通过流式细胞仪测量附件和流感的内在病毒在A549细胞
Summary
We present a protocol describing a semi-quantitative method for measuring both, the attachment of influenza A virus to A549 cells, as well as the internalization of virus particles into the target cells by flow cytometry.
Abstract
Attachment to target cells followed by internalization are the very first steps of the life cycle of influenza A virus (IAV). We provide here a detailed protocol for measuring relative changes in the amount of viral particles that attach to A549 cells, a human lung epithelial cell line, as well as in the amount of particles that are internalized into the cell. We use biotinylated virus which can be easily detected following staining with Cy3-labeled streptavidin (STV-Cy3). We describe the growth, purification and biotinylation of A/WSN/33, a widely used IAV laboratory strain. Cold-bound biotinylated IAV particles on A549 cells are stained with STV-Cy3 and measured using flow cytometry. To investigate uptake of viral particles, cold-bound virus is allowed to internalize at 37 °C. In order to differentiate between external and internalized viral particles, a blocking step is applied: Free binding spots on the biotin of attached virus on the cell surface are bound by unlabeled streptavidin (STV). Subsequent cell permeabilization and staining with STV-Cy3 then enables detection of internalized viral particles. We present a calculation to determine the relative amount of internalized virus. This assay is suitable to measure effects of drug-treatments or other manipulations on attachment or internalization of IAV.
Introduction
A型流感病毒（IAV）的条目是与病毒的受体对靶细胞1的质膜的结合将启动一个多步骤的过程。的受体IAV是唾液酸其存在于大量的各种糖蛋白和糖脂的。 IAV的血凝素（HA）蛋白质，其存在于病毒包膜结合唾液酸，从而介导附着病毒颗粒到靶细胞2的质膜。病毒进入经由网格蛋白介导的内吞作用的细胞，而且还可选条目途径，如巨吞，已经描述3-6。 HA与唾液酸之间的相互作用似乎是足够用于介导两个，附着并触发病毒颗粒7的内化。然而，替代的条目受体已经提出和它们在IAV条目角色目前正在研究1,8,9。
姜黄素rently，均努力确定涉及在病毒的生命周期，与显影急需新的抗病毒疗法10-12中的目的宿主因素。病毒进入将是针对IAV增长，以阻断病毒感染，在其最早的点了有利的一步。测量IAV进入不同阶段的实验是挑战，因为通常大量的病毒都需要检测发来的病毒。此外，检测范围的变化病毒内容只线性由于缺乏病毒复制。这强调了需要检测灵敏度高。
我们在这里介绍的测定法允许附加的病毒的检测在细胞表面以及检测内化病毒的有关细胞相关病毒的总量。使用生物素化的野生型病毒能够通过染色STV-Cy3和读出用流式细胞仪测量方便。生物素化病毒是冷结合到细胞s到允许附件，但阻止病毒颗粒的内化。可将细胞固定，透，并用STV-Cy3的测量附加的病毒。从细胞外，附着病毒体的信号可以如果一个封闭步骤是固定在其中的细胞与非标记的链霉（STV）之前应用废止。在下一步骤中，以下附着生物素化IAV的，温度被转移到37℃和病毒颗粒的内化被允许发生。内化颗粒免受STV结合从而使预算外和细胞内的病毒之间的歧视。
每个实验条件下，四个样品是必需的：“0分钟”：第一个样品，标记为“0分钟”，是在细胞表面以测量冷结合的病毒。 '0分钟+ STV'：第二样品，标为“0分钟+ STV'，给出了实验的基线信号强度。附加的病毒被阻止机智相比于“0分钟”样品H STV和信号下列染色STV-Cy3标记应该是低得多。 '30分钟'：第三样品，'30分钟'包含附着和内在病毒由于温度偏移至37℃30分钟。 '30分钟+ STV“：第四个样品，'30分钟+ STV”措施病毒的细胞内部分。在30分钟培养期之后将STV封闭步骤被应用。其结果是，在细胞表面的病毒颗粒通过STV绑定留下可用于与STV-Cy3的染色只内在病毒。内化病毒的相对量可被计算为内化的病毒（在'30分钟+ STV测定“样品）由病毒的总量（由'30分钟描述划分'样品）的比例。
作为对照，我们建议包括模拟感染的细胞。以下模拟感染的细胞的STV-Cy3的染色的信号给出了背景所得从染色方案。为IAV附件的控制是预处理细胞细菌神经氨酸酶（NA）的。 NA切割开来细胞糖蛋白的唾液酸，从而从细胞表面除去附着受体。叠氮化钠（SA）是一种强效的代谢抑制剂，并由此阻止细胞内吞作用13。用叠氮化钠处理的细胞应该是病毒结合阳性阴性用于内化。
Protocol
Representative Results
Discussion
我们的协议描述测量病毒的附件和内在化流式细胞仪的一种简单的方法。它允许使用标记的野生型病毒其模拟更加紧密病毒感染相比，使用的病毒样颗粒（VLP）组成。尽管我们的协议进行了优化，以测量附件IAV和内在它可以很容易地适用于其他病毒。此外，如流式细胞术，用于读出，共染色可以很容易地添加到协议如以下击倒的目的蛋白质或过表达来测试表达水平。因此，该测定允许根据?…
Offenlegungen
The authors have nothing to disclose.
Acknowledgements
这项工作是由瑞士国家科学基金会（31003A_135278）海面温度的赠款支持。 MOP是一个博士生津贴从安盛研究基金的受益人。我们感谢帕特里夏·尼格的帮助， 图1的设计。
Materials
| DMEM | Life Technologies | 41966-052 | |
| FBS | Life Technologies | 10270-106 | |
| Penicillin-Streptomycin | Life Technologies | 15140-163 | |
| PBS | Life Technologies | 14190-169 | |
| BSA | VWR Calbiochem | 126579 | |
| HEPES | Life Technologies | 15630-100 | |
| D-Sucrose | Fluka | 84100 | |
| TRIS | Biosolve BV | 20092391 | |
| EDTA | Sigma-Aldrich | 3680 | |
| EZ-LINK NHS-SS-BIOTIN kit | Fisher Scientific | W9971E | |
| Bio Rad Protein Bio Assay | Bio Rad | 500-0006 | |
| deepwell tubes (1.2 ml microtubes) | Milian | 82 00 001 | |
| PFA | Lucerna chem Electron microscopy sciences | 15710 | |
| ultracentrifuge tubes | Hemotec HmbH | 253070 | |
| Triton X-100 | Fluka | 93420 | |
| STV-Cy3 | Life Technologies | 43-4315 | |
| STV | Life Technologies | 43-4302 | |
| sodium azide | Fluka | 71290 | |
| bacterial neuraminidase/sialidase | Sigma-Aldrich | N6514-1UN |
Referenzen
- Edinger, T. O., Pohl, M. O., Stertz, S. Entry of influenza A virus: host factors and antiviral targets. J Gen Virol. 95, 263-277 (2014).
- Palese, P., Shaw, M. L., Knipe, D. M., Howley, P. M. . Fields Virolog. 2, (2007).
- Matlin, K. S., Reggio, H., Helenius, A., Simons, K. Infectious entry pathway of influenza virus in a canine kidney cell line. J Cell Biol. 91, 601-613 (1981).
- Sieczkarski, S. B., Whittaker, G. R. Influenza virus can enter and infect cells in the absence of clathrin-mediated endocytosis. J Virol. 76, 10455-10464 (2002).
- Rust, M. J., Lakadamyali, M., Zhang, F., Zhuang, X. Assembly of endocytic machinery around individual influenza viruses during viral entry. Nat Struct Mol Biol. 11, 567-573 (2004).
- Vries, E., et al. Dissection of the influenza A virus endocytic routes reveals macropinocytosis as an alternative entry pathway. PLoS Pathog. 7, e1001329 (2011).
- Vries, E., et al. Influenza A virus entry into cells lacking sialylated N-glycans. Proc Natl Acad Sci USA. 109, 7457-7462 (2012).
- Londrigan, S. L., et al. N-linked glycosylation facilitates sialic acid-independent attachment and entry of influenza A viruses into cells expressing DC-SIGN or L-SIGN. J Virol. 85, 2990-3000 (2011).
- Wang, S. F., et al. DC-SIGN mediates avian H5N1 influenza virus infection in cis and in trans. Biochem Biophys Res Commun. 373, 561-566 (2008).
- Pohl, M. O., Edinger, T. O., Stertz, S. Prolidase is required for early trafficking events during influenza A virus entry. J Virol. 88, 11271-11283 (2014).
- Konig, R., et al. Human host factors required for influenza virus replication. Nature. 463, 813-817 (2010).
- Ludwig, S. Targeting cell signalling pathways to fight the flu: towards a paradigm change in anti-influenza therapy. J Antimicrob Chemothe. 64, 1-4 (2009).
- Simoes, S., Slepushkin, V., Duzgunes, N., Pedroso de Lima, M. C. On the mechanisms of internalization and intracellular delivery mediated by pH-sensitive liposomes. Biochim Biophys Acta. 1515, 23-37 (2001).
- Tran, A. T., et al. Knockdown of specific host factors protects against influenza virus-induced cell death. Cell death, and disease. 4, e769 (2013).
- Bradford, M. M. A rapid and sensitive method for the quantitation of microgramm quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 72, 248-254 (1976).
- Macey, M. G., Macey, M. G. . Flow Cytometry Principles and Applications. , (2007).
- Burness, A. T., Pardoe, I. U. Effect of enzymes on the attachment of influenza and encephalomyocarditis viruses to erythrocytes. J Gen Viro. 55, 275-288 (1981).
