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

• Published in: Journal of virology 2020-07, Vol.94 (14)
• Main Authors: Gonzales, Baptiste, de Rocquigny, Hugues, Beziau, Anne, Durand, Stephanie, Burlaud-Gaillard, Julien, Lefebvre, Antoine, Krull, Sandra, Emond, Patrick, Brand, Denys, Piver, Eric
• Format: Article
• Language: English
• Subjects: Cell Membrane - genetics; Cell Membrane - metabolism; Cell Membrane - virology; Down-Regulation; Endosomes - genetics; Endosomes - metabolism; Endosomes - virology; Gene Expression Regulation, Enzymologic; HeLa Cells; HIV-1 - genetics; HIV-1 - metabolism; Humans; Lysosomes - genetics; Lysosomes - metabolism; Lysosomes - virology; Phosphatidylinositol 4,5-Diphosphate - genetics; Phosphatidylinositol 4,5-Diphosphate - metabolism; Phosphotransferases (Alcohol Group Acceptor) - biosynthesis; Phosphotransferases (Alcohol Group Acceptor) - genetics; Proteasome Endopeptidase Complex - genetics; Proteasome Endopeptidase Complex - metabolism; Protein Precursors - genetics; Protein Precursors - metabolism; Proteolysis
• ISSN: 0022-538X; 1098-5514
• DOI: 10.1128/JVI.00189-20

HIV-1 assembly occurs principally at the plasma membrane (PM) of infected cells. Gag polyprotein precursors (Pr55 ) 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)P ]. The major synthesis pathway of PI(4,5)P 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 localization at the PM, we compared the cellular behavior of Pr55 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 to the PM with a rerouting of the polyprotein within intracellular compartments. The efficiency of Pr55 release was thus impaired through the silencing of these two isoforms, while PIP5K1β is dispensable for Pr55 targeting to the PM. The PM mistargeting due to the silencing of PIP5K1α leads to Pr55 hydrolysis through lysosome and proteasome pathways, while the silencing of PIP5K1γ leads to Pr55 accumulation in late endosomes. Our findings demonstrated that, within the PIP5K1 family, only the PI(4,5)P pools produced by PIP5K1α and PIP5K1γ are involved in the Pr55 PM targeting process. PM specificity of Pr55 membrane binding is mediated through the interaction of PI(4,5)P with the matrix (MA) basic residues. It was shown that overexpression of a PI(4,5)P -depleting enzyme strongly impaired PM localization of Pr55 However, cellular factors that control PI(4,5)P production required for Pr55 -PM targeting have not yet been characterized. In this study, by individually inhibiting PIP5K1 isoforms, we elucidated a correlation between PI(4,5)P metabolism pathways mediated by PIP5K1 isoforms and the targeting of Pr55 to the PM of TZM-bl HeLa cells. Confocal microscopy analyses of cells depleted from PIP5K1α and PIP5K1γ show a rerouting of Pr55 to various intracellular compartments. Notably, Pr55 is degraded by the proteasome and/or by the lysosomes in PIP5K1α-depleted cells, while Pr55 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 targeting to the PM.