Erwann Loret

Acetylation of Tat Defines a CyclinT1-Independent Step in HIV Transactivation

The HIV transcriptional activator Tat is acetylated by p300 at a single lysine residue in the TAR RNA binding domain. We have generated monoclonal and polyclonal antibodies specific for the acetylated form of Tat (AcTat). Microinjection of anti-AcTat antibodies inhibited Tat-mediated transactivation in cells. Similarly, the p300 inhibitor Lys-CoA and siRNA specific for p300 suppressed Tat transcriptional activity. Full-length synthetic AcTat bound to TAR RNA with the same affinity as unacetylated Tat, but formation of a Tat-TAR-CyclinT1 ternary complex was completely inhibited in the presence of AcTat. We propose that Tat acetylation may help in dissociating the Tat cofactor CyclinT1 from TAR RNA and serve to transfer Tat onto the elongating RNA polymerase II.

The Putative Bioactive Surface of Insect-selective Scorpion Excitatory Neurotoxins

Scorpion neurotoxins of the excitatory group show total specificity for insects and serve as invaluable probes for insect sodium channels. However, despite their significance and potential for application in insect-pest control, the structural basis for their bioactivity is still unknown. We isolated, characterized, and expressed an atypically long excitatory toxin, Bj-xtrIT, whose bioactive features resembled those of classical excitatory toxins, despite only 49% sequence identity. With the objective of clarifying the toxic site of this unique pharmacological group, Bj-xtrIT was employed in a genetic approach using point mutagenesis and biological and structural assays of the mutant products. A primary target for modification was the structurally unique C-terminal region. Sequential deletions of C-terminal residues suggested an inevitable significance of Ile73 and Ile74 for toxicity. Based on the bioactive role of the C-terminal region and a comparison of Bj-xtrIT with a Bj-xtrIT-based model of a classical excitatory toxin, AaHIT, a conserved surface comprising the C terminus is suggested to form the site of recognition with the sodium channel receptor.

Phosphatidylinositol-(4,5)-bisphosphate enables efficient secretion of HIV-1 Tat by infected T-cells.

Human immunodeficiency virus type 1 (HIV‐1) transcription relies on its transactivating Tat protein. Although devoid of a signal sequence, Tat is released by infected cells and secreted Tat can affect uninfected cells, thereby contributing to HIV‐1 pathogenesis. The mechanism and the efficiency of Tat export remained to be documented. Here, we show that, in HIV‐1‐infected primary CD4+ T‐cells that are the main targets of the virus, Tat accumulates at the plasma membrane because of its specific binding to phosphatidylinositol‐4,5‐bisphosphate (PI(4,5)P2). This interaction is driven by a specific motif of the Tat basic domain that recognizes a single PI(4,5)P2 molecule and is stabilized by membrane insertion of Tat tryptophan side chain. This original recognition mechanism enables binding to membrane‐embedded PI(4,5)P2 only, but with an unusually high affinity that allows Tat to perturb the PI(4,5)P2‐mediated recruitment of cellular proteins. Tat–PI(4,5)P2 interaction is strictly required for Tat secretion, a process that is very efficient, as ∼2/3 of Tat are exported by HIV‐1‐infected cells during their lifespan. The function of extracellular Tat in HIV‐1 infection might thus be more significant than earlier thought.

Mechanism of calcite crystal growth inhibition by the N-terminal undecapeptide of lithostathine.

Pancreatic juice is supersaturated with calcium carbonate. Calcite crystals therefore may occur, obstruct pancreatic ducts, and finally cause a lithiasis. Human lithostathine, a protein synthesized by the pancreas, inhibits the growth of calcite crystals by inducing a habit modification: the rhombohedral {10 1̄4} usual habit is transformed into a needle-like habit through the {112̄0} crystal form. A similar observation was made with the N-terminal undecapeptide (pE1R11) of lithostathine. We therefore aimed at discovering how peptides inhibit calcium salt crystal growth. We solved the complete x-ray structure of lithostathine, including the flexible N-terminal domain, at 1.3 Å. Docking studies of pE1R11 with the (101̄4) and (11 2̄0) faces through molecular dynamics simulation resulted in three successive steps. First, the undecapeptide progressively unfolded as it approached the calcite surface. Second, mobile lateral chains of amino acids made hydrogen bonds with the calcite surface. Last, electrostatic bonds between calcium ions and peptide bonds stabilized and anchored pE1R11 on the crystal surface. pE1R11-calcite interaction was stronger with the (11 2̄0) face than with the (10 1̄4) face, confirming earlier experimental observations. Energy contributions showed that the peptide backbone governed the binding more than did the lateral chains. The ability of peptides to inhibit crystal growth is therefore essentially based on backbone flexibility.

What does the structure-function relationship of the HIV-1 Tat protein teach us about developing an AIDS vaccine?

The human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription protein Tat is an important factor in viral pathogenesis. In addition to its function as the key trans-activator of viral transcription, Tat is also secreted by the infected cell and taken up by neighboring cells where it has an effect both on infected and uninfected cells. In this review we will focus on the relationship between the structure of the Tat protein and its function as a secreted factor. To this end we will summarize some of the exogenous functions of Tat that have been implicated in HIV-1 pathogenesis and the impact of structural variations and viral subtype variants of Tat on those functions. Finally, since in some patients the presence of Tat-specific antibodies or CTL frequencies are associated with slow or non-progression to AIDS, we will also discuss the role of Tat as a potential vaccine candidate, the advances made in this field, and the importance of using a Tat protein capable of eliciting a protective or therapeutic immune response to viral challenge.

1H-13C nuclear magnetic resonance assignment and structural characterization of HIV-1 Tat protein.

Tat is a viral protein essential for activation of the HIV genes and plays an important role in the HIV-induced immunodeficiency. We chemically synthesized a Tat protein (86 residues) with its six glycines C alpha labelled with 13C. This synthetic protein has the full Tat activity. Heteronuclear nuclear magnetic resonance (NMR) spectra and NOE back-calculation made possible the sequential assignment of the 86 spin systems. Consequently, 915 NMR restraints were identified and 272 of them turned out to be long range ([i-j] > 4), providing structural information on the whole Tat protein. The poor spectral dispersion of Tat NMR spectra does not allow an accurate structure to be determined as for other proteins studied by 2D NMR. Nevertheless, we were able to determine the folding for Tat protein at a 1-mM protein concentration in a 100 mM, pH 4.5 phosphate buffer. The two main Tat functional regions, the basic region and the cysteine-rich region, are well exposed to solvent while a part of the N-terminal region and the C-terminal region constitute the core of Tat Bru. The basic region adopts an extended structure while the cysteine-rich region is made up of two loops. Resolution of this structure was determinant to develop a drug design approach against Tat. The chemical synthesis of the drugs allowed the specific binding and the inhibition of Tat to be verified.

HIV-1 Tat protein enhances Microtubule polymerization

BackgroundHIV infection and progression to AIDS is characterized by the depletion of T cells, which could be due, in part, to apoptosis mediated by the extra-cellular HIV-encoded Tat protein as a consequence of Tat binding to tubulin. Microtubules are tubulin polymers that are essential for cell structure and division. Molecules that target microtubules induce apoptosis and are potent anti-cancer drugs. We studied the effect on tubulin polymerization of three Tat variants: Tat HxB2 and Tat Eli from patients who are rapid progressors (RP) and Tat Oyi from highly exposed but persistently seronegative (HEPS) patients. We compared the effect on tubulin polymerization of these Tat variants and peptides corresponding to different parts of the Tat sequence, with paclitaxel, an anti-cancer drug that targets microtubules.ResultsWe show that Tat, and specifically, residues 38–72, directly enhance tubulin polymerization. We demonstrate that Tat could also directly trigger the mitochondrial pathway to induce T cell apoptosis, as shown in vitro by the release of cytochrome c from isolated mitochondria.ConclusionsThese results show that Tat directly acts on microtubule polymerization and provide insights into the mechanism of T cell apoptosis mediated by extra-cellular Tat.

Human immunodeficiency virus type 1 subtype C tat fails to induce intracellular calcium flux and induces reduced tumor necrosis factor production from monocytes

ABSTRACT Over 50% of all human immunodeficiency virus type 1 (HIV-1) infections worldwide are caused by subtype C strains, yet most research to date focuses on subtype B, the subtype most commonly found in North America and Europe. The HIV-1 trans-acting regulatory protein (Tat) is essential for regulating productive replication of HIV-1. Tat is secreted by HIV-infected cells and alters several functions of uninfected bystander cells. One such function is that, by acting at the cell membrane, subtype B Tat stimulates the production of tumor necrosis factor (TNF) and chemokine (C-C motif) ligand 2 (CCL2) from human monocytes and can act as a chemoattractant. In this study, we show that the mutation of a cysteine to a serine at residue 31 of Tat commonly found in subtype C variants significantly inhibits the abilities of the protein to bind to chemokine (C-C motif) receptor 2 (CCR2), induce intracellular calcium flux, stimulate TNF and CCL2 production, and inhibit its chemoattractant properties. We also show that TNF is important in mediating some effects of extracellular Tat. This report therefore demonstrates the important functional differences between subtype C and subtype B Tat and highlights the need for further investigation into the different strains of HIV-1.

The C-terminus of HIV-1 Tat modulates the extent of CD178 mediated apoptosis of T cells

HIV infection and the progression to AIDS are characterized by the depletion of CD4+ T cells through apoptosis of the uninfected bystander cells and the direct killing of HIV-infected cells. This is mediated in part by the human immunodeficiency virus, type 1 Tat protein, which is secreted by virally infected cells and taken up by uninfected cells and CD178 gene expression, which is critically involved in T cell apoptosis. The differing ability of HIV strains to induce death of infected and uninfected cells may play a role in the clinical and biological differences displayed by HIV strains. We chemically synthesized the 86-residue truncated short variant of Tat and its full-length form. We show that the trans-activation ability of Tat at the long terminal repeat does not correlate with T cell apoptosis but that the ability of Tat to up-regulate CD178 mRNA expression and induce apoptosis in T cells is critically dependent on the C terminus of Tat. Moreover, the greater 86-residue Tat-induced apoptosis is via the extrinsic pathway of CD95-CD178.

Antiretroviral therapy does not block the secretion of the human immunodeficiency virus tat protein.

Tat is a viral protein secreted from HIV infected cells and extra cellular Tat is suspected to prevent destruction of HIV infected cells from cells of the cellular immunity. The effect of anti retroviral therapy (ART) on Tat secretion has never been investigated. In this study, we tested for antibody reactivity against Tat variants representative of the main HIV subtypes in HIV positive patients receiving ART with undetectable viral loads ( < 40 copies/mL) over the course of one year with a blood sampling every three months. For each of theses five blood sampling, an average of 50 % of patients had Anti-Tat IgG, it turned out that 86% of patients could recognize Tat at least in one blood sampling during the course of the study. Amazingly, anti-Tat IgG appeared and/or disappeared in 66 % of patients. Only 20% had anti-Tat IgG remaining persistently while 14% were consistently without anti Tat IgG in the five blood sampling. No significant correlation was found between anti-Tat IgG and CD4+ T cell, CD8+ T cell and B cell counts revealing the incapacity of these anti Tat IgG to neutralize extra cellular Tat. Interestingly the absence and then the appearance of anti-Tat IgG in patients suggest the presence of HIV infected cells in the blood that may constitute a significant reservoir of HIV infected cells. As a conclusion antiretroviral therapy does not block the secretion of Tat and may explain why HIV infected cells can survive in spite of an effective ART treatment.