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.