HIV-1 TAT AND APOPTOSIS

Tat is produced early after infection and is essential for viral gene expression and virus production. Work conducted in the last few years by several laboratories has shed light on the numerous interconnected functions of this striking small protein, both intra- and extracellularly.

By the early 1990s, Tat was shown to inhibit proliferation and induce apoptosis in activated uninfected cells.^31,39-41 Depending on the cell type and conditions (in particular, Tat concentrations and source), apoptosis of uninfected “bystander” cells was also described. Tat-dependent apoptosis was described in several cell types, including T and B cells, erythroid cells, endothelial cells, and neuronal cells. For example, Tat was found to display apoptotic properties on CD4⁺ Jurkat T lym- phoblastoid cells but was ineffective when unstimulated PBMCs or PBMCs activated by anti-CD3 antibodies (OKT3) were incubated with up to 5 μM synthetic Tat Lai or Tat fragments (Figure 9.2). In the case of Jurkat cells, apoptosis was demonstrated by different techniques, including flow

FIGURE 9.2 Tat promotes apoptosis of CD3-stimulated Jurkat cells but does not kill PBMCs. (A) PBMCs were exposed up to 72 h to indicated doses of synthetic Tat protein or Tat fragments, labeled with 7 amino­actinomycin D (7-AAD), and submitted to FACS analysis. Curves are the means of three independent experiments (SD < 5%). Note that, in these experimental conditions, there is no significant cellular uptake of 7-AAD, thus indicating the absence of Tat-induced PBMC death. (B) Jurkat cells were stimulated for 24 h in the presence of OKT3 (anti-CD3), were then submitted or not to synthetic Tat Lai, were colabeled with the ∆Ψm-sensitive probe JC-1 and with the DNA-interacting dye Hoechst 33342 and with 7-AAD, and were submitted to both FACS analysis and fluorescence microscopy observations. A representative ∆Ψm time response (after OKT3 plus Tat treatment) is shown in the upper panel. Inserted fluorescent micrographs show Jurkat cells incubated (upper insert) or not (lower insert) with synthetic Tat (5 μM) for 24 h and stained with 1 μM ∆Ψ_m-sensitive dye JC-1 (light dots in bottom image fluorescence shows mitochondria with high ∆Ψ_m, light area on the upper right side of the top image fluorescence shows mitochondria with low ∆Ψ_m) and with Hoechst 33342 (base color of top and bottom images fluorescence).

FIGURE 9.3 Tat enhances caspase-3 activation and PARP cleavage in CD3-stimulated Jurkat cells. Experi­mental conditions were similar to those described in the legend of Figure 9.2. At 24 h post-Tat treatment, cells were subjected to permeabilization and labeled with either a polyclonal FITC-tagged antibody directed to activated caspase-3 (A) or a polyclonal FITC-tagged antibody directed to cleaved PARP (B). A representative experiment is shown. Note that pretreatment with the pan-caspase inhibitor zVAD-fmk (50 μM) fully inhibited Tat-induced caspase-3 activation.

cytometry after propidium iodide labeling of treated cells, the TUNEL (terminal deoxynucleoti­dyltransferase-mediated dUTP-biotin nick-end labeling) method, the cleavage of caspase-3 and poly(ADP ribose polymer)ase (PARP) revealed by FITC-labeled specific antibodies and FACS analysis, and the evaluation of mitochondrial transmembrane potential (∆Ψm) state (Figure 9.2 and Figure 9.3)⁴² (Lecoeur and Jacotot, personal communication). Tat was also shown to induce cell death by apoptosis in primary cortical neurons (Lecoeur, Chauvier et al.⁴³) and cultured human fetal neurons⁴⁴ and rat neurons. For example, it was shown recently with striatal neurons that both HIV-1 gp120 and Tat fragment 1-72 significantly increased caspase-3 activation in a concentration­dependent manner, but that even though both components induced significant neurotoxicity, the nature of the apoptotic pathways preceding death differed.⁴⁵

Mechanisms by which Tat was reported to induce apoptosis include upregulation of the apop- totic effector molecule Fas ligand in a pathway that might involve NF-κB,^31,46 inhibition of the expression of manganese-dependent superoxide dismutase (Mn-SOD),⁴⁷ and the activation of cyclin-dependent kinases.⁴¹ These studies were performed in the context of uninfected cells.

Tat binds tubulin and polymerized microtubules through a four-amino-acid residue subdomain located in the core region, leading to the alteration of microtubule dynamics and activation of a mitochondria-dependent apoptotic pathway.⁴⁸

Tat-dependent apoptosis may also be explained by the effects of Tat on Bcl-2 and related proteins. Tat-mediated downregulation of Bcl-2 was effectively observed at both the transcriptional and translational levels. Also, Tat-transfected cells expressed increased amounts of Bax, a Bcl-2 family protein known to induce apoptosis.⁴⁹ In contrast, however, stable and transient transfections of Jurkat cells with the cDNA of Tat and a plasmid containing Bcl-2 promoter in front of CAT (Bcl-2 Pr/CAT) were found to stimulate CAT activity and lead to an increase of Bcl-2 mRNA and protein expression. This effect was specifically related to Tat, because Jurkat cells transfected with the cDNA of Tat in antisense orientation, Tat carrying a mutation at the position 22 (mutant C22G), or the control vector alone (pRPneo-SL3) did not show any significant difference in Bcl-2 promoter activity with respect to parental Jurkat cells.⁵⁰

In the context of infected cells, Tat was found to induce apoptosis by upregulating caspase-8, a protease that cleaves downstream caspases such as caspase-3, thus initiating the caspase cascade and increasing the sensitivity of cells to apoptotic signals.⁵¹ The induction of apoptosis could be prevented by treating cells with the caspase-8 inhibitor 2-IETD-fmk.

Controversies remain, however, because Tat can either induce apoptosis^{31,39-49,51-53} or inhibit apoptosis, depending on context.^50,54,55 Furthermore, certain cells, such as chimpanzee T cells, have been shown to be resistant to Tat-enhanced apoptosis. A number of studies were devoted to the molecular mechanisms by which Tat might directly or indirectly regulate apoptosis. Several cofac­tors mediating Tat-induced apoptosis were characterized, and models of Tat-mediated apoptosis were proposed. At this stage, however, it seems apparent that the observed ability of Tat to activate or inhibit apoptosis is highly dependent on the cell type, its stage of activation and infection, the use of tumoral or primary cells, their environments (presence of co-stimulatory signals and other cells), and the method used to reveal apoptosis.^54,56,57 Significant differences were also observed in the presence of recombinant and synthetic Tat according to their integrity, their degree of purity, and their proper folding. The length (86 versus 106 residues) of the protein might also be important. This suggests that the results of some studies would need to be revisited.