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Type I phosphatidylinositol-4-phosphate 5-kinase α and γ play a key role in targeting HIV-1 Pr55 Gag to the plasma membrane

Abstract : HIV-1 assembly occurs principally at the plasma membrane (PM) of infected cells. Gag polyprotein precursors (Pr55Gag) 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, β and γ). To examine whether the activity of a specific PIP5K1 isoform determines proper Pr55Gag localization at the PM, we compared cellular behavior of Pr55Gag 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 Pr55Gag to the PM with a rerouting of the polyprotein within intracellular compartments. The efficiency of Pr55Gag release was thus impaired through the silencing of these two isoforms while PIP5K1β is dispensable for Pr55Gag targeting to PM. The PM-mistargeting due to the silencing of PIP5K1α leads to Pr55Gag hydrolysis through lysosome and proteasome pathways while the silencing of PIP5K1γ leads to Pr55Gag accumulation in late endosomes. Our findings demonstrated that, within the PIP5K1s family, only the PI(4,5)P2 pools produced by PIP5K1α and γ are involved in Pr55Gag PM targeting process.IMPORTANCE PM specificity of Pr55Gag membrane binding is mediated through the interaction of PI(4,5)P2 with the MA basic residues. It was shown that overexpression of a PI(4,5)P2-depleting enzyme strongly impaired plasma membrane (PM) localization of Pr55Gag However, cellular factors that control PI(4,5)P2-production required for Pr55Gag-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 Pr55Gag to the PM of TZM-bl HeLa cells. Confocal microscopy analyses of cells depleted from PIP5K1α and γ show a rerouting of Pr55Gag to various intracellular compartments. Notably, Pr55Gag is degraded by the proteasome and/or by the lysosomes in PIP5K1α-depleted cells while Pr55Gag is targeted to endosomal vesicles in PIP5K1γ-depleted cells. Thus, our results highlight for the first time the roles of PIP5K1α and γ as determinants of Pr55Gag targeting to the PM.
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Baptiste Gonzales, Hugues de Rocquigny, Anne Beziau, Stéphanie Durand, Julien Burlaud-Gaillard, et al.. Type I phosphatidylinositol-4-phosphate 5-kinase α and γ play a key role in targeting HIV-1 Pr55 Gag to the plasma membrane. Journal of Virology, American Society for Microbiology, In press, pp.JVI.00189-20. ⟨10.1128/JVI.00189-20⟩. ⟨hal-02872300⟩

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