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.