Extracellular Tat Protein Research Articles

Vaccination with tat toxoid attenuates disease in simian/HIV-challenged macaques.

The Tat protein is essential for HIV type 1 (HIV-1) replication and may be an important virulence factor in vivo. We studied the role of Tat in viral pathogenesis by immunizing rhesus macaques with chemically inactivated Tat toxoid and challenging these animals by intrarectal inoculation with the simian/human immunodeficiency virus 89.6PD. Immune animals had significantly attenuated disease with lowered viral RNA, interferon-alpha, and chemokine receptor expression (CXCR4 and CCR5) on CD4(+) T cells; these features of infection have been linked to in vitro effects of Tat and respond similarly to extracellular Tat protein produced during infection. Immunization with Tat toxoid inhibits key steps in viral pathogenesis and should be included in therapeutic or preventive HIV-1 vaccines.

Extracellular HIV-1 Tat protein differentially activates the JNK and ERK/MAPK pathways in CD4 T cells.

To investigate the intracellular signals elicited by extracellular HIV-1 Tat protein in lymphoid CD4 T cells. CD4 Jurkat T cells were treated with a series of glutathione S-transferase (GST)-Tat fusion proteins: full-length two-exon GST-Tat (GST-Tat2E); one-exon Tat, in which the second exon of Tat was deleted (GST-Tat1E); two-exon Tat, in which the seven arginine residues have been changed to alanine residues (GST-TatArg(mut)), GST-TatdeltaN, which shows a deletion of the N-terminal 21 amino acids. The cells were either treated with soluble GST-Tat proteins or seeded on plates coated with GST-Tat proteins immobilized on plastic. At various time points, Jurkat cells were lysed and examined for c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) activity. Soluble and immobilized GST-Tat2E, but not GST-Tat1E, GST-TatArg(mut) and GST-TatdeltaN, activated JNK in a dose-dependent manner, induced a rapid phosphorylation of c-Jun on Ser63 and promoted the de novo synthesis of c-Jun protein. Moreover, both GST-Tat2E and GST-Tat1E also stimulated ERK/MAPK. However, the activation of JNK was maximal at concentrations of 100 nM of GST-Tat2E and was blocked by the S6-kinase inhibitor rapamycin, whereas the activation of ERK/MAPK was already maximal at 1 nM of GST-Tat2E and was enhanced by rapamycin. Tat-mediated activation of JNK requires the second exon of Tat, which is dispensable for the activation of ERK/MAPK. The ability to stimulate JNK and ERK/MAPK does not require Tat internalization.

Intercellular traffic of human immunodeficiency virus type 1 transactivator protein defined by monoclonal antibodies

Monoclonal antibodies (mAbs) directed against the amino-terminal region (N-terminal sequence 2–19) of transactivator protein (tat) of HIV-1 have been shown to inhibit intercellular transactivation mediated by the extracellular tat protein. The intracellular transactivation was not significantly affected by anti-tat mAbs. The specificity of anti-tat mAbs in abolishing the transactivating potential of extracellular tat is documented by studies with mAbs to HIV-1 reverse transcriptase, or to a human mammary cancer protein. None of these antibodies showed any inhibitory effect on intercellular transactivation. Specific interaction of anti-tat IgG with tat protein expressed in Jurkat cells is further supported by experiments on immunoblotting. Extracellular tat is responsible for signals which induce a variety of biological responses in HIV-infected cells, as well as in uninfected cells. The fact that anti-tat mAbs can abolish the intercellular traffic of tat protein offers a unique strategy in the development of vaccines against AIDS.

Extracellular HIV-1 Tat Protein Up-Regulates the Expression of Surface CXC-Chemokine Receptor 4 in Resting CD4+ T Cells

Here we report that synthetic HIV-1 Tat protein, immobilized on a solid substrate, up-regulates the surface expression of the CXC-chemokine receptor 4 (CXCR4), but not of the CC-chemokine receptor 5 in purified populations of primary resting CD4+ T cells. The Tat-mediated increase of CXCR4 occurred in a well-defined range of concentrations (1-10 nM of immobilized Tat) and time period (4-8 h postincubation). Moreover, the increase of CXCR4 was accompanied by an increased entry of the HXB2 T cell line-tropic (X4-tropic), but not of the BaL macrophage-tropic strain of HIV-1. The ability of Tat to up-regulate CXCR4 expression was abrogated by the protein synthesis inhibitor cycloheximide, clearly indicating the requirement of de novo synthesis. As Tat protein is actively released by HIV-1 infected cells, our data indicate a potentially important role for extracellular Tat in rendering bystander CD4+ T cells more susceptible to infection with X4-tropic HIV-1 isolates.

Characterization of the effects of two polysulfonated distamycin A derivatives, PNU145156E and PNU153429, on HIV type 1 Tat protein.

We examined whether two sulfonated distamycin A derivatives, PNU145156E and PNU153529, inhibit the trans-activating and angiogenic effects of HIV-1 Tat protein. The study was carried out by analyzing the activity of the two drugs on: (1) extracellular and intracellular Tat protein, introduced into HL3T1 cells containing an integrated HIV-1 LTR/CAT plasmid; (2) binding of Tat to 3H-labeled heparin and to 14C-labeled PNU145156E; and (3) the angiogenic response induced in vivo by culture medium conditioned by T53c14 cells, which release extracellular Tat. PNU145156E and PNU153429 interacted with extracellular Tat in the culture medium and physically bound the Tat protein, most likely sequestering it in the extracellular space. As a consequence, the two drugs inhibited trans-activation of the HIV-1 LTR on addition of the free Tat protein to HL3T1 cells. However, the two compounds inhibited the activity of intracellular Tat when they were introduced into the cells by lipofection. In vivo experiments showed that the two drugs blocked the neoangiogenesis induced by Tat released in the conditioned medium of T53c14 cells. Owing to the critical role of intracellular and extracellular Tat in HIV-1 replication, these drugs show promise as a means to control the progression of HIV-1 infection as well as the neoplastic and angiogenic effects induced by Tat in the course of AIDS.

HIV-1 Tat induces tyrosine phosphorylation of p125FAK and its association with phosphoinositide 3-kinase in PC12 cells.

To evaluate the signal transduction potential of HIV-1 Tat in a neuronal cell model. The tyrosine phosphorylation levels of the focal adhesion kinase p125FAK and its association with phosphoinositide 3-kinase (PI 3-K) were evaluated in serum-starved rat pheochromocytoma PC12 cells, either treated with low concentrations (0.1-1 nM) of extracellular HIV-1 Tat protein or stably transfected with Tat cDNA. Extracellular Tat induced a rapid increase of p125FAK tyrosine phosphorylation and p125FAK-associated PI 3-K activity. By using recombinant mutated Tat proteins, it was found that deletion of amino acids 73-86 encoded by the second exon of the tat gene resulted in a significant decrease of the ability of Tat to induce p125FAK tyrosine phosphorylation. Paradoxically, mutations in the basic region encoded by the first exon of tat, which is essential for nuclear localization and HIV-1 LTR transactivation, increased the ability of Tat to stimulate p125FAK tyrosine phosphorylation. Moreover, in comparison with cells transfected with a control vector, PC12 cells stably transfected with tat cDNA showed greater amounts of p125FAK protein, an increase in p125FAK tyrosine phosphorylation and higher levels of p125FAK-associated PI 3-K activity. The addition of anti-Tat neutralizing antibody to tat-transfected PC12 cells in culture blocked both the p125FAK tyrosine phosphorylation and its association with PI 3-K but did not affect the total amount of p125FAK. HIV-1 Tat protein enhanced both the expression and the functionality of p1 25FAK in PC12 neuronal cells. Whereas the first event required intracellular Tat, the increased p125FAK phosphorylation was strictly dependent upon extracellular Tat.

The Basic Domain in HIV-1 Tat Protein as a Target for Polysulfonated Heparin-mimicking Extracellular Tat Antagonists

Heparin binds extracellular HIV-1 Tat protein and modulates its HIV long terminal repeat (LTR)-transactivating activity (M. Rusnati, D. Coltrini, P. Oreste, G. Zoppetti, A. Albini, D. Noonan, F. d'Adda di Fagagna, M. Giacca, and M. Presta (1997) J. Biol. Chem. 272, 11313-11320). On this basis, the glutathione S-transferase (GST)-TatR49/52/53/55/56/57A mutant, in which six arginine residues within the basic domain of Tat were mutagenized to alanine residues, was compared with GST-Tat for its capacity to bind immobilized heparin. Dissociation of the GST-TatR49/52/53/55/56/57A.heparin complex occurred at ionic strength significantly lower than that required to dissociate the GST-Tat.heparin complex. Accordingly, heparin binds immobilized GST-Tat and GST-TatR49/52/53/55/56/57A with a dissociation constant equal to 0.3 and 1.0 microM, respectively. Also, the synthetic basic domain Tat-(41-60) competes with GST-Tat for heparin binding. Suramin inhibits [3H]heparin/Tat interaction, 125I-GST-Tat internalization, and the LTR-transactivating activity of extracellular Tat in HL3T1 cells and prevents 125I-GST-Tat binding and cell proliferation in Tat-overexpressing T53 cells. The suramin derivative 14C-PNU 145156E binds immobilized GST-Tat with a dissociation constant 5 times higher than heparin and is unable to bind GST-TatR49/52/53/55/56/57A. Although heparin was an antagonist more potent than suramin, modifications of the backbone structure in selected suramin derivatives originated Tat antagonists whose potency was close to that shown by heparin. In conclusion, suramin derivatives bind the basic domain of Tat, prevent Tat/heparin and Tat/cell surface interactions, and inhibit the biological activity of extracellular Tat. Our data demonstrate that tailored polysulfonated compounds represent potent extracellular Tat inhibitors of possible therapeutic value.

Extracellular HIV-1 Tat Protein Induces the Rapid Ser133 Phosphorylation and Activation of CREB Transcription Factor in Both Jurkat Lymphoblastoid T Cells and Primary Peripheral Blood Mononuclear Cells

Extracellular HIV-1 Tat protein (0.1-100 ng/ml) induced a rapid (peak at 30 min) increase in the Ser133 phosphorylation levels of the transcription factor CREB in serum-starved Jurkat cells, as revealed by Western blot and indirect immunofluorescence analyses. Nuclear cAMP-responsive element (CRE) binding activity in electrophoretic mobility shift assays was constitutive in unstimulated Jurkat cells, showing only a small increase upon Tat treatment. However, transient transfection experiments performed with various chloramphenicol acetyl-transferase (CAT) constructs showed that Tat produced a fourfold induction of CAT activity only in the presence of a CRE-dependent CAT construct. Moreover, the use of plasmids encoding for GAL4-CREB fusion proteins demonstrated that Tat induction of pG4-CAT reporter gene required the CREB moiety of the GAL4-CREB fusion protein and that Ser133 CREB was essential for Tat activity. Extracellular Tat also stimulated Ser133 CREB phosphorylation in freshly isolated PBMC; this effect was completely blocked by either staurosporin, a broad-spectrum inhibitor of various protein kinases, or PD 98059, a specific inhibitor of mitogen-activated protein kinases (MAPK). Furthermore, extracellular Tat induced a rapid (peak at 5-15 min) stimulation of the MAPK catalytic activity in primary PBMC. Altogether, these findings suggest that HIV-1 Tat protein activates CREB in lymphoid cells through a signal cascade involving the MAPK pathway.

Extracellular HIV-1 Tat Protein Induces a Rapid and Selective Activation of Protein Kinase C (PKC)-α, -ϵ, and -ζ Isoforms in PC12 Cells

The addition in culture of extracellular HIV-1 Tat protein (0.1-1 nM) to PC12 cells induced a rapid increase of the bulk protein kinase C (PKC) catalytic activity. Among various PKC isoforms (α, βI, βII, δ, ϵ, η, θ, and ζ) expressed in PC12 cells, Tat selectively stimulated α, ϵ, and ζ, as judged by activities in immunoprecipitates. Activation of these isoforms was suppressed by the tyrosine kinase inhibitor genistein. Moreover, PKC-ζ showed the fastest kinetics of activation in response to Tat, but PKC-α and PKC-ϵ showed the highest levels of activation. PKC-α activation was accompanied by a rise of intracellular IP3, while the PI 3-kinase inhibitors wortmannin and LY294002 suppressed PKC-ϵ activation. Taken together, these findings demonstrate that extracellular Tat shows a cytokine-like activity in PC12 cells, being able to trigger an intracellular signalling cascade which involves PKC-α, -ϵ, and -ζ.

Extracellular human immunodeficiency virus type 1 Tat protein promotes aggregation and adhesion of cerebellar neurons

Recombinant human immunodeficiency virus (HIV-1) Tat protein added to the culture medium of rat cerebellar neurons promoted aggregation and formation of spoke-like neurites in a dose-dependent manner. Tat proteins containing mutations in the Arg-Gly-Asp (RGD) cell adhesion motif or a deletion of the cysteine-rich domain had no effect on neuronal morphology. In contrast, a Tat protein that contained a deletion of the proline-rich domain promoted neuronal aggregation. Aggregation of neurons was inhibited by the addition of monoclonal antibodies directed against the RGD and basic domains of Tat, but not against the proline-rich domain. The same domains of Tat required to induce aggregation also mediated adhesion of neurons to Tat-coated substrates. The HIV-2 Tat protein, which lacks an RGD sequence but contains cysteine-rich and basic domains similar to HIV-1 Tat, induced aggregation and acted as a substrate for adhesion when added at higher concentrations than HIV-1 Tat. Vitronectin, fibronectin, and RGD-containing peptides did not induce morphological changes in neurons or act as substrates for adhesion. The ability of Tat to induce morphological changes and promote adhesion was independent of the ability of Tat to transactivate HIV gene expression. Our results suggest that extracellular Tat protein most likely alters neuronal morphology and mediates adhesion by acting in a manner similar to an extracellular matrix protein.

Extracellular human immunodeficiency virus type 1 Tat protein is associated with an increase in both NF-kappa B binding and protein kinase C activity in primary human astrocytes

Human immunodeficiency virus type 1 (HIV-1) infection has been associated with an increase in the binding of the transcription factor NF-kappa B to its consensus sequence in the viral promoter. Using cultures of primary human fetal astrocytes, we show that exogenous HIV-1 Tat protein, which has been demonstrated to be released from infected cells, is associated with an increase in the binding of this transcription factor to an HIV-1 long terminal repeat kappa B sequence. This effect occurs rapidly and is independent of new protein synthesis. We also demonstrate that extracellular Tat protein is associated with an increase in protein kinase C activity. If Tat functions similarly in other cell types, such findings could relate to some of this protein's previously described physiological effects. These effects include Tat's ability to upregulate the synthesis of specific cytokines and to act as a growth factor.

In vivo and in vitro modulatory effect of human immunodeficiency virus type-1 (HIV-1) Tat protein on protein kinase C activity

Extracellular HIV-1 Tat protein shows a pleiotropic activity on the survival/proliferation of different cell types, which may be relevant to the pathogenesis of the immune suppression as well as of the frequent neoplastic disorders observed during the course of HIV-1 disease. Therefore, we investigated the effect of recombinant Tat on the protein kinase C (PKC) activity in Jurkat CD4(+) T lymphoma cells by using a serine substituted specific PKC peptide substrate, which allowed the evaluation of the whole catalytic activity of both Ca++-dependent and Ca++-independent PKC isoforms. High concentrations of recombinant Tat (1 mu g/ml) induced an early (5 min) stimulation followed by a secondary (30-60 min) inhibition of PKC in whole Jurkat cell homogenates. Immuno-localization experiments showed that recombinant Tat protein was rapidly taken up by Jurkat cells within the first 5 min from the addition in culture, thus suggesting the possibility that the secondary inhibitory phase of Tat on PKC activity in Jurkat cells could be due to a direct interaction between the two proteins. Consistently, PKC immunoprecipitated from Jurkat cells or purified from rat brain was significantly inhibited by the addition of high (0.1-1 mu g) but not low (1-10 ng) doses of Tat in a cell-free in vitro assay. The inhibition of PKC catalytic activity mediated by 1 mu g of Tat was at least partially due to competition among substrates. The present data may help in understanding the opposite effects on the survival/proliferation of different cell types observed in the presence of picomolar (stimulation) vs nanomolar (inhibition) concentrations of recombinant Tat.

Effect of Antibody to HIV-1 Tat Protein on Viral Replication in Vitro and Progression of HIV-1 Disease in Vivo

In HIV-1-infected cell cultures, a relatively low concentration (5 micrograms/ml) of monoclonal antibody (mAb) against HIV-1-transactivating Tat protein was an efficient inhibitor of HIV-1 replication both in HIV-1(IIIB)-infected Jurkat cell and peripheral blood mononuclear cell (PBMC) cultures and significantly reduced the expression of a Tat-responsive CAT-reporter construct in HIV-1(IIIB)-infected Jurkat cells. Anti-Tat mAb also caused a significant reduction and a consistent delay in HIV-1 replication when added to PBMCs from HIV-1-infected patients cocultivated with phytohemagglutinin (PHA)-stimulated normal PBMCs. These data indicate that an autocrine-paracrine loop sustained by extracellular Tat protein, which is actively released by HIV-1-infected cells, may affect HIV-1 replication in cell cultures in vitro. An inverse relationship between natural anti-Tat antibody levels and p24 antigenemia was demonstrated by retrospective analysis of serial serum samples obtained from 10 HIV-1-seropositive hemophiliac patients followed over a 7-9-year period. This datum points to a possible influence of anti-Tat antibody on the progression of HIV-1 disease in vivo. These findings have strong implications for Tat protein as a possible target for specific immunotherapy in HIV-1-infected patients.

Exogenous human immunodeficiency virus type‐1 Tat protein selectively stimulates a phosphatidylinositol‐specific phospholipase C nuclear pathway in the Jurkat T cell line

We investigated the effect of extracellular Tat protein of human immunodeficiency virus-type 1 (HIV-1) on the phosphatidylinositol (PI) cycle, which represents a major signal transduction pathway in lymphoid cells. Recombinant Tat, recombinant HIV-1 p24 and cross-linked anti-CD3 monoclonal antibody (mAb) were added in culture for 1-60 min to Jurkat lymphoblastoid CD4+ T cells. The stimulation of T cell receptor by cross-linked anti-CD3 mAb resulted in a rapid increase of the phosphatidylinositol-specific phospholipase C (PI-PLC) activity in whole cell lysates. On the other hand, Tat protein, either alone or in combination with anti-CD3 mAb, showed little effect on the PI turnover of whole cell extracts. Tat, however, selectively stimulated a nuclear-specific PI-PLC with a peak of activity after 30 min from the addition in culture to Jurkat cells. Interestingly, this time corresponded to that required for the uptake and nuclear localization of recombinant Tat protein, as demonstrated by electron microscope immunocytochemistry experiments with anti-Tat mAb. Moreover, exogenous Tat reached the nucleus of Jurkat cells in a bioactive form, as shown in a HIV-1 long terminal repeat-chloramphenicol acetyl transferase transactivation assay. The specific increase of a nuclear PI-PLC activity was further demonstrated by the ability of Tat to stimulate PI turnover also when added directly to isolated nuclei. As a whole, these data demonstrate that Tat selectively stimulates a nuclear polyphosphoinositide hydrolysis, which appears to be independent of the cellular PI turnover. The relevance of these findings for a better understanding of the biological functions of extracellular Tat is discussed.

HIV Type 1 Extracellular Tat Protein Stimulates Growth and Protects Cells of BK Virus/tatTransgenic Mice from Apoptosis

Cells from BKV/tat transgenic mice were characterized for their tumorigenic phenotype in nude and syngeneic BDF mice. The results indicate that the BKV/tat recombinant transgene has a weak tumorigenic potential, mostly predisposing to oncogenesis, and that second events are required for the development of tumorigenicity. Tat is endogenously produced and released by tumor cells. It is taken up by recipient cells directly from the culture medium, without need of cell to cell contact. Extracellular Tat stimulates proliferation of cells from BKV/tat transgenic mice and protects them from apoptosis under conditions of serum starvation. Our results are in agreement with a model in which Tat induces its effects on target cells in two different ways. Growth promotion may require interaction of extracellular Tat with surface receptors eliciting a signal for cell proliferation, whereas intranuclear localization of Tat is necessary for transactivation of viral and cellular genes.

Cytokines from activated T cells induce normal endothelial cells to acquire the phenotypic and functional features of AIDS-Kaposi's sarcoma spindle cells.

Kaposi's sarcoma (KS) is a proliferative disease of vascular origin particularly frequent in HIV-1-infected homosexual men (AIDS-KS) and characterized by proliferating spindle-shaped cells, angiogenesis, and inflammatory cell infiltration. Previous work has suggested that KS spindle cells are of endothelial cell origin and that chronic immune activation via the release of inflammatory cytokines may cooperate with basic fibroblast growth factor (bFGF) and the HIV-1 Tat protein in the induction and progression of AIDS-KS. Here we show that KS spindle cells have features of activated endothelial cells, and that conditioned media from activated T cells, rich in the same inflammatory cytokines increased in HIV-1-infected individuals, induce normal endothelial cells to acquire the phenotypic and functional features of KS cells. These include (a) acquisition of a similar pattern of cell surface antigen expression; (b) similar proliferative response to bFGF; (c) induction of the responsiveness to the mitogenic effect of extracellular HIV-1 Tat protein that is now able to promote the G1-S transition of endothelial cell cycle; and (d) induction in nude mice of vascular lesions closely resembling early KS as well as the lesions induced by inoculation of KS cells. These results suggest that chronic immune activation, via release of inflammatory cytokines, may play a role in the induction of KS.

Human immunodeficiency virus type 1 Tat activity in human neuronal cells: uptake and trans-activation.

Neurological dysfunction in AIDS occurs in the absence of productive infection of neurons, and may involve modulation of neuronal cell function by viral or cellular products released from surrounding infected cells. The human immunodeficiency virus type 1 (HIV-1) trans-activator protein Tat may be one such factor, as it can act as a neurotoxin, induces marked morphological changes in neurons and astrocytes in primary embryonic rodent brain cultures, and is released by certain HIV-1-infected cells. In addition, Tat can alter expression of cellular genes in several non-neuronal cell types. To explore the possibility that Tat may also mediate neuronal dysfunction in AIDS through non-lethal effects on neurons, we determined the trans-activating ability of Tat in human neuronal cells. We generated human neuronal cell lines stably expressing several HIV-1 tat genes, and also tested human neuronal cells exposed to extracellular recombinant Tat protein. Both endogenously expressed Tat as well as exogenous recombinant Tat protein up-regulated HIV-1 long terminal region (LTR)-driven gene expression by several hundred-fold. Only brief exposure to recombinant Tat was necessary and no toxic effects were seen at levels sufficient for trans-activation. Furthermore, Tat significantly enhanced virus expression in neuronal cells transfected with molecular clones of HIV-1. These results show that Tat is trans-activationally active in human neuronal cells, and can be taken up from the extracellular compartment by these cells in a biologically active form. Neurons represent an important potential target for Tat-mediated cellular dysfunction.

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.

Exogenous tat Protein Activates Human Endothelial Cells

Abstracttat protein, a human immunodeficiency virus (HIV) gene product that functions as a transactivator for HIV replication, is known to be secreted extracellularly by infected cells. To determine the potential role of tat in the dissemination of HIV into extravascular tissue, this protein was examined for its ability to activate human endothelial cells. The results show that tat does indeed stimulate endothelial cells. This is evidenced by their expression of the endo-thelial-leukocyte adhesion molecules, E-selectin, critical for the initial binding of leukocytes to the blood vessel wall, and their increased synthesis of interleukin-6 (IL-6), a cytokine known to enhance endothelial cell permeability. Furthermore, tat acts synergistically with low concentrations of tumor necrosis factor-α to enhance IL-6 secretion. These data suggest that extracellular tat protein secreted or released into the microenvironment may contribute significantly to the determination of specific sites of leukocyte binding to blood vessels, to transmigration into tissue, and to eventual dissemination of HIV-infected cells or free virions into tissue.

Exogenous tat protein activates human endothelial cells [see comments

tat protein, a human immunodeficiency virus (HIV) gene product that functions as a transactivator for HIV replication, is known to be secreted extracellularly by infected cells. To determine the potential role of tat in the dissemination of HIV into extravascular tissue, this protein was examined for its ability to activate human endothelial cells. The results show that tat does indeed stimulate endothelial cells. This is evidenced by their expression of the endothelial- leukocyte adhesion molecules, E-selectin, critical for the initial binding of leukocytes to the blood vessel wall, and their increased synthesis of interleukin-6 (IL-6), a cytokine known to enhance endothelial cell permeability. Furthermore, tat acts synergistically with low concentrations of tumor necrosis factor-alpha to enhance IL-6 secretion. These data suggest that extracellular tat protein secreted or released into the microenvironment may contribute significantly to the determination of specific sites of leukocyte binding to blood vessels, to transmigration into tissue, and to eventual dissemination of HIV-infected cells or free virions into tissue.