Human immunodeficiency virus type 1 envelope-mediated neuronal death: uncoupling of viral replication and neurotoxicity

Abstract

Although brain tissue from patients with human immunodeficiency virus (HIV) and/or AIDS is consistently infected by HIV type 1 (HIV-1), only 20 to 30% of patients exhibit clinical or neuropathological evidence of brain injury. Extensive HIV-1 sequence diversity is present in the brain, which may account in part for the variability in the occurrence of HIV-induced brain disease. Neurological injury caused by HIV-1 is mediated directly by neurotoxic viral proteins or indirectly through excess production of host molecules by infected or activated glial cells. To elucidate the relationship between HIV-1 infection and neuronal death, we examined the neurotoxic effects of supernatants from human 293T cells or macrophages expressing recombinant HIV-1 virions or gp120 proteins containing the V1V3 or C2V3 envelope region from non-clade B, brain-derived HIV-1 sequences. Neurotoxicity was measured separately as apoptosis or total neuronal death, with apoptosis representing 30 to 80% of the total neuron death observed, depending on the individual virus. In addition, neurotoxicity was dependent on expression of HIV-1 gp120 and could be blocked by anti-gp120 antibodies, as well as by antibodies to the human CCR5 and CXCR4 chemokine receptors. Despite extensive sequence diversity in the recombinant envelope region (V1V3 or C2V3), there was limited variation in the neurotoxicity induced by supernatants from transfected 293T cells. Conversely, supernatants from infected macrophages caused a broader range of neurotoxicity levels that depended on each virus and was independent of the replicative ability of the virus. These findings underscore the importance of HIV-1 envelope protein expression in neurotoxic pathways associated with HIV-induced brain disease and highlight the envelope as a target for neuroprotective therapeutic interventions.

FIG. 1.

Recombinant and pseudotyped virus strains express gp160 and gp120 env-encoded proteins. env-encoded protein expression was detected in transfected 293T cells (A to F) and supernatants (G) by immunocytochemistry and Western blotting analyses, respectively. Representative clones for different clades and replication-competent and -incompetent recombinant virus strains, together with JRFL-env pseudotyped pNL4-3-Luc-E⁻R⁻ virus (JRFL-env-PV), were tested. Panels: A, mock-transfected 293T cells (negative control); B, B118-C2V3 (clade A); C, B99-C2V3 (clade A); D, B100-C2V3 (clade D); E, JRFL-C2V3 (clade B); F, JRFL-env-PV. Arrows indicate immunopositive cells, while arrowheads highlight syncytium formation and a plus sign indicates a replication-competent virus. Original magnification, ×400; bar, 50 μm for all panels.

FIG. 2.

Replication kinetics and cell entry by recombinant virus strains. A subset of recombinant virus strains showed high RT levels in PBMCs (A); all of the virus strains tested (except pNL4-3) that replicated in PBMCs also exhibited infection of MDMs, as measured by p24 ELISA (B). PBMCs or MDMs were infected with equivalent amounts of p24 antigen of each virus. Replication-competent virus strains entered and expressed luciferase in one or more of the cell lines, depending on their use of chemokine receptors (C). Three brain-derived chimeric virus strains (B118-C2V3, JRFL-C2V3, and JRFL-V1V3) and the JRFL-env pseudotyped virus (JRFL-env-PV) used CCR5 for infections, while the spleen-derived chimeric virus (NL-S100-V1V3) and the parent virus, pNL4-3, used CXCR4 as a coreceptor for virus entry. The replication-incompetent recombinant virus strains did not enter any of these cell lines, on the basis of luciferase activity. The results represent the means and SEM of data from three experiments (triplicate wells in each experiment). The virus clade is indicated, as are paired C2V3 and V1V3 virus strains (brackets). A plus sign denotes a replication-competent virus. The coreceptors used by the virus strains are shown at the top (R5 for CCR5 and X4 for CXCR4). Luc, pNL4-3-LucE⁻R⁻; control, cell background control without infection.

FIG. 3.

Recombinant and pseudotyped virus strains induce neuronal necrosis and apoptosis. Representative images of neuronal (LAN-2) cells treated with supernatants from mock-infected macrophages (A) or macrophages infected with a pseudotyped virus strain (JRFL-env-PV) (B), showing apoptotic (TUNEL-positive) cells with a bright green signal (arrows) and live nonreactive cells exhibiting dull green (arrowheads). Comparisons were made of the mean cell death percentages (±SEM) for total neuronal cell death and neuronal apoptosis cells in direct (C) and indirect (D) neurotoxicity assays. Original magnification, ×200. The statistical significance of differences from the controls was determined by ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The letters A and B in parentheses indicate virus clades, and a plus sign indicates a replication-competent virus strain.

FIG. 4.

Comparison of direct and indirect neurotoxicity assays. Supernatants from transfected 293T cells (direct neurotoxicity) or from infected macrophages (indirect neurotoxicity) were applied to the human neuronal cell line LAN-2, followed by examination of the mean fold increase (±SEM) in total neuronal death (A) and the mean enrichment factor (±SEM) in neuronal apoptosis (B). Minimal cell death was observed for the luciferase-expressing vector (Luc), mock-infected 293T-derived supernatants (293T), and medium alone (Control), but staurosporine (SP, 0.2 μM) caused apoptotic cell death. (C) Comparison of mean direct and indirect neurotoxicity for both total neuronal death and apoptotic death for all of the viruses tested. (D) Correlation between direct and indirect neuronal apoptosis for all of the virus strains tested. (E) Correlation of direct neurotoxicity between apoptotic and total neuronal death for all of the virus strains tested. (F) Comparison of neurotoxicity between replication-competent and -incompetent viruses for both apoptotic and total neuronal death. Virus clades are underlined, and paired C2V3 and V1V3 virus strains are indicated by brackets. A plus sign denotes a replication-competent virus strain. The background neuronal death determined by trypan blue exclusion in the medium control varied in individual experiments (ranging from 2.3 to 5.1%). The statistical significance of differences from the medium control was determined by the Student t test (*, P < 0.05; **, P < 0.01, ***; P < 0.001).

FIG. 5.

Comparison of neurotoxicity in neuronal LAN-2 cells and primary HFNs. Direct and indirect total neuronal death was analyzed for LAN-2 cells (A) and HFNs (B). (C) Comparison of mean direct and indirect neurotoxicity among all of the virus strains tested for each cell type. (D) Correlation of direct and indirect total neuronal death between LAN-2 cells and primary neurons. The background neuronal death determined by trypan blue exclusion in the medium control varied in the individual experiments (ranging from 3.1 to 4.6%). The letters A, B, and D indicate virus clades, and a plus sign indicates a replication-competent virus. The statistical significance of differences from the Luc control was determined by the Student t test (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

FIG. 6.

Envelope- and chemokine receptor-mediated neurotoxicity. (A) Anti-envelope antibodies significantly reduced the mean percentage (±SEM) of direct and indirect neurotoxicity caused by recombinant virus strains in total neuronal death. (B) Anti-R5 and anti-X4 monoclonal antibodies and the G-protein-coupled receptor inhibitor PTX also reduced the mean percentage of direct neurotoxicity mediated by recombinant virus strains in both total neuronal-death (B) and apoptosis (C) assays. The statistical significance of differences from the untreated control was determined by ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The letters A, B, and D in parentheses indicate virus clades, and a plus sign indicates a replication-competent virus.

FIG. 7.

Chemokine receptor expression and activation. Human neuronal LAN-2 cells (A to C) and primary HFNs (D to F) expressed both the CCR5 (B and E) and CXCR4 (C and F) chemokine receptors, respectively, on neuronal perikarya (arrows) and neurites (arrowheads). Panels A and D represent immunostaining with isotype-matched antibodies.

FIG. 8.

Induction of STAT-1 nuclear translocation and expression of TNF-α, IL-1β, and iNOS. Representative images of human macrophages mock infected (A) or infected with a replication-competent (B118-C2V3) (B) or a replication-incompetent (B100-C2V3) (C) recombinant virus strain, showing STAT-1 immunoreactivity in cytoplasm (arrowheads) and in nuclei following translocation (arrows) 2 h after exposure of cells to neurotoxic supernatants. (D) Real time RT-PCR was performed with primary human macrophage cultures, and the results are expressed relative to the GAPDH mRNA level, revealing that all of the virus strains tested induced mean fold increases in TNF-α, IL-1β, and iNOS mRNA levels that were independent of viral replicative properties. The statistical significance of differences from the uninfected control was determined by ANOVA (*, P < 0.05; **, P < 0.01; ***, P < 0.001). The letters A, B, and D in parentheses indicated virus clades, and a plus sign indicates a replication-competent virus. Original magnification, ×400.