Application of virus-like particles (VLP) to NMR characterization of viral membrane protein interactions.
Application of virus-like particles (VLP) to NMR characterization of viral membrane protein interactions.
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DOI：
10.1007/s10858-016-0025-1
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发表时间：
2016-03
影响因子：
2.7
通讯作者：
Caffrey M
中科院分区：
文献类型：
--
作者：
Antanasijevic A;Kingsley C;Basu A;Bowlin TL;Rong L;Caffrey M
病毒的膜蛋白在病毒生命周期中起着关键作用，是治疗干预的重要靶点。病毒样颗粒(VLP)为研究病毒膜蛋白在天然环境中的生化和生物物理性质提供了可能。具体地说，VLP结构包含整个蛋白质序列，并由天然的膜成分组成，包括脂类、胆固醇、碳水化合物和细胞蛋白。在这项研究中，我们从流感中制备了含有全长血凝素(HA)或神经氨酸酶(NA)的VLP，并研究了它们与小分子抑制剂的相互作用。使用HA-VLP，我们首先证明了使用标准蔗糖梯度纯化方案制备的VLP样品含有大量的血清蛋白质，这些蛋白质显示出很高的非特异性相互作用的潜力，从而使配体-靶相互作用的核磁共振研究复杂化。然后，我们证明，通过添加凝胶过滤层析步骤，血清污染物可以被很大程度上去除。接下来，利用HA-VLP，我们证明了在研究配体与膜结合靶的相互作用时，WaterLOGSY核磁共振比饱和转移差分(STD)核磁共振更灵敏。此外，我们还用标准核磁共振方法比较了VLP包埋HA和重组HA的配基取向。在接下来的步骤中，我们使用NA-VLP表征了NA底物类似物和抑制剂的动力学和结合特性，包括对H274Y-NA突变体的研究，该突变体导致对当前流感抗病毒药物的广泛耐药性。综上所述，我们的工作表明VLP有很高的潜力成为病毒膜蛋白的生化和生物物理研究的标准工具，特别是当VLP被高度纯化并与含有天然膜蛋白的对照VLP结合时。
The membrane proteins of viruses play critical roles in the virus life cycle and are attractive targets for therapeutic intervention. Virus-like particles (VLP) present the possibility to study the biochemical and biophysical properties of viral membrane proteins in their native environment. Specifically, the VLP constructs contain the entire protein sequence and are comprised of native membrane components including lipids, cholesterol, carbohydrates and cellular proteins. In this study we prepare VLP containing full-length hemagglutinin (HA) or neuraminidase (NA) from influenza and characterize their interactions with small molecule inhibitors. Using HA-VLP, we first show that VLP samples prepared using the standard sucrose gradient purification scheme contain significant amounts of serum proteins, which exhibit high potential for non-specific interactions, thereby complicating NMR studies of ligand-target interactions. We then show that the serum contaminants may be largely removed with the addition of a gel filtration chromatography step. Next, using HA-VLP we demonstrate that WaterLOGSY NMR is significantly more sensitive than Saturation Transfer Difference (STD) NMR for the study of ligand interactions with membrane bound targets. In addition, we compare the ligand orientation to HA embedded in VLP with that of recombinant HA by STD NMR. In a subsequent step, using NA-VLP we characterize the kinetic and binding properties of substrate analogs and inhibitors of NA, including study of the H274Y-NA mutant, which leads to wide spread resistance to current influenza antivirals. In summary, our work suggests that VLP have high potential to become standard tools in biochemical and biophysical studies of viral membrane proteins, particularly when VLP are highly purified and combined with control VLP containing native membrane proteins.