Human immunodeficiency virus type 1 protease cleaves procaspase 8 in vivo

Abstract

Human immunodeficiency virus type 1 (HIV-1) infection causes apoptosis of infected CD4 T cells as well as uninfected (bystander) CD4 and CD8 T cells. It remains unknown what signals cause infected cells to die. We demonstrate that HIV-1 protease specifically cleaves procaspase 8 to create a novel fragment termed casp8p41, which independently induces apoptosis. casp8p41 is specific to HIV-1 protease-induced death but not other caspase 8-dependent death stimuli. In HIV-1-infected patients, casp8p41 is detected only in CD4(+) T cells, predominantly in the CD27(+) memory subset, its presence increases with increasing viral load, and it colocalizes with both infected and apoptotic cells. These data indicate that casp8p41 independently induces apoptosis and is a specific product of HIV-1 protease which may contribute to death of HIV-1-infected cells.

FIG. 1.

HIV-1 protease is present in the cytosol of infected cells and cleaves procaspase 8 into a novel p41 fragment. (A) HIV-1-infected Jurkat cells and uninfected Jurkat cells were fractionated into cytosolic and membrane fractions and assessed for purity by immunoblotting for the membrane-specific protein LAT (insert), and equal amounts of cytosol or membrane fractions were assessed for protease activity using a fluorogenic Gag-Pol substrate. (B) Determination of HIV-1 PR cleavage site in procaspase 8. Procaspase 8 with an amino-terminal Flag epitope and a carboxyl-terminal Myc epitope was constructed. The F355, F356 site is indicated by an arrow. (C) Recombinant ³⁵S-labeled Flag-procaspase 8-Myc was reacted with HIV-1 PR or renin and analyzed by autoradiography (right panel). PR-digested Flag-procaspase 8-Myc was immunoblotted with Flag (left panel, middle) or Myc (left panel, bottom). The 41-kDa peptide produced by protease digestion that contains the Flag epitope was sequenced. (D) Wild-type procaspase 8 or procaspase 8 with mutations F355R, F356N [FF(RN)] was reacted with various amounts of protease and analyzed by autoradiography. Wild-type procaspase 8 incubated with HIV-1 PR resulted in a 41-kDa band that was much less evident in the RN mutant. Results are representative of three independent experiments.

FIG. 2.

Casp8p41 induces apoptosis. (A) Cytosolic extracts from Jurkat T cells were reacted with recombinant HIV-1 PR, glutathione S-transferase (GST) alone, or recombinant GST-casp8p41, or with cytosolic extracts from CPT-treated cells, and analyzed for events of apoptosis; both HIV-1 PR and casp8p41 induce Bid cleavage into p15 tBid, cytochrome c release into the cytosol, and procaspase 9 cleavage. The cytosolic and mitochondrial extracts were blotted for the mitochondria-specific protein HSP70 to confirm purity. (B) HeLa cells were trans- fected with GFP alone, GFP-HIV protease, or GFPcasp8p41 and stained with 4′,6′-diamidino-2-phenylindole (DAPI) to identify cell nuclei and TUNEL to identify apoptosis. As controls, campothecin-treated cells and DNase I-treated cells were included. (C) Primary human CD4 T cells were transfected with GFP alone or GFPcasp8p41 and analyzed by flow cytometry, gating only on the GFP-positive cells, and analyzed by TUNEL and for active caspase 3 (D)or annexin V-PE staining (E). Results are representative of three independent experiments.

FIG. 3.

casp8p41 antibody specifically recognizes casp8p41 but not other caspase 8 fragments. (A) 293T cells were transfected with GFP, GFP full-length (FL) caspase 8, GFPcasp8p43, or GFPcasp8p41 and immunoblotted with anti-casp8p41 antibodies or anti-GFP. Only cells transfected with GFPcasp8p41 contained protein that was recognized by the casp8p41 antibody, whereas the GFP antibody identified transfected protein in all cells. (B) HeLa cells were transfected with either GFPcasp8p41, GFP FL caspase 8, or GFPcasp8p43 and analyzed by immunofluorescence with either casp8p41 monoclonal antibody or isotype control antibody with secondary anti-mouse PE. Only those cells transfected with casp8p41 and stained with the casp8p41 antibody were PE positive. (C) Jurkat T cells were transfected with HIV-1 PR alone (0), in the presence of the HIV-1 PR inhibitor nelfinavir (Nfv), or in the presence of the pancaspase inhibitor Z-VAD fmk (z-VAD) and analyzed for casp8p41 production. (D) Jurkat T cells were treated with the indicated stimuli and analyzed by flow cytometry for casp8p41 and active caspase 3. The percentages of active caspase 3⁺ cells and casp8p41-positive cells were determined.

FIG. 4.

HIV-1 infection results in casp8p41. Jurkat T cells acutely infected with HIV-1 IIIB were analyzed at the indicated days by flow cytometry for intracellular p24 and casp8p41 or an isotype control. (A) Only p24-positive cells contained casp8p41, and the proportion of casp8p41⁺ cells increased as a function of time. (B) Jurkat T cells were infected with HIV-1 as above and analyzed for casp8p41 positivity, as well as cell death as assessed by trypan positivity. Results are representative of three independent experiments.

FIG. 5.

casp8p41 is present in infected and apoptotic cells from HIV-1 patients and colocalizes with the memory subset. (A) Peripheral blood mononuclear cells from an HIV-1⁺ individual were surface stained with monoclonal antibodies against CD3, CD4, CD8, CD27, and CD45RO and then fixed and permeabilized, followed by intracellular staining for casp8p41. The casp8p41-positive CD4 T cells (red) were assessed for CD27 and CD45RO expression and compared to casp8p41-negative CD4 T cells. (B) Peripheral blood lymphocytes from HIV-1-infected patients, with a range of viral replication, were analyzed by flow cytometry for casp8p41. (C) PBL from HIV-1-infected patients or uninfected donors were analyzed for p24 and casp8p41. In the representative results shown, neither p24 nor casp8p41 was observed in uninfected donors. In patients with suppressed levels of viral replication, low levels of p24 and casp8p41 were seen. However, in patients with a high viral burden, high levels of p24 positivity and casp8p41 positivity were observed that in most cases were colocalized. (D) Cells from a patient with a viral load of >50,000 were costained for active caspase 3 and for casp8p41.