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Evidence that Gag facilitates HIV-1 envelope association both in GPI-enriched plasma membrane and detergent resistant membranes and facilitates envelope incorporation onto virions in primary CD4+ T cells.

Patil A, Gautam A, Bhattacharya J - Virol. J. (2010)

Bottom Line: HIV-1 particle assembly mediated by viral Gag protein occurs predominantly at plasma membrane.While colocalization of HIV-1 envelope with lipid rich microenvironment have been shown in T cells, the significance of viral proteins modulating envelope association in such microdomains in plasma membrane enriched in glycosylphosphatidylinositol-anchored proteins in primary CD4+ T cells that are natural targets of HIV-1 is poorly understood.Here we show that in primary CD4+ T cells that are natural targets of HIV-1 in vivo, Gag modulates HIV-1 envelope association with GM1 ganglioside and CD59 rich cellular compartments as well as with detergent resistant membranes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular Virology, National AIDS Research Institute, Bhosari, Pune, India.

ABSTRACT
HIV-1 particle assembly mediated by viral Gag protein occurs predominantly at plasma membrane. While colocalization of HIV-1 envelope with lipid rich microenvironment have been shown in T cells, the significance of viral proteins modulating envelope association in such microdomains in plasma membrane enriched in glycosylphosphatidylinositol-anchored proteins in primary CD4+ T cells that are natural targets of HIV-1 is poorly understood. Here we show that in primary CD4+ T cells that are natural targets of HIV-1 in vivo, Gag modulates HIV-1 envelope association with GM1 ganglioside and CD59 rich cellular compartments as well as with detergent resistant membranes. Our data strengthen evidence that Gag-Env interaction is important in envelope association with lipid rafts containing GPI-anchored proteins for efficient assembly onto mature virions resulting in productive infection of primary CD4+ T cells.

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Envelope association with DRM in primary CD4+ T cells. A. CD4+ T cells infected with VSV-G pseudotyped pNL4.3 and pNL4.3 (L30E) were treated with cold Triton-X 100 and fractionated in sucrose density gradients as described in the text. Gradient fractions were subsequently probed for envelope with gp41 antibodies 2F5 and 4E10, anti-human CD59 for CD59 and CTxB-HRP for GM1 by SDS-PAGE followed by Western blotting. B. Density (g/cm3) of fractions showing DRM and DSM (detergent soluble membrane) fractions [39].
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Figure 2: Envelope association with DRM in primary CD4+ T cells. A. CD4+ T cells infected with VSV-G pseudotyped pNL4.3 and pNL4.3 (L30E) were treated with cold Triton-X 100 and fractionated in sucrose density gradients as described in the text. Gradient fractions were subsequently probed for envelope with gp41 antibodies 2F5 and 4E10, anti-human CD59 for CD59 and CTxB-HRP for GM1 by SDS-PAGE followed by Western blotting. B. Density (g/cm3) of fractions showing DRM and DSM (detergent soluble membrane) fractions [39].

Mentions: We next assessed if there is any correlation between disruptions of envelope association with CD59+ compartment in primary CD4+ T cells and its association with DRM of the same cell type. The DRM assay was carried out essentially as described previously [8]. Briefly, primary CD4+ T cells infected with VSV-G pseudotyped pNL4.3 WT and pNL4.3 (L30E) were lyzed with cold 0.5% Triton-X-100 and fractionated through sucrose density gradient centrifugation in an ultracentrifuge (Beckman Coulter Inc) at 1,00,000 × g for 8-12 hours at 4°C. Ten equal fractions were collected and were examined for presence of gp160, CD59 and GM-1 ganglioside by Western blot using human anti-gp41 monoclonal antibody (2F5) [31] and 4E10 [33], mouse anti-human CD59 (BD Biosciences Inc) and cholera toxin conjugated to horse radish peroxidase (HRP) (Sigma, Inc.) respectively. As shown in Figure 2, L30E mutation in Gag restricted envelope association with DRM fractions of CD4+ T cells in contrast to the wild type. Our results were further substantiated by the presence of both CD59 and GM1 in DRM fractions under same experimental conditions.


Evidence that Gag facilitates HIV-1 envelope association both in GPI-enriched plasma membrane and detergent resistant membranes and facilitates envelope incorporation onto virions in primary CD4+ T cells.

Patil A, Gautam A, Bhattacharya J - Virol. J. (2010)

Envelope association with DRM in primary CD4+ T cells. A. CD4+ T cells infected with VSV-G pseudotyped pNL4.3 and pNL4.3 (L30E) were treated with cold Triton-X 100 and fractionated in sucrose density gradients as described in the text. Gradient fractions were subsequently probed for envelope with gp41 antibodies 2F5 and 4E10, anti-human CD59 for CD59 and CTxB-HRP for GM1 by SDS-PAGE followed by Western blotting. B. Density (g/cm3) of fractions showing DRM and DSM (detergent soluble membrane) fractions [39].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2820015&req=5

Figure 2: Envelope association with DRM in primary CD4+ T cells. A. CD4+ T cells infected with VSV-G pseudotyped pNL4.3 and pNL4.3 (L30E) were treated with cold Triton-X 100 and fractionated in sucrose density gradients as described in the text. Gradient fractions were subsequently probed for envelope with gp41 antibodies 2F5 and 4E10, anti-human CD59 for CD59 and CTxB-HRP for GM1 by SDS-PAGE followed by Western blotting. B. Density (g/cm3) of fractions showing DRM and DSM (detergent soluble membrane) fractions [39].
Mentions: We next assessed if there is any correlation between disruptions of envelope association with CD59+ compartment in primary CD4+ T cells and its association with DRM of the same cell type. The DRM assay was carried out essentially as described previously [8]. Briefly, primary CD4+ T cells infected with VSV-G pseudotyped pNL4.3 WT and pNL4.3 (L30E) were lyzed with cold 0.5% Triton-X-100 and fractionated through sucrose density gradient centrifugation in an ultracentrifuge (Beckman Coulter Inc) at 1,00,000 × g for 8-12 hours at 4°C. Ten equal fractions were collected and were examined for presence of gp160, CD59 and GM-1 ganglioside by Western blot using human anti-gp41 monoclonal antibody (2F5) [31] and 4E10 [33], mouse anti-human CD59 (BD Biosciences Inc) and cholera toxin conjugated to horse radish peroxidase (HRP) (Sigma, Inc.) respectively. As shown in Figure 2, L30E mutation in Gag restricted envelope association with DRM fractions of CD4+ T cells in contrast to the wild type. Our results were further substantiated by the presence of both CD59 and GM1 in DRM fractions under same experimental conditions.

Bottom Line: HIV-1 particle assembly mediated by viral Gag protein occurs predominantly at plasma membrane.While colocalization of HIV-1 envelope with lipid rich microenvironment have been shown in T cells, the significance of viral proteins modulating envelope association in such microdomains in plasma membrane enriched in glycosylphosphatidylinositol-anchored proteins in primary CD4+ T cells that are natural targets of HIV-1 is poorly understood.Here we show that in primary CD4+ T cells that are natural targets of HIV-1 in vivo, Gag modulates HIV-1 envelope association with GM1 ganglioside and CD59 rich cellular compartments as well as with detergent resistant membranes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Molecular Virology, National AIDS Research Institute, Bhosari, Pune, India.

ABSTRACT
HIV-1 particle assembly mediated by viral Gag protein occurs predominantly at plasma membrane. While colocalization of HIV-1 envelope with lipid rich microenvironment have been shown in T cells, the significance of viral proteins modulating envelope association in such microdomains in plasma membrane enriched in glycosylphosphatidylinositol-anchored proteins in primary CD4+ T cells that are natural targets of HIV-1 is poorly understood. Here we show that in primary CD4+ T cells that are natural targets of HIV-1 in vivo, Gag modulates HIV-1 envelope association with GM1 ganglioside and CD59 rich cellular compartments as well as with detergent resistant membranes. Our data strengthen evidence that Gag-Env interaction is important in envelope association with lipid rafts containing GPI-anchored proteins for efficient assembly onto mature virions resulting in productive infection of primary CD4+ T cells.

Show MeSH
Related in: MedlinePlus