Angelika Chandra

Molecular interactions of the type 1 human immunodeficiency virus transregulatory protein Tat with N-methyl-d-aspartate receptor subunits

We investigated the effect of type 1 human immunodeficiency virus (HIV-1) regulatory protein Tat on N-methyl-d-aspartate (NMDA) receptors expressed in Xenopus oocytes by voltage-clamp recording and its role in NMDA-mediated neurotoxicity using cultured rat hippocampal neurons. Tat (0.01-1muM) potentiated NMDA-induced currents of recombinant NMDA receptors. However, in the presence of Zn(2+), the potentiating effect of Tat was much more pronounced, indicating an additional Zn(2+)-related effect on NMDA receptors. Consistently, Tat potentiated currents of the particularly Zn(2+)-sensitive NR1/NR2A NMDA receptor with a higher efficacy, whereas currents from a Zn(2+)-insensitive mutant were only marginally augmented. In addition, chemical-modified Tat, deficient for metal binding, did not reverse Zn(2+)-mediated inhibition of NMDA responses, demonstrating that Tat disinhibits NMDA receptors from Zn(2+)-mediated antagonism by complexing the cation. We therefore investigated the interplay of Tat and Zn(2+) in NMDA-mediated neurotoxicity using cultures of rat hippocampal neurons. Zn(2+) exhibited a prominent rescuing effect when added together with the excitotoxicant NMDA, which could be reverted by the Zn(2+)-chelator tricine. Similar to tricine, Tat enhanced NMDA-mediated neurotoxicity in the presence of neuroprotective Zn(2+) concentrations. Double-staining with antibodies against Tat and the NR1 subunit of the NMDA receptor revealed partial colocalization of the immunoreactivities in membrane patches of hippocampal neurons, supporting the idea of a direct interplay between Tat and glutamatergic transmission. We therefore propose that release of Zn(2+)-mediated inhibition of NMDA receptors by HIV-1 Tat contributes to the neurotoxic effect of glutamate and may participate in the pathogenesis of AIDS-associated dementia.

Inhibition of SIRT1 by HIV-1 viral protein Tat results in activation of p53 pathway

Human immunodeficiency virus-1 (HIV-1) disease is characterized by a relentless decline in CD4(+) T cells, resulting in the development of AIDS. Extracellular Tat secreted from the HIV-1 infected cells, enters non-infected T cells to induce apoptosis. A number of mechanisms, none of which is mutually exclusive, have been attributed to the cell depletion property of Tat protein. In the present communication, we provide evidence that the cell-killing effect of Tat is mediated by the activation of p53 pathway via inhibition of SIRT1, an NAD(+)-dependent deacetylase belonging to class III histone deacetylases. This evidence is based on the following experimental facts reported herein: (1) Overexpression of Tat protein decreases both the deacetylase and promoter activity of SIRT1, (2) SIRT1 inhibition by Tat involves increased levels of acetylated p53 and (3) The activation of p53 leads to subsequent increases in the expression of p53 target genes, p21 and BAX.

Sequence Variation within the Dominant Amino Terminus Epitope Affects Antibody Binding and Neutralization of Human Immunodeficiency Virus Type 1 Tat Protein

ABSTRACT Tat is among the required regulatory genes of human immunodeficiency virus type 1 (HIV-1). Tat functions both within infected cells as a transcription factor and as an extracellular factor that binds and alters bystander cells. Some functions of extracellular Tat can be neutralized by immune serum or monoclonal antibodies. In order to understand the antibody response to Tat, we are defining antibody epitopes and the effects of natural Tat sequence variation on antibody recognition. The dominant Tat epitope in macaque sera is within the first 15 amino acids of the protein amino terminus. Together with a subdominant response to amino acids 57 to 60, these two regions account for most of the macaque response to linear Tat epitopes and both regions are also sites for the binding of neutralizing antibodies. However, the dominant and subdominant epitope sequences differ among virus strains, and this natural variation can preclude antibody binding and Tat neutralization. We also examined serum samples from 31 HIV-positive individuals that contained Tat binding antibodies; 23 of the 31 sera recognized the amino terminus peptide. Similar to binding in macaques, human antibody binding to the amino terminus was affected by variations at positions 7 and 12, sequences that are distinct for clade B compared to other viral clades. Tat-neutralizing antibodies to the dominant amino terminus epitope are affected by HIV clade variation.

Detection of distinct patterns of anti-tat antibodies in HIV-infected individuals with or without Kaposi's sarcoma.

Patterns of antibody response to recombinant transactivator protein (HIV-1 tat) in serum samples from HIV-1-negative persons (n = 60), HIV-1-infected asymptomatic persons (n = 20), HIV-1-infected people with Kaposis sarcoma (n = 25) and of people with Kaposis sarcoma without HIV-1 infection have been analyzed. None of the healthy people had anti-tat IgG in their serum. All asymptomatic patients with HIV-1 infection were anti-tat IgG-positive. Epitope mapping revealed that these sera have anti-tat IgG to all the functional domains of tat protein. Four of the 25 HIV-1-infected patients with Kaposis sarcoma were anti-tat IgG-positive; however, epitope analysis revealed that IgG to functional domains of tat protein, in particular to TAR-binding site, were absent. All patients with Kaposis sarcoma without HIV-1 infection were anti-tat IgG-negative. Presence or absence of anti-tat IgG, and prevalence of different antibody profiles in different groups of patients suggest the pathophysiologic role of tat protein. Thus, a passive immunization with anti-tat IgG could be a useful strategy to influence the pathophysiologic state of the disease.

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.

Docking studies reveal a selective binding of D-penicillamine to the transactivator protein of human immunodeficiency virus type 1

DOCK and Affinity studies were carried out to study the binding of D‐ and L‐penicillamine to the transactivator protein (tat) of human immunodeficiency virus type 1 (HIV‐1). These studies reveal a selective binding of D‐penicillamine to the cysteine‐rich region covering amino acid residues 20–38 of the tat protein. A careful analysis of the components of the binding energy of the D‐ and L‐isomers reveals that the D‐isomer has a more favorable van der Waals interaction resulting from an optimal placement of the dimethylthiomethyl side chain in the binding site. This observation matches the experimental data that D‐penicillamine is a more potent inhibitor of tat‐mediated transactivation than the L‐isomer. The docking and experimental data offer an interesting approach to design structural molecules with potential application to block signal functions of the tat protein in HIV‐1 pathogenesis.