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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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Polarization and transfer of HIV antigens. Conjugates were formed between effector and target cells in the presence of mAbs specific for CD4 (green) and Env (blue). (A) Cells were fixed, permeabilized, and stained for Gag (red); arrows show Gag staining within the target cell. (B) Conjugates were prepared as for A, but incubated at 37°C for 3 h before fixation, permeabilization, and Gag staining. (C) Conjugates were prepared for 1 h as aforementioned, except that the effector cells were prelabeled with CellTracker green. After permeabilization, cells were stained for Gag (blue) and EEA1 (red); a three-dimensional reconstruction of a z series of images is shown.
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fig5: Polarization and transfer of HIV antigens. Conjugates were formed between effector and target cells in the presence of mAbs specific for CD4 (green) and Env (blue). (A) Cells were fixed, permeabilized, and stained for Gag (red); arrows show Gag staining within the target cell. (B) Conjugates were prepared as for A, but incubated at 37°C for 3 h before fixation, permeabilization, and Gag staining. (C) Conjugates were prepared for 1 h as aforementioned, except that the effector cells were prelabeled with CellTracker green. After permeabilization, cells were stained for Gag (blue) and EEA1 (red); a three-dimensional reconstruction of a z series of images is shown.

Mentions: The formation of adhesive junctions at the target–effector cell interface may facilitate HIV-1 infection. To investigate the movement of virus in our system, we analyzed conjugates for viral Gag and Env localization. Conjugates formed in the presence of mAbs L120 and 50-69 were fixed, permeabilized, and stained with antisera against Gagp17/p24. Gag and Env frequently colocalized with CD4 at the interface, attaining a maximum level of clustering within 1 h (Fig. 5 A). Gag could be observed within the target cell from 1 h after conjugate initiation onwards (Fig. 5 A, arrows), demonstrating rapid and, by 3 h, massive (Fig. 5 B) translocation of viral core protein across the interface into the target cell. Env could also be seen located on and within the target cell, although to a lesser extent than Gag. At 1 h after conjugate formation, much of the Gag colocalized with Env at the interface. However, by 3 h, the majority of Gag was uncoupled from Env. The presence of Env stained with 50-69 on and in the target cell strongly suggests that Env-mediated fusion with the target cell has taken place, depositing Env in the target cell membrane and potentially leading to its subsequent internalization by endocytosis (36). To exclude the possibility that Gag and Env, possibly in the form of virions, were being taken into the target cell by endocytosis (a potentially nonproductive route of viral entry), we costained for Gag and EEA1, an early endosome marker. Fig. 5 C shows negligible colocalization between Gagp24 staining in the target cell and the EEA1 marker, strongly suggesting that most Gag enters cells via a nonendocytic route.


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)

Polarization and transfer of HIV antigens. Conjugates were formed between effector and target cells in the presence of mAbs specific for CD4 (green) and Env (blue). (A) Cells were fixed, permeabilized, and stained for Gag (red); arrows show Gag staining within the target cell. (B) Conjugates were prepared as for A, but incubated at 37°C for 3 h before fixation, permeabilization, and Gag staining. (C) Conjugates were prepared for 1 h as aforementioned, except that the effector cells were prelabeled with CellTracker green. After permeabilization, cells were stained for Gag (blue) and EEA1 (red); a three-dimensional reconstruction of a z series of images is shown.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: Polarization and transfer of HIV antigens. Conjugates were formed between effector and target cells in the presence of mAbs specific for CD4 (green) and Env (blue). (A) Cells were fixed, permeabilized, and stained for Gag (red); arrows show Gag staining within the target cell. (B) Conjugates were prepared as for A, but incubated at 37°C for 3 h before fixation, permeabilization, and Gag staining. (C) Conjugates were prepared for 1 h as aforementioned, except that the effector cells were prelabeled with CellTracker green. After permeabilization, cells were stained for Gag (blue) and EEA1 (red); a three-dimensional reconstruction of a z series of images is shown.
Mentions: The formation of adhesive junctions at the target–effector cell interface may facilitate HIV-1 infection. To investigate the movement of virus in our system, we analyzed conjugates for viral Gag and Env localization. Conjugates formed in the presence of mAbs L120 and 50-69 were fixed, permeabilized, and stained with antisera against Gagp17/p24. Gag and Env frequently colocalized with CD4 at the interface, attaining a maximum level of clustering within 1 h (Fig. 5 A). Gag could be observed within the target cell from 1 h after conjugate initiation onwards (Fig. 5 A, arrows), demonstrating rapid and, by 3 h, massive (Fig. 5 B) translocation of viral core protein across the interface into the target cell. Env could also be seen located on and within the target cell, although to a lesser extent than Gag. At 1 h after conjugate formation, much of the Gag colocalized with Env at the interface. However, by 3 h, the majority of Gag was uncoupled from Env. The presence of Env stained with 50-69 on and in the target cell strongly suggests that Env-mediated fusion with the target cell has taken place, depositing Env in the target cell membrane and potentially leading to its subsequent internalization by endocytosis (36). To exclude the possibility that Gag and Env, possibly in the form of virions, were being taken into the target cell by endocytosis (a potentially nonproductive route of viral entry), we costained for Gag and EEA1, an early endosome marker. Fig. 5 C shows negligible colocalization between Gagp24 staining in the target cell and the EEA1 marker, strongly suggesting that most Gag enters cells via a nonendocytic route.

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