Blockade of BFA-mediated apoptosis in macrophages by the HIV-1 Nef protein

Abstract

HIV-1 Nef protein has key roles at almost all stages of the viral life cycle. We assessed the role of Nef and of the translation elongation factor eEF1A in primary human macrophages. Nuclear retention experiments and inhibition of the exportin-t (Exp-t) pathway suggested that cytoplasmic relocalization of eEF1A, mediated by Exp-t occurs in Nef-treated monocyte-derived macrophages (MDMs). We observed the presence of tRNA in the Nef/eEF1A complexes. Nucleocytoplasmic relocalization of the Nef/eEF1A complexes prevented stress-induced apoptosis of MDMs treated with brefeldin A. Blockade of stress-induced apoptosis of MDMs treated with HIV-1 Nef resulted from enhanced nucleocytoplasmic transport of eEF1A with decreased release of mitochondrial cytochrome c, and from increased tRNA binding to cytochrome c, ultimately leading to an inhibition of caspase activation. Our results indicate that HIV-1 Nef, through the nucleocytoplasmic relocalization of eEF1A and tRNAs, enhances resistance to stress-induced apoptosis in primary human macrophages.

Figure 1

eEF1A interacts with HIV-1 Nef protein in vitro and in vivo. (a) Binding of HIV-1 Nef to eEF1A was measured in GST pull-down assays using U937 cells as a source of lysates. β-actin detection represents input loading controls of the lysates that were used in binding reactions. Results are representative of three independent experiments. The right panel represents coomassie staining of expressed proteins that were used in the binding reaction of GST pull-down. (b) Cytoplasmic and nuclear extracts from several cell lines (Vero, MRC5, and U937 cells), PBLs, and MDMs treated with rNef (100 ng/ml) for 0.5 h were immunoprecipitated with an anti-eEF1A antibody, or Nef (100 ng/ml)-treated total cellular lysates were immunoprecipitated with an isotype control antibody. Immunoprecipitated material was analyzed by western blotting with an anti-Nef monoclonal antibody. Recombinant Nef-treated total cell lysates were used as a positive control. Results are representative of three independent experiments. (c) Cellular extracts of U937 cells transfected for 48 h with a nef-expressing plasmid (pNef) were immunoprecipitated with an anti-eEF1A antibody, anti-Nef antibody, or isotype control antibody. Immunoprecipitated material was analyzed by western blotting with an anti-Nef monoclonal antibody. Results are representative of two independent experiments. (d) Cellular extracts of PBMCs infected in vitro with HIV-1_89.6 or mock infected were immunoprecipitated with an anti-eEF1A antibody or anti-Nef monoclonal antibody. Immunoprecipitated material was analyzed by western blotting with an anti-Nef monoclonal antibody or anti-eEF1A antibody. Results are representative of two independent experiments. (e and f) eEF1A and HIV-1 Nef interact in vitro in a mammalian two-hybrid assay. (e) Schematic representation of expression constructs used in co-transfection experiments in the mammalian two-hybrid model. (f) Mammalian two-hybrid analysis with eEF1A fused to the VP16 activator domain and HIV-1 Nef fused to the GAL4 domain. Luciferase assays were conducted on total extracts from U937 cells transfected with the luciferase expression construct pG5-Luc, pBIND-Nef, pACT-eEF1A, or control plasmids. As a positive control, two plasmids, pACT-MyoD and pBIND-Id, were co-transfected, and co-transfection of empty vectors was used as a negative control. Results represent the mean of three independent experiments. ***P<0.001

Figure 2

The N-terminal 74 amino acids of eEF1A bind to the C-terminal region (aa 55–206) of HIV-1 Nef. (a) The N-terminal region of eEF1A is sufficient for binding to HIV-1 Nef. Left panel: schematic diagram of eEF1A mutants expressed in bacteria as HA-tagged fusion proteins and their Nef-binding properties. Numbers indicate the amino-acid residues retained by the deletion mutants. In vitro Nef binding of these mutants are reported qualitatively as + or − to the right of the figure. N/A, not applicable; WT, wild type. Right panel: eEF1A interacts with HIV-1 Nef via its N-terminus extremity (aa 1–74). Using wild-type GST–Nef constructs, binding of purified WT eEF1A, eEF1A 1–74, eEF1A 14–74 and eEF1A 298–463 were measured in GST pull-down assays. Input corresponds to 10% of the material used for pull-down. Results are representative of two independent experiments. (b) The C-terminal region (aa 55–206) of HIV-1 Nef is sufficient for binding to eEF1A. Left panel: schematic diagram of WT HIV-1 Nef and mutants expressed in bacteria as GST-tagged fusion proteins. The names of the mutants are shown to the left of the figure; numbers refer to the amino-acid residues retained by the deletion mutants. The bacterial expression status and in vitro binding of these mutants are reported qualitatively as + or − to the right of the figure. Right panel represents the interaction of HIV-1 Nef with eEF1A via its C-terminal region (aa 55–206) and coomassie staining of expressed proteins that were used in the binding reaction of GST pull-down. The binding of eEF1A present in lysates of MDMs was measured in GST pull-down assays using WT and mutated GST–Nef constructs. Results are representative of two independent experiments

Figure 3

Nuclear–cytoplasmic relocalization of eEF1A/rNef occurs in MDMs treated with rNef and depends on Exp-t. (a) Kinetics of eEF1A/rNef/Exp-t in nuclear and cytoplasmic compartments of MDMs. Nuclear and cytoplasmic extracts of MDMs treated with rNef (100 ng/ml) were prepared and the expression of eEF1A, Exp-t and rNef were assessed in both cellular compartments up to 12 h post treatment using western blotting. Similarly, nuclear and cytoplasmic extracts of untreated MDMs (mock) were prepared and tested for the expression of eEF1A, Exp-t and rNef. β-actin and TBP were used as loading controls. Results are representative of three independent experiments. (b) Using wild-type GST–Nef constructs, the binding of Exp-t present in MDM lysates was assessed in GST pull-down assays. Input corresponds to 10% of the material used for pull-down. Results are representative of two independent experiments. (c) eEF1A interacts with Exp-t in MDM lysates. Total MDM extracts were prepared and the eEF1A/Exp-t interaction assessed by immunoprecipitation with an anti-eEF1A antibody and western blotting with an anti-Exp-t monoclonal antibody. Results are representative of three independent experiments. (d) Knockdown of the Exp-t protein by siRNA in MDMs. MDM cultures were transfected with a scrambled control or Exp-t siRNA and total cellular extracts were prepared 48 h post transfection. Protein expression was analyzed by western blot. β-actin was used as a loading control. (e) Effect of Exp-t siRNA on nuclear–cytoplasmic transport of eEF1A and rNef in MDMs. MDM cultures were transfected with a scrambled control or Exp-t siRNA for 48 h before treatment with rNef (100 ng/ml) for 3 h. Nuclear and cytoplasmic extracts were prepared and analyzed by western blot using anti-Nef and anti-eEF1A antibodies. β-actin and TBP were used as loading controls. Results are representative of two independent experiments. Protein levels of eEF1A and Nef after siRNA transfection were quantified by densitometry using ImageJ 1.40 software (protein levels in cells transfected with scrambled siRNAs were arbitrarily established at 1)

Figure 4

rNef-mediated inhibition of BFA-induced apoptosis in MDMs parallels cytoplasmic accumulation of eEF1A and is dependent on Exp-t. (a) rNef prevents BFA-induced apoptosis in MDMs. MDMs were treated with BFA (10 μg/ml) for 12 or 15 h or left untreated in the presence of increasing concentrations of rNef (0, 125, 750 ng/ml). Apoptosis was detected by annexin-V flow cytometric analysis. The histogram summarizes the survival of MDMs following treatment with BFA (10 μg/ml) for 12 or 15 h in the presence of increasing concentrations of rNef. The results represent means of three independent experiments. *P<0.05. (b) rNef prevents BFA-induced apoptosis, but neither TM-induced apoptosis nor TG-induced apoptosis in MDMs. MDMs were treated with BFA (10 μg/ml), TM (10 μg/ml) or TG (10 μg/ml) for 5 or 12 h or left untreated in the presence of rNef (1 μg/ml). Apoptosis was measured by the TUNEL assay. Results are representative of data observed in three independent experiments. The mock panel is shown in triplicate to the left of the BFA, TM and TG panels to facilitate the results reading. (c) The C-terminal extremity of Nef prevents BFA-induced apoptosis in MDMs. MDMs were treated with BFA (10 μg/ml) for 12 h or left untreated in the presence of WTNef, Nef1-60 or Nef55-206 (1 μg/ml). Apoptosis was measured by the TUNEL assay. Results are representative of data observed in two independent experiments. (d) Blockade of BFA-induced apoptosis in MDMs by rNef is dependent on Exp-t. MDM cultures were transfected with a scrambled control, Exp-t siRNA, Exp-1 siRNA, or Exp-5 siRNA for 48 h before treatment, with BFA (10 μg/ml) for 12 h or left untreated in the presence (1 μg/ml) or absence of rNef. Apoptosis was detected by flow cytometric analysis of annexin-V. The histogram shows the survival percentage of MDMs treated with mock, BFA, or BFA+Nef in the presence of exportin siRNAs. Results are representative of three independent experiments. *P<0.05. (e) Blockage of BFA-induced apoptosis in MDMs by rNef is dependent on eEF1A. MDMs were treated with control siRNA or eEF1A siRNA for 48 h before treatment, with BFA (10 μg/ml) for 12 h or left untreated in the presence (1 μg/ml) or absence of rNef. Apoptosis was measured by the TUNEL assay. Results are representative of two independent experiments. EF1A knockdown in MDMs was monitored by western blot (upper right panel). The histogram shows the percentage of TdT positive cells

Figure 4

rNef-mediated inhibition of BFA-induced apoptosis in MDMs parallels cytoplasmic accumulation of eEF1A and is dependent on Exp-t. (a) rNef prevents BFA-induced apoptosis in MDMs. MDMs were treated with BFA (10 μg/ml) for 12 or 15 h or left untreated in the presence of increasing concentrations of rNef (0, 125, 750 ng/ml). Apoptosis was detected by annexin-V flow cytometric analysis. The histogram summarizes the survival of MDMs following treatment with BFA (10 μg/ml) for 12 or 15 h in the presence of increasing concentrations of rNef. The results represent means of three independent experiments. *P<0.05. (b) rNef prevents BFA-induced apoptosis, but neither TM-induced apoptosis nor TG-induced apoptosis in MDMs. MDMs were treated with BFA (10 μg/ml), TM (10 μg/ml) or TG (10 μg/ml) for 5 or 12 h or left untreated in the presence of rNef (1 μg/ml). Apoptosis was measured by the TUNEL assay. Results are representative of data observed in three independent experiments. The mock panel is shown in triplicate to the left of the BFA, TM and TG panels to facilitate the results reading. (c) The C-terminal extremity of Nef prevents BFA-induced apoptosis in MDMs. MDMs were treated with BFA (10 μg/ml) for 12 h or left untreated in the presence of WTNef, Nef1-60 or Nef55-206 (1 μg/ml). Apoptosis was measured by the TUNEL assay. Results are representative of data observed in two independent experiments. (d) Blockade of BFA-induced apoptosis in MDMs by rNef is dependent on Exp-t. MDM cultures were transfected with a scrambled control, Exp-t siRNA, Exp-1 siRNA, or Exp-5 siRNA for 48 h before treatment, with BFA (10 μg/ml) for 12 h or left untreated in the presence (1 μg/ml) or absence of rNef. Apoptosis was detected by flow cytometric analysis of annexin-V. The histogram shows the survival percentage of MDMs treated with mock, BFA, or BFA+Nef in the presence of exportin siRNAs. Results are representative of three independent experiments. *P<0.05. (e) Blockage of BFA-induced apoptosis in MDMs by rNef is dependent on eEF1A. MDMs were treated with control siRNA or eEF1A siRNA for 48 h before treatment, with BFA (10 μg/ml) for 12 h or left untreated in the presence (1 μg/ml) or absence of rNef. Apoptosis was measured by the TUNEL assay. Results are representative of two independent experiments. EF1A knockdown in MDMs was monitored by western blot (upper right panel). The histogram shows the percentage of TdT positive cells

Figure 5

rNef-mediated cytoplasmic accumulation of eEF1A in BFA-treated MDMs inhibits caspase activation and decreases the cytoplasmic release of cytochrome c. (a) Inhibition of caspase-3 activation in BFA-stimulated MDMs treated with rNef (1000 ng/ml). (b) Inhibition of caspase-3 activation in BFA-stimulated MDMs treated with rNef is dose-dependent. Mitochondrial cytochrome c release in BFA-treated MDMs is blocked by rNef in a dose-dependent manner and positively correlates with cytoplasmic accumulation of eEF1A. (c) Knockdown of Exp-1 and Exp-5 proteins by siRNA in MDMs. MDM cultures were transfected with a scrambled control, Exp-1 siRNA, or Exp-5 siRNA. Total cellular extracts were prepared 48 h post transfection. Protein expression was analyzed by western blot. β-actin was used as a loading control. (d) Inhibition of caspase-3 activation in BFA-stimulated MDMs treated with rNef is dependent on Exp-t. (e) Mitochondrial cytochrome c release in BFA-treated MDMs is blocked by rNef. (f) Inhibition of caspase-9 activation in BFA-stimulated MDMs treated with rNef (1000 ng/ml). Protein levels of caspase-9 were quantified by densitometry using ImageJ 1.40 software (the level of caspase-9 in mock cells was arbitrarily established at 1). (g) Inhibition of caspase-9 activation in BFA-stimulated MDMs treated with rNef is dose-dependent. Protein levels of caspase-9 were quantified by densitometry using ImageJ 1.40 software (the level of caspase-9 in mock cells was arbitrarily established at 1). (h) Inhibition of caspase activation in BFA-stimulated MDMs treated with rNef, but neither in TM-stimulated MDMs nor in TG-stimulated MDMs treated with rNef. Left panel: time–course of caspase activation in BFA-stimulated MDMs, TM-stimulated MDMs or TG-stimulated MDMs. Right panel: inhibition of caspase activation in BFA-stimulated MDMs treated with rNef, but neither in TM-stimulated MDMs nor in TG-stimulated MDMs treated with rNef. MDM cultures were treated with BFA (10 μg/ml), TM (10 μg/ml), or TG (10 μg/ml) for 12 h in the presence of rNef (1 μg/ml), and caspase-3 and -9 activation was measured in total cellular lysates. Results are representative of data obtained in three independent experiments

Figure 5

rNef-mediated cytoplasmic accumulation of eEF1A in BFA-treated MDMs inhibits caspase activation and decreases the cytoplasmic release of cytochrome c. (a) Inhibition of caspase-3 activation in BFA-stimulated MDMs treated with rNef (1000 ng/ml). (b) Inhibition of caspase-3 activation in BFA-stimulated MDMs treated with rNef is dose-dependent. Mitochondrial cytochrome c release in BFA-treated MDMs is blocked by rNef in a dose-dependent manner and positively correlates with cytoplasmic accumulation of eEF1A. (c) Knockdown of Exp-1 and Exp-5 proteins by siRNA in MDMs. MDM cultures were transfected with a scrambled control, Exp-1 siRNA, or Exp-5 siRNA. Total cellular extracts were prepared 48 h post transfection. Protein expression was analyzed by western blot. β-actin was used as a loading control. (d) Inhibition of caspase-3 activation in BFA-stimulated MDMs treated with rNef is dependent on Exp-t. (e) Mitochondrial cytochrome c release in BFA-treated MDMs is blocked by rNef. (f) Inhibition of caspase-9 activation in BFA-stimulated MDMs treated with rNef (1000 ng/ml). Protein levels of caspase-9 were quantified by densitometry using ImageJ 1.40 software (the level of caspase-9 in mock cells was arbitrarily established at 1). (g) Inhibition of caspase-9 activation in BFA-stimulated MDMs treated with rNef is dose-dependent. Protein levels of caspase-9 were quantified by densitometry using ImageJ 1.40 software (the level of caspase-9 in mock cells was arbitrarily established at 1). (h) Inhibition of caspase activation in BFA-stimulated MDMs treated with rNef, but neither in TM-stimulated MDMs nor in TG-stimulated MDMs treated with rNef. Left panel: time–course of caspase activation in BFA-stimulated MDMs, TM-stimulated MDMs or TG-stimulated MDMs. Right panel: inhibition of caspase activation in BFA-stimulated MDMs treated with rNef, but neither in TM-stimulated MDMs nor in TG-stimulated MDMs treated with rNef. MDM cultures were treated with BFA (10 μg/ml), TM (10 μg/ml), or TG (10 μg/ml) for 12 h in the presence of rNef (1 μg/ml), and caspase-3 and -9 activation was measured in total cellular lysates. Results are representative of data obtained in three independent experiments

Figure 6

rNef-mediated cytoplasmic accumulation of eEF1A in BFA-treated MDMs parallels tRNA binding to cytochrome c. (a) Direct detection of tRNAs in rNef/eEF1A complexes in MDMs treated with rNef. Lysates from MDMs treated with rNef (100 ng/ml) for 3 h or left untreated (mock) were prepared, immunoprecipitated with anti-eEF1A and anti-Nef antibodies, and tRNALys in the eEF1A/rNef complexes was amplified by RT-PCR. Lysates were also immunoprecipitated with an isotype control antibody. The presence of HIV-1 Nef protein in the immune complex was determined by western blot. As an internal control, lysates from MDMs treated with rNef (100 ng/ml) for 3 h were prepared, treated with RNAse A (10 μg/ml) for 30 min, immunoprecipitated with an anti-eEF1A antibody, and tRNALys in the eEF1A/Nef complexes was amplified by RT-PCR. (b) Binding of tRNA to cytochrome c is eEF1A dependent. MDMs were mock treated or treated with BFA (10 μg/ml) or BFA (10 μg/ml)+Nef (1000 ng/ml) for 12 h in the presence of control or eEF1A siRNA. The lysates were immunoprecipitated with anti-cytochrome c, and presence and amount of tRNALys were determined by qRT-PCR and further samples were resolved on 3% agarose gel and stained with EtBr. (c and d) MDMs were treated with recombinant Nef (100 ng/ml); the lysates were immunoprecipitated with an anti-Nef Ab (c) or anti-eEF1A Ab (d) and the presence of tRNAMet, tRNALys, tRNATrp, and tRNAPhe in the Nef/eEF1A complex was determined by qRT-PCR. (e and f) MDMs were treated with BFA for 12 h in the presence or absence of rNef (0–1500 ng/ml); the lysates were immunoprecipitated with an anti-eEF1A Ab (e) or anti-cytochrome c Ab (f) and the presence of tRNAMet, tRNALys, tRNATrp, and tRNAPhe was determined by qRT-PCR. (g and h) The presence of tRNAs associated with eEF1A and cytochrome c in BFA-stimulated MDMs treated with rNef is dependent on Exp-t. MDM cultures were transfected with a scrambled control siRNA, Exp-t siRNA, Exp-1 siRNA, or Exp-5 siRNA. Cytoplasmic extracts of MDMs treated with BFA (10 μg/ml) for 12 h in the absence or presence of rNef (1000 ng/ml) were prepared 48 h post transfection, immunoprecipitated with an anti-eEF1A Ab (g) or anti-cytochrome c Ab (h), and the presence of tRNALys, tRNAMet, tRNAPhe, and tRNATrp binding to eEF1A and cytochrome c was determined by qRT-PCR. Results representative of three independent experiments are shown. *P<0.05. (i) The presence of tRNAs associated with cytochrome c in BFA-stimulated MDMs treated with rNef is dependent on eEF1A. MDM cultures were transfected with control siRNA or eEF1A siRNA. Cytoplasmic extracts of MDMs treated with BFA (10 μg/ml) for 12 h in the absence or presence of rNef (1000 ng/ml) were prepared 48 h post transfection and were immunoprecipitated with an anti-cytochrome c Ab. The presence of tRNAMet, tRNALys, tRNAPhe, and tRNATrp binding to cytochrome c was determined using qRT-PCR. Results representative of two independent experiments are shown

Figure 7

Potential inhibitory effect of the eEF1A/rNef/tRNA complex on the intrinsic apoptotic pathway in BFA-treated MDMs. HIV-1 Nef protein favors translocation and accumulation of eEF1A from the nucleus toward the cytoplasm of the cell. tRNA is present in the Nef/eEF1A complex that is exported by Exp-t and buffers the cytochrome c released under oxidative stress conditions. At the same time the Nef/eEF1A complex inhibits caspase activation. Furthermore, eEF1A could stabilize the microtubules and give relief to the cell to survive under stress conditions. BFA, brefeldin A; Exp-t, exportin-t