Figure 1.
HIV-1 RNA activates GCN2 and the kinase is proteolytically cleaved during HIV-1 infection.
(A) Purified mouse GCN2 was subjected to in vitro eIF2alpha kinase assay in the presence of increasing concentrations of in vitro transcribed RNAs corresponding to the mouse GCN2 open reading frame (as a negative control), or to the genomic RNAs of Sindbis virus or HIV-1, respectively, as indicated. In this experiment 32P-γ-ATP was included in the kinase reaction mix and phosphorylation of eIF2alpha was visualized after analysis by SDS-PAGE and autoradiography. (B) In vitro eIF2alpha kinase assay of purified wild type GCN2 (WT), GCN2-K618R (K618R) or GCN2-m2 (m2) in the presence of Sindbis virus (SV) or HIV-1 RNA, as indicated. Results were obtained by western blot analysis of the samples using different antisera to detect eIF2alpha phosphorylated on serine 51, total eIF2alpha and phosphorylated and total GCN2. (C) MT-2 cells were mock infected (−) or infected with 10 or 100 ng of p24gag per 106 cells of HIV-1NL4–3 and harvested at the indicated days post-infection (D.P.I.). Aliquots of cell lysates containing equal amount of proteins were analyzed by western blot (8% SDS-PAGE) using specific antibodies as indicated. (D) MT-2 cells were mock-infected (−) or infected with 100 ng of HIV-1 NL4-3 per 106 cells, in the absence or the presence of the indicated concentrations of saquinavir (SQ) and harvested at day 3 post-infection. Aliquots of cell lysates containing equal amounts of proteins were analyzed by immunoblot (10% SDS-PAGE) using specific antibodies as indicated. (E) PHA-stimulated PBMCs were mock-infected (−) or infected with 100 ng of HIV-1 NL4-3 per 106 cells, in the absence or the presence of 0.5 µM SQ and harvested at the indicated D.P.I. Aliquots of cell lysates containing equal amounts of proteins were analyzed by immunoblot using specific antibodies as indicated. Results are representative of at least three independent experiments.
Figure 2.
HIV-1 protease directly cleaves GCN2.
(A) BHK-21 cells were transfected by electroporation with in vitro transcribed capped Sindbis virus RNAs from plasmids pT7SV-HIV-1PR (C+HIV-1Pro), pT7SV-2Apro (C+2APro), pT7SVwt (C) for the expression of HIV-1 and PV-2A proteases. After 4 h cells were metabolically labeled with [35S]-Met-Cys. Equivalent amounts of total protein were subjected to 12% SDS-PAGE, transferred to PDVF membranes and subjected to autoradiography (lower panel) to visualize protease expression. The membranes were then probed with specific antisera for detection of endogenous GCN2 and eIF4GI as indicated. (B) COS-7 cells were subjected to coupled infection/DNA transfection with recombinant vaccinia virus (vvT7), pTM1 (−) or pTM1-derived plasmid encoding HIV-1Pro and pcDNA3.1/Myc-His plasmid, empty (−) or encoding human or mouse GCN2. At 18 h post-transfection cells were lysed and equivalent amounts of total protein were analyzed by western blot using a specific antiserum for detection of GCN2. (C) Affinity purified mouse or human GCN2 was incubated for 3 h at 30°C in the absence or the presence of HIV-1 or HIV-2 proteases. Incubation was stopped by addition of SDS-PAGE sample buffer and proteins were analyzed by western blot using a specific antiserum for detection of GCN2. (D) HeLa cell-free extracts were incubated with recombinant HIV-1Pro in the absence or the presence of 2.5 µM SQ for the indicated times. Proteins were analyzed by western blot using specific antisera for detection of GCN2, eIF4GI, PABP or actin as indicated. (E) Purified mouse and human GCN2 GST-fusion proteins were incubated in the presence of recombinant HIV-1Pro for 3 h at 30°C. Proteins were resolved in SDS-PAGE and stained with Coomassie blue. Shown in the lower box is the GCN2 sequence obtained by Edman degradation of the indicated C-terminal fragments. Results are representative of at least three independent experiments.
Figure 3.
HIV-1 cleavage inhibits GCN2 eIF2alpha kinase activity.
(A) Affinity purified human GCN2 was incubated for 30 min at 30°C in the absence or the presence of HIV-1Pro and in the absence or the presence of 2.5 µM SQ, as indicated, prior to being assayed in an eIF2alpha kinase assay (lanes 1–5). In lane 6, SQ was added after incubation of GCN2 with the protease, just before initiating the eIF2alpha kinase assay. HIV-1 RNA was only present during the kinase assay. Incubation was stopped by addition of SDS-PAGE sample buffer and proteins were analyzed by western blot using specific antisera to detect eIF2alpha phosphorylated on serine 51, total eIF2alpha, and phosphorylated and total GCN2. Similar results were obtained from a duplicate experiment. (B) Schematic representation of the structural domains of human GCN2 and the generated GCN2 fragments according to the HIV-1Pro cleavage site. The 1649 amino acid GCN2 sequence is illustrated by a larger box and the figure is drawn to scale. Highlighted domains include the N-terminal (black); the ‘Pseudokinase’ (dark grey) that is related to sub-domains I–XI of eukaryotic protein kinases; the conserved two lobes of the eIF2alpha kinase domain (black), separated by a large insert (light grey); the HisRS-like domain (dark grey); and a C-terminal domain (light grey). The numbers refer to the amino acid residues. (C) In vitro eIF2alpha kinase assay of purified full-length human GCN2 (1–1649) and the generated GCN2 fragments according to the HIV-1Pro cleavage site (1–560 and 561–1649) in the absence or the presence of HIV-1 RNA as indicated. Proteins were analyzed by western blot using specific antisera to detect eIF2alpha phosphorylated on serine 51, total eIF2alpha, and full-length GCN2 or fragments (anti-myc). Similar results were obtained from a duplicate experiment.
Figure 4.
Absence of GCN2 favor HIV-1 protein expression.
(A, B) HeLa cells were co-transfected with plasmids encoding wild type GCN2 or the inactive mutant GCN2-K618R and also with a mixture of HIV-1 encoding plasmids pNL4-3.Luc.R-E- and pNL4-3.GFP.R-E-. Cells were harvested 24 h after transfection. Aliquots of cell lysates containing equal amounts of proteins were analyzed by western blot using specific antisera to detect GCN2, eIF2alpha and GFP (A). Luciferase activity was measured, and results are shown as the ratio between the activity in cells over-expressing GCN2-K618R mutant and in cells over-expressing wild type GCN2. * P<0.05 vs. WT (B). (C, D) Normal HeLa cells (no siRNA) and HeLa cells stably expressing a control siRNA (siRNA 3) or a specific siRNA for GCN2 knock down (siRNA 4) were transiently transfected with a mixture of HIV-1 encoding plasmids, pNL4-3.Luc.R-E- and pNL4-3.GFP.R-E-. Cells were harvested 24 h after transfection. Aliquots of cell lysates containing equal amounts of proteins were analyzed by western blot using specific antisera to detect GCN2, eIF2alpha, GFP and HIV-1-p24 (C). Luciferase activity was also measured, and results are shown as the ratio between the activity in cells expressing siRNA 3 or siRNA 4 and in normal cells (no siRNA). * P<0.01 vs. no RNAi and RNAi 3 (D). (E) Wild type (WT) and GCN2 knock out (GCN2−/−) MEF were transiently transfected by electroporation with a pcDNA-based plasmid encoding luciferase protein (pcDNA-Luc), as a control, or with an HIV-1 encoding plasmid, pNL4-3.Luc.R-E-. Cells were harvested 24 h after transfection and luciferase activity was measured in cell extracts. Luciferase activity produced in cells from the HIV-1-encoding plasmid was normalized dividing it by the activity produced in cells from pcDNA-Luc plasmid. Results represent the ratio of normalized luciferase activity between wild type MEFs (set as one) and GCN2−/−. * P<0.005 vs. WT. Results represent the mean of three to four independent experiments and error bars indicate the standard error (SE).
Figure 5.
GCN2 is activated in cells expressing HIV-1 RNA.
(A) BHK-C and BHK-A1 cells were transfected with a pcDNA-based plasmid encoding luciferase protein (pcDNA-Luc) or with a mixture of HIV-1 encoding plasmids, pNL4-3.Luc.R-E- and pNL4-3.GFP.R-E-. Aliquots of cell lysates containing equal amounts of proteins were analyzed by western blot using specific antisera to detect eIF2alpha phosphorylated on serine 51, phosphorylated or total GCN2, GFP and actin. (B) The same as in (A), but using wild type (WT) and GCN2 knock out (GCN2-/-) MEFs. The membrane was probed with different antisera to detect eIF2alpha phosphorylated on serine 51, total eIF2alpha, phosphorylated or total GCN2 and GFP. (C) The same as in (A) and (B), but using HeLa cells. In this case the transfection was done by electroporation. The membrane was probed with different antisera to detect eIF2alpha phosphorylated on serine 51, total eIF2alpha, phosphorylated or total GCN2, GFP and actin. Similar results were obtained from duplicate experiments. (D and E) In samples equivalent to those of the experiments described in (A) and (B) where cells were transfected with pNL4-3.Luc.R-E- plasmid, luciferase activity was measured and HIV-1 RNA quantified. Relative values of both are shown as the ratio between GCN2-/- MEFs or BHK-A1 and the corresponding control cells (wild type MEFs or BHK-C), whose value was set as one. Data are expressed as mean ± SE. * P < 0.05 vs. BHK-C (D) or WT (E). (F) Wild type (WT) and S51A MEFs were transiently transfected by electroporation with a pcDNA-based plasmid encoding luciferase protein (pcDNA-Luc), as a control, or with an HIV-1 encoding plasmid, pNL4-3.Luc.R-E-. Cells were harvested 24 hr after electroporation and luciferase activity was measured in cell extracts. Luciferase activity produced in cells from the HIV-1-encoding plasmid was normalized dividing it by the activity produced in cells from pcDNA-Luc plasmid. Results represent the ratio of normalized luciferase activity between wild type MEFs (set as one) and S51A MEFs. * P < 0.01 vs. WT.