Resistance of chimpanzee T cells to human immunodeficiency virus type 1 Tat-enhanced oxidative stress and apoptosis.

CD4+ T-cell depletion in AIDS patients involves induction of apoptosis in human immunodeficiency virus (HIV)-infected and noninfected T cells. The HIV type 1 (HIV-1)-transactivating protein Tat enhances apoptosis and activation-induced cell death (AICD) of human T cells. This effect is mediated by the CD95 (APO-1/Fas) receptor-CD95 ligand (CD95L) system and may be linked to the induction of oxidative stress by Tat. Here we show that HIV-1 Tat-induced oxidative stress is necessary for sensitized AICD in T cells caused by CD95L expression. Tat-enhanced apoptosis and CD95L expression in T cells are inhibited by neutralizing anti-Tat antibodies, antioxidants, and the Tat inhibitor Ro24-7429. Chimpanzees infected with HIV-1 show viral replication resembling early infection in humans but do not show T-cell depletion or progression towards AIDS. The cause for this discrepancy is unknown. Here we show that unlike Tat-treated T cells in humans, Tat-treated chimpanzee T cells do not show downregulation of manganese superoxide dismutase or signs of oxidative stress. Chimpanzee T cells are also resistant to Tat-enhanced apoptosis, AICD, and CD95L upregulation.

Cell surface sialylation plays a role in modulating sensitivity towards APO-1-mediated apoptotic cell death.

APO-1/Fas(CD95), a member of the tumour necrosis factor (TNF)/nerve growth factor (NGF) receptor superfamily transduces apoptotic signals into apoptosis sensitive cells. In metabolic labelling experiments using the highly APO-1 positive cell lines HUT78 (adultT cell leukemia) and SKW6.4 (Blymphoblastoid cell line) APO-1 was characterised as a long living protein with a complex glycosylation pattern involving terminal sialic acid groups which account for 8-kDa of its apparent molecular weight on SDS-PAGE. APO-1 expression and the degree of sialylation were determined in additionalT and B cell lines. On the group I Burkitt's lymphoma cell line BL60 transfected with human APO-1 (K50) low sialylated species were detected only on the cell surface, suggesting that sialylation might be functionally important. Removal of terminal sialic acid groups by treatment of B and T cell lines with Vibrio cholerae neuraminidase (VCN) augmented sensitivity towards anti-APO-1 and human APO-1 ligand induced apoptosis. Similarly, VCN-treated U937 cells were rendered more sensitive to TNFalpha-induced cell death. Thus, sialylation may be one mechanism to regulate sensitivity towards ligand-mediated cell death in this receptor family.

Molecular mechanisms of APO-1/Fas(CD95)-mediated apoptosis in tolerance and AIDS.

The cell surface molecule APO-1/Fas(CD95), a member of the Tumor Necrosis Factor (TNF) receptor/Nerve Growth Factor (NGF) receptor superfamily mediates apoptosis upon cross-linking by agonistic antibodies or its ligand. Recent findings suggest that APO-1/Fas(CD95) and its ligand are the key molecules for antigen receptor-induced apoptosis in activated mature T cells. Here we propose a mechanism for antigen receptor-induced apoptosis of activated T cells via APO-1 ligand mediated autocrine suicide. This mechanism may also contribute to the depletion of CD4+ T cells in AIDS.

Sensitization of T cells to CD95-mediated apoptosis by HIV-1 Tat and gp120.

The depletion of CD4+ T cells in AIDS is correlated with high turnover of the human immunodeficiency virus HIV-1 and associated with apoptosis. The molecular mechanism of apoptosis in HIV infection, however, is largely unknown. T-cell apoptosis might be affected by viral proteins such as HIV-1 Tat and gp120 (refs 10, 11). T-cell-receptor (TCR)-induced apoptosis was recently shown to involve the CD95 (APO-1/Fas) receptor. We show here that HIV-1 Tat strongly sensitizes TCR- and CD4(gp120)-induced apoptosis by upregulation of CD95 ligand expression. Concentrations of Tat found to be effective in cultures of HIV-1-infected cells were also observed in sera from HIV-1-infected individuals. Taken together, our results indicate that HIV-1 Tat and gp120 accelerate CD95-mediated, activation-induced T-cell apoptosis, a mechanism that may contribute to CD4+ T-cell depletion in AIDS.

HIV-1 Tat potentiates TNF-induced NF-kappa B activation and cytotoxicity by altering the cellular redox state.

This study demonstrates that human immunodeficiency virus type 1 (HIV-1) Tat protein amplifies the activity of tumor necrosis factor (TNF), a cytokine that stimulates HIV-1 replication through activation of NF-kappa B. In HeLa cells stably transfected with the HIV-1 tat gene (HeLa-tat cells), expression of the Tat protein enhanced both TNF-induced activation of NF-kappa B and TNF-mediated cytotoxicity. A similar potentiation of TNF effects was observed in Jurkat T cells and HeLa cells treated with soluble Tat protein. TNF-mediated activation of NF-kappa B and cytotoxicity involves the intracellular formation of reactive oxygen intermediates. Therefore, Tat-mediated effects on the cellular redox state were analyzed. In both T cells and HeLa cells HIV-1 Tat suppressed the expression of Mn-dependent superoxide dismutase (Mn-SOD), a mitochondrial enzyme that is part of the cellular defense system against oxidative stress. Thus, Mn-SOD RNA protein levels and activity were markedly reduced in the presence of Tat. Decreased Mn-SOD expression was associated with decreased levels of glutathione and a lower ratio of reduced:oxidized glutathione. A truncated Tat protein (Tat1-72), known to transactivate the HIV-1 long terminal repeat (LTR), no longer affected Mn-SOD expression, the cellular redox state or TNF-mediated cytotoxicity. Thus, our experiments demonstrate that the C-terminal region of HIV-1 Tat is required to suppress Mn-SOD expression and to induce pro-oxidative conditions reflected by a drop in reduced glutathione (GSH) and the GSH:oxidized GSH (GSSG) ratio.(ABSTRACT TRUNCATED AT 250 WORDS)

Human immunodeficiency virus type 1 Tat upregulates interleukin-2 secretion in activated T cells.

Dysregulation of cytokines secreted by T cells may play an important role in the pathogenesis of AIDS. To investigate the effects of human immunodeficiency virus type 1 (HIV-1) Tat on interleukin-2 (IL-2) expression, we used IL-2 promoter-chloramphenicol acetyltransferase constructs and IL-2-secreting Jurkat T cells as a model system. Transient expression of HIV-1 Tat induced a five- to eightfold increase in IL-2 promoter activity in Jurkat T cells stimulated with phytohemagglutinin and phorbol myristate acetate. IL-2 secretion was increased more than twofold in both Jurkat T cells and primary T cells stimulated by extracellular HIV-1 Tat protein. Analysis of mRNA suggested that Tat exerts its effect on IL-2 primarily at the transcriptional level. The NF-kappa B site at positions -206 to -195 of the IL-2 promoter was required but not sufficient for the Tat effect. The Tat-mediated increase in IL-2 promoter activity could selectively be blocked by antisense tat or-unlike the analogous effect of human T-cell lymphotropic virus type 1 Tax-by cyclosporin A. The observed increase in IL-2 levels might facilitate virus spread from or to T cells. Furthermore, it might contribute to the hypergammaglobulinemia or, together with other cytokines found to be dysregulated, the T-helper cell dysfunctions observed in AIDS patients.

Construction and overexpression in Escherichia coli of genetically engineered derivatives of penicillin-binding protein 2' of Staphylococcus epidermidis.

Removal of the putative amino-terminal membrane spanning region of penicillin-binding protein 2' (PBP-2') of Staphylococcus epidermidis WT55 was carried out by truncating the amino terminus-coding end of the mecA gene. PCR and site directed mutagenesis were used to introduce unique restriction sites at position 68 (HindIII) and at position 80 (NcoI) of the mecA gene, respectively. The coupling of the shortened coding regions to the trc promoter and gene fusion to the lacZ gene, aimed to facilitate subsequent protein purifications, resulted in strong expression in the cytoplasm of Escherichia coli and partial sequestration into insoluble protein granules. The truncated PBP-2' retained its penicillin-binding ability and also bound the monoclonal antibody directed against PBP-2' of Staphylococcus aureus.