Phosphatidylinositol-(4,5)-Bisphosphate Acyl Chains Differentiate Membrane Binding of HIV-1 Gag from That of the Phospholipase Cδ1 Pleckstrin Homology Domain.
Phosphatidylinositol-(4,5)-Bisphosphate Acyl Chains Differentiate Membrane Binding of HIV-1 Gag from That of the Phospholipase Cδ1 Pleckstrin Homology Domain.
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磷脂酰肌醇-(4,5)-二磷酸酰基链区分 HIV-1 Gag 的膜结合与磷脂酶 Cδ1 Pleckstrin 同源域的膜结合。
DOI：
10.1128/jvi.00794-15
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发表时间：
2015
影响因子：
5.4
通讯作者：
Ono,Akira
中科院分区：
文献类型：
--
作者：
Olety,Balaji;Veatch,SarahL;Ono,Akira
HIV-1 Gag驱动病毒粒子组装，与质膜特异性的磷脂酰肌醇-(4，5)-二磷酸[PI(4，5)P2]相互作用。虽然细胞酸性磷脂结合蛋白/结构域，如磷脂酶Cδ1的PI(4，5)P2特异性Pleckstrin同源结构域(PHPLδ1)介导了头基特异的与相应磷脂的相互作用，但Gag-PI(4，5)P相互作用的确切性质仍未确定。在这项研究中，我们使用巨大的单层囊泡(GUV)来研究含有不饱和或饱和酰基链的PI(4，5)P对高效液相δ1和GAG膜结合的影响。不饱和二油酰基-PI(4，5)P2[DO-PI(4，5)P2]和饱和二棕榈酰基-PI(4，5)P2[DP-PI(4，5)P2]均成功地将高效液相δ1固定到单相瓜尔豆膜上。相反，DO-PI(4，5)P而不是DP-PI(4，5)P向GUV募集GAG，表明PI(4，5)P酰链有助于GAG与膜的稳定结合。含有PI(4，5)P2、胆固醇和二棕榈酰磷脂酰丝氨酸的GUV分离为两个共存相：一个是液体相，另一个似乎是磷脂酰丝氨酸富集凝胶相。在这些囊泡中，无论PI(4，5)磷脂酰基链是什么，液相中都聚集了高效液相δ1。同样，当PI(4，5)P含有DO-酰基链时，GAG也会结合到液相中。含有DP-PI(4，5)P2的GUV没有检测到GAG与液相结合。然而，出乎意料的是，DP-PI(4，5)P仍然促进GAG的募集，而不是PHPLδ1，以促进这些GUV的二棕榈酰磷脂酰丝氨酸富集凝胶相。总之，这些结果揭示了PI(4，5)P2acyl链在两种PI(4，5)P2结合蛋白GAG和PHPLδ1膜结合中的不同作用。值得注意的是，我们观察到非肉豆蔻化的Gag保持了对含有不饱和酰链的PI(4，5)P的偏好，这表明GAG对PI(4，5)P酰链饱和的敏感性是由基质-PI(4，5)P相互作用直接决定的，而不是由一种依赖于多种状态的机制间接决定的。许多细胞蛋白也通过以pHPLδ1为代表的PI(4，5)P2相互作用结构域被募集到质膜上。然而，这些宿主蛋白与病毒Gag蛋白在PI(4，5)P相互作用性质上的差异和/或相似性仍有待确定，特别是在膜结合的背景下。利用一个新的基于大分子单层囊泡的体系，我们发现含有不饱和酰链的PI(4，5)P类似地招募了高效液相δ1和GAG，而含有饱和酰链的PI(4，5)P既招募了高效液相δ1，又没有聚集或将这些蛋白分类到囊泡的不同阶段。据我们所知，这是第一次研究表明PI(4，5)P2acyl链对PI(4，5)P2结合蛋白的膜结合有不同的调节作用。由于Gag膜结合对后代病毒粒子的产生是必不可少的，PI(4，5)P2acyl链的性质可能成为抗HIV治疗策略的潜在靶点。
HIV-1 Gag, which drives virion assembly, interacts with a plasma membrane (PM)-specific phosphoinositide, phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2]. While cellular acidic phospholipid-binding proteins/domains, such as the PI(4,5)P2-specific pleckstrin homology domain of phospholipase Cδ1 (PHPLCδ1), mediate headgroup-specific interactions with corresponding phospholipids, the exact nature of the Gag-PI(4,5)P2interaction remains undetermined. In this study, we used giant unilamellar vesicles (GUVs) to examine how PI(4,5)P2with unsaturated or saturated acyl chains affect membrane binding of PHPLCδ1and Gag. Both unsaturated dioleoyl-PI(4,5)P2[DO-PI(4,5)P2] and saturated dipalmitoyl-PI(4,5)P2[DP-PI(4,5)P2] successfully recruited PHPLCδ1to membranes of single-phase GUVs. In contrast, DO-PI(4,5)P2but not DP-PI(4,5)P2recruited Gag to GUVs, indicating that PI(4,5)P2acyl chains contribute to stable membrane binding of Gag. GUVs containing PI(4,5)P2, cholesterol, and dipalmitoyl phosphatidylserine separated into two coexisting phases: one was a liquid phase, and the other appeared to be a phosphatidylserine-enriched gel phase. In these vesicles, the liquid phase recruited PHPLCδ1regardless of PI(4,5)P2acyl chains. Likewise, Gag bound to the liquid phase when PI(4,5)P2had DO-acyl chains. DP-PI(4,5)P2-containing GUVs showed no detectable Gag binding to the liquid phase. Unexpectedly, however, DP-PI(4,5)P2still promoted recruitment of Gag, but not PHPLCδ1, to the dipalmitoyl-phosphatidylserine-enriched gel phase of these GUVs. Altogether, these results revealed different roles for PI(4,5)P2acyl chains in membrane binding of two PI(4,5)P2-binding proteins, Gag and PHPLCδ1. Notably, we observed that nonmyristylated Gag retains the preference for PI(4,5)P2containing an unsaturated acyl chain over DP-PI(4,5)P2, suggesting that Gag sensitivity to PI(4,5)P2acyl chain saturation is determined directly by the matrix-PI(4,5)P2interaction, rather than indirectly by a myristate-dependent mechanism.IMPORTANCEBinding of HIV-1 Gag to the plasma membrane is promoted by its interaction with a plasma membrane-localized phospholipid, PI(4,5)P2. Many cellular proteins are also recruited to the plasma membrane via PI(4,5)P2-interacting domains represented by PHPLCδ1. However, differences and/or similarities between these host proteins and viral Gag protein in the nature of their PI(4,5)P2interactions, especially in the context of membrane binding, remain to be determined. Using a novel giant unilamellar vesicle-based system, we found that PI(4,5)P2with an unsaturated acyl chain recruited PHPLCδ1and Gag similarly, whereas PI(4,5)P2with saturated acyl chains either recruited PHPLCδ1but not Gag or sorted these proteins to different phases of vesicles. To our knowledge, this is the first study to show that PI(4,5)P2acyl chains differentially modulate membrane binding of PI(4,5)P2-binding proteins. Since Gag membrane binding is essential for progeny virion production, the PI(4,5)P2acyl chain property may serve as a potential target for anti-HIV therapeutic strategies.