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Clinical Trial
. 2013;9(5):e1003328.
doi: 10.1371/journal.ppat.1003328. Epub 2013 May 2.

DRAM triggers lysosomal membrane permeabilization and cell death in CD4(+) T cells infected with HIV

Mireille Laforge  1 , Sophie Limou, Francis Harper, Nicoletta Casartelli, Vasco Rodrigues, Ricardo Silvestre, Houda Haloui, Jean-Francois Zagury, Anna Senik, Jerome Estaquier
Affiliations
Clinical Trial

DRAM triggers lysosomal membrane permeabilization and cell death in CD4(+) T cells infected with HIV

Mireille Laforge et al. PLoS Pathog. 2013.

"V体育官网入口" Abstract

Productive HIV infection of CD4(+) T cells leads to a caspase-independent cell death pathway associated with lysosomal membrane permeabilization (LMP) and cathepsin release, resulting in mitochondrial outer membrane permeabilization (MOMP). Herein, we demonstrate that HIV infection induces damage-regulated autophagy modulator (DRAM) expression in a p53-dependent manner. Knocking down the expression of DRAM and p53 genes with specific siRNAs inhibited autophagy and LMP. However, inhibition of Atg5 and Beclin genes that prevents autophagy had a minor effect on LMP and cell death VSports手机版. The knock down of DRAM gene inhibited cytochrome C release, MOMP and cell death. However, knocking down DRAM, we increased viral infection and production. Our study shows for the first time the involvement of DRAM in host-pathogen interactions, which may represent a mechanism of defense via the elimination of infected cells. .

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"V体育官网" Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. HIV-1 infection induces DRAM expression in CD4+ T cells.
Primary CD4+ T cells in the absence (NI) or presence of HIV-1LAI (HIV-1) have been analyzed. (A) Lysates of CD4+ T cells were prepared on days 3, 4 and 5 post-infection and separated by western blots, and then immunoblotted with specific antibody against DRAM. Actin was used as a control for protein loading. (B) DRAM protein levels were quantified in three independent experiments. Non-infected cells on day 3 were considered arbitrarily equivalent to 1 for normalization. Bars show the mean ± SD. The asterisk (*) indicates statistically significant difference, p<0.05. (C) DRAM mRNA level was quantified by RT-PCR on days 3, 4 and 5 post-infection. Non-infected cells were considered arbitrarily equivalent to 1. Bars show the mean ± SD of three independent experiments. The asterisk (*) indicates statistically significant difference, p<0.05. (D) HIV-infected CD4+ T lymphocytes were transfected with either control siRNA (mock) or siRNAs specific for p53 or DRAM. Cells extracts at day 5 post-transfection were analyzed and immunoblotted with specific p53 and DRAM antibodies. Actin was used as a control of loading. (E) Bars show the mean ± SD of four independent experiments. (F) Cells were stained with antibodies against p53 or DRAM (in red) or with an antibody against Gag (in green). Nuclei were counterstained with DAPI (blue).
Figure 2
Figure 2. HIV-1 infection induces autophagic machinery in CD4+ T cells.
(A) Western-blot analysis of LC3-I and LC3-II proteins in CD4+ T cells in the absence (NI) or presence of HIV-1LAI (HIV-1) on days 4, 5 and 6 post-infection. Actin was used as a control for protein loading. Ratio of LC3-II/LC3-I was calculated and bars show the mean ± SD of four independent experiments; *, p<0.05. (B) Cells were stained on day 5 with mAbs recognizing the LC3 antigen (red) and the Gag antigen (green), and examined by confocal microscopy. The insets show magnified images of LC3-II puncta in infected cells. (C) Percentages of CD4+ T cells displaying punctate LC3-II staining on days 4, 5 and 6 post-infection. Histograms show the means ± SD of five individual experiments; *, p<0.05. More than 200 cells were counted for each condition. (D) Within infected samples, quantification of Gag+ and Gag− cells with punctate LC3-II accumulation. More than 200 cells were counted for each staining, 5 days post-infection, and the results presented are means ± SD of five independent experiments; *, p<0.05.
Figure 3
Figure 3. DRAM regulates autophagy in CD4+ T cells infected with HIV-1.
(A) Immunoblots of lysates from CD4+ T cells in the absence (NI) or presence of HIV-1LAI (HIV-1) at day 5 after infection. Membranes were probed for LC3, beclin 1, Atg5, and actin. (B) The amount of each protein was measured and normalized with respect to the loading control (Actin) (relative expression). Bars show the mean ± SD of five independent experiments. *, p<0.05. (C) Beclin 1 mRNA level was quantified by RT-PCR on days 4 and 5 post-infection. Non-infected cells (NI) on day 4 were considered arbitrarily equivalent to 1 for normalization. Bars show the mean ± SD of three independent experiments (*, p<0.05). (D) Samples from uninfected (NI) and HIV-infected CD4+ T cells incubated in the absence or presence of Pepstatin A (PA). At day 5 the cells were dissolved in SDS. Membranes were probed for LC3-II and actin. Bars show the mean ± SD of three independent experiments; *, p<0.05. (E) Western-blot analysis of p62 protein in CD4+ T cells in the absence (NI) or presence of HIV-1LAI (HIV-1) on days 4, 5 and 6 post-infection. Actin was used as a control for protein loading. The level of p62 was calculated, and the levels in non-infected cells (NI) on day 4 were considered arbitrarily equivalent to 1. (F) CD4+ T cells were transfected with siRNA specific for p53 and DRAM or the control siRNA (mock) and then infected with HIV-1. On day 5 post-infection, cells were analyzed by immunoblotting using LC-3 antibody. Actin was a control of loading. Values represent LC3-II/LC3-I ratio. (G) LC3-II staining (number of puncta per cell ≥6) was examined by fluorescence microscopy in Gag+ target cells. The values shown are means ± SD of three independent experiments (≥200 cells were examined per experiment).
Figure 4
Figure 4. DRAM induces lysosomal membrane permeabilization (LMP) in HIV-infected CD4 T cells.
CD4+ T cells were transfected with siRNA specific for p53 and DRAM or the control siRNA (mock). (A) Cells were loaded with fluorescent dextran molecules of 40 kDa and examined by laser scanning confocal microscopy. Pictures show gag+ and gag− cells from infected samples. Diffuse staining revealed lysosomal permeabilization. (B) The values shown are means ± SD of two independent experiments (≥200 cells were examined).
Figure 5
Figure 5. DRAM induces cathepsin D and cytochrome C release in HIV-infected CD4+ T cells.
HIV-infected CD4+ T cells were transfected with siRNA specific for p53 and DRAM or the control siRNA (mock) and then infected with HIV-1. Non-infected cells were used as a control. (A) At day 5 post-infection, cells were stained with specific antibodies against cathepsin D (Cat D), cytochrome C (Cyt c) and Gag antigen. (B) The subcellular distribution of Cat D and Cyt c in the Gag+ cells was analyzed. More than 200 cells were counted for each staining and the results shown are the means ± SD of three independent experiments; * p<0.05. (C) Heavy membrane and cytosolic fractions derived from CD4+ T cells, five days after infection, were analyzed by western blotting for the presence of Cat D and Cyt c. Anti-HSP60 and anti-Lamp-1 were used to verify the absence of mitochondrial and lysosomal contamination, respectively. Actin was used as a control of loading. One representative experiment out to three performed giving similar results is shown.
Figure 6
Figure 6. DRAM induces cell death in HIV-infected CD4+ T cells.
CD4+ T cells were transfected with siRNAs specific for p53 or DRAM or the control siRNA (mock), and then infected with HIV. (A) ΔΨm loss was assessed using a DioC6 probe at day 5 post-infection. A representative experiment out five independent experiments is shown. (B) The values shown are means ± SD; *, p<0.05. (C) Cell death was assessed using a propidium iodide (PI) probe at day 5 post-infection. A representative experiment is shown. (D) The values shown are means ± SD of three independent experiments; *, p<0.05. (E) Absolute numbers of infected cells were calculated on days 5 (black square) and 6 (white square) post-infection. Histograms show means ± SD of three independent experiments; * p<0.05. (F) Viral production was followed by measuring p24 release in the supernatants of CD4+ T cells. Results are the mean ± SD of three independent experiments; * p<0.05.
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References

    1. Jaattela M (2004) Multiple cell death pathways as regulators of tumour initiation and progression. Oncogene 23: 2746–2756. - PubMed
    1. Guicciardi ME, Leist M, Gores GJ (2004) Lysosomes in cell death. Oncogene 23: 2881–2890. - PubMed
    1. Stoka V, Turk B, Schendel SL, Kim TH, Cirman T, et al. (2001) Lysosomal protease pathways to apoptosis. Cleavage of bid, not pro-caspases, is the most likely route. J Biol Chem 276: 3149–3157. - PubMed (V体育官网)
    1. Bidere N, Lorenzo HK, Carmona S, Laforge M, Harper F, et al. (2003) Cathepsin D triggers Bax activation, resulting in selective apoptosis-inducing factor (AIF) relocation in T lymphocytes entering the early commitment phase to apoptosis. J Biol Chem 278: 31401–31411. - PubMed
    1. Moutouh L, Estaquier J, Richman DD, Corbeil J (1998) Molecular and cellular analysis of human immunodeficiency virus-induced apoptosis in lymphoblastoid T-cell-line-expressing wild-type and mutated CD4 receptors. J Virol 72: 8061–8072. - PMC - PubMed

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