Glycosylphosphatidylinositol Anchor Deficiency Attenuates the Production of Infectious HIV-1 and Renders Virions Sensitive to Complement Attack

Abstract

Human immunodeficiency virus type 1 (HIV-1) escapes complement-mediated lysis (CML) by incorporating host regulators of complement activation (RCA) into its envelope. CD59, a key member of RCA, is incorporated into HIV-1 virions at levels that protect against CML. Since CD59 is a glycosylphosphatidylinositol-anchored protein (GPI-AP), we used GPI anchor-deficient Jurkat cells (Jurkat-7) that express intracellular CD59, but not surface CD59, to study the molecular mechanisms underlying CD59 incorporation into HIV-1 virions and the role of host proteins in virus replication. Compared to Jurkat cells, Jurkat-7 cells were less supportive to HIV-1 replication and more sensitive to CML. Jurkat-7 cells exhibited similar capacities of HIV-1 binding and entry to Jurkat cells, but were less supportive to viral RNA and DNA biosynthesis as infected Jurkat-7 cells produced reduced amounts of HIV-1 RNA and DNA. HIV-1 virions produced from Jurkat-7 cells were CD59 negative, suggesting that viral particles acquire CD59, and probably other host proteins, from the cell membrane rather than intracellular compartments. As a result, CD59-negative virions were sensitive to CML. Strikingly, these virions exhibited reduced activity of virus binding and were less infectious, implicating that GPI-APs may be also important in ensuring the integrity of HIV-1 particles. Transient expression of the PIG-A gene restored CD59 expression on the surface of Jurkat-7 cells. After HIV-1 infection, the restored CD59 was colocalized with viral envelope glycoprotein gp120/gp41 within lipid rafts, which is identical to that on infected Jurkat cells. Thus, HIV-1 virions acquire RCA from the cell surface, likely lipid rafts, to escape CML and ensure viral infectivity.

Keywords: GPI-AP; HIV-1; antibody; complement; virus binding; virus replication.

FIG. 1.

Characterization of intracellular and surface expressions of GPI anchor and CD59 by Jurkat versus Jurkat-7 cells. (A) Expression of total GPI-APs on the surface of Jurkat versus Jurkat-7 cells was determined using FACS analysis of FLAER (an Alexa488-labeled inactive variant of aerolysin that does not cause lysis of cells) staining. Control, Jurkat cells were not stained with FLAER. (B) Intracellular and cell surface expressions of CD59. For CSS, cells were directly incubated with FITC-conjugated anti-human CD59 mAb (MEM-43) for 30 min, followed by washing and fixation before FACS analysis. For ICS, cells were fixed and permeabilized for 30 min before incubation with FITC-conjugated anti-human CD59 mAb (MEM-43). After washing and fixation, cells were subjected to FACS analysis. (C) Colocalization of CD59 and lipid rafts on the surface of Jurkat versus Jurkat-7 cells. After surface staining with CD59 and CTB, Jurkat and Jurkat-7 cells were subjected to confocal microscopy to determine CD59 and lipid raft colocalization. Yellow color in Merge panel indicates the colocalization of CD59 (green) with lipid rafts (red) on the surface of Jurkat and Jurkar-7 cells. Cellular DNA content and nuclei were stained with DAPI (blue). Control, control staining with isotype-matched Abs. CTB is a marker for lipid rafts; CD59 indicates surface staining with FITC-conjugated anti-human CD59 mAb (MEM-43); Scale bars, 10 μm. The data represent the results from at least three independent experiments. Abs, antibodies; CSS, cell surface staining; CTB, cholera toxin subunit B; DAPI, 4′,6-diamidino-2-phenylindole; FACS, flow cytometric analysis; FLAER, fluorescein-labeled proaerolysin; GPI-APs, glycosylphosphatidylinositol-anchored proteins; ICS, intracellular staining; mAb, monoclonal antibody.

FIG. 2.

GPI anchor–deficient Jurkat-7 cells were sensitive to complement attack. Jurkat and Jurkat-7 cells were treated with C (complement competent) or iC (inactivated complement) in the presence or absence of anti-human CD3 Ab or LPS. Cells were subjected to PI staining to determine cytolysis. (A) A representative experiment of FACS analysis shows cytolysis of Jurkat versus Jurkat-7 cells in response to complement attack through classical and alternative pathways of complement activation triggered by anti-human CD3 Ab and LPS, respectively. (B) Pooled data from three separate experiments of FACS analysis show the percentage of PI-positive Jurkat versus Jurkat-7 cells in response to complement attack through classical and alternative pathways of complement activation. C, complement; iC, heat-inactivated complement; LPS+C, 10 μg/ml of LPS plus complement; Ab+C, 1 μg/ml of anti-human CD3 Ab plus complement. The data represent the results from at least three independent experiments. LPS, lipopolysaccharide; ns, no significance; PI, propidium iodide. *p < .05; **p < .01.

FIG. 3.

GPI anchor–deficient Jurkat-7 cells were less supportive to HIV-1 replication. (A) Cell-free culture supernatants were collected from HIV-1 NL4-3-infected Jurkat and Jurkat-7 cells on every other day during an infection course of 20 days. The supernatants were subjected to p24 ELISA measurement of HIV-1 titers. (B) HIV-1-infected Jurkat and Jurkat-7 cells were subjected to p24 ICS and FACS to determine the percentage of HIV-1-positive cells. (C) Semiquantitative one-step RT-PCR detection of HIV-1 Gag RNA from HIV-1-infected Jurkat versus Jurkat-7 cells on days 7 and 14 postinfection. Human GAPDH RNA was detected as an internal control of cellular RNA loading. (D) Pooled data from five separate experiments of RT-PCR detection of HIV-1 Gag RNA. (E) Total DNA extracted from HIV-1-infected Jurkat and Jurkat-7 cells on day 7 and 14 postinfection was subjected to PCR detection of HIV-1 Gag DNA. PCR detection of human GAPDH was used as an internal control of cellular DNA loading. (F) Pooled data from five separate experiments of PCR detection of HIV-1 Gag DNA. ELISA, enzyme-linked immunosorbent assay; HIV-1, human immunodeficiency virus type 1; J, Jurkat cells; J7, Jurkat-7 cells; PCR, polymerase chain reaction; RT-PCR, reverse transcriptase PCR; **p < .01.

FIG. 4.

GPI anchor deficiency did not affect HIV-1 binding and entry into the host cells. (A) Semiquantitative one-step RT-PCR detection of HIV-1 Gag RNA from virus-bound Jurkat and Jurkat-7 cells. Human GAPDH RNA was detected as an internal control of cellular RNA loading. (B) Jurkat and Jurkat-7 cells were subjected to surface staining and FACS analysis of CD4, CCR5, and CXCR4. The data represent the results from at least three independent experiments. J, Jurkat cells; J7, Jurkat-7 cells.

FIG. 5.

Virions from infected Jurkat-7 cells were less infectious, CD59 defective, and vulnerable to complement attack. (A) HIV-1 virions from supernatants of HIV-1 NL4-3-infected Jurkat and Jurkat-7 cells were used at the same amount of p24 (20 ng/ml) to infect Jurkat cells. Cell-free culture supernatants were collected on every other day postinfection and subjected to p24 measurement to determine HIV-1 production. (B) HIV-1 virions from supernatants of HIV-1 NL4-3-infected Jurkat and Jurkat-7 cells were used at the same amount of p24 (20 ng/ml) to infect TZM-bl cells for 2 days. After infection, TZM-bl cells were lysed with the passive lysis buffer and subjected to measurement of luminescence to determine viral infectivity. (C) HIV-1 virions (20 ng/ml of p24) from infected Jurkat and Jurkat-7 cells were incubated with 1 × 10⁶ Jurkat cells at the virus binding condition (4°C for 30 min). After extensive washings with PBS, cells were subjected to extraction of total RNA for one-step RT-PCR to determine virus binding. (D) HIV-1 pellets prepared from cell-free supernatants of HIV-1 NL4-3-infected Jurkat and Jurkat-7 cells were subjected to Western blot analysis of CD59 and Gag protein abundance. (E) Virolysis of HIV-1 virions from cell-free supernatants of HIV-1 NL4-3-infected Jurkat versus Jurkat-7 cells. HIV-1 virions were exposed to NHS or HIS in the presence or absence of anti-HIV-1 gp120 mAb (2G12, 20 μg/ml) or LPS (10 μg/ml) at 37°C for 30 min. Virions were subjected to p24 ELISA measurement to determine virolysis. Jurkat-sup, supernatants from HIV-1-infected Jurkat cells; Jurkat-7-sup, supernatants from HIV-1-infected Jurkat-7 cells; PBS, phosphate-buffered saline; NHS, normal human sera; HIS, human heat-inactivated sera. The data represent the results from at least three independent experiments.

FIG. 6.

Restoration of GPI anchor biosynthesis rendered CD59 expression on the surface of Jurkat-7 cells. (A) After transfection with either pPIG-A1 or pCtrl plasmid constructs, Jurkat-7 cells were subjected to surface staining with FLAER and BRIC229, followed by FACS analysis of GPI-APs and CD59, respectively. (B) Colocalization of CD59 and lipid rafts (CTB) on the surface of Jurkat-7 cells posttransfection with either pPIG-A1 or pCtrl plasmid constructs. (C) Colocalization of gp120/gp41 and CD59 in lipid rafts (CTB) on the surface of HIV-1-infected Jurkat-7 cells that were transfected with pPIG-A1 or pCtrl plasmid constructs. Jurkat-7 cells were transfected with either pPIG-A1 or pCtrl plasmid constructs for 24 h. Cells were infected with HIV-1 NL4-3 for 48 h. After infection, cells were subjected to confocal microscopy analysis of gp120 (2G12 staining) and CD59 (BRIC229 staining) in lipid rafts (CTB staining) on the cell surface. Yellow color in Merge panel indicates the colocalization of CD59 (red) and/or gp120 (magenta) in lipid rafts (green) on the surface of transfected Jurkar-7 cells. Cellular DNA content and nuclei were stained with DAPI (blue). Scale bars, 10 μm; CD59 indicates surface staining with BRIC229; CTB is a marker for lipid rafts; pCtrl, control plasmid; pPIG-A1, PIG-A-expressing plasmid. The data represent results from at least three independent experiments.