Structural Insights into the Mechanism of HIV-1 Tat Secretion from the Plasma Membrane

Human immunodeficiency virus type 1 (HIV-1) trans-activator of transcription (Tat) is a small, intrinsically disordered basic protein that plays diverse roles in the HIV-1 replication cycle, including promotion of efficient viral RNA transcription. Tat is released by infected cells and subsequently absorbed by healthy cells, thereby contributing to HIV-1 pathogenesis including HIV-associated neurocognitive disorder. It has been shown that, in HIV-1-infected primary CD4 T-cells, Tat accumulates at the plasma membrane (PM) for secretion, a mechanism mediated by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). However, the structural basis for Tat interaction with the PM and thereby secretion is lacking. Herein, we employed NMR and biophysical methods to characterize Tat86 (86 amino acids) interactions with PI(4,5)P2 and lipid nanodiscs (NDs). Our data revealed that Arg49, Lys50 and Lys51 (RKK motif) constitute the PI(4,5)P2 binding site, that Tat86 interaction with lipid NDs is dependent on PI(4,5)P2 and phosphatidylserine (PS), and that the arginine-rich motif (RRQRRR) preferentially interacts with PS. Furthermore, we show that Trp11, previously implicated in Tat secretion, penetrates deeply in the membrane; substitution of Trp11 severely reduced Tat86 interaction with membranes. Deletion of the entire highly basic region and Trp11 completely abolished Tat86 binding to lipid NDs. Our data support a mechanism by which HIV-1 Tat secretion from the PM is mediated by a tripartite signal consisting of binding of the RKK motif to PI(4,5)P2, arginine-rich motif to PS, and penetration of Trp11 in the membrane. Altogether, these findings provide new insights into the molecular requirements for Tat binding to membranes during secretion. (c) 2022 Elsevier Ltd. All rights reserved.