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. 2005 Apr;79(7):4257-69.
doi: 10.1128/JVI.79.7.4257-4269.2005.

Human Immunodeficiency virus type 1 Nef potently induces apoptosis in primary human brain microvascular endothelial cells via the activation of caspases

Edward A Acheampong  1 , Zahida Parveen, Lois W Muthoga, Mehrnush Kalayeh, Muhammad Mukhtar, Roger J Pomerantz
Affiliations

Human Immunodeficiency virus type 1 Nef potently induces apoptosis in primary human brain microvascular endothelial cells via the activation of caspases

Edward A Acheampong et al. J Virol. 2005 Apr.

Abstract

The lentiviral protein Nef plays a major role in the pathogenesis of human immunodeficiency virus type I (HIV-1) infection. Although the exact mechanisms of its actions are not fully understood, Nef has been shown to be essential for the maintenance of high-titer viral replication and disease pathogenesis in in vivo models of simian immunodeficiency virus infection of monkeys. Nef has also been suggested to play a pivotal role in the depletion of T cells by promoting apoptosis in bystander cells. In this context, we investigated the ability of extracellular and endogenously expressed HIV-1 Nef to induce apoptosis in primary human brain microvascular endothelial cells (MVECs). Human brain MVECs were exposed to baculovirus-expressed HIV-1 Nef protein, an HIV-1-based vector expressing Nef, spleen necrosis virus (SNV)-Nef virus (i.e., SNV vector expressing HIV-1 Nef as a transgene), and the HIV-1 strain ADA and its Nef deletion mutant, ADADeltaNef. We observed that ADA Nef, the HIV-1 vector expressing Nef, and SNV-Nef were able to induce apoptosis in a dose-dependent manner. The mutant virus with a deletion in Nef was able to induce apoptosis in MVECs to modest levels, but the effects were not as pronounced as with the wild-type HIV-1 strain, ADA, the HIV-1-based vector expressing Nef, or SNV-Nef viruses. We also demonstrated that relatively high concentrations of exogenous HIV-1 Nef protein were able to induce apoptosis in MVECs. Gene microarray analyses showed increases in the expression of several specific proapoptotic genes. Western blot analyses revealed that the various caspases involved with Nef-induced apoptosis are processed into cleavage products, which occur only during programmed cell death. The results of this study demonstrate that Nef likely contributes to the neuroinvasion and neuropathogenesis of HIV-1, through its effects on select cellular processes, including various apoptotic cascades.

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Figures

FIG. 1.
FIG. 1.
Schematic representations of the triple-vector plasmid systems employed for the recombinant HIV-1 Nef, SNV-Nef, and LacZ viruses. Replication-competent retroviral particles were generated by triple transfection of transfer vector (A) pHR′CMVNef (HIV-1-based vector expressing Nef) or (B) SNV-Nef (SNV-based vector expressing Nef) or pHR′CMVLacZ, the packaging construct pCMVΔR8.2, and the VSV envelope-encoding plasmid pMD.G.
FIG. 2.
FIG. 2.
MVECs transduced with HIV-1 Nef and SNV-Nef vector virions expressing Nef. Primary isolated human brain endothelial cells were transduced with either HIV-1 LacZ, HIV-1 Nef, or SNV-Nef vector virions and then cultured on two-well chamber slides for 48 h. The cells were fixed with 2% paraformaldehyde and incubated with primary antibodies against Nef, and later with Cy2-conjugated secondary antibodies, after permeabilization. These results are representative of those from experiments performed in triplicate and repeated at least twice.
FIG. 3.
FIG. 3.
Recombinant HIV-1 Nef protein-mediated apoptosis of human brain MVECs. Representative TUNEL staining of MVECs exposed to GST- and baculovirally expressed HIV-1 Nef protein is shown. MVECs were exposed to either GST protein or HIV-1 Nef protein expressed from Sf9 insect cells for 72 h. The concentrations of protein used were 10, 50, and 100 ng/ml. As a positive control, MVECs were exposed to DNase at a concentration of 10 μg/ml for 10 min at room temperature. TUNEL assays were performed with the in situ cell death detection kit, TMR Red (Roche Diagnostics). The cells were analyzed by fluorescence microscopy (Olympus System microscope, model BX60, with fluorescence attachment BX-FLA). These results are representative of those from experiments performed in triplicate and repeated at least twice.
FIG. 4.
FIG. 4.
Nef expressed from the retroviral vector pHR′CMVNef is sufficient to induce apoptosis in MVECs in a dose-dependent manner. Representative TUNEL staining of MVECs infected with HIV-1-based vectors expressing either Nef or LacZ for 72 h, after which cells were subjected to TUNEL staining, is shown. The amount of virus used varied from 1 to 10 ng of HIV-1 p24 antigen equivalents per ml. TUNEL assays were performed with the in situ cell death detection kit, TMR Red (Roche Diagnostics). The cells were analyzed by fluorescence microscopy (Olympus System microscope, model BX60, with fluorescence attachment BX-FLA). These results are representative of those from experiments performed in triplicate and repeated at least twice.
FIG. 5.
FIG. 5.
HIV-1 Nef expressed from the spleen necrosis viral vector, SNV-Nef, induces apoptosis in MVECs in a dose-dependent manner. Representative TUNEL staining of SNV-LacZ and SNV-Nef+ vector virion-treated MVECs, infected for 72 h, is shown. The quantities of virions used to infect cells were 10, 100, 500, and 1,000 CFU/ml. As a positive control, MVECs were exposed to DNase at a concentration of 10 μg/ml for 10 min at room temperature. TUNEL assays were performed with the in situ cell death detection kit, TMR Red (Roche Diagnostics). The cells were analyzed by fluorescence microscopy (Olympus System microscope, model BX60, with fluorescence attachment BX-FLA). These results are representative of those from experiments performed in triplicate and repeated at least twice.
FIG. 6.
FIG. 6.
HIV-1 R5 strain ADA induces apoptosis in MVECs in a dose-dependent manner. Representative TUNEL staining of HIV-1 ADA-treated MVECs is shown. Cells were infected with HIV-1 ADA for 72 h, after which cells were subjected to TUNEL staining. The amount of virus used ranged from 1 to 100 ng of HIV-1 p24 antigen equivalents per ml. As a positive control, MVECs were exposed to DNase at a concentration of 10 μg/ml for 10 min at room temperature. TUNEL assays were performed with the in situ cell death detection kit, TMR Red (Roche Diagnostics). The cells were analyzed by fluorescence microscopy (Olympus System microscope, model BX60, with fluorescence attachment BX-FLA). These results are representative of those from experiments performed in triplicate and repeated at least twice.
FIG. 7.
FIG. 7.
An HIV-1 R5 strain with a deletion in the Nef gene (ADAΔNef) induces apoptosis in MVECs at lower levels than wild-type HIV-1 ADA. Representative TUNEL staining of HIV-1 ADAΔNef-treated MVECs is shown. Cells were infected with HIV-1 ADAΔNef for 72 h, after which they were subjected to TUNEL staining. The amount of virus used ranged from 1 to 100 ng of HIV-1 Gag p24 equivalents per ml. As a positive control, MVECs were exposed to DNase at a concentration of 10 μg/ml for 10 min at room temperature. TUNEL assays were performed with the in situ cell death detection kit, TMR Red (Roche Diagnostics). The cells were analyzed with fluorescence microscopy (Olympus System microscope, model BX60, with fluorescence attachment BX-FLA). These results are representative of those from experiments performed in triplicate and repeated at least twice.
FIG. 8.
FIG. 8.
Human apoptosis gene microarrays of MVECs transduced with HIV-1 LacZ virus (top panel) and HIV-1-based vector Nef+ virus (bottom panel). These results are representative of those from two independent experiments.
FIG. 9.
FIG. 9.
Western blotting analyses of PARP (A), caspase-3 (B), and caspase-9 (C) during HIV-1 Nef-induced apoptosis in MVECs. Cells were either left untreated, exposed to 100 ng of GST protein per ml, transduced with 10 ng of p24 antigen equivalents of HIV-1-based vector LacZ virus per ml, treated with 100 ng of recombinant Nef protein per ml, transduced with 10 ng of p24 antigen equivalents of HIV-1-based vector Nef virus per ml, or pretreated with 100 nM specific caspase inhibitor and then transduced with HIV-1-based vector Nef virus. Twenty-five micrograms of total protein extract was loaded in each lane and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Caspase-3 and -9 and PARP were analyzed by Western blotting with specific caspase-3, caspase-9 and PARP monoclonal antibodies. These results are representative of those from experiments repeated at least twice. (A) PARP. Lanes: M, protein marker; 1, MVECs treated with 100 ng of recombinant Nef protein per ml; 2, MVECs transduced with 10 ng of p24 antigen equivalents of HIV-1-based vector Nef virus per ml; 3, untreated MVECs; 4, MVECs exposed to 100 ng of GST protein per ml; 5, MVECs pretreated with 100 nM broad-spectrum caspase inhibitor Z-VAD-FMK (BD PharMingen) and then transduced with HIV-1-based vector Nef virus; 6, MVECs transduced with 10 ng of p24 antigen equivalents of HIV-1-based vector LacZ virus per ml. The upper arrow depicts 116-kDa PARP, and the lower arrow depicts approximately the 85-kDa cleaved PARP. (B) Caspase-3. Lanes: 1, untreated MVECs; 2, MVECs exposed to 100 ng of GST protein per ml; 3, MVECs transduced with 10 ng of p24 antigen equivalents of HIV-1-based vector LacZ virus per ml; 4, MVECs treated with 100 ng of recombinant Nef protein per ml; 5, MVECs transduced with 10 ng of p24 antigen equivalents of HIV-1-based vector Nef virus per ml; 6, MVECs pretreated with 100 nM of Ac-DEVD-CHO (BD PharMingen), a specific caspase 3 inhibitor, and then transduced with HIV-1-based Nef virus. The top arrow depicts 35-kDa intact caspase-3, and the bottom arrow depicts the 17- to 19-kDa cleaved caspase-3 fragment. (C) Caspase-9. Lanes: M, protein marker; 1, untreated MVECs; 2, MVECs exposed to 100 ng of GST protein per ml; 3, MVECs transduced with 10 ng of p24 antigen equivalents of HIV-1-based vector LacZ virus per ml; 4, MVECs pretreated with 100 nM Z-LEHD-FMK, a specific caspase 9 inhibitor (BD PharMingen), and then transduced with HIV-1-based Nef virus; 5, MVECs treated with 100 ng of recombinant Nef protein per ml; 6, MVECs transduced with 10 ng of p24 antigen equivalents of HIV-1-based vector Nef virus per ml. The top arrow depicts 45-kDa pro-caspase-9, and the bottom arrow depicts 35- and 37-kDa cleaved caspase-9 fragments.
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