
Direct cell-cell transfer is an efficient mechanism of viral dissemination within an infected host, and human immunodeficiency virus 1 (HIV-1) can exploit this mode of spread. Receptor recognition by HIV-1 occurs via interactions between the viral surface envelope glycoprotein (Env), gp120, and CD4 and a chemokine receptor, CCR5 or CXCR4. Here, we demonstrate that the binding of CXCR4-using HIV-1-infected effector T cells to primary CD4(+)/CXCR4(+) target T cells results in rapid recruitment to the interface of CD4, CXCR4, talin, and lymphocyte function-associated antigen 1 on the target cell, and of Env and Gag on the effector cell. Recruitment of these membrane molecules into polarized clusters was dependent on Env engagement of CD4 and CXCR4 and required remodelling of the actin cytoskeleton. Transfer of Gag from effector to target cell was observed by 1 h after conjugate formation, was independent of cell-cell fusion, and was probably mediated by directed virion fusion with the target cell. We propose that receptor engagement by Env directs the rapid, actin-dependent recruitment of HIV receptors and adhesion molecules to the interface, resulting in a stable adhesive junction across which HIV infects the target cell.

Show MeSH
Related in: MedlinePlus