Ulva lactuca, A Source of Troubles and Potential Riches.

Ulva lactuca is a green macro alga involved in devastating green tides observed worldwide. These green tides or blooms are a consequence of human activities. Ulva blooms occur mainly in shallow waters and the decomposition of this alga can produce dangerous vapors. Ulva lactuca is a species usually resembling lettuce, but genetic analyses demonstrated that other green algae with tubular phenotypes were U. lactuca clades although previously described as different species or even genera. The capacity for U. lactuca to adopt different phenotypes can be due to environment parameters, such as the degree of water salinity or symbiosis with bacteria. No efficient ways have been discovered to control these green tides, but the Mediterranean seas appear to be protected from blooms, which disappear rapidly in springtime. Ulva contains commercially valuable components, such as bioactive compounds, food or biofuel. The biomass due to this alga collected on beaches every year is beginning to be valorized to produce valuable compounds. This review describes different processes and strategies developed to extract these different valuable components.

Didehydro-Cortistatin A Inhibits HIV-1 by Specifically Binding to the Unstructured Basic Region of Tat.

The intrinsically disordered HIV-1 Tat protein binds the viral RNA transactivation response structure (TAR), which recruits transcriptional cofactors, amplifying viral mRNA expression. Limited Tat transactivation correlates with HIV-1 latency. Unfortunately, Tat inhibitors are not clinically available. The small molecule didehydro-cortistatin A (dCA) inhibits Tat, locking HIV-1 in persistent latency, blocking viral rebound. We generated chemical derivatives of dCA that rationalized molecular docking of dCA to an active and specific Tat conformer. These revealed the importance of the cycloheptene ring and the isoquinoline nitrogen's positioning in the interaction with specific residues of Tat's basic domain. These features are distinct from the ones required for inhibition of cyclin-dependent kinase 8 (CDK8), the only other known ligand of dCA. Besides, we demonstrated that dCA activity on HIV-1 transcription is independent of CDK8. The binding of dCA to Tat with nanomolar affinity alters the local protein environment, rendering Tat more resistant to proteolytic digestion. dCA thus locks a transient conformer of Tat, specifically blocking functions dependent of its basic domain, namely the Tat-TAR interaction; while proteins with similar basic patches are unaffected by dCA. Our results improve our knowledge of the mode of action of dCA and support structure-based design strategies targeting Tat, to help advance development of dCA, as well as novel Tat inhibitors.IMPORTANCE Tat activates virus production, and limited Tat transactivation correlates with HIV-1 latency. The Tat inhibitor dCA locks HIV in persistent latency. This drug class enables block-and-lock functional cure approaches, aimed at reducing residual viremia during therapy and limiting viral rebound. dCA may also have additional therapeutic benefits since Tat is also neurotoxic. Unfortunately, Tat inhibitors are not clinically available. We generated chemical derivatives and rationalized binding to an active and specific Tat conformer. dCA features required for Tat inhibition are distinct from features needed for inhibition of cyclin-dependent kinase 8 (CDK8), the only other known target of dCA. Furthermore, knockdown of CDK8 did not impact dCA's activity on HIV-1 transcription. Binding of dCA to Tat's basic domain altered the local protein environment and rendered Tat more resistant to proteolytic digestion. dCA locks a transient conformer of Tat, blocking functions dependent on its basic domain, namely its ability to amplify viral transcription. Our results define dCA's mode of action, support structure-based-design strategies targeting Tat, and provide valuable information for drug development around the dCA pharmacophore.

A Low Molecular Weight Protein from the Sea Anemone Anemonia viridis with an Anti-Angiogenic Activity.

Sea anemones are a remarkable source of active principles due to a decentralized venom system. New blood vessel growth or angiogenesis is a very promising target against cancer, but the few available antiangiogenic compounds have limited efficacy. In this study, a protein fraction, purified from tentacles of Anemonia viridis, was able to limit endothelial cells proliferation and angiogenesis at low concentration (14 nM). Protein sequences were determined with Edman degradation and mass spectrometry in source decay and revealed homologies with Blood Depressing Substance (BDS) sea anemones. The presence of a two-turn alpha helix observed with circular dichroism and a trypsin activity inhibition suggested that the active principle could be a Kunitz-type inhibitor, which may interact with an integrin due to an Arginine Glycin Aspartate (RGD) motif. Molecular modeling showed that this RGD motif was well exposed to solvent. This active principle could improve antiangiogenic therapy from existing antiangiogenic compounds binding on the Vascular Endothelial Growth Factor (VEGF).

Intradermal injection of a Tat Oyi-based therapeutic HIV vaccine reduces of 1.5 log copies/mL the HIV RNA rebound median and no HIV DNA rebound following cART interruption in a phase I/II randomized controlled clinical trial.

BACKGROUND: A Tat Oyi vaccine preparation was administered with informed consent to 48 long-term HIV-1 infected volunteers whose viral loads had been suppressed by antiretroviral therapy (cART). These volunteers were randomized in double-blind method into four groups (n = 12) that were injected intradermally with 0, 11, 33, or 99 µg of synthetic Tat Oyi proteins in buffer without adjuvant at times designated by month 0 (M0), M1 and M2, respectively. The volunteers then underwent a structured treatment interruption between M5 and M7. RESULTS: The primary outcomes of this phase I/IIa clinical trial were the safety and lowering the extent of HIV RNA rebound after cART interruption. Only one undesirable event possibly due to vaccination was observed. The 33 µg dose was most effective at lowering the extent of HIV RNA and DNA rebound (Mann and Whitney test, p = 0.07 and p = 0.001). Immune responses against Tat were increased at M5 and this correlated with a low HIV RNA rebound at M6 (p = 0.01). CONCLUSION: This study suggests in vivo that extracellular Tat activates and protects HIV infected cells. The Tat Oyi vaccine in association with cART may provide an efficient means of controlling the HIV-infected cell reservoir.

HIV extracellular Tat: myth or reality?

The human immunodeficiency virus type 1 (HIV) eradication will require elimination of HIV infected cells. No antiretroviral treatments (ART) or vaccine approaches have been able to reduce significantly the level of HIV infected cells in peripheral blood. This inefficacy is generally explained by the presence of a major reservoir of latent HIV infected cells in the central nervous system (CNS) that would be a sanctuary where Cytotoxic T Lymphocytes (CTL) have no access and would refresh peripheral blood with activated HIV infected cells. In this review, the presence of a major reservoir in the CNS appears to be inconsistent with recent clinical studies measuring HIV DNA. The major reservoirs are gut tissue, rectal tissue and the peripheral blood where HIV infected cells survive in an environment containing CTL. Extracellular Tat might protect HIV infected cells from CTL due to its capacity to cross CTL membranes and trigger apoptosis. Evidences of Tat secretion from HIV infected cells are shown with the detection of Tat antibodies in different clinical studies. Presence of neutralizing Tat antibodies in cohorts of patients who were exposed to HIV but who are now seronegative is described. The conclusion of this review is that a vaccine eliciting neutralizing antibodies against Tat might significantly reduce the level of HIV infected cells, what ART or other vaccine approaches have been unable to achieve now. It could be a first step towards HIV eradication.

Multimodality vaccination against clade C SHIV: partial protection against mucosal challenges with a heterologous tier 2 virus.

We sought to test whether vaccine-induced immune responses could protect rhesus macaques (RMs) against upfront heterologous challenges with an R5 simian-human immunodeficiency virus, SHIV-2873Nip. This SHIV strain exhibits many properties of transmitted HIV-1, such as tier 2 phenotype (relatively difficult to neutralize), exclusive CCR5 tropism, and gradual disease progression in infected RMs. Since no human AIDS vaccine recipient is likely to encounter an HIV-1 strain that exactly matches the immunogens, we immunized the RMs with recombinant Env proteins heterologous to the challenge virus. For induction of immune responses against Gag, Tat, and Nef, we explored a strategy of immunization with overlapping synthetic peptides (OSP). The immune responses against Gag and Tat were finally boosted with recombinant proteins. The vaccinees and a group of ten control animals were given five low-dose intrarectal (i.r.) challenges with SHIV-2873Nip. All controls and seven out of eight vaccinees became systemically infected; there was no significant difference in viremia levels of vaccinees vs. controls. Prevention of viremia was observed in one vaccinee which showed strong boosting of virus-specific cellular immunity during virus exposures. The protected animal showed no challenge virus-specific neutralizing antibodies in the TZM-bl or A3R5 cell-based assays and had low-level ADCC activity after the virus exposures. Microarray data strongly supported a role for cellular immunity in the protected animal. Our study represents a case of protection against heterologous tier 2 SHIV-C by vaccine-induced, virus-specific cellular immune responses.

Identification of a highly conserved surface on Tat variants.

Extracellular Tat is suspected to protect HIV-1-infected cells from cellular immunity. Seropositive patients are unable to produce neutralizing antibodies against Tat, and Tat is still secreted under antiviral treatment. In mice, the Tat OYI vaccine candidate generates neutralizing antibodies such as the mAb 7G12. A peptide called MIMOOX was designed from fragments of Tat OYI identified as the possible binding site for mAb 7G12. MIMOOX was chemically synthesized, and its structure was stabilized with a disulfide bridge. Circular dichroism spectra showed that MIMOOX had mainly ß turns but no α helix as Tat OYI. MIMOOX was recognized by mAb 7G12 in ELISA only in reduced conditions. Moreover, a competitive recognition assay with mAb 7G12 between MIMOOX and Tat variants showed that MIMOOX mimics a highly conserved surface in Tat variants. Rat immunizations with MIMOOX induce antibodies recognizing Tat variants from the main HIV-1 subtypes and confirm the Tat OYI vaccine approach.

A monoclonal antibody directed against a conformational epitope of the HIV-1 trans-activator (Tat) protein neutralizes cross-clade.

The identification of a neutralizing mAb against extracellular HIV-1 transactivator of transcription (Tat) is important for the development of an efficient HIV-1 treatment. Tat plays an essential role in HIV-1 pathogenesis, not only for HIV-1 replication but also as an extracellular toxin able to disrupt the immune system. We showed previously that immunization of rabbits with Tat Oyi, a variant cloned from an African woman who did not develop AIDS following HIV-1 infection, raised antibodies able to recognize different Tat variants. We carried out mice immunization with Tat Oyi and selected a mAb named 7G12, which had the capacity to cross-recognize heterologous Tat variants by a common three-dimensional epitope. These results highlighted that Tat variants were able to acquire a structure, in contrast to a number of studies showing Tat as an unfolded protein. mAb 7G12 also had the capacity to neutralize the biological activities of these Tat variants by blocking the cellular uptake of extracellular Tat. This is the first study using Tat Oyi to produce a mAb able to neutralize effectively activities of extracellular Tats from different HIV-1 subtypes. This mAb has an important potential in therapeutic passive immunization and could help HIV-1 infected patients to restore their immunity.

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.

HIV-1 infected patients have antibodies recognizing folded Tat.

Tat is a regulatory viral protein known as transactivator of HIV-1 genes but Tat is also secreted in the blood from HIV-1 infected cells. Extra cellular Tat can cross cellular membranes to trigger apoptosis and might explain the incapacity of the cellular immunity to eliminate HIV-1 infected cells. There is a controversy regarding Tat structure with studies suggesting that Tat would be a naturally unfolded protein. Here, we show that synthetic Tat variants need to be folded to have a transactivation activity in a cellular assay but this folding is unstable regarding the buffers and/or pH used as solvent. We show also that the recognition of a Tat variant versus peptides, covering its sequence, was different. Using an indirect ELISA method with 40 sera from volunteer HIV-1 infected patients, we show that Tat was recognized by 19 human sera either exclusively (n=8) or with Tat peptides (n=11). Dot Blot showed that unfolded Tat was no longer detectable by sera of the first group (n=8) compared to folded Tat. As a conclusion, this study suggests that Tat could be a naturally folded protein in the blood of HIV infected patients.

Differential induction of rat neuronal excitotoxic cell death by human immunodeficiency virus type 1 clade B and C tat proteins.

In the absence of effective antiretroviral therapy, infection with clade B human immunodeficiency virus (HIV-1) infection commonly progresses to AIDS dementia. However, in India, where clade C infection is most prevalent, severe cognitive impairment due to HIV-1 is reported to be less prevalent. The Tat protein of HIV-1, which is released from HIV-1-infected macrophages, is thought to play a major role in the disruption of neuronal function as well as in the infiltration of macrophages associated with advanced neuropathogenesis. Clade B Tat is excitotoxic to hippocampal neurons by potentiating N-methyl-d-aspartate-induced currents of the zinc-sensitive NR1/NR2A N-methyl-d-aspartate receptor in a zinc-binding-dependent mechanism. This study characterizes the zinc-binding properties of clade C Tat protein. Using ultraviolet spectroscopy and the Ellman reaction, we show that clade C Tat protein binds just one zinc ion per monomer. We then investigated the ability of clade C Tat to block the inhibition of N-methyl-d-aspartate receptors from zinc antagonism through ion chelation. Although clade C Tat enhanced N-methyl-d-aspartate-mediated rat hippocampus neuronal toxicity in the presence of zinc, the increase was significantly less than that observed with clade B Tat. These findings suggest that the observed differences in neuropathogenesis found with HIV-1 clade C infection compared to clade B may, in part, be due to a decrease in Tat-mediated neurotoxicity.

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)P(2)). This interaction is driven by a specific motif of the Tat basic domain that recognizes a single PI(4,5)P(2) molecule and is stabilized by membrane insertion of Tat tryptophan side chain. This original recognition mechanism enables binding to membrane-embedded PI(4,5)P(2) only, but with an unusually high affinity that allows Tat to perturb the PI(4,5)P(2)-mediated recruitment of cellular proteins. Tat-PI(4,5)P(2) interaction is strictly required for Tat secretion, a process that is very efficient, as approximately 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.

HIV-1 clade B Tat, but not clade C Tat, increases X4 HIV-1 entry into resting but not activated CD4+ T cells.

CXCR4-using human immunodeficiency virus, type 1 (HIV-1) variants emerge late in the course of infection in >40% of individuals infected with clade B HIV-1 but are described less commonly with clade C isolates. Tat is secreted by HIV-1-infected cells where it acts on both uninfected bystander cells and infected cells. In this study, we show that clade B Tat, but not clade C Tat, increases CXCR4 surface expression on resting CD4+ T cells through a CCR2b-dependent mechanism that does not involve de novo protein synthesis. The expression of plectin, a cytolinker protein that plays an important role as a scaffolding platform for proteins involved in cellular signaling including CXCR4 signaling and trafficking, was found to be significantly increased following B Tat but not C Tat treatment. Knockdown of plectin using RNA interference showed that plectin is essential for the B Tat-induced translocation of CXCR4 to the surface of resting CD4+ T cells. The increased surface CXCR4 expression following B Tat treatment led to increased function of CXCR4 including increased chemoattraction toward CXCR4-using-gp120. Moreover, increased CXCR4 surface expression rendered resting CD4+ T cells more permissive to X4 but not R5 HIV-1 infection. However, neither B Tat nor C Tat was able to up-regulate surface expression of CXCR4 on activated CD4+ T cells, and both proteins inhibited the infection of activated CD4+ T cells with X4 but not R5 HIV-1. Thus, B Tat, but not C Tat, has the capacity to render resting, but not activated, CD4+ T cells more susceptible to X4 HIV-1 infection.

Two purified and characterized phospholipases A2 from Cerastes cerastes venom, that inhibit cancerous cell adhesion and migration.

Two non-toxic PLA2s were purified to homogeneity from Cerastes cerastes Tunisian snake venom. The purification process employed gel filtration on Sephadex G-75 followed by C18 reverse phase high-pressure liquid chromatography. These two acidic enzymes, namely CC-PLA2-1 and CC-PLA2-2, have a molecular weight of 13,737.52 and 13,705.63 Da, respectively. These two PLA2 are the first reported glycosylated phospholipases A2 purified from snake venom. The rates of glycosylation are 2.5% and 0.5% (w/w), respectively. Specific activities of 1800 U/mg and 2400 U/mg for CC-PLA2-1 and CC-PLA2-2, respectively, were measured at optimal conditions. CC-PLA2-1 and CC-PLA2-2 strongly inhibited coagulation. They also exhibited a marked dose-dependent inhibitory effect on platelet aggregation induced by ADP and arachidonic acid in platelet-rich plasma. Interestingly, CC-PLA2-1 and CC-PLA2-2 inhibited in a dose-dependent manner adhesion of IGR39 melanoma and HT1080 fibrosarcoma cells to fibrinogen and fibronectin. Furthermore, both CC-PLA2-1 and CC-PLA2-2 abolished HT1080 cell migration towards fibrinogen and fibronectin. This activity is reported for the first time for PLA2 enzymes.

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.

Homonuclear 1H NMR and circular dichroism study of the HIV-1 Tat Eli variant.

BACKGROUND: The HIV-1 Tat protein is a promising target to develop AIDS therapies, particularly vaccines, due to its extracellular role that protects HIV-1-infected cells from the immune system. Tat exists in two different lengths, 86 or 87 residues and 99 or 101 residues, with the long form being predominant in clinical isolates. We report here a structural study of the 99 residue Tat Eli variant using 2D liquid-state NMR, molecular modeling and circular dichroism. RESULTS: Tat Eli was obtained from solid-phase peptide synthesis and the purified protein was proven biologically active in a trans-activation assay. Circular dichroism spectra at different temperatures up to 70 degrees C showed that Tat Eli is not a random coil at 20 degrees C. Homonuclear 1H NMR spectra allowed us to identify 1639 NMR distance constraints out of which 264 were interresidual. Molecular modeling satisfying at least 1474 NMR constraints revealed the same folding for different model structures. The Tat Eli model has a core region composed of a part of the N-terminus including the highly conserved Trp 11. The extra residues in the Tat Eli C-terminus protrude from a groove between the basic region and the cysteine-rich region and are well exposed to the solvent. CONCLUSION: We show that active Tat variants share a similar folding pattern whatever their size, but mutations induce local structural changes.

Tat mutations in an African cohort that do not prevent transactivation but change its immunogenic properties.

Humoral responses against extra-cellular HIV-1 Tat may be beneficial as Tat has been implicated in the viral pathogenesis associated with HIV-1 disease progression. We determined the levels of anti-Tat IgG in sera of HIV-1 seropositive individuals from the Rural Clinical Cohort in Uganda using nine different Tat proteins representative of the major subtypes presently accounting for 97% of infections worldwide. We observed the presence of anti-Tat IgG able to react against the various subtypes tested, although none cross-reacted against all nine variants. We show that 46.25% of seropositive patients were able to recognise at least one Tat variant with 1:1000 sera dilution. We also show that the C terminus of Tat is the most variable region and an important epitope that might explain the limitation of cross-recognition of Tat antibodies regarding Tat variants. This study shows in seropositive patients that Tat can tolerate mutations without modification of its primary function but with changes in its immunogenic properties. These findings should be considered when designing Tat-based HIV-1 vaccines.

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

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.

Reservoir cells no longer detectable after a heterologous SHIV challenge with the synthetic HIV-1 Tat Oyi vaccine.

BACKGROUND: Extra-cellular roles of Tat might be the main cause of maintenance of HIV-1 infected CD4 T cells or reservoir cells. We developed a synthetic vaccine based on a Tat variant of 101 residues called Tat Oyi, which was identified in HIV infected patients in Africa who did not progress to AIDS. We compared, using rabbits, different adjuvants authorized for human use to test on ELISA the recognition of Tat variants from the five main HIV-1 subtypes. A formulation was tested on macaques followed by a SHIV challenge with a European strain. RESULTS: Tat Oyi with Montanide or Calcium Phosphate gave rabbit sera able to recognize all Tat variants. Five on seven Tat Oyi vaccinated macaques showed a better control of viremia compared to control macaques and an increase of CD8 T cells was observed only on Tat Oyi vaccinated macaques. Reservoir cells were not detectable at 56 days post-challenge in all Tat Oyi vaccinated macaques but not in the controls. CONCLUSION: The Tat Oyi vaccine should be efficient worldwide. No toxicity was observed on rabbits and macaques. We show in vivo that antibodies against Tat could restore the cellular immunity and make it possible the elimination of reservoir cells.