Type I Phosphatidylinositol-4-Phosphate 5-Kinases α and γ Play a Key Role in Targeting HIV-1 Pr55^Gag to the Plasma...

HIV-1 assembly occurs principally at the plasma membrane (PM) of infected cells. Gag polyprotein precursors (Pr55^Gag) are targeted to the PM, and their binding is mediated by the interaction of myristoylated matrix domain and a PM-specific phosphoinositide, the phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P₂]. The major synthesis pathway of PI(4,5)P₂ involves the activity of phosphatidylinositol-4-phosphate 5-kinase family type 1 composed of three isoforms (PIP5K1α, PIP5K1β, and PIP5K1γ). To examine whether the activity of a specific PIP5K1 isoform determines proper Pr55^Gag localization at the PM, we compared the cellular behavior of Pr55^Gag in the context of PIP5K1 inhibition using siRNAs that individually targeted each of the three isoforms in TZM-bl HeLa cells. We found that downregulation of PIP5K1α and PIP5K1γ strongly impaired the targeting of Pr55^Gag to the PM with a rerouting of the polyprotein within intracellular compartments. The efficiency of Pr55^Gag release was thus impaired through the silencing of these two isoforms, while PIP5K1β is dispensable for Pr55^Gag targeting to the PM. The PM mistargeting due to the silencing of PIP5K1α leads to Pr55^Gag hydrolysis through lysosome and proteasome pathways, while the silencing of PIP5K1γ leads to Pr55^Gag accumulation in late endosomes. Our findings demonstrated that, within the PIP5K1 family, only the PI(4,5)P₂ pools produced by PIP5K1α and PIP5K1γ are involved in the Pr55^Gag PM targeting process.IMPORTANCE PM specificity of Pr55^Gag membrane binding is mediated through the interaction of PI(4,5)P₂ with the matrix (MA) basic residues. It was shown that overexpression of a PI(4,5)P₂-depleting enzyme strongly impaired PM localization of Pr55^Gag However, cellular factors that control PI(4,5)P₂ production required for Pr55^Gag-PM targeting have not yet been characterized. In this study, by individually inhibiting PIP5K1 isoforms, we elucidated a correlation between PI(4,5)P₂ metabolism pathways mediated by PIP5K1 isoforms and the targeting of Pr55^Gag to the PM of TZM-bl HeLa cells. Confocal microscopy analyses of cells depleted from PIP5K1α and PIP5K1γ show a rerouting of Pr55^Gag to various intracellular compartments. Notably, Pr55^Gag is degraded by the proteasome and/or by the lysosomes in PIP5K1α-depleted cells, while Pr55^Gag is targeted to endosomal vesicles in PIP5K1γ-depleted cells. Thus, our results highlight, for the first time, the roles of PIP5K1α and PIP5K1γ as determinants of Pr55^Gag targeting to the PM.