Human immunodeficiency virus type 1 Vpr induces apoptosis in human neuronal cells

Abstract

Human immunodeficiency virus type 1 (HIV-1) infection of the central nervous system (CNS) causes AIDS dementia complex (ADC) in certain infected individuals. Recent studies have suggested that patients with ADC have an increased incidence of neuronal apoptosis leading to neuronal dropout. Of note, a higher level of the HIV-1 accessory protein Vpr has been detected in the cerebrospinal fluid of AIDS patients with neurological disorders. Moreover, extracellular Vpr has been shown to form ion channels, leading to cell death of cultured rat hippocampal neurons. Based on these previous findings, we first investigated the apoptotic effects of the HIV-1 Vpr protein on the human neuronal precursor NT2 cell line at a range of concentrations. These studies demonstrated that apoptosis induced by both Vpr and the envelope glycoprotein, gp120, occurred in a dose-dependent manner compared to protein treatment with HIV-1 integrase, maltose binding protein (MBP), and MBP-Vpr in the undifferentiated NT2 cells. For mature, differentiated neurons, apoptosis was also induced in a dose-dependent manner by both Vpr and gp120 at concentrations ranging from 1 to 100 ng/ml, as demonstrated by both the terminal deoxynucleotidyltransferase (Tdt)-mediated dUTP-biotin nick end labeling and Annexin V assays for apoptotic cell death. In order to clarify the intracellular pathways and molecular mechanisms involved in Vpr- and gp120-induced apoptosis in the NT2 cell line and differentiated mature human neurons, we then examined the cellular lysates for caspase-8 activity in these studies. Vpr and gp120 treatments exhibited a potent increase in activation of caspase-8 in both mature neurons and undifferentiated NT2 cells. This suggests that Vpr may be exerting selective cytotoxicity in a neuronal precursor cell line and in mature human neurons through the activation of caspase-8. These data represent a characterization of Vpr-induced apoptosis in human neuronal cells, and suggest that extracellular Vpr, along with other lentiviral proteins, may increase neuronal apoptosis in the CNS. Also, identification of the intracellular activation of caspase-8 in Vpr-induced apoptosis of human neuronal cells may lead to therapeutic approaches which can be used to combat HIV-1-induced neuronal apoptosis in AIDS patients with ADC.

FIG. 1

(A) Plasmid map of MBP-Vpr fusion protein and its cleaved counterparts. HIV-1 Vpr (strain 89.6) was cloned into the pMAL-c expression vector (New England Biolabs) using DNA fragments amplified by PCR. Vpr 89.6 was inserted between the XbaI and HindIII sites in the polylinker region of the pMAL-c vector. Both the forward and backward primers were 28-bp oligonucleotides, and their sequences were 5′-ACGTCTAGAATGGAACAAGCCCCAGAAG-3′ and 5′-ATGCCAAGCTTTAGGATCTACTGGCTCC-3′, respectively. When the PCR products were obtained, the insert was cloned into the vector, and all recombinant plasmids were verified by restriction enzyme cleavage and DNA sequence analysis. (B) Left panel, SDS-PAGE of purified MBP-Vpr. MBP alone is shown in lane 2, and MBP-Vpr is shown in lane 3. Protein molecular mass standards were run for lanes 1 and 4 (molecular masses are shown to the left). Right panel, Western blot analysis of recombinant HIV-1 Vpr. MBP-Vpr, cleaved by factor Xa, is located in lanes 1 and 2, alongside MBP alone, treated with factor Xa, in lane 3. The molecular mass of MBP-Vpr was 58 kDa, and that of free Vpr was 14 kDa. Purified Vpr was identified in the Western blot analysis using polyclonal anti-Vpr antibodies raised in rabbits.

FIG. 1

(A) Plasmid map of MBP-Vpr fusion protein and its cleaved counterparts. HIV-1 Vpr (strain 89.6) was cloned into the pMAL-c expression vector (New England Biolabs) using DNA fragments amplified by PCR. Vpr 89.6 was inserted between the XbaI and HindIII sites in the polylinker region of the pMAL-c vector. Both the forward and backward primers were 28-bp oligonucleotides, and their sequences were 5′-ACGTCTAGAATGGAACAAGCCCCAGAAG-3′ and 5′-ATGCCAAGCTTTAGGATCTACTGGCTCC-3′, respectively. When the PCR products were obtained, the insert was cloned into the vector, and all recombinant plasmids were verified by restriction enzyme cleavage and DNA sequence analysis. (B) Left panel, SDS-PAGE of purified MBP-Vpr. MBP alone is shown in lane 2, and MBP-Vpr is shown in lane 3. Protein molecular mass standards were run for lanes 1 and 4 (molecular masses are shown to the left). Right panel, Western blot analysis of recombinant HIV-1 Vpr. MBP-Vpr, cleaved by factor Xa, is located in lanes 1 and 2, alongside MBP alone, treated with factor Xa, in lane 3. The molecular mass of MBP-Vpr was 58 kDa, and that of free Vpr was 14 kDa. Purified Vpr was identified in the Western blot analysis using polyclonal anti-Vpr antibodies raised in rabbits.

FIG. 2

(A) HIV-1 Vpr- and gp120-induced apoptosis in undifferentiated NT2 cells, as measured by the TUNEL assay. Protein treatments were at concentrations of 1, 10, 50, and 100 ng/ml on the undifferentiated NT2 cell line (left sides of panels, fluorescent staining with the TUNEL assay; right side, phase-contrast photomicrograph of the same slide section). The microscope used was an Olympus System microscope, model BX60, with fluorescence attachment BX-FLA. TUNEL assays were performed using the in situ cell death detection kit, TMR red (Boehringer-Mannheim). Magnification, ×13.6. (B) HIV-1 Vpr- and gp20-induced apoptosis in mature neurons, as measured by TUNEL assay. Protein treatments were administered at concentrations of 1, 50, and 100 ng/ml on the mature, differentiated neurons. Left sides of panels show fluorescent staining with the TUNEL assay in situ cell death detection kit, TMR red (Boehringer-Mannheim); right sides show a phase-contrast photomicrograph of the same slide section. The lowest panels (line of four photomicrographs) show confirmatory data using a different TUNEL assay kit (Oncogene Research Products) for all protein treatments given at 100 ng/ml. Magnification, ×13.6.

FIG. 2

(A) HIV-1 Vpr- and gp120-induced apoptosis in undifferentiated NT2 cells, as measured by the TUNEL assay. Protein treatments were at concentrations of 1, 10, 50, and 100 ng/ml on the undifferentiated NT2 cell line (left sides of panels, fluorescent staining with the TUNEL assay; right side, phase-contrast photomicrograph of the same slide section). The microscope used was an Olympus System microscope, model BX60, with fluorescence attachment BX-FLA. TUNEL assays were performed using the in situ cell death detection kit, TMR red (Boehringer-Mannheim). Magnification, ×13.6. (B) HIV-1 Vpr- and gp20-induced apoptosis in mature neurons, as measured by TUNEL assay. Protein treatments were administered at concentrations of 1, 50, and 100 ng/ml on the mature, differentiated neurons. Left sides of panels show fluorescent staining with the TUNEL assay in situ cell death detection kit, TMR red (Boehringer-Mannheim); right sides show a phase-contrast photomicrograph of the same slide section. The lowest panels (line of four photomicrographs) show confirmatory data using a different TUNEL assay kit (Oncogene Research Products) for all protein treatments given at 100 ng/ml. Magnification, ×13.6.

FIG. 3

(A) Apoptosis in undifferentiated NT2 cells, as determined by an Annexin V assay. Protein treatments were administered at concentrations of 1, 10, 50, and 100 ng/ml on the undifferentiated NT2 cell line. Left sides of panels show fluorescent staining with the Annexin V assay (Oncogene Research Products); right sides show a phase-contrast photomicrograph of the same slide section. The microscope used was an Olympus System microscope, model BX60, with fluorescence attachment BX-FLA. Magnification, ×7.5. (B) Apoptosis in mature human neurons, as determined by an Annexin V assay. Protein treatments were administered at concentrations of 1, 50, and 100 ng/ml on the mature, differentiated neurons. Left sides of panels show fluorescent staining with the Annexin V assay (Oncogene Research Products); right sides show a phase-contrast photomicrograph of the same slide section. Magnification, ×15.

FIG. 3

(A) Apoptosis in undifferentiated NT2 cells, as determined by an Annexin V assay. Protein treatments were administered at concentrations of 1, 10, 50, and 100 ng/ml on the undifferentiated NT2 cell line. Left sides of panels show fluorescent staining with the Annexin V assay (Oncogene Research Products); right sides show a phase-contrast photomicrograph of the same slide section. The microscope used was an Olympus System microscope, model BX60, with fluorescence attachment BX-FLA. Magnification, ×7.5. (B) Apoptosis in mature human neurons, as determined by an Annexin V assay. Protein treatments were administered at concentrations of 1, 50, and 100 ng/ml on the mature, differentiated neurons. Left sides of panels show fluorescent staining with the Annexin V assay (Oncogene Research Products); right sides show a phase-contrast photomicrograph of the same slide section. Magnification, ×15.

FIG. 4

(A) Caspase-8 activity in undifferentiated NT2 cells. Treatments with 1, 10, 50, or 100 ng/ml of each respective protein (x axis) and percent caspase-8 activity for each treatment relative to caspase-8 activity in gp120 (100 ng/ml)-treated cell cultures, which were considered as 100% (y axis), are indicated. Jurkat T cells treated with 0.2-μg/ml doxorubicin for 48 h (positive control) exhibited a 17-fold increase in caspase-8 activity over that of control cultures. (B) Caspase-8 activity in mature, differentiated human neurons. Treatment with 1, 50, or 100 ng/ml of respective protein (x axis) and percent caspase-8 activity for each treatment relative to caspase-8 activity in gp120 (100 ng/ml)-treated cell cultures, which were considered as 100% (y axis), are shown. Jurkat T cells treated with 0.2-μg/ml doxorubicin for 48 h exhibited a 9.4-fold increase in caspase-8 activity over that of control cultures (positive control).