HIV-1 Tat is present in the serum of people living with HIV-1 despite viral suppression.

OBJECTIVES: Despite successful human immunodeficiency virus (HIV) control with combination antiretroviral therapy (cART), individuals with HIV still face health risks, including cancers, cardiovascular and neurocognitive diseases. An HIV protein, Tat, is potentially involved in these HIV-related diseases. Previous studies demonstrated circulating Tat in the blood of untreated people with HIV. Here, we measured Tat levels in the serum of cART-treated people with HIV to examine the effect of cART on Tat production. METHODS: Serum samples from 63 HIV-positive and 20 HIV-seronegative individuals were analyzed using an ELISA assay that detected Tat concentrations above 2.5 ng/mL. RESULTS: Among HIV-positive individuals, the Tat level ranged from 0 to 14 ng/mL. 25.4% (16 out of 63) exceeded the 2.5 ng/mL cut-off, with a median HIV Tat level of 4.518 [3.329-8.120] ng/mL. No correlation was revealed between Tat levels and CD4+ T cell counts, serum HIV RNA, p24 antigen, or anti-Tat levels. CONCLUSIONS: Despite cART, circulating HIV Tat persists and may contribute to HIV-related diseases. This emphasizes the need for further research on the mechanisms of Tat action in non-infected cells where it can penetrate upon circulation in the blood.

A Tripartite Complex HIV-1 Tat-Cyclophilin A-Capsid Protein Enables Tat Encapsidation That Is Required for HIV-1 Infectivity.

HIV-1 Tat is a key viral protein that stimulates several steps of viral gene expression. Tat is especially required for the transcription of viral genes. Nevertheless, it is still not clear if and how Tat is incorporated into HIV-1 virions. Cyclophilin A (CypA) is a prolyl isomerase that binds to HIV-1 capsid protein (CA) and is thereby encapsidated at the level of 200 to 250 copies of CypA/virion. Here, we found that a Tat-CypA-CA tripartite complex assembles in HIV-1-infected cells and allows Tat encapsidation into HIV virions (1 Tat/1 CypA). Biochemical and biophysical studies showed that high-affinity interactions drive the assembly of the Tat-CypA-CA complex that could be purified by size exclusion chromatography. We prepared different types of viruses devoid of transcriptionally active Tat. They showed a 5- to 10 fold decrease in HIV infectivity, and conversely, encapsidating Tat into ΔTat viruses greatly enhanced infectivity. The absence of encapsidated Tat decreased the efficiency of reverse transcription by ~50% and transcription by more than 90%. We thus identified a Tat-CypA-CA complex that enables Tat encapsidation and showed that encapsidated Tat is required to initiate robust viral transcription and thus viral production at the beginning of cell infection, before neosynthesized Tat becomes available. IMPORTANCE The viral transactivating protein Tat has been shown to stimulate several steps of HIV gene expression. It was found to facilitate reverse transcription. Moreover, Tat is strictly required for the transcription of viral genes. Although the presence of Tat within HIV virions would undoubtedly favor these steps and therefore enable the incoming virus to boost initial viral production, whether and how Tat is present within virions has been a matter a debate. We here described and characterized a tripartite complex between Tat, HIV capsid protein, and the cellular chaperone cyclophilin A that enables efficient and specific Tat encapsidation within HIV virions. We further showed that Tat encapsidation is required for the virus to efficiently initiate infection and viral production. This effect is mainly due to the transcriptional activity of Tat.

How palmitoylation affects trafficking and signaling of membrane receptors.

S-acylation (or palmitoylation) is a reversible post-translational modification (PTM) that modulates protein activity, signalization and trafficking. Palmitoylation was found to significantly impact the activity of various membrane receptors involved in either pathogen entry, such as CCR5 (for HIV) and anthrax toxin receptors, cell proliferation (epidermal growth factor receptor), cardiac function (ß-Adrenergic receptor), or synaptic function (AMPA receptor). Palmitoylation of these membrane receptors indeed affects not only their internalization, localization, and activation, but also other PTMs such as phosphorylation. In this review, we discuss recent results showing how palmitoylation differently affects the biology of these membrane receptors.

Cyclophilin A enables specific HIV-1 Tat palmitoylation and accumulation in uninfected cells.

Most HIV-1 Tat is unconventionally secreted by infected cells following Tat interaction with phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) at the plasma membrane. Extracellular Tat is endocytosed by uninfected cells before escaping from endosomes to reach the cytosol and bind PI(4,5)P2. It is not clear whether and how incoming Tat concentrates in uninfected cells. Here we show that, in uninfected cells, the S-acyl transferase DHHC-20 together with the prolylisomerases cyclophilin A (CypA) and FKBP12 palmitoylate Tat on Cys31 thereby increasing Tat affinity for PI(4,5)P2. In infected cells, CypA is bound by HIV-1 Gag, resulting in its encapsidation and CypA depletion from cells. Because of the lack of this essential cofactor, Tat is not palmitoylated in infected cells but strongly secreted. Hence, Tat palmitoylation specifically takes place in uninfected cells. Moreover, palmitoylation is required for Tat to accumulate at the plasma membrane and affect PI(4,5)P2-dependent membrane traffic such as phagocytosis and neurosecretion.

Unconventional secretion of viral proteins.

Although largely less numerous and characterized than bacterial secreted effectors, several viral virulence factors are secreted by virus infected cells. However, their mode of secretion only starts to be studied at the molecular level. Several of these viral effectors are secreted using an unconventional secretion pathway, i.e. despite the lack of signal sequence. We here review recent results illustrating the diversity of these pathways. In the case of HIV-1 proteins Tat and matrix (p17) proteins, secretion directly takes place at the plasma membrane level following binding to PI(4,5)P2. The secretion of HTLV-I Tax was found to partly rely on exocytic pathway intermediates. The secretion pathways of VP22 of Herpes simplex virus type I and VP40 of the Ebola virus are less well characterized but VP40 can be recruited to the plasma membrane by PI(4,5)P2 that thus appears as a key partner enabling the unconventional secretion of many viral proteins. Several studies indicated that circulating retroviral transactivating proteins Tat and Tax are involved in the development of AIDS and HTLV-I associated myelopathy/tropical spastic paraparesis, respectively.

Phosphatidylinositol (4,5)-bisphosphate-mediated pathophysiological effect of HIV-1 Tat protein.

Human immunodeficiency virus (HIV)-infected cells actively release the transcriptional activator (Tat) viral protein that is required for efficient HIV gene transcription. Extracellular Tat is able to enter uninfected cells. We recently reported that internalized Tat escapes endosomes to reach the cytosol and is then recruited to the plasma membrane by phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). As a consequence, Tat strongly impairs different critical cellular functions in several cell types. Here we will review recent evidences showing that Tat, by affecting the interaction of key cellular effectors with PtdIns(4,5)P2, blocks exocytosis from neuroendocrine cells, perturbs the synaptic vesicle exo-endocytosis cycle, prevents efficient phagocytosis by macrophages, and alters potassium channel activity in cardiac cells. Potential mechanistic aspects of Tat effects on these cellular processes will be discussed.

HIV-Tat induces a decrease in IKr and IKsvia reduction in phosphatidylinositol-(4,5)-bisphosphate availability.

Patients with HIV present with a higher prevalence of QT prolongation, of which molecular bases are still not clear. Among HIV proteins, Tat serves as a transactivator that stimulates viral genes expression and is required for efficient HIV replication. Tat is actively secreted into the blood by infected T-cells and affects organs such as the heart. Tat has been shown to alter cardiac repolarization in animal models but how this is mediated and whether this is also the case in human cells is unknown. In the present study, we show that Tat transfection in heterologous expression systems led to a decrease in hERG (underlying cardiac IKr) and human KCNE1-KCNQ1 (underlying cardiac IKs) currents and to an acceleration of their deactivation. This is consistent with a decrease in available phosphatidylinositol-(4,5)-bisphosphate (PIP2). A mutant Tat, unable to bind PIP2, did not reproduce the observed effects. In addition, WT-Tat had no effect on a mutant KCNQ1 which is PIP2-insensitive, further confirming the hypothesis. Twenty-four-hour incubation of human induced pluripotent stem cells-derived cardiomyocytes with Wild-type Tat reduced IKr and accelerated its deactivation. Concordantly, this Tat incubation led to a prolongation of the action potential (AP) duration. Events of AP alternans were also recorded in the presence of Tat, and were exacerbated at a low pacing cycle length. Altogether, these data obtained on human K+ channels both in heterologous expression systems and in human cardiomyocytes suggest that Tat sequesters PIP2, leading to a reduction of IKr and IKs, and provide a molecular mechanism for QT prolongation in HIV-infected patients.

Bee venom phospholipase A2 as a membrane-binding vector for cell surface display or internalization of soluble proteins.

We showed that bee venom phospholipase A2 can be used as a membrane-binding vector to anchor to the surface of cells a soluble protein fused to its C-terminus. ZZ, a two-domain derivative of staphylococcal protein A capable of binding constant regions of antibodies was fused to the C-terminus of the phospholipase or to a mutant devoid of enzymatic activity. The fusion proteins bound to the surface of cells and could themselves bind IgGs. Their fate depended on the cell type to which they bound. On the A431 carcinoma cell line the proteins remained exposed on the cell surface. In contrast, on human dendritic cells the proteins were internalized into early endosomes.

Detecting HIV-1 Tat in Cell Culture Supernatants by ELISA or Western Blot.

HIV-1 Tat is efficiently secreted by HIV-1-infected or Tat-transfected cells. Accordingly, Tat concentrations in the nanomolar range have been measured in the sera of HIV-1-infected patients, and this protein acts as a viral toxin on bystander cells. Nevertheless, assaying Tat concentration in media or sera is not that straightforward because extracellular Tat is unstable and particularly sensitive to oxidation. Moreover, most anti-Tat antibodies display limited affinity. Here, we describe methods to quantify extracellular Tat using a sandwich ELISA or Western blotting when Tat is secreted by suspension or adherent cells, respectively. In both cases it is important to capture exported Tat using antibodies before any Tat oxidation occurs; otherwise it will become denatured and unreactive toward antibodies.

Autophagy in Mycobacterium tuberculosis and HIV infections.

Human Immunodeficiency Virus (HIV) and Mycobacterium tuberculosis (M.tb) are among the most lethal human pathogens worldwide, each being responsible for around 1.5 million deaths annually. Moreover, synergy between acquired immune deficiency syndrome (AIDS) and tuberculosis (TB) has turned HIV/M.tb co-infection into a major public health threat in developing countries. In the past decade, autophagy, a lysosomal catabolic process, has emerged as a major host immune defense mechanism against infectious agents like M.tb and HIV. Nevertheless, in some instances, autophagy machinery appears to be instrumental for HIV infection. Finally, there is mounting evidence that both pathogens deploy various countermeasures to thwart autophagy. This mini-review proposes an overview of the roles and regulations of autophagy in HIV and M.tb infections with an emphasis on microbial factors. We also discuss the role of autophagy manipulation in the context of HIV/M.tb co-infection. In future, a comprehensive understanding of autophagy interaction with these pathogens will be critical for development of autophagy-based prophylactic and therapeutic interventions for AIDS and TB.

HIV-1 Tat inhibits phagocytosis by preventing the recruitment of Cdc42 to the phagocytic cup.

Most macrophages remain uninfected in HIV-1-infected patients. Nevertheless, the phagocytic capacity of phagocytes from these patients is impaired, favouring the multiplication of opportunistic pathogens. The basis for this phagocytic defect is not known. HIV-1 Tat protein is efficiently secreted by infected cells. Secreted Tat can enter uninfected cells and reach their cytosol. Here we found that extracellular Tat, at the subnanomolar concentration present in the sera of HIV-1-infected patients, inhibits the phagocytosis of Mycobacterium avium or opsonized Toxoplasma gondii by human primary macrophages. This inhibition results from a defect in mannose- and Fcγ-receptor-mediated phagocytosis, respectively. Inhibition relies on the interaction of Tat with phosphatidylinositol (4,5)bisphosphate that interferes with the recruitment of Cdc42 to the phagocytic cup, thereby preventing Cdc42 activation and pseudopod elongation. Tat also inhibits FcγR-mediated phagocytosis in neutrophils and monocytes. This study provides a molecular basis for the phagocytic defects observed in uninfected phagocytes following HIV-1 infection.

Autophagy restricts HIV-1 infection by selectively degrading Tat in CD4+ T lymphocytes.

UNLABELLED: Autophagy is a ubiquitous mechanism involved in the lysosomal-mediated degradation of cellular components when they are engulfed in vacuoles called autophagosomes. Autophagy is also recognized as an important regulator of the innate and adaptive immune responses against numerous pathogens, which have, therefore, developed strategies to block or use the autophagy machinery to their own benefit. Upon human immunodeficiency virus type 1 (HIV-1) infection, viral envelope (Env) glycoproteins induce autophagy-dependent apoptosis of uninfected bystander CD4(+) T lymphocytes, a mechanism likely contributing to the loss of CD4(+) T cells. In contrast, in productively infected CD4(+) T cells, HIV-1 is able to block Env-induced autophagy in order to avoid its antiviral effect. To date, nothing is known about how autophagy restricts HIV-1 infection in CD4(+) T lymphocytes. Here, we report that autophagy selectively degrades the HIV-1 transactivator Tat, a protein essential for viral transcription and virion production. We demonstrated that this selective autophagy-mediated degradation of Tat relies on its ubiquitin-independent interaction with the p62/SQSTM1 adaptor. Taken together, our results provide evidence that the anti-HIV effect of autophagy is specifically due to the degradation of the viral transactivator Tat but that this process is rapidly counteracted by the virus to favor its replication and spread. IMPORTANCE: Autophagy is recognized as one of the most ancient and conserved mechanisms of cellular defense against invading pathogens. Cross talk between HIV-1 and autophagy has been demonstrated depending on the virally challenged cell type, and HIV-1 has evolved strategies to block this process to replicate efficiently. However, the mechanisms by which autophagy restricts HIV-1 infection remain to be elucidated. Here, we report that the HIV-1 transactivator Tat, a protein essential for viral replication, is specifically degraded by autophagy in CD4(+) T lymphocytes. Both Tat present in infected cells and incoming Tat secreted from infected cells are targeted for autophagy degradation through a ubiquitin-independent interaction with the autophagy receptor p62/SQSTM1. This study is the first to demonstrate that selective autophagy can be an antiviral process by degrading a viral transactivator. In addition, the results could help in the design of new therapies against HIV-1 by specifically targeting this mechanism.

Preliminary characterisation of nanotubes connecting T-cells and their use by HIV-1.

BACKGROUND INFORMATION: Cells, especially those of the immune system, can form long and thin connections termed tunnelling nanotubes (TNTs). These structures can reach >100 µm in length and, in T-cells, contain actin but no tubulin and are not open ended. T-cell TNTs were found to form following cell contact and to enable the transfer of HIV-1 from an infected- to a connected-T-cell. TNTs are poorly characterised at molecular level. RESULTS: We found Rab11 and tetraspanins, especially CD81, all along T-cells TNTs, whereas Rab4 and Rab35 were absent from these structures. Regarding actin cytoskeleton regulators, Exo70, N-WASP and especially ezrin accumulated at the level of the TNT tip that contacts the connected cell. Phosphoinositides such as PI(4,5)P2 were also concentrated at this level together with HIV-1 Gag. Gag spots on cells and TNTs were essentially immobile, and likely correspond to area of Gag multimerisation for budding to form virus-like particles. Mobility of PHPLCδ , a specific probe for PI(4,5)P2 , was reduced > threefold at the level of TNT basis or tip compared with the cell body. CONCLUSION: Our study identified the TNT tip as an active zone of actin cytoskeleton reorganisation with the presence of ezrin, Exo70, N-WASP and PI(4,5)P2 . The latter is also known to enable HIV-1 Gag recruitment for viral budding, and the presence of Gag at this level, contacting the connected cell, indicates that the TNT tip is also a favourite place for HIV-1 assembly and budding.

HIV-1 Tat protein perturbs diacylglycerol production at the plasma membrane of neurosecretory cells during exocytosis.

Human immunodeficiency virus (HIV)-infected cells actively release the transcriptional activator (Tat) viral protein that is required for efficient HIV gene transcription. We recently reported that extracellular Tat is able to enter uninfected neurosecretory cells. Internalized Tat escapes endosomes to reach the cytosol and is then recruited to the plasma membrane by phosphatidylinositol 4,5-bisphophate (PtdIns(4,5)P 2). Tat strongly impairs exocytosis from chromaffin and PC12 cells and perturbs synaptic vesicle exo-endocytosis cycle through its ability to interact with PtdIns(4,5)P 2. Among PtdIns(4,5)P 2-dependent processes required for neurosecretion, we found that Tat impairs annexin A2 recruitment involved in the organization of exocytotic sites at the plasma membrane. Moreover Tat perturbs the actin cytoskeleton reorganization necessary for the movement of secretory vesicles toward their plasma membrane fusion sites during the exocytotic process.    Here, we investigated whether extracellular Tat affects PtdIns(4,5)P 2 metabolism in PC12 cells. Using a diacylglycerol (DAG) sensor, we found that ATP stimulation of exocytosis triggers the production of DAG at the plasma membrane as seen by the relocation of the DAG probe from the cytosol to the plasma membrane. Exposure to Tat strongly delayed the recruitment of the DAG sensor, suggesting a reduced level of DAG production at the early phase of ATP stimulation. These observations indicate that Tat reduces the hydrolysis rate of PtdIns(4,5)P 2 by phospholipase C during exocytosis. Thus, the neuronal disorders often associated with HIV-1 infection may be linked to the capacity of Tat to interact with PtdIns(4,5)P 2, and alter both its metabolism and functions in neurosecretion.

HIV-1 Tat protein inhibits neurosecretion by binding to phosphatidylinositol 4,5-bisphosphate.

HIV-1 transcriptional activator (Tat) enables viral transcription and is also actively released by infected cells. Extracellular Tat can enter uninfected cells and affect some cellular functions. Here, we examine the effects of Tat protein on the secretory activity of neuroendocrine cells. When added to the culture medium of chromaffin and PC12 cells, Tat was actively internalized and strongly impaired exocytosis as measured by carbon fiber amperometry and growth hormone release assay. Expression of Tat mutants that do not bind to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] did not affect secretion, and overexpression of phosphatidylinositol 4-phosphate 5-kinase (PIP5K), the major PtdIns(4,5)P2 synthesizing enzyme, significantly rescued the Tat-induced inhibition of neurosecretion. This suggests that the inhibition of exocytosis may be the consequence of PtdIns(4,5)P2 sequestration. Accordingly, expression of Tat in PC12 cells interfered with the secretagogue-dependent recruitment of annexin A2 to the plasma membrane, a PtdIns(4,5)P2-binding protein that promotes the formation of lipid microdomains that are required for exocytosis. In addition Tat significantly prevented the reorganization of the actin cytoskeleton necessary for the movement of secretory vesicles towards plasma membrane fusion sites. Thus, the capacity of extracellular Tat to enter neuroendocrine cells and sequester plasma membrane PtdIns(4,5)P2 perturbs several PtdIns(4,5)P2-dependent players of the exocytotic machinery, thereby affecting neurosecretion. We propose that Tat-induced inhibition of exocytosis is involved in the neuronal disorders associated with HIV-1 infection.

HIV-1 antisense transcription is preferentially activated in primary monocyte-derived cells.

In this study, an antisense luciferase-expressing human immunodeficiency virus type 1 (HIV-1) molecular clone was used to infect primary cells. We found that antisense transcription activity from the 3' long terminal repeat (LTR) was significantly more abundant in monocyte-derived cells than in activated T lymphocytes. Moreover, by analyzing antisense transcription in infected monocyte-derived dendritic cells (MDDCs), we observed that the majority of HIV-1-infected MDDCs with significant antisense transcription activity did not produce Gag. We also confirmed that the negative-strand-encoded antisense protein (ASP) was expressed in monocyte-derived cells.