HIV-1 Tat protein concomitantly down-regulates apical caspase-10 and up-regulates c-FLIP in lymphoid T cells: a potential molecular mechanism to escape TRAIL cytotoxicity

In this study, we showed the existence of a positive correlation between the amount of human immunodeficiency virus-type 1 (HIV-1) RNA in HIV-1 seropositive subjects and the plasma levels of TRAIL. Since it has been previously demonstrated that HIV-1 Tat protein up-regulates the expression of TRAIL in monocytic cells whereas tat-expressing lymphoid cells are more resistant to TRAIL cytotoxicity, we next investigated the effect of Tat on the expression/activity of both apical caspase-8 and -10, which play a key role in mediating the initial phases of apoptosis by TRAIL, and c-FLIP. Jurkat lymphoblastoid human T cell lines stably transfected with a plasmid expressing wild-type (HIV-1) tat gene showed normal levels of caspase-8 but significantly decreased levels of caspase-10 at both mRNA and protein levels with respect to Jurkat transfected with the control plasmid or with a mutated (cys22) non-functional tat cDNA. A significant decrease of caspase-10 expression/activity was also observed in transient transfection experiments with plasmid carrying tat cDNA. Moreover, c-FLIP(L) and c-FLIP(S) isoforms were up-regulated in tat-expressing cells at both mRNA and protein level in comparison with control cells. Taken together, these results provide a molecular basis to explain the resistance of tat-expressing Jurkat cells to apoptosis induced by TRAIL and, possibly, to other death-inducing ligands.     

DNA immunization with HIV-1 tat mutated in the trans activation domain induces humoral and cellular immune responses against wild-type Tat

Intramuscular immunization of mice with plasmids encoding two transdominant negative mutants of the HIV-1 Tat protein (Tat22 and Tat22/37) elicited a humoral response to wild-type Tat that is comparable to that induced by inoculation of wild-type tat DNA or Tat protein. The percentage of the responders and the Ab titers continued to increase after three additional DNA boosts and pretreatment with bupivacaine at the site of inoculation, without a significant difference (p > 0.05) among the three groups of mice immunized with mutant and wild-type tat genes. By utilizing synthetic peptides representing the amino acid sequence of Tat, one major B cell epitope was defined within the cysteine-rich domain of Tat. Anti-Tat IgG Abs directed against this epitope were found in mice immunized with all tat DNA constructs, whereas different Tat epitopes were detected in mice immunized with the Tat protein. Similarly, IgG2a was the predominant isotype in DNA-immunized mice, with both mutants and wild-type tat genes, as compared with protein immunization, which induced mostly IgG1 and IgG3. Sera from most immunized mice neutralized the effect of extracellular Tat in activating HIV-1 replication. A cellular response was also elicited as indicated by the proliferation of splenocytes when stimulated with wild-type Tat. These results indicate that the wild-type Tat Ag is recognized by Abs and T cells induced by DNA immunization with mutated tat genes, suggesting the possible use of these Tat transdominant mutants, lacking viral trans activation activity and capable of blocking wild-type Tat activity, in the development of an anti-HIV-1 vaccine.     

Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation.

During acute human immunodeficiency virus type 1 (HIV-1) infection or after transfection of the tat gene, Tat protein is released into the cell culture supernatant. In this extracellular form, Tat stimulates both HIV-1 gene expression and the growth of cells derived from Kaposi's sarcoma (KS) lesions of HIV-1-infected individuals (AIDS-KS cells). Tat protein and its biological activities appear in the cell supernatants at the peak of Tat expression, when the rate of cell death is low (infection) or cell death is undetectable (transfection) and increased levels of cytoplasmic Tat are present. Tat-containing supernatants stimulate maximal AIDS-KS cell growth but only low to moderate levels of HIV-1 gene expression. This is due to the different concentrations of exogenous Tat required for the two effects. The cell growth-promoting effects of Tat peak at between 0.1 and 1 ng of purified recombinant protein per ml in the cell growth medium and do not increase with concentration. In contrast, both the detection of nuclear-localized Tat taken up by cells and the induction of HIV-1 gene expression or replication require higher Tat concentrations (> or = 100 ng/ml), and all increase linearly with increasing amounts of the exogenous protein. These data suggest that Tat can be released by a mechanism(s) other than cell death and that the cell growth-promoting activity and the virus-transactivating effect of extracellular Tat are mediated by different pathways.     

Modifications in the human T cell proteome induced by intracellular HIV-1 Tat protein expression

The effects of the human immunodeficiency virus type 1 (HIV-1) Tat protein on cellular gene expression were analysed using a Jurkat cell line that was stably transfected with tat gene in a doxycycline-repressible expression system. Expressed Tat protein (aa 1-101) was proved to present basically a nuclear localisation, and to be fully functional to induce HIV LTR transactivation. Tat expression also resulted in protection from Tunicamycin-induced apoptosis as determined by DNA staining and TUNEL assays. We applied proteomics methods to investigate changes in differential protein expression in the transfected Jurkat-Tat cells. Protein identification was performed using 2-D DIGE followed by MS analysis. We identified the down-regulation of several cytoskeletal proteins such as actin, beta-tubulin, annexin II, as well as gelsolin, cofilin and the Rac/Rho-GDI complex. Down-expression of these proteins could be involved in the survival of long-term reservoirs of HIV-infected CD4+ T cells responsible for continuous viral production. In conclusion, in addition to its role in viral mRNA elongation, the proteomic approach has provided insight into the way that Tat modifies host cell gene expression.     

Suppression of interleukin-2 and interleukin-2 receptor expression in Jurkat cells stably expressing the human immunodeficiency virus Tat protein.

The Jurkat T cell line was stably transfected with an Epstein-Barr virus-based episomal replicon designed to express high levels of the HIV-1 Tat protein. After selection in hygromycin B, high-level Tat activity was detected in 3 of 18 transfected cell lines. After stimulation with phytohemagglutinin (PHA) and phorbol myristate acetate (PMA), Tat transfectants with high Tat expression showed diminished expression of interleukin-2 (IL-2) and the interleukin-2 receptor alpha chain (IL-2R) when compared to untransfected Jurkat cells or Jurkat cell lines transfected with the parent control plasmid. Sublines derived from the high-level Tat transfectants with reduced Tat activity showed normalization of PHA/PMA-induced IL-2 expression. Northern analysis showed diminished expression of IL-2 and IL-2R mRNA in the stimulated Tat transfectants. Inhibition of IL-2 and IL-2R expression by the HIV-1 Tat protein may contribute to the immune suppression that characterizes HIV-1 infection.     

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)     

Death of CD4(+) T-cell lines caused by human immunodeficiency virus type 1 does not depend on caspases or apoptosis

A critical aspect of AIDS pathogenesis that remains unclear is the mechanism by which human immunodeficiency virus type 1 (HIV-1) induces death in CD4(+) T lymphocytes. A better understanding of the death process occurring in infected cells may provide valuable insight into the viral component responsible for cytopathicity. This would aid the design of preventive treatments against the rapid decline of CD4(+) T cells that results in AIDS. Previously, apoptotic cell death has been reported in HIV-1 infections in cultured T cells, and it has been suggested that this could affect both infected and uninfected cells. To evaluate the mechanism of this effect, we have studied HIV-1-induced cell death extensively by infecting several T-cell lines and assessing the level of apoptosis by using various biochemical and flow cytometric assays. Contrary to the prevailing view that apoptosis plays a prominent role in HIV-1-mediated T-cell death, we found that Jurkat and H9 cells dying from HIV-1 infection fail to exhibit the collective hallmarks of apoptosis. Among the parameters investigated, Annexin V display, caspase activity and cleavage of caspase substrates, TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) signal, and APO2.7 display were detected at low to negligible levels. Neither peptide caspase inhibitors nor the antiapoptotic proteins Bcl-x(L) or v-FLIP could prevent cell death in HIV-1-infected cultures. Furthermore, Jurkat cell lines deficient in RIP, caspase-8, or FADD were as susceptible as wild-type Jurkat cells to HIV-1 cytopathicity. These results suggest that the primary mode of cytopathicity by laboratory-adapted molecular clones of HIV-1 in cultured cell lines is not via apoptosis. Rather, cell death occurs most likely via a necrotic or lytic form of death independent of caspase activation in directly infected cells.     

Targeting Tat and IFN(alpha) as a therapeutic AIDS vaccine

Evolution to AIDS is characterized by a progressive cellular immune suppression. Although there is substantial evidence for several mechanisms involved in disrupting the immune response by induction of apoptosis in responder cells by contact with infected cells, we propose that humoral factors also play a role, and that one such factor is the extracellular form of the human immunodeficiency virus (HIV)-1 Tat protein and another is IFN(alpha). Both Tat and interferon-alpha (IFN(alpha)) inhibit antigen-stimulate T-cell proliferation, and specific anti-Tat and/or anti-IFN(alpha) Abs prevent generation of HIV-1-induced suppressor cells. We propose that high titer anti-Tat and/or anti-IFN(alpha) Abs, neutralizing extracellular Tat, and/or IFN(alpha), induced by vaccines described here, antagonize HIV-1-induced immunosuppression. Innocuous vaccines were prepared by using inactivated but immunogenic Tat (Toxoid) and inactivated and immunogenic IFN(alpha) (kinoid) derivatives. Both Tat Toxoid and IFN(alpha) kinoid were well tolerated and elicited specific neutralizing antibodies (Abs) in mice, monkeys, and seronegative and HIV-1-infected individuals.     

D-penicillamine inhibits transactivation of human immunodeficiency virus type-1 (HIV-1) LTR by transactivator protein

D-Penicillamine, an amino acid analogue of cysteine, has been shown to inhibit the transactivation of HIV-1 LTR by the transactivator protein, tat protein. The transactivation was studied in Jurkat cells co-transfected with plasmids containing HIV-LTR sequences fused to the bacterial chloramphenicol acetyltransferase (CAT) gene and HIV tat gene. The expression of CAT activity was a measure of transactivation of LTR by the tat protein. Incubation of transfected Jurkat cells with D-penicillamine led to inhibition of CAT activity. This inhibition was found to be concentration-dependent; more than 90% inhibition of chloramphenicol acetylation was seen in extracts prepared from cultures incubated with 40 micrograms/ml of D-penicillamine. Earlier experiments have shown that D-penicillamine at 40 micrograms/ml can completely inhibit HIV-1 (HTLV-III B) replication in H9 cells [(1986) Drug Res. 36, 184-186]. These results suggest that inhibition of transactivation may be the molecular mechanism involved in the inhibition of HIV-1 replication by D-penicillamine.     

Tumorigenesis by human herpesvirus 8 vGPCR is accelerated by human immunodeficiency virus type 1 Tat

Human herpesvirus 8 (HHV-8), also called Kaposi's sarcoma (KS) herpesvirus, can cause KS but is inefficient. Untreated human immunodeficiency virus type 1 (HIV-1) coinfection is a powerful risk factor. The HHV-8 chemokine receptor, vGPCR (ORF74), activates NF-kappaB and NF-AT, and their levels of activation are synergistically increased by HIV-1 Tat. Transgenic vGPCR mice develop KS-like tumors. A cell line derived from one such tumor expresses vGPCR and forms tumors in nude mice. Here we show that transfection of DNA encoding HIV-1 tat (but not a transactivation-defective mutant) into these tumor cells increases NF-kappaB and NF-AT activation levels and accelerates tumor formation. Tumorigenesis was also accelerated when Tat DNA was transfected into normal cells and the transfected cells were mixed with the tumor cells and injected into a single site. Tumorigenesis was also increased when the two cell types were injected at separate sites, suggesting that tumorigenesis is accelerated by Tat through soluble factors.     

Human immunodeficiency virus type 1 inhibits DNA damage-triggered apoptosis by a Nef-independent mechanism

It is controversial whether the accessory human immunodeficiency virus type 1 (HIV-1) Nef protein inhibits or enhances apoptosis. To address this issue, we investigated the effect of Nef on programmed cell death with vectors or proviral HIV-1 constructs coexpressing Nef and green fluorescent protein from single bicistronic RNAs. This approach allows us to readily identify transfected or infected cells and to correlate cell death directly with Nef expression levels. We demonstrate that Nef does not significantly affect apoptosis in transfected or HIV-1-infected Jurkat T cells or primary human peripheral blood mononuclear cells. Unexpectedly, however, both nef⁺ and nef-defective HIV-1 infection blocked apoptosis in cells treated with UV light or etoposide but not cell death induced by CD95 antibody, TRAIL, Ly294002, or serum starvation. Our results show that HIV-1 infection inhibits DNA damage-induced but not death receptor-dependent cell death by a Nef-independent mechanism.     

Human Immunodeficiency Virus Type 1 Tat Induces Apoptosis and Increases Sensitivity to Apoptotic Signals by Up-Regulating FLICE/Caspase-8

Apoptosis contributes to the loss of CD4 cells during human immunodeficiency virus type 1 (HIV-1) infection. Although the product of the env gene, gp160/gp120, is known to play a role in cell death mediated by HIV-1, the role of other HIV-1 genes in the process is unclear. We found that HIV-1 lacking the env gene (HIVΔenv) still induced apoptosis in T-cell lines and primary CD4 T cells. The ability to induce apoptosis was attributable to Tat, a viral regulatory protein. Tat induction of apoptosis was separate from the transactivation function of Tat, required expression of the second exon of Tat, and was associated with the increased expression and activity of caspase-8 (casp-8), a signaling molecule in apoptotic pathways. Moreover, induction of apoptosis could be prevented by treating cells with an inhibitor of casp-8. In addition, we show that HIV-1Δenv infection and Tat expression increased the sensitivity of cells to Fas-mediated apoptosis, an apoptotic pathway that signals via casp-8. The up-regulation of casp-8 by HIV-1 Tat expression may contribute to the increased apoptosis and sensitivity to apoptotic signals observed in the cells of HIV-1-infected persons.     

Effects of the human immunodeficiency virus type 1 Tat protein on the expression of inflammatory cytokines.

Increased levels of inflammatory cytokines, including tumor necrosis factor (TNF), interleukin-1 (IL-1), and IL-6, have been detected in specimens from human immunodeficiency virus type 1 (HIV-1)-infected individuals. Here we demonstrate that HIV-1 activates the expression of TNF but not of IL-1 and IL-6 in acutely and chronically infected T cells. The increase in TNF gene expression is due to activation of the TNF promoter by the viral gene product Tat. Transactivation of TNF gene expression requires the product of the first exon of the tat gene and is cell type independent. T cells chronically infected with pol-defective HIV-1 provirus constitutively express both Tat and TNF at levels significantly higher (fivefold) than those seen in control cells, and treatment with phorbol myristate acetate greatly enhances Tat expression and TNF production. As TNF can increase the production of IL-1 and IL-6 and these inflammatory cytokines all enhance HIV-1 gene expression and affect the immune, vascular, and central nervous systems, the activation of TNF by Tat may be part of a complex pathway in which HIV-1 uses viral products and host factors to increase its own expression and infectivity and to induce disease.     

人免疫缺陷病毒1型TAT突变蛋白及反义TAT RNA对TAT反式激活作用的抑制

Ｔａｔ是人免疫缺陷病毒（ＨＩＶ）基因组编码的反式激活因子,突变分析表明它含有几个重要的功能域。为寻找控制ＨＩＶ复制的途径,构建了以ＨＩＶ－１ＬＴＲ（－１５８－＋８０）为启动子的Ｔａｔ ｃＤＮＡ全长反义表达质粒ｐＡＳ－Ｔａｔ,并用已经构建的ＨＩＶ ＬＴＲ－１５８到＋８０为启动子,具有不同突变点的突变Ｔａｔ基因表达质粒,以荧光酶基因为报告基因,共转染Ｊｕｒｋａｔ细胞,结果发现无论是反义Ｔａｔ表达质粒还