Membrane Binding of the Rous Sarcoma Virus Gag Protein Is Cooperative and Dependent on the Spacer Peptide Assembly Domain.
Membrane Binding of the Rous Sarcoma Virus Gag Protein Is Cooperative and Dependent on the Spacer Peptide Assembly Domain.
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劳斯肉瘤病毒 Gag 蛋白的膜结合是协同的并且依赖于间隔肽组装结构域。
DOI：
10.1128/jvi.02733-15
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发表时间：
2015
影响因子：
5.4
通讯作者：
Vogt,VolkerM
中科院分区：
文献类型：
--
作者：
Dick,RobertA;Barros,Marilia;Jin,Danni;Lösche,Mathias;Vogt,VolkerM
逆转录病毒 Gag 蛋白的膜结合和组装成晶格的原理已被了解。然而，人们对这些过程之间的关系知之甚少。使用纯化的劳斯肉瘤病毒 Gag 和 Gag 截短体，我们研究了 Gag-Gag 相互作用和 Gag-膜相互作用的相互关系。通过脂质体结合和支持双层上的表面等离振子共振，Gag 与膜的结合比基质 (MA)（分离的膜结合域）更紧密。原则上，这种差异可以通过导致膜结合协同性的蛋白质-蛋白质相互作用来解释，或者通过 Gag 的 N 端 MA 和 C 端核衣壳 (NC) 与双层的同时相互作用来解释，因为两者都是高度碱性的。然而，我们发现 NC 并不是强膜结合所必需的。相反，需要间隔肽组装结构域 (SPA)，一种推定的 24 个残基螺旋序列，包含 Gag 的 12 个残基 SP 片段，并与衣壳 (CA) C 末端和 NC N 末端重叠。众所周知，SPA 对于未成熟 Gag 晶格的正确组装至关重要。 SPA 中的单个氨基酸突变消除了体外组装，从而显着降低了 Gag 与脂质体的结合。在体内，质膜定位依赖于 SPA。基于异位Cys残基的二硫键交联表明膜上Gag蛋白之间的接触与病毒样颗粒中已知的接触相似。综上所述，我们将这些结果解释为 Gag 膜相互作用是协同的，因为它取决于 Gag 多聚化的能力。重要性逆转录病毒结构蛋白 Gag 具有三个主要结构域。 N 端 MA 结构域直接与细胞质膜 (PM) 相互作用。中央 CA 结构域以及紧邻的序列有助于将数千个 Gag 分子组装成晶格。 C 端 NC 结构域与基因组相互作用，导致病毒 RNA 的包装。对于纯化的 Gag 的体外组装，在没有膜的情况下，NC 与核酸的结合以某种方式促进进一步的 Gag-Gag 相互作用，从而导致 Gag 晶格的形成。 MA 介导的膜结合对病毒颗粒组装的贡献尚不清楚。在这里，我们报告说，在没有核酸的情况下，膜提供了一个促进 Gag-Gag 相互作用的平台。这项研究表明，Gag（而非 MA）与膜的结合是协同的，并将 SPA 确定为控制这种协同性的主要因素。
The principles underlying membrane binding and assembly of retroviral Gag proteins into a lattice are understood. However, little is known about how these processes are related. Using purified Rous sarcoma virus Gag and Gag truncations, we studied the interrelation of Gag-Gag interaction and Gag-membrane interaction. Both by liposome binding and by surface plasmon resonance on a supported bilayer, Gag bound to membranes much more tightly than did matrix (MA), the isolated membrane binding domain. In principle, this difference could be explained either by protein-protein interactions leading to cooperativity in membrane binding or by the simultaneous interaction of the N-terminal MA and the C-terminal nucleocapsid (NC) of Gag with the bilayer, since both are highly basic. However, we found that NC was not required for strong membrane binding. Instead, the spacer peptide assembly domain (SPA), a putative 24-residue helical sequence comprising the 12-residue SP segment of Gag and overlapping the capsid (CA) C terminus and the NC N terminus, was required. SPA is known to be critical for proper assembly of the immature Gag lattice. A single amino acid mutation in SPA that abrogates assemblyin vitrodramatically reduced binding of Gag to liposomes.In vivo, plasma membrane localization was dependent on SPA. Disulfide cross-linking based on ectopic Cys residues showed that the contacts between Gag proteins on the membrane are similar to the known contacts in virus-like particles. Taken together, we interpret these results to mean that Gag membrane interaction is cooperative in that it depends on the ability of Gag to multimerize.IMPORTANCEThe retroviral structural protein Gag has three major domains. The N-terminal MA domain interacts directly with the plasma membrane (PM) of cells. The central CA domain, together with immediately adjoining sequences, facilitates the assembly of thousands of Gag molecules into a lattice. The C-terminal NC domain interacts with the genome, resulting in packaging of viral RNA. For assemblyin vitrowith purified Gag, in the absence of membranes, binding of NC to nucleic acid somehow facilitates further Gag-Gag interactions that lead to formation of the Gag lattice. The contributions of MA-mediated membrane binding to virus particle assembly are not well understood. Here, we report that in the absence of nucleic acid, membranes provide a platform that facilitates Gag-Gag interactions. This study demonstrates that the binding of Gag, but not of MA, to membranes is cooperative and identifies SPA as a major factor that controls this cooperativity.