在甲型流感病毒的衣壳的体外拆卸梯度离心
Summary
Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.
Abstract
Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.
Introduction
甲型流感病毒（IAV）是包膜病毒属于正粘病毒科的。它的基因编码的一个分段，负义单链RNA基因组。在人类中，IAV引起呼吸道感染，发生的季节性疫情和承担全球性流行病1的潜力。在结合于宿主细胞表面2上的唾液酸残基，IAV是通过网格蛋白依赖的内吞作用和网格蛋白独立的途径3-8内化。在胞吞液泡的酸性环境（pH值<5.5）触发在IAV穗糖蛋白血凝素（HA），这导致在病毒融合和已故体膜9的一个主要的构象变化。一旦IAV衣壳（这里也被称为病毒“核心”）已经从晚期内涵体（LES），它在胞质溶胶中，随后通过病毒核糖核蛋白（vRNPs）转运到细胞核未涂覆逃脱 – 的部位O˚F病毒复制和转录10-13。之前的HA的酸活化的病毒经历在内吞系统，素数为随后的拆卸14-16芯pH的逐渐减少。在此“启动”步骤中的病毒膜M2离子通道介导的质子和K +10,14,16的流入。该病毒内部的离子浓度的变化扰乱了相互作用的基质蛋白M1和八个vRNP束建立，并促进IAV脱壳在以下膜融合10,14-17细胞质中。
起动步骤的直接和定量的分析已经阻碍了一个事实，即内涵体难以接近实验。入门过程，而且高度非同步的。此外，终点测定法，如释放的病毒RNA或感染性测量的定量RT-PCR，没有提供关于病毒capsi的生化状态的详细的图片ð在进入任何给定的一步。同时通过siRNA或药物治疗的内体扰动已显著贡献IAV进入18-20的理解，微调是困难的，容易出现非特异性副作用，在严格控制的内吞体成熟方案。
为了避免这些问题，我们已经适于基于使用速度梯度离心17的以前开发体外协议。相对于其他的尝试21,22，23,24，其中大多数是基于蛋白水解裂解和清洁剂处理，然后进行EM分析的组合，这种方法使一个容易量化的结果。离心通过不同的梯度层允许沉降颗粒被暴露，并与以受控的方式变化的情况作出反应。在所提出的协议，IAV从澄清尿囊液的或纯化的病毒颗粒衍生的被沉淀成两层甘油梯度，其中所述底层包含非离子型洗涤剂NP-40（ 图1）。作为病毒进入第二，含去污剂的层，病毒脂质包膜和包膜糖蛋白轻轻溶解并留下。核心，八个vRNP束的组成和由一个基质层包围，沉积物作为一个稳定的结构进沉淀部分。病毒核心蛋白，例如M1和vRNP相关核蛋白（NP），可以在通过SDS-PAGE和考马斯染色的沉淀来识别。特别是，考虑市售梯度凝胶的优点，并与高度敏感的胶体考马斯25染色，使高精度，即使是少量的病毒核心相关蛋白的检测。
此设置的基础用于测试是否不同的条件，例如pH，盐浓度，和推定脱壳因素上的核心部件和芯STABIL的沉淀行为的影响性。到这个目的，在甘油梯度只有较低的，含去污剂的层是通过将因子或感兴趣的条件进行修改。该技术已在研究的不同的pH值和盐浓度的IAV芯16的完整性的影响特别有价值的。 IAV脱壳的中间步骤可以在随后vRNP离解在pH 5.5和下部16,17（ 图1）温和的酸性pH值（<6.5）进行监测，包括在基质层的离解。后者步骤是由高K +浓度的甘油层的存在进一步增强，反映了晚内吞环境16。因此，当病毒和芯通过梯度沉降他们经历了改变环境中核内体，模仿的条件。结果是在体外病毒核心的逐步解体，补充细胞生物学实验得出的结果。
T“>这里介绍的方法，使快速和IAV的高度重复性分析（X31和A / WSN / 33）和IBV（B /李/ 40）脱壳由酸性pH值触发增钾+16,17以及拆卸的在碱性pH曝光17副粘病毒核心。可以想象，该方法可以适于其它包膜病毒进入细胞过程中深入了解病毒衣壳的结构和衣壳拆卸的生化特性。Protocol
Representative Results
Discussion
病毒衣壳是亚稳大分子复合物。虽然病毒粒子组装需要病毒基因组的衣壳和冷凝，下一轮感染的启动取决于这个紧凑的衣壳的结构拆卸。病毒已经进化到利用各种细胞机制，以控制涂层脱壳周期，包括细胞受体，伴侣分子，蛋白水解酶，由马达蛋白或解旋酶，以及pH和离子开关26,27提供物理力。在这里，我们描述了基于甘油梯度离心特异性测试的促进IAV芯拆卸因子的效果的体外方法?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
我们感谢河野洋平山内和罗伯塔·曼奇尼为我们提供了试剂。 ，欧洲研究委员会（ERC），以及由瑞士国家科学基金会（Sinergia）的AH实验室是由居里夫人初始培训网络（ITN）的支持。
Materials
| cOmplete™, EDTA-free protease inhibitor tablets | Sigma-Aldrich | 11873580001 | The stock solution can be stored at 2 to 8 °C for 1 to 2 weeks |
| Glacial acetic acid | Merck Millipore | 100063 | |
| Glycerol anhydrous BioChemica | AppliChem | A1123 | |
| Hydrochloric acid | Merck Millipore | 100317 | |
| Long injection needle (21 GA, 9 cm, bevel or blunt-end) | |||
| MES hydrate | Sigma-Aldrich | M8250 | |
| Methanol | Merck Millipore | 106009 | |
| NP-40 | Sigma-Aldrich | I8896 | Now commercially available as IGEPAL® CA-630 |
| NuPAGE® 4-12% Bis-Tris mini gels, 10 wells, 1.0 mm | Life Technologies | NP0321 | |
| NuPAGE® LDS sample buffer (4X) | Life Technologies | NP0008 | |
| NuPAGE® MOPS SDS running buffer (20X) | Life Technologies | NP0001 | |
| pH indicator strips, pH 4.0 – 7.0 | Merck Millipore | 109542 | |
| QC Colloidal Coomassie Stain | BIO RAD | 1610803 | |
| Sodium chloride | Merck Millipore | 106406 | |
| Sodiumhydroxide | Merck Millipore | 106498 | |
| Steritop™ filter unit | Merck Millipore | SCGPT05RE | |
| SW41 Ti, ultracentrifuge rotor set | Beckman Coulter | 331336 | |
| Thinwall, Ultra-clear centrifuge tubes, 13.2 ml, 14 x 89 mm | Beckman Coulter | 344059 | |
| Tris hydrochloride | AppliChem | A1087 | |
| X31 Influenza A virus (H3N2), egg-grown, clarified allantoic fluid | Virapur | Freshly thawed on 4°C |
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