The immunologic and virologic mechanisms that lead to bystander CD4+
-T-cell death during HIV-1 infection remain poorly understood. In this study, we investigated HIV-1-induced bystander apoptosis in HVS-immortalized CD4+
T cells, which resemble activated mature primary CD4+
T cells (10
). We found that infection of CD4/HVS T cells with X4 or R5X4 strains caused high levels of bystander apoptosis. Furthermore, exposure of CD4/HVS T cells to HIV-1 virions induced apoptosis in the absence of productive infection. Changes in the Env that increase envelope/receptor affinity or coreceptor binding site exposure enhanced the capacity of HIV-1 virions to induce bystander apoptosis. These results suggest that HIV-1 variants with increased envelope/receptor affinity and/or enhanced coreceptor binding site exposure may promote T-cell depletion in vivo by accelerating bystander CD4+
The relative importance of direct cytopathicity versus bystander apoptosis during HIV-1 infection in vitro and in vivo has been the subject of much debate (15
). Direct cytopathic effects are the predominant cause of CD4+
-T-cell death in T-cell lines infected with lab-adapted HIV-1 strains (15
), whereas apoptosis of both infected and uninfected CD4+
T cells occurs in lymphoid tissue explants infected with lab-adapted or primary strains (37
). Several groups have reported that HIV-1-induced bystander apoptosis in primary T cells is highly dependent on the presence of monocytes/macrophages (3
), which can prime T cells to undergo apoptosis induced by Fas, tumor necrosis factor alpha, or gp120 (3
) and are present in human lymphoid tissue explants (35
). Remarkably, we found that high levels of bystander apoptosis are induced by infection of CD4/HVS T cells with primary X4 HIV-1 isolates (SG3 and ELI1), or GFP-expressing X4 and R5X4 recombinant strains (NL4-3-GFP and 89.6-GFP), in the absence of monocytes/macrophages and other antigen-presenting cells. CD4/HVS T cells are highly activated (10
) and therefore may not require priming by antigen-presenting cells to undergo bystander apoptosis. Thus, apparent discrepancies in the literature on HIV-1-mediated killing of infected versus uninfected cells may in part reflect differences between experimental systems, such as the level of T-cell activation and the cell culture microenvironment.
The fusion inhibitor T20 prevented virus entry but did not inhibit the induction of apoptosis by HIV-1 virions. Moreover, the levels of apoptosis induced by HIV-1 virions pseudotyped with a fusion-defective mutant Env were similar to those induced by virions with wild-type Env. These results imply that membrane fusion is not required for HIV-1 to induce bystander apoptosis and argue against a “fusion from without” mechanism in which HIV-1 particles induce massive cell membrane fusion leading to membrane permeability and cell death. We used flow cytometric analysis to detect p24 antigen or GFP as a marker for productively infected cells. One concern is whether all of the presumed bystander cells are truly uninfected, since this method may not detect infected cells in the early stages of the replication cycle. A similar concern applies to studies using cultures treated with AZT where viruses enter cells but replication is blocked during reverse transcription. However, our experiments using T20 and a fusion-defective mutant indicate that virus entry and the early steps of infection are not required for HIV-1-induced bystander apoptosis and suggest a critical role for events prior to membrane fusion.
HIV-1 virions with Envs defective for CXCR4 binding were also defective for inducing apoptosis in CD4/HVS T cells. Furthermore, AMD3100 and an anti-CXCR4 monoclonal antibody inhibited apoptosis induced by X4 HIV-1 virions. Likewise, TAK-779 and an anti-CCR5 monoclonal antibody inhibited apoptosis induced by R5 HIV-1 virions. These findings suggest that binding of the Env to CXCR4 or CCR5 is required for the induction of bystander apoptosis. CXCR4- and CCR5-dependent signal transduction pathways include activation of G proteins and the focal adhesion tyrosine kinases Pyk2 and FAK along with the proapoptotic p38 mitogen-activated protein kinase (22
). To test whether the proapoptotic signal is Gα
i-dependent, we incubated CD4/HVS T cells with pertussis toxin (1 μg/ml) prior to the addition of HIV-1 virions (G.H.H. and D.G., unpublished observation). However, the results could not be interpreted because pertussis toxin treatment caused high background levels of apoptosis. Further studies are required to determine the role of specific signaling pathways in bystander apoptosis induced by HIV-1 virions.
In contrast to the requirement for coreceptor binding, CD4 binding was not intrinsically required for HIV-1-induced bystander apoptosis. In particular, HIV-1 virions with envelope glycoproteins that interact directly with CXCR4 but are defective for CD4 binding retained the ability to induce apoptosis. Furthermore, bystander apoptosis was inhibited by AMD3100 and an anti-CXCR4 monoclonal antibody, which do not interfere with the binding of gp120 to CD4. These results contrast with previous studies, which suggested that the HIV-1 Env induces proapoptotic signals through CD4 (1
). However, Env binding to CD4 leads to subsequent Env-coreceptor binding by triggering exposure of the coreceptor binding site (25
). Thus, Env binding to CD4 indirectly leads to coreceptor engagement. CD4-mediated signaling can prime T cells for activation-induced apoptosis and/or enhance coreceptor-mediated signaling pathways (5
). However, our studies suggest that Env-CD4 binding is not sufficient to induce apoptosis in the absence of Env-coreceptor binding.
Changes in the envelope glycoproteins that increase envelope/receptor affinity or coreceptor binding site exposure enhanced the capacity of HIV-1 to induce bystander apoptosis. These strain-dependent differences were independent of effects on replication efficiency, as they were also observed in experiments using nonreplicating virions. Mechanistically, these properties of the envelope glycoproteins can impact bystander apoptosis by enhancing Env-coreceptor interactions and thereby increasing the intensity of coreceptor-mediated proapoptotic signaling. To this end, increased viral affinity for CD4 could indirectly enhance coreceptor-mediated signaling by increasing virion attachment and coreceptor binding site exposure (25
). Alternatively, HIV-1 virions with increased affinity for CD4 may have a greater capacity to induce apoptosis in cells that express CD4 at low levels (25
). Bystander apoptosis can also be influenced by certain host cell proteins, such as MHC class II and ICAM-1, which are incorporated into HIV-1 virions (12
; G.H.H. and D.G., unpublished results). Consistent with these findings, we found that virions produced in 293T cells did not induce apoptosis in CD4/HVS T cells unless provirus plasmids were cotransfected with MHC class II isoforms. Incorporation of MHC class II molecules into HIV-1 virions may enhance binding to target cells via interactions with CD4 and/or induce signaling events that prime T cells to undergo apoptosis.
In summary, our experiments suggest that HIV-1 virions induce a proapoptotic signal in CD4+
T cells through a CXCR4- or CCR5-mediated pathway that does not require CD4 signaling or membrane fusion. The ability of HIV-1 to induce bystander apoptosis is influenced by properties of the Env that enhance envelope/coreceptor interactions, including envelope/receptor affinity, exposure of the coreceptor binding site, and host cell membrane proteins on the virion surface. Previous studies have estimated that only 0.00001 to 0.01% of HIV-1 virions are infectious in vitro and in vivo (24
). Thus, noninfectious virions may contribute to HIV-1 pathogenicity in vivo by inducing bystander apoptosis mediated by Env-coreceptor interactions. Therapeutic strategies to block these interactions may reduce CD4+
-cell depletion in AIDS patients.