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APOBEC3G-depleted resting CD4+ T cells remain refractory to HIV1 infection.

Santoni de Sio FR, Trono D - PLoS ONE (2009)

Bottom Line: They further suggested that T cell activation abrogates the A3G-mediated block by directing this protein to a high molecular mass complex.However, we found that effective suppression of A3G by combined RNA interference and expression of HIV1 Vif does not relieve the restrictive phenotype of post-activation resting T cells.We also failed to find a correlation between HIV resistance and the presence of A3G in a low molecular complex in primary T cells.

View Article: PubMed Central - PubMed

Affiliation: Global Health Institute, School of Life Sciences, Frontiers in Genetics National Center of Competence in Research, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

ABSTRACT

Background: CD4+ T lymphocytes are the primary targets of HIV1 but cannot be infected when fully quiescent, due to a post-entry block preventing the completion of reverse transcription. Chiu et al. recently proposed that this restriction reflects the action of APOBEC3G (A3G). They further suggested that T cell activation abrogates the A3G-mediated block by directing this protein to a high molecular mass complex.

Methodology/principal findings: In the present work, we sought to explore further this model. However, we found that effective suppression of A3G by combined RNA interference and expression of HIV1 Vif does not relieve the restrictive phenotype of post-activation resting T cells. We also failed to find a correlation between HIV resistance and the presence of A3G in a low molecular complex in primary T cells.

Conclusions/significance: We conclude that A3G is unlikely to play a role in the HIV restrictive phenotype of quiescent T lymphocytes.

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Related in: MedlinePlus

Experimental approach.A, CD4+ cells were purified from human peripheral blood (resting), exposed to high-dose IL2 and PHA for two days (activated) and then maintained for 11–14 days in low-dose IL2 (resting post-act.). B, the three cell populations were analyzed for expression of activation markers expression (CD25, CD69) (left) and GFP production 3 days after infection with a GFP-containing HIV1 derivative.
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pone-0006571-g001: Experimental approach.A, CD4+ cells were purified from human peripheral blood (resting), exposed to high-dose IL2 and PHA for two days (activated) and then maintained for 11–14 days in low-dose IL2 (resting post-act.). B, the three cell populations were analyzed for expression of activation markers expression (CD25, CD69) (left) and GFP production 3 days after infection with a GFP-containing HIV1 derivative.

Mentions: In their work leading to the proposal that A3G is responsible for restricting HIV in quiescent CD4+ cells, Chiu et al. down-regulated the protein by siRNA tranfection [8]. In order to facilitate subsequent mechanistic studies, we decided to use an alternative model, in which activated CD4+ T lymphocytes were transduced with lentiviral vectors carrying effector sequences, and analyzed for HIV1 permissiveness once back to a resting and restrictive state. For this, CD4+ T cells were purified from human PB (resting cells), stimulated by IL2 and phytohemagglutinin (PHA) (activated cells) and led to rest again by decreasing IL-2 concentration in the culture medium (resting post-activation cells) (Figure 1a). To validate our model, we first compared the level of activation and susceptibility to infection of the three populations, which were analyzed by flow cytometry for the presence of activation markers and for GFP expression following exposure to a GFP-expressing HIV reporter virus. As expected, activated T cells expressed high levels of the activation markers CD25 and CD69, and were susceptible to HIV infection (68% of GFP+ cells). In contrast, resting post-activation cells expressed negligible levels of activation markers and were fully restrictive to HIV infection (0.1% of GFP+ cells), as were un-activated resting cells (1% GFP+ cells) (Figure 1b).


APOBEC3G-depleted resting CD4+ T cells remain refractory to HIV1 infection.

Santoni de Sio FR, Trono D - PLoS ONE (2009)

Experimental approach.A, CD4+ cells were purified from human peripheral blood (resting), exposed to high-dose IL2 and PHA for two days (activated) and then maintained for 11–14 days in low-dose IL2 (resting post-act.). B, the three cell populations were analyzed for expression of activation markers expression (CD25, CD69) (left) and GFP production 3 days after infection with a GFP-containing HIV1 derivative.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2719058&req=5

pone-0006571-g001: Experimental approach.A, CD4+ cells were purified from human peripheral blood (resting), exposed to high-dose IL2 and PHA for two days (activated) and then maintained for 11–14 days in low-dose IL2 (resting post-act.). B, the three cell populations were analyzed for expression of activation markers expression (CD25, CD69) (left) and GFP production 3 days after infection with a GFP-containing HIV1 derivative.
Mentions: In their work leading to the proposal that A3G is responsible for restricting HIV in quiescent CD4+ cells, Chiu et al. down-regulated the protein by siRNA tranfection [8]. In order to facilitate subsequent mechanistic studies, we decided to use an alternative model, in which activated CD4+ T lymphocytes were transduced with lentiviral vectors carrying effector sequences, and analyzed for HIV1 permissiveness once back to a resting and restrictive state. For this, CD4+ T cells were purified from human PB (resting cells), stimulated by IL2 and phytohemagglutinin (PHA) (activated cells) and led to rest again by decreasing IL-2 concentration in the culture medium (resting post-activation cells) (Figure 1a). To validate our model, we first compared the level of activation and susceptibility to infection of the three populations, which were analyzed by flow cytometry for the presence of activation markers and for GFP expression following exposure to a GFP-expressing HIV reporter virus. As expected, activated T cells expressed high levels of the activation markers CD25 and CD69, and were susceptible to HIV infection (68% of GFP+ cells). In contrast, resting post-activation cells expressed negligible levels of activation markers and were fully restrictive to HIV infection (0.1% of GFP+ cells), as were un-activated resting cells (1% GFP+ cells) (Figure 1b).

Bottom Line: They further suggested that T cell activation abrogates the A3G-mediated block by directing this protein to a high molecular mass complex.However, we found that effective suppression of A3G by combined RNA interference and expression of HIV1 Vif does not relieve the restrictive phenotype of post-activation resting T cells.We also failed to find a correlation between HIV resistance and the presence of A3G in a low molecular complex in primary T cells.

View Article: PubMed Central - PubMed

Affiliation: Global Health Institute, School of Life Sciences, Frontiers in Genetics National Center of Competence in Research, Ecole Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

ABSTRACT

Background: CD4+ T lymphocytes are the primary targets of HIV1 but cannot be infected when fully quiescent, due to a post-entry block preventing the completion of reverse transcription. Chiu et al. recently proposed that this restriction reflects the action of APOBEC3G (A3G). They further suggested that T cell activation abrogates the A3G-mediated block by directing this protein to a high molecular mass complex.

Methodology/principal findings: In the present work, we sought to explore further this model. However, we found that effective suppression of A3G by combined RNA interference and expression of HIV1 Vif does not relieve the restrictive phenotype of post-activation resting T cells. We also failed to find a correlation between HIV resistance and the presence of A3G in a low molecular complex in primary T cells.

Conclusions/significance: We conclude that A3G is unlikely to play a role in the HIV restrictive phenotype of quiescent T lymphocytes.

Show MeSH
Related in: MedlinePlus