Mechanism for HIV-1 Tat Insertion into the Endosome Membrane

Abstract

The human immunodeficiency virus, type 1, transactivating protein Tat is a small protein that is strictly required for viral transcription and multiplication within infected cells. The infected cells actively secrete Tat using an unconventional secretion pathway. Extracellular Tat can affect different cell types and induce severe cell dysfunctions ranging from cell activation to cell death. To elicit most cell responses, Tat needs to reach the cell cytosol. To this end, Tat is endocytosed, and low endosomal pH will then trigger Tat translocation to the cytosol. Although this translocation step is critical for Tat cytosolic delivery, how Tat could interact with the endosome membrane is unknown, and the key residues involved in this interaction require identification. We found that, upon acidification below pH 6.0 (i.e. within the endosomal pH range), Tat inserts into model membranes such as monolayers or lipid vesicles. This insertion process relies on Tat single Trp, Trp-11, which is not needed for transactivation and could be replaced by another aromatic residue for membrane insertion. Nevertheless, Trp-11 is strictly required for translocation. Tat conformational changes induced by low pH involve a sensor made of its first acidic residue (Glu/Asp-2) and the end of its basic domain (residues 55-57). Mutation of one of these elements results in membrane insertion above pH 6.5. Tat basic domain is also required for efficient Tat endocytosis and membrane insertion. Together with the strict conservation of Tat Trp among different virus isolates, our results point to an important role for Tat-membrane interaction in the multiplication of human immunodeficiency virus type 1.

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

“…After this time, Tat co-localization with Lamp-1 strongly decreased, while that of Tat-W11Y with Lamp-1 remained stable. These results are consistent with the identification of late endosomes as the elements of the endocytic pathway from which Tat escapes to reach the cytosol 19 , and the inability of Tat-W11Y to escape from endosomes 18 . The kinetics of Tat intracellular transport in MDMs is consistent with those of phagocytosis inhibition (Fig.…”

“…Tat-W11Y mutant that cannot access the cytosol 18 did not affect phagocytosis, indicating that Tat cytosolic delivery is required to inhibit the FcgR-mediated phagocytosis. Similar dose-response and kinetics curves were obtained when we examined the effect of Tat on the phagocytosis of zymosan (Fig.…”

“…This interaction apparently disorganizes the phagocytosis machinery, probably by affecting ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms7211 the recruitment of one of its key components. It is noteworthy that the absence of effect of transfected Tat-W11Y, which transactivates LTR-driven genes as efficiently as WT Tat on transfection into cells 18 , shows that the transcriptional activity of Tat is not involved in its capacity to inhibit phagocytosis.…”

“…Circulating Tat is recognized by various cellular receptors that will enable it to enter uninfected cells by endocytosis before crossing the endosome membrane to reach the cytosol 17 . This transmembrane transport is triggered by endosomal low pH that induces Tat conformational modifications, allowing the exposure of Tat-single Trp (Trp11) that will initiate membrane insertion 18 . Tat translocation to the cytosol is catalysed by the molecular chaperone Hsp90 (ref.…”

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

“…In neurons, extracellular Tat interacts with the low-density lipoprotein receptor-related protein (LRP) and is then internalized into endosomal structures (Liu et al, 2000), where Tat concentration can reach 2 pg/ml (Deshmane et al, 2011). Upon delivery to endosomes, the amino acid tryptophan 11 initiates Tat insertion into the late endosome membrane (Yezid et al, 2009). Tat transmembrane transport is then catalyzed by the cytosolic chaperone Hsp90 that helps Tat to refold on the trans side of the endosomal membrane (Vendeville et al, 2004).…”