Limits...
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.

Show MeSH

Related in: MedlinePlus

Kinetics of receptor polarization. Target and effector cells were mixed and incubated for 30 min on ice to synchronize cell adhesion to the poly-l-lysine–coated coverslips. Coverslips were transferred to 37°C and incubated for 10, 30, or 60 min before transfer to ice with the addition of NaN3-containing buffer. Cells were either stained during conjugate formation with mAbs to Env and CD4 or to Env alone followed by incubation on ice to detect CD4 (using rabbit polyclonal serum), CXCR4, and LFA-1. Conjugates were fixed, stained with secondary antibodies, and analyzed by confocal microscopy. Conjugates were scored for copolarization of Env and CD4 (black bars), Env and LFA-1 (white bars), or polarization of CXCR4 alone (diagonally striped bars) The error bars show the standard error of the mean.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2211771&req=5

fig3: Kinetics of receptor polarization. Target and effector cells were mixed and incubated for 30 min on ice to synchronize cell adhesion to the poly-l-lysine–coated coverslips. Coverslips were transferred to 37°C and incubated for 10, 30, or 60 min before transfer to ice with the addition of NaN3-containing buffer. Cells were either stained during conjugate formation with mAbs to Env and CD4 or to Env alone followed by incubation on ice to detect CD4 (using rabbit polyclonal serum), CXCR4, and LFA-1. Conjugates were fixed, stained with secondary antibodies, and analyzed by confocal microscopy. Conjugates were scored for copolarization of Env and CD4 (black bars), Env and LFA-1 (white bars), or polarization of CXCR4 alone (diagonally striped bars) The error bars show the standard error of the mean.

Mentions: We estimated the rate of receptor and Env recruitment to the interface. Conjugate formation was synchronized at 4°C followed by 37°C for various times, after which cells were placed on ice, stained in the presence of NaN3, fixed, and analyzed. Copolarization with Env was defined as cocapping of the molecules to the interface with little or no residual staining around the cell periphery as seen in Figs. 1, B and C, and 2 B. From a theoretical value of ∼0%, CD4-Env copolarization at the interface at T = 0, copolarization was observed in ∼40% of conjugates by 10 min, increasing to 65% at 30 min and 75% at 60 min (Fig. 3) . LFA-1 copolarization with Env was analyzed in the same manner. By 10 min, ∼30% of LFA-1 was copolarized with Env, increasing to 50% at 30 min and 57% at 60 min. CXCR4 polarization was analyzed in the absence of coclustering with Env because the CXCR4 mAb does not bind gp120–CXCR4 complexes and, hence, did not consistently demonstrate strong colocalization between these molecules. Approximately 45% of conjugates had polarized CXCR4 staining at 10 min, decreasing to 42% at 30 min and 25% at 60 min. The decrease in CXCR4 staining was probably the result of progressive CXCR4 engagement by gp120, preventing binding of the detection mAb. This method of analysis overestimates the time required for receptor polarization because of the lag implicit in raising the temperature from 4 to 37°C, but demonstrates that receptor recruitment to the interface is a rapid and efficient process.


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 receptor polarization. Target and effector cells were mixed and incubated for 30 min on ice to synchronize cell adhesion to the poly-l-lysine–coated coverslips. Coverslips were transferred to 37°C and incubated for 10, 30, or 60 min before transfer to ice with the addition of NaN3-containing buffer. Cells were either stained during conjugate formation with mAbs to Env and CD4 or to Env alone followed by incubation on ice to detect CD4 (using rabbit polyclonal serum), CXCR4, and LFA-1. Conjugates were fixed, stained with secondary antibodies, and analyzed by confocal microscopy. Conjugates were scored for copolarization of Env and CD4 (black bars), Env and LFA-1 (white bars), or polarization of CXCR4 alone (diagonally striped bars) The error bars show the standard error of the mean.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: Kinetics of receptor polarization. Target and effector cells were mixed and incubated for 30 min on ice to synchronize cell adhesion to the poly-l-lysine–coated coverslips. Coverslips were transferred to 37°C and incubated for 10, 30, or 60 min before transfer to ice with the addition of NaN3-containing buffer. Cells were either stained during conjugate formation with mAbs to Env and CD4 or to Env alone followed by incubation on ice to detect CD4 (using rabbit polyclonal serum), CXCR4, and LFA-1. Conjugates were fixed, stained with secondary antibodies, and analyzed by confocal microscopy. Conjugates were scored for copolarization of Env and CD4 (black bars), Env and LFA-1 (white bars), or polarization of CXCR4 alone (diagonally striped bars) The error bars show the standard error of the mean.
Mentions: We estimated the rate of receptor and Env recruitment to the interface. Conjugate formation was synchronized at 4°C followed by 37°C for various times, after which cells were placed on ice, stained in the presence of NaN3, fixed, and analyzed. Copolarization with Env was defined as cocapping of the molecules to the interface with little or no residual staining around the cell periphery as seen in Figs. 1, B and C, and 2 B. From a theoretical value of ∼0%, CD4-Env copolarization at the interface at T = 0, copolarization was observed in ∼40% of conjugates by 10 min, increasing to 65% at 30 min and 75% at 60 min (Fig. 3) . LFA-1 copolarization with Env was analyzed in the same manner. By 10 min, ∼30% of LFA-1 was copolarized with Env, increasing to 50% at 30 min and 57% at 60 min. CXCR4 polarization was analyzed in the absence of coclustering with Env because the CXCR4 mAb does not bind gp120–CXCR4 complexes and, hence, did not consistently demonstrate strong colocalization between these molecules. Approximately 45% of conjugates had polarized CXCR4 staining at 10 min, decreasing to 42% at 30 min and 25% at 60 min. The decrease in CXCR4 staining was probably the result of progressive CXCR4 engagement by gp120, preventing binding of the detection mAb. This method of analysis overestimates the time required for receptor polarization because of the lag implicit in raising the temperature from 4 to 37°C, but demonstrates that receptor recruitment to the interface is a rapid and efficient process.

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