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In COS cells Vpu can both stabilize tetherin expression and counteract its antiviral activity.

Waheed AA, Kuruppu ND, Felton KL, D'Souza D, Freed EO - PLoS ONE (2014)

Bottom Line: While the antagonism of human tetherin by Vpu has been associated with both proteasomal and lysosomal degradation, the link between Vpu-mediated tetherin degradation and the ability of Vpu to counteract the antiviral activity of tetherin remains poorly understood.This stabilization of tetherin by Vpu requires the transmembrane sequence of human tetherin.This study demonstrates that COS cells provide a model system in which Vpu-mediated enhancement of HIV-1 release is uncoupled from Vpu-mediated tetherin degradation.

View Article: PubMed Central - PubMed

Affiliation: Virus-Cell Interaction Section, HIV Drug Resistance Program, NCI-Frederick, Frederick, Maryland, United States of America.

ABSTRACT
The interferon-inducible cellular protein tetherin (CD317/BST-2) inhibits the release of a broad range of enveloped viruses. The HIV-1 accessory protein Vpu enhances virus particle release by counteracting this host restriction factor. While the antagonism of human tetherin by Vpu has been associated with both proteasomal and lysosomal degradation, the link between Vpu-mediated tetherin degradation and the ability of Vpu to counteract the antiviral activity of tetherin remains poorly understood. Here, we show that human tetherin is expressed at low levels in African green monkey kidney (COS) cells. However, Vpu markedly increases tetherin expression in this cell line, apparently by sequestering it in an internal compartment that bears lysosomal markers. This stabilization of tetherin by Vpu requires the transmembrane sequence of human tetherin. Although Vpu stabilizes human tetherin in COS cells, it still counteracts the ability of tetherin to suppress virus release. The enhancement of virus release by Vpu in COS cells is associated with a modest reduction in cell-surface tetherin expression, even though the overall expression of tetherin is higher in the presence of Vpu. This study demonstrates that COS cells provide a model system in which Vpu-mediated enhancement of HIV-1 release is uncoupled from Vpu-mediated tetherin degradation.

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Vpu counteracts the virus release inhibition mediated by tetherin in COS cells.293T or COS cells were co-transfected with WT pNL4-3 or Vpu-defective (pNL4-3/delVpu) molecular clones and human or agm tetherin expression vectors in the absence or presence of Vpu expression plasmid (15∶1∶5 NL4-3:tetherin:Vpu DNA ratio). (A) One day posttransfection, virus-containing supernatants were harvested and virions were pelleted by ultracentrifugation. Cell and virus lysates were subjected to western blot analysis with HIV-Ig. (B) One day posttransfection with pNL4-3/delVpu without (−Vpu) or with (+Vpu) Vpu expression plasmid and either human or agm tetherin expression vector, cells were metabolically labeled for 2 h, HIV proteins from cell and virus lysates were immunoprecipitated with HIV-Ig and analyzed by SDS-PAGE followed by fluorography. Relative virus release efficiency was calculated as the amount of virion-associated p24 relative to total Gag (cell + virion), normalized to 1 for release in the absence of Vpu. Data shown are means ± SD from four independent experiments.
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pone-0111628-g004: Vpu counteracts the virus release inhibition mediated by tetherin in COS cells.293T or COS cells were co-transfected with WT pNL4-3 or Vpu-defective (pNL4-3/delVpu) molecular clones and human or agm tetherin expression vectors in the absence or presence of Vpu expression plasmid (15∶1∶5 NL4-3:tetherin:Vpu DNA ratio). (A) One day posttransfection, virus-containing supernatants were harvested and virions were pelleted by ultracentrifugation. Cell and virus lysates were subjected to western blot analysis with HIV-Ig. (B) One day posttransfection with pNL4-3/delVpu without (−Vpu) or with (+Vpu) Vpu expression plasmid and either human or agm tetherin expression vector, cells were metabolically labeled for 2 h, HIV proteins from cell and virus lysates were immunoprecipitated with HIV-Ig and analyzed by SDS-PAGE followed by fluorography. Relative virus release efficiency was calculated as the amount of virion-associated p24 relative to total Gag (cell + virion), normalized to 1 for release in the absence of Vpu. Data shown are means ± SD from four independent experiments.

Mentions: To examine the impact of tetherin stabilization on the ability of Vpu to counteract tetherin-mediated restriction of virus release, we expressed NL4-3delVpu with human or agm tetherin in the absence or presence of Vpu and measured virus release efficiency by radio-immunoprecipitation analysis. 293T cells were examined in parallel. As reported [1], [68], the release efficiency of delVpu virus is comparable to that of the WT in both 293T and COS cells, as these cell lines are deficient for tetherin expression. In contrast, in the presence of human or agm tetherin, HIV-1 release is severely inhibited in both of these cell lines (Fig. 4A and Fig. S2). Vpu co-expression rescued HIV-1 release by ∼10-fold in 293T cells (Fig. 4B). In COS cells, although Vpu increased the steady-state levels of tetherin (Fig. 1B) it was still able to rescue HIV-1 release by ∼5-fold. As expected [26], Vpu showed minimal effect on virus release in the presence of agm tetherin independent of cell line (Fig. 4B and Fig. S2).


In COS cells Vpu can both stabilize tetherin expression and counteract its antiviral activity.

Waheed AA, Kuruppu ND, Felton KL, D'Souza D, Freed EO - PLoS ONE (2014)

Vpu counteracts the virus release inhibition mediated by tetherin in COS cells.293T or COS cells were co-transfected with WT pNL4-3 or Vpu-defective (pNL4-3/delVpu) molecular clones and human or agm tetherin expression vectors in the absence or presence of Vpu expression plasmid (15∶1∶5 NL4-3:tetherin:Vpu DNA ratio). (A) One day posttransfection, virus-containing supernatants were harvested and virions were pelleted by ultracentrifugation. Cell and virus lysates were subjected to western blot analysis with HIV-Ig. (B) One day posttransfection with pNL4-3/delVpu without (−Vpu) or with (+Vpu) Vpu expression plasmid and either human or agm tetherin expression vector, cells were metabolically labeled for 2 h, HIV proteins from cell and virus lysates were immunoprecipitated with HIV-Ig and analyzed by SDS-PAGE followed by fluorography. Relative virus release efficiency was calculated as the amount of virion-associated p24 relative to total Gag (cell + virion), normalized to 1 for release in the absence of Vpu. Data shown are means ± SD from four independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4216104&req=5

pone-0111628-g004: Vpu counteracts the virus release inhibition mediated by tetherin in COS cells.293T or COS cells were co-transfected with WT pNL4-3 or Vpu-defective (pNL4-3/delVpu) molecular clones and human or agm tetherin expression vectors in the absence or presence of Vpu expression plasmid (15∶1∶5 NL4-3:tetherin:Vpu DNA ratio). (A) One day posttransfection, virus-containing supernatants were harvested and virions were pelleted by ultracentrifugation. Cell and virus lysates were subjected to western blot analysis with HIV-Ig. (B) One day posttransfection with pNL4-3/delVpu without (−Vpu) or with (+Vpu) Vpu expression plasmid and either human or agm tetherin expression vector, cells were metabolically labeled for 2 h, HIV proteins from cell and virus lysates were immunoprecipitated with HIV-Ig and analyzed by SDS-PAGE followed by fluorography. Relative virus release efficiency was calculated as the amount of virion-associated p24 relative to total Gag (cell + virion), normalized to 1 for release in the absence of Vpu. Data shown are means ± SD from four independent experiments.
Mentions: To examine the impact of tetherin stabilization on the ability of Vpu to counteract tetherin-mediated restriction of virus release, we expressed NL4-3delVpu with human or agm tetherin in the absence or presence of Vpu and measured virus release efficiency by radio-immunoprecipitation analysis. 293T cells were examined in parallel. As reported [1], [68], the release efficiency of delVpu virus is comparable to that of the WT in both 293T and COS cells, as these cell lines are deficient for tetherin expression. In contrast, in the presence of human or agm tetherin, HIV-1 release is severely inhibited in both of these cell lines (Fig. 4A and Fig. S2). Vpu co-expression rescued HIV-1 release by ∼10-fold in 293T cells (Fig. 4B). In COS cells, although Vpu increased the steady-state levels of tetherin (Fig. 1B) it was still able to rescue HIV-1 release by ∼5-fold. As expected [26], Vpu showed minimal effect on virus release in the presence of agm tetherin independent of cell line (Fig. 4B and Fig. S2).

Bottom Line: While the antagonism of human tetherin by Vpu has been associated with both proteasomal and lysosomal degradation, the link between Vpu-mediated tetherin degradation and the ability of Vpu to counteract the antiviral activity of tetherin remains poorly understood.This stabilization of tetherin by Vpu requires the transmembrane sequence of human tetherin.This study demonstrates that COS cells provide a model system in which Vpu-mediated enhancement of HIV-1 release is uncoupled from Vpu-mediated tetherin degradation.

View Article: PubMed Central - PubMed

Affiliation: Virus-Cell Interaction Section, HIV Drug Resistance Program, NCI-Frederick, Frederick, Maryland, United States of America.

ABSTRACT
The interferon-inducible cellular protein tetherin (CD317/BST-2) inhibits the release of a broad range of enveloped viruses. The HIV-1 accessory protein Vpu enhances virus particle release by counteracting this host restriction factor. While the antagonism of human tetherin by Vpu has been associated with both proteasomal and lysosomal degradation, the link between Vpu-mediated tetherin degradation and the ability of Vpu to counteract the antiviral activity of tetherin remains poorly understood. Here, we show that human tetherin is expressed at low levels in African green monkey kidney (COS) cells. However, Vpu markedly increases tetherin expression in this cell line, apparently by sequestering it in an internal compartment that bears lysosomal markers. This stabilization of tetherin by Vpu requires the transmembrane sequence of human tetherin. Although Vpu stabilizes human tetherin in COS cells, it still counteracts the ability of tetherin to suppress virus release. The enhancement of virus release by Vpu in COS cells is associated with a modest reduction in cell-surface tetherin expression, even though the overall expression of tetherin is higher in the presence of Vpu. This study demonstrates that COS cells provide a model system in which Vpu-mediated enhancement of HIV-1 release is uncoupled from Vpu-mediated tetherin degradation.

Show MeSH
Related in: MedlinePlus