INSERM U1259, University of Tours, Tours, France.
INSERM U1253, University of Tours, Tours, France.
J Virol. 2020 Jul 1;94(14). 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.
HIV-1 的组装主要发生在受感染细胞的质膜（PM）上。Gag 多蛋白前体（Pr55）被靶向到 PM，其结合由豆蔻酰化基质结构域与 PM 特异性的磷酸肌醇-(4,5)-二磷酸 [PI(4,5)P]之间的相互作用介导。PI(4,5)P 的主要合成途径涉及由三个同工型（PIP5K1α、PIP5K1β 和 PIP5K1γ）组成的磷酸肌醇-4-磷酸 5-激酶家族 1 的活性。为了研究特定的 PIP5K1 同工型的活性是否决定了 Pr55 在 PM 上的正确定位，我们使用靶向三种同工型的 siRNA 在 TZM-bl HeLa 细胞中比较了 Pr55 在 PIP5K1 抑制背景下的细胞行为。我们发现，下调 PIP5K1α 和 PIP5K1γ 强烈地破坏了 Pr55 向 PM 的靶向，导致多蛋白在细胞内隔室中重新路由。因此，通过这些两种同工型的沉默，Pr55 的释放效率受到损害，而 PIP5K1β 对于 Pr55 靶向 PM 是可有可无的。由于 PIP5K1α 的沉默导致 PM 靶向错误，导致 Pr55 通过溶酶体和蛋白酶体途径水解，而 PIP5K1γ 的沉默导致 Pr55 在晚期内体中积累。我们的研究结果表明，在 PIP5K1 家族中，只有 PIP5K1α 和 PIP5K1γ 产生的 PI(4,5)P 池参与了 Pr55 PM 靶向过程。Pr55 膜结合的 PM 特异性是通过 PI(4,5)P 与基质（MA）碱性残基的相互作用介导的。研究表明，过表达一种 PI(4,5)P 耗竭酶强烈地损害了 Pr55 在 PM 上的定位。然而，控制 Pr55-PM 靶向所需的用于 Pr55-PM 靶向的细胞因子尚未得到表征。在这项研究中，通过单独抑制 PIP5K1 同工型，我们阐明了由 PIP5K1 同工型介导的 PI(4,5)P 代谢途径与 Pr55 向 TZM-bl HeLa 细胞 PM 的靶向之间的相关性。用 PIP5K1α 和 PIP5K1γ 耗竭的细胞进行共焦显微镜分析显示，Pr55 重新路由到各种细胞内隔室。值得注意的是，在 PIP5K1α 耗竭的细胞中，Pr55 通过蛋白酶体和/或溶酶体降解，而在 PIP5K1γ 耗竭的细胞中，Pr55 被靶向到内体囊泡。因此，我们的结果首次强调了 PIP5K1α 和 PIP5K1γ 作为 Pr55 靶向 PM 的决定因素的作用。
