Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY 14853, USA.
Proc Natl Acad Sci U S A. 2012 Nov 13;109(46):18761-6. doi: 10.1073/pnas.1209408109. Epub 2012 Sep 24.
Membrane binding of the HIV-1 group-specific antigen (Gag) structural protein, a critical step in viral assembly at the plasma membrane, is mediated by the myristoylated, highly basic matrix (MA) domain, which interacts with negatively charged lipids in the inner leaflet. According to a popular model, virus particles bud from membrane rafts, microdomains enriched in cholesterol and high-melting phospholipids with higher order than found outside rafts. How Gag might recognize membrane rafts, if they exist in the inner leaflet, is unknown. Using a liposome flotation assay with proteins translated in vitro, we investigated whether Gag can sense the composition of the hydrophobic part of the bilayer, by fixing lipid head group composition and varying hydrophobic properties. In liposomes composed solely of phosphatidylserine and phosphatidylcholine, and with the same overall membrane negative charge, Gag strongly preferred lipids with both acyl chains unsaturated over those with only one chain unsaturated. Adding cholesterol increased Gag binding and led to closer packing of phospholipids. However, higher membrane order, as measured by electron spin resonance, was not correlated with increased Gag binding. Gag proteins from two other retroviruses gave similar results. These liposome binding preferences were qualitatively recapitulated by purified myristoylated HIV-1 MA. Phosphatidylinositol 4,5-bisphosphate and cholesterol enhanced binding in an additive manner. Taken together, these results show that Gag is sensitive both to the acyl chains of phosphatidylserine and to cholesterol concentration and other details of the membrane environment. These observations may help explain how retroviruses acquire a raft-like lipid composition.
HIV-1 群特异性抗原 (Gag) 结构蛋白与质膜的病毒装配有关，其膜结合由豆蔻酰化的高度碱性基质 (MA) 结构域介导，该结构域与质膜内层中的带负电荷的脂质相互作用。根据一个流行的模型，病毒颗粒从膜筏（富含胆固醇和高熔点磷脂的微区，其有序性高于筏外）中出芽。如果质膜内层存在膜筏，Gag 如何识别它们是未知的。我们使用体外翻译的蛋白质进行的脂质体浮选测定，通过固定脂质头部基团组成并改变疏水性来研究 Gag 是否可以感知双层疏水区的组成。在仅由磷脂酰丝氨酸和磷脂酰胆碱组成的脂质体中，并且具有相同的总膜负电荷，Gag 强烈优先选择具有两个不饱和酰基链的脂质，而不是仅具有一个不饱和酰基链的脂质。添加胆固醇增加了 Gag 的结合，并导致磷脂的紧密堆积。然而，更高的膜有序性（如通过电子自旋共振测量）与增加的 Gag 结合没有相关性。两种其他逆转录病毒的 Gag 蛋白给出了类似的结果。纯化的 HIV-1 MA 定性地再现了这种脂质体结合偏好。磷脂酰肌醇 4,5-二磷酸和胆固醇以累加的方式增强了结合。综上所述，这些结果表明 Gag 既对磷脂酰丝氨酸的酰基链敏感，也对胆固醇浓度和膜环境的其他细节敏感。这些观察结果可能有助于解释逆转录病毒如何获得类似筏的脂质组成。
