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.

Increasing stability and toxicity of Pseudomonas exotoxin by attaching an antiproteasic Peptide.

Trypsin-like activities are present within the endocytic pathway and allow cells to inactivate a fraction of incoming toxins, such as Pseudomonas exotoxin (PE), that require endocytic uptake before reaching the cytosol to inactivate protein synthesis. PE is a favorite toxin for building immunotoxins. The latter are promising molecules to fight cancer or transplant rejection, and producing more active toxins is a key challenge. More broadly, increasing protein stability is a potentially useful approach to improve the efficiency of therapeutic proteins. We report here that fusing an antiproteasic peptide (bovine pancreatic trypsin inhibitor, BPTI) to PE increases its toxicity to human cancer cell lines by 20-40-fold. Confocal microscopic examination of toxin endocytosis, digestion, and immunoprecipitation experiments showed that the fused antiproteasic peptide specifically protects PE from trypsin-like activities. Hence, the attached BPTI acts as a bodyguard for the toxin within the endocytic pathway. Moreover, it increased the PE elimination half-time in mice by 70%, indicating that the fused BPTI stabilizes the toxin in vivo. This BPTI-fusion approach may be useful for protecting other circulating or internalized proteins of therapeutic interest from premature degradation.

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.

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.

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.

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.