Name: in vitro disassembly of influenza a virus capsids by gradient centrifugation
Uploaded: 2016-03-27T15:00:00.000+00:00
Duration: 7 min 24 s
Description: Watch this Scientific Journal Video about In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation at JoVE.com

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。

1.缓冲区和股票溶液的制备<ol><li>由470毫克的Tris盐酸，390毫克的MES水合物和580毫克氯化钠溶解在80毫升的DDH 2 O制备的MNT缓冲液（20mM MES，30毫摩尔Tris和100mM NaCl）中调整缓冲至pH 7.4，并将其与的DDH 2 O带来至100ml的最终体积</li><li>通过在80毫升的dh每2 O溶解9.76克MES水合物准备500毫米MES缓冲三个相同的解决方案。通过用浓NaOH滴定调节缓冲剂至pH 5.8，5.4和5.0，分别。带来各缓冲器的100毫升的dh 2 O的最终体积</li><li>通过在80ml各溶解7.88克Tris盐酸的dh 2 O制备的500mM Tris缓冲液两个相同的解决方案，并调节pH值至分别7.4和6.4，通过用浓HCl滴定。使缓冲至100ml与卫生署2 O的最终体积</li><li>用蒸馏水2 O准备50％的甘油溶液，搅拌均匀，直到GLYC埃罗尔是均匀混合。过滤通过一个Steritop滤波器单元（0.22微米孔径）成玻璃瓶的溶液。 </li><li>制备10％NP-40溶液和1M NaCl的卫生署2 O. </li><li>通过在4毫升的DDH 2 O溶解两片准备25倍浓缩的蛋白酶抑制剂</li></ol> 2.甘油梯度的制备<ol><li>制备5毫升洗涤剂缓冲主混合物（300毫摩尔NaCl，2％NP-40，2倍浓缩的蛋白酶抑制剂），用于每一pH条件下的溶液至试验（pH为7.4，6.4，5.8，5.4和5.0）。为了这个目的，混合9毫升1M NaCl的的6毫升10％的NP-40，2.4毫升25×蛋白酶抑制剂的股票，并且9毫升的DDH 2 0。 </li><li>对于每一pH条件下吸管4.4毫升的主混合物到50毫升锥形管中。 </li><li>用于pH值高于5.8添加0.6毫升相应调节pH的500毫摩尔Tris原液到管。用于pH 5.8和更低的添加0.6毫升相应调节pH的500mM的MES原液。 </li><li>制作5毫升含有300mM NaCl的缓冲液，2×蛋白酶抑制剂，60毫摩尔Tris调节至pH 7.4和的DDH 2 O这将作为不含去污剂的控制梯度缓冲液。 </li><li>如果需要，通过分别添加浓HCl或NaOH溶液，精细调节溶液至pH 7.4，6.4，5.8，5.4和5.0的pH值。 </li><li>将5ml的50％甘油原液添加到5ml导致六个不同的25％甘油溶液含洗涤剂和不含去污剂的缓冲混合物。通过用pH试纸检查的pH值。 注意:如果所测量的pH值从所需值显著不同，各自的溶液应该再次制备。 </li><li> 7比:通过在3混合50％甘油的库存与卫生署2 O制备15％甘油溶液中。 </li></ol> 3. IAV的超速离心<ol><li>通过使用5ml的注射器和娘家姓添加3毫升15％甘油溶液到超清晰离心管（13.2毫升14毫米×89毫米）DLE（21g的9厘米长）。在管的内壁不离开滴因为这可能会扰乱梯度的完整性。重复此一共有六个离心管，每个的五个pH条件来测试和一个用于控制的样品。 </li><li>小心地将15％甘油层下3.4毫升25％甘油溶液，通过使用5ml的注射器和长针。小心两层不能混用。重复此为所有六个条件与相应的调节pH的甘油溶液进行测试。 注:II级生物安全柜工作，为下面的步骤。 </li><li>轻轻覆盖用30微升澄清含有IAV（X31，H3N2）在1ml MNT缓冲液稀释的尿囊液的甘油梯度（相当于总病毒蛋白的约20-30微克）对于每个梯度。 </li><li>平衡对立管，将它们放入一个SW41超速离心转子。离心150分钟，55,000 XG和12℃。 </li><li>离心后，小心通过使用干净巴斯德吸管和一抽吸器除去上清液， 即 ，既甘油层。重悬在40微升（1×）的非还原性LDS样品缓冲液沉淀。它到吸管上下数次以完全溶解沉淀是​​重要的。将样品转移到1.5 ml离心管。 </li></ol>沉淀部分和考马斯染色的4 SDS-PAGE <ol><li>加热所有样品在95℃下进行10分钟。此时的样品可以贮存于-20℃，直到它们通过SDS-PAGE进行分析。 </li><li>负载20微升溶解丸粒到一个预制梯度（4-12％）的Bis-Tris微型凝胶在1×MOPS SDS 200伏运行1小时运行缓冲液。 </li><li>使用40％的甲醇和10％冰醋酸在DDH 2 O定影溶液</li><li>孵育在定影溶液的凝胶1小时，并在15厘米的细胞培养皿用胶体考马斯溶液足够体积，同时轻轻染色过夜在室温下摇动。 注:以关闭盘以避免染色溶液的蒸发是很重要的。 </li><li>退色在DDH 2 O.凝胶直至凝胶背景变得清晰更换DDH 2 O的每次15-20分钟。存储在DDH 2 O的凝胶在4°C，直到它被扫描带量化。 </li><li>扫描高分辨率的凝胶，并使用市售的或定制的软件，用于蛋白质谱带强度的定量。减去从靠近相应频带的区域中的背景信号和（在pH 7.4）正常化这些值到不含去污剂的对照样品。 </li></ol>

<ol>
	<li>Taubenberger, J. K., Kash, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+Virus+Evolution,+Host+Adaptation,+and+Pandemic+Formation.">Influenza Virus Evolution, Host Adaptation, and Pandemic Formation.</a> Cell host &amp; microbe. 7 (6), 440-451 (2010).</li><li>Skehel, J. J., Wiley, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+binding+and+membrane+fusion+in+virus+entry:+the+influenza+hemagglutinin.">Receptor binding and membrane fusion in virus entry: the influenza hemagglutinin.</a> Annual review of biochemistry. 69, 531-569 (2000).</li><li>de Vries, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+the+influenza+a+virus+endocytic+routes+reveals+macropinocytosis+as+an+alternative+entry+pathway.">Dissection of the influenza a virus endocytic routes reveals macropinocytosis as an alternative entry pathway.</a> PLoS pathogens. 7 (3), e1001329 (2011).</li><li>Matlin, K. S., Reggio, H., Helenius, A., Simons, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectious+entry+pathway+of+influenza+virus+in+a+canine+kidney+cell+line.">Infectious entry pathway of influenza virus in a canine kidney cell line.</a> J Cell Biol. 91 (3 Pt 1), 601-613 (1981).</li><li>Patterson, S., Oxford, J. S., Dourmashkin, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+mechanism+of+influenza+virus+entry+into+cells.">Studies on the mechanism of influenza virus entry into cells.</a> J Gen Virol. 43 (1), 223-229 (1979).</li><li>Rust, M. J., Lakadamyali, M., Zhang, F., Zhuang, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assembly+of+endocytic+machinery+around+individual+influenza+viruses+during+viral+entry.">Assembly of endocytic machinery around individual influenza viruses during viral entry.</a> Nat Struct Mol Biol. 11 (6), 567-573 (2004).</li><li>Sieczkarski, S. B., Whittaker, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+virus+entry+via+endocytosis.">Dissecting virus entry via endocytosis.</a> J Gen Virol. 83 (Pt 7), 1535-1545 (2002).</li><li>Yoshimura, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infectious+Cell+Entry+Mechanism+of+Influenza+Virus.">Infectious Cell Entry Mechanism of Influenza Virus.</a> Journal of Virology. 43 (1), 284-293 (1982).</li><li>White, J., Kartenbeck, J., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-fusion+activity+of+influenza+virus.">Membrane-fusion activity of influenza virus.</a> Embo Journal. 1 (2), 217-222 (1982).</li><li>Martin, K., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+transport+of+influenza+virus+ribonucleoproteins:+The+viral+matrix+protein+(M1)+promotes+export+and+inhibits+import.">Nuclear transport of influenza virus ribonucleoproteins: The viral matrix protein (M1) promotes export and inhibits import.</a> Cell. 67 (1), 117-130 (1991).</li><li>Palese, P., Shaw, M. L., Knipe, D. M., Howley, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+47.">Chapter 47.</a> Fields Virology. , 1647-1690 (2006).</li><li>Chou, Y. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colocalization+of+Different+Influenza+Viral+RNA+Segments+in+the+Cytoplasm+before+Viral+Budding+as+Shown+by+Single-molecule+Sensitivity+FISH+Analysis.">Colocalization of Different Influenza Viral RNA Segments in the Cytoplasm before Viral Budding as Shown by Single-molecule Sensitivity FISH Analysis.</a> PLoS Pathog. 9 (5), e1003358 (2013).</li><li>Martin, K., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transport+of+incoming+influenza+virus+nucleocapsids+into+the+nucleus.">Transport of incoming influenza virus nucleocapsids into the nucleus.</a> Journal of Virology. 65 (1), 232-244 (1991).</li><li>Bui, M., Whittaker, G., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+M1+protein+and+low+pH+on+nuclear+transport+of+influenza+virus+ribonucleoproteins.">Effect of M1 protein and low pH on nuclear transport of influenza virus ribonucleoproteins.</a> Journal of Virology. 70 (12), 8391-8401 (1996).</li><li>Li, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=pH-Controlled+Two-Step+Uncoating+of+Influenza+Virus.">pH-Controlled Two-Step Uncoating of Influenza Virus.</a> Biophysical Journal. 106 (7), 1447-1456 (2014).</li><li>Stauffer, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stepwise+priming+by+acidic+pH+and+high+K++is+required+for+efficient+uncoating+of+influenza+A+virus+cores+after+penetration.">Stepwise priming by acidic pH and high K+ is required for efficient uncoating of influenza A virus cores after penetration.</a> Journal of Virology. , (2014).</li><li>Zhirnov, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solubilization+of+matrix+protein+M1/M+from+virions+occurs+at+different+pH+for+orthomyxo-+and+paramyxoviruses.">Solubilization of matrix protein M1/M from virions occurs at different pH for orthomyxo- and paramyxoviruses.</a> Virology. 176 (1), 274-279 (1990).</li><li>Banerjee, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influenza+A+virus+uses+the+aggresome+processing+machinery+for+host+cell+entry.">Influenza A virus uses the aggresome processing machinery for host cell entry.</a> Science. 346 (6208), 473-477 (2014).</li><li>Huotari, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cullin-3+regulates+late+endosome+maturation.">Cullin-3 regulates late endosome maturation.</a> Proceedings of the National Academy of Sciences. 109 (3), 823-828 (2012).</li><li>Yamauchi, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+Deacetylase+8+Is+Required+for+Centrosome+Cohesion+and+Influenza+A+Virus+Entry.">Histone Deacetylase 8 Is Required for Centrosome Cohesion and Influenza A Virus Entry.</a> PLoS Pathog. 7 (10), e1002316 (2011).</li><li>Bachmayer, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+Solubilization+of+Hemagglutinin+and+Neuraminidase+from+Influenza+Viruses.">Selective Solubilization of Hemagglutinin and Neuraminidase from Influenza Viruses.</a> Intervirology. 5 (5), 260-272 (1975).</li><li>Reginster, M., Nermut, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+Characterization+of+Influenza+Virus+Cores.">Preparation and Characterization of Influenza Virus Cores.</a> Journal of General Virology. 31 (2), 211-220 (1976).</li><li>Nermut, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Further+Investigation+on+the+Fine+Structure+of+Influenza+Virus.">Further Investigation on the Fine Structure of Influenza Virus.</a> Journal of General Virology. 17 (3), 317-331 (1972).</li><li>Schulze, I. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+influenza+virus:+II.+A+model+based+on+the+morphology+and+composition+of+subviral+particles.">The structure of influenza virus: II. A model based on the morphology and composition of subviral particles.</a> Virology. 47 (1), 181-196 (1972).</li><li>Candiano, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blue+silver:+A+very+sensitive+colloidal+Coomassie+G-250+staining+for+proteome+analysis.">Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis.</a> Electrophoresis. 25 (9), 1327-1333 (2004).</li><li>Greber, U. F., Singh, I., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+virus+uncoating.">Mechanisms of virus uncoating.</a> Trends in microbiology. 2 (2), 52-56 (1994).</li><li>Yamauchi, Y., Helenius, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus+entry+at+a+glance.">Virus entry at a glance.</a> Journal of Cell Science. 126 (6), 1289-1295 (2013).</li><li>Zhirnov, O. P., Grigoriev, V. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disassembly+of+Influenza+C+Viruses,+Distinct+from+That+of+Influenza+A+and+B+Viruses+Requires+Neutral-Alkaline+pH.">Disassembly of Influenza C Viruses, Distinct from That of Influenza A and B Viruses Requires Neutral-Alkaline pH.</a> Virology. 200 (1), 284-291 (1994).</li></ol>

The authors have nothing to disclose.

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?...

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&lt;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R​​T-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<html>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（&lt;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"&gt;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。

<table><tbody><tr><td>cOmplete&amp;trade;, EDTA-free protease inhibitor tablets</td><td>Sigma-Aldrich</td><td>11873580001</td><td>The stock solution can be stored at 2 to 8 &amp;deg;C for 1 to 2 weeks</td></tr><tr><td>Glacial acetic acid</td><td>Merck Millipore</td><td>100063</td><td></td></tr><tr><td>Glycerol anhydrous BioChemica</td><td>AppliChem</td><td>A1123</td><td></td></tr><tr><td>Hydrochloric acid</td><td>Merck Millipore</td><td>100317</td><td></td></tr><tr><td>Long injection needle (21&amp;nbsp;G, 9 cm, bevel or blunt-end)</td><td></td><td></td><td></td></tr><tr><td>MES hydrate</td><td>Sigma-Aldrich</td><td>M8250</td><td></td></tr><tr><td>Methanol</td><td>Merck Millipore</td><td>106009</td><td></td></tr><tr><td>NP-40</td><td>Sigma-Aldrich</td><td>I8896</td><td>Now commercially available as IGEPAL&amp;reg; CA-630</td></tr><tr><td>NuPAGE 4-12% Bis-Tris mini gels, 10&amp;nbsp;wells, 1.0 mm</td><td>Life Technologies</td><td>NP0321</td><td></td></tr><tr><td>NuPAGE LDS sample buffer (4x)</td><td>Life Technologies</td><td>NP0008</td><td></td></tr><tr><td>NuPAGE MOPS SDS running buffer (20x)</td><td>Life Technologies</td><td>NP0001</td><td></td></tr><tr><td>pH indicator strips, pH 4.0-7.0</td><td>Merck Millipore</td><td>109542</td><td></td></tr><tr><td>QC Colloidal Coomassie Stain</td><td>BIO RAD</td><td>1610803</td><td></td></tr><tr><td>Sodium chloride</td><td>Merck Millipore</td><td>106406</td><td></td></tr><tr><td>Sodiumhydroxide</td><td>Merck Millipore</td><td>106498</td><td></td></tr><tr><td>Steritop filter unit</td><td>Merck Millipore</td><td>SCGPT05RE</td><td></td></tr><tr><td>SW41 Ti, ultracentrifuge rotor set</td><td>Beckman Coulter</td><td>331336</td><td></td></tr><tr><td>Thinwall, Ultra-clear centrifuge tubes, 13.2&amp;nbsp;ml, 14 mm x 89 mm</td><td>Beckman Coulter</td><td>344059</td><td></td></tr><tr><td>Tris hydrochloride&amp;nbsp;</td><td>AppliChem</td><td>A1087</td><td></td></tr><tr><td>X31 Influenza A virus (H3N2), egg-grown, clarified allantoic fluid</td><td>Virapur</td><td></td><td>Freshly thawed on 4 &amp;deg;C</td></tr></tbody></table>

in vitro disassembly of influenza a virus capsids by gradient centrifugation

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.

如已经讨论的，在核内体内吸需要呈现IAV芯脱壳胜任。芯的质子化削弱M1和vRNPs的相互作用（病毒RNA，NP组成，和聚合酶复合PB1 / PB2 / PA）。当传入病毒暴露于pH为在早期内涵体（EES）6.5（或更低），并继续进行，直到在的LE在pH5.0周围的病毒熔丝此过程开始。 以模仿，pH降低，甘油梯度的底层以恒定的盐浓度被调节至pH 7.4和5.0之?...

Watch this Scientific Journal Video about In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation at JoVE.com

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

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.

在甲型流感病毒的衣壳的体外拆卸梯度离心

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

in-vitro-disassembly-influenza-virus-capsids-gradient

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

8.9K 次观看.  ETH Zurich. 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.

视频: In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Preparation of Viral DNA from Nucleocapsids

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. Several downstream applications require large quantities of pure viral DNA, which is provided by this protocol. These applications include viral genome sequencing, where the removal of host DNA is crucial to optimize data output for viral sequences, and the production of new viral recombinant strains, where co-transfection of purified plasmid and linear viral DNA facilitates recombination.1,2,3 
This procedure utilizes a combination of extractions and density-based centrifugation to isolate purified linear herpesvirus nucleocapsid DNA from infected cells.4,5 The initial purification steps aim to isolate purified viral capsids, which contain and protect the viral DNA during the extractions and centrifugation steps that remove cellular proteins and DNA. Lysis of nucleocapsids then releases viral DNA, and two final phenol-chloroform steps remove remaining proteins. The final DNA captured from solution is highly concentrated and pure, with an average OD260/280 of 1.90. Depending on the quantity of infected cells used, yields of viral DNA range from 150-800 &mu;g or more. The purity of this DNA makes it stable during long-term storage at 4C. This DNA is thus ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections. 
Prior to beginning the protocol, it is important to know the average number of cells per dish (e.g. an average of 8 x 106 PK-15 cells in a confluent 15 cm dish), and the titer of the viral stock to be used (e.g. 1 x 108 plaque-forming units per ml). These are necessary to calculate the appropriate multiplicity of infection (MOI) for the protocol.6 For instance, to infect one 15 cm dish of PK-15 cells with the above viral stock, at an MOI of 5, you would use 400 &mu;l of viral stock and dilute it with 3.6 ml of medium (total inoculation volume of 4 ml for one 15 cm plate).
Multiple viral DNA preparations can be prepared at the same time. The number of simultaneous preparations is limited only by the number of tubes held by the ultracentrifuge rotor (one per virus; see step 3.9 below). Here we describe the procedure as though being done for one virus.

We describe the process of isolating high purity herpesvirus nucleocapsid DNA from infected cells. The final DNA captured from solution is of high concentration and purity, making it ideally suited for high-throughput sequencing, high fidelity PCR reactions, and transfections to produce new viral recombinants.

从核衣壳的病毒DNA的制备

Viruses are obligate cellular parasites, and thus the study of their DNA requires isolating viral material away from host cell contaminants and DNA. ...

科研

免疫与感染

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

在体外对HIV - 1核心脱壳

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

简单而强大在体内和在体外方法研究病毒大会

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

流感病毒从感染细胞的染色质的核糖核蛋白复合物的亲和纯化

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

A型流感病毒的核蛋白复合物的分离纯化及可视化

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

生物学

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

替代体外测定病毒衣壳结构完整性的方法

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

Assembly and Purification of Prototype Foamy Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an integrase (IN) enzyme that catalyzes the covalent joining of viral to host DNA, which is known as strand transfer. Integration may be modeled in vitro with recombinant retroviral IN and DNA oligomers mimicking the ends of the viral genome. In order to more closely recapitulate the integration reaction that occurs in vivo, integration complexes are assembled from recombinant IN and synthetic oligomers by dialysis in a reduced salt concentration buffer. The integration complex, called an intasome, may be purified by size exclusion chromatography. In the case of prototype foamy virus (PFV), the intasome is a tetramer of IN and two DNA oligomers and is readily separated from monomeric IN and free oligomer DNA. The integration efficiency of PFV intasomes may be assayed under a variety of experimental conditions to better understand the dynamics and mechanics of retroviral integration.

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

泡沫病毒 Intasomes 原型的组装与纯化

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

Efficient Adeno-Associated Virus Vector Production Using a Cell Factory Platform

A Streamlined and Standardized Procedure for Generating High-Titer, High-Quality Adeno-Associated Virus Vectors Utilizing a Cell Factory Platform

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. Traditional approaches in research laboratories often involve extensive use of cell culture dishes to cultivate HEK293T cells, a process that can be both laborious and problematic. Here, a unique in-house method is presented, which simplifies this process with a specific cell factory (or cell stacks, CF10) platform. An integration of polyethylene glycol/aqueous two-phase partitioning with iodixanol gradient ultracentrifugation improves both the yield and purity of the generated AAV vectors. The purity of the AAV vectors is verified through SDS-PAGE and silver staining, while the ratio of full to empty particles is determined using transmission electron microscopy (TEM). This approach offers an efficient cell factory platform for the production of AAV vectors at high yields, coupled with an improved purification method to meet the quality demands for in vivo studies.

Author Spotlight: Advancing Gene Therapy Research with High-Titer Adeno-Associated Virus Vector Production

As the field of gene therapy continues to evolve, there is a growing need for innovative methods that can address these challenges. Here, a unique method is presented, which streamlines the process of generating high-yield and high-purity AAV vectors using a cell factory platform, meeting the quality standards for in vivo studies.

利用 Cell Factory 平台生成高滴度、高质量腺相关病毒载体的简化和标准化程序

Preclinical gene therapy research, particularly in rodent and large animal models, necessitates the production of AAV vectors with high yield and purity. ...

生物工程

Single Cell Micro-aspiration as an Alternative Strategy to Fluorescence-activated Cell Sorting for Giant Virus Mixture Separation

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or fluorescence activated cell sorting (FACS) applied to the viral population. However, when the viruses in the mixture have similar morphologic properties and one of the viruses multiplies slowly, the presence of two viruses is discovered at the stage of genome assembly and the viruses cannot be separated for further characterization. To solve this problem, we developed a single cell micro-aspiration procedure that allows for separation and cloning of highly similar viruses. In the present work, we present how this alternative strategy allowed us to separate the small viral subpopulations of Clandestinovirus ST1 and Usurpativirus LCD7, giant viruses that grow slowly and do not lead to amoebal lysis compared to the lytic and fast-growing Faustovirus. Purity control was assessed by specific gene amplification and viruses were produced for further characterization.

Here, we describe a single cell micro-aspiration method for the separation of infected amoebae. In order to separate viral subpopulations in Vermamoeba vermiformis infected by Faustoviruses and unknown giant viruses, we developed the protocol detailed below and demonstrated its ability to separate two low-abundance novel giant viruses.

单细胞微吸气作为荧光激活细胞分类的替代策略，用于巨型病毒混合物分离

During the amoeba co-culture process, more than one virus may be isolated in a single well. We previously solved this issue by end point dilution and/or ...

通过双碘沙醇密度梯度离心分离 AAV

Suspension Culture Production and Purification of Adeno-Associated Virus by Iodixanol Density Gradient Centrifugation for In Vivo Applications

This protocol describes recombinant adeno-associated virus (rAAV) production and purification by iodixanol density gradient centrifugation, a serotype-agnostic method of purifying AAV first described in 1999. rAAV vectors are widely used in gene therapy applications to deliver transgenes to various human cell types. In this work, the recombinant virus is produced by transfection of Expi293 cells in suspension culture with plasmids encoding the transgene, vector capsid, and adenoviral helper genes. Iodixanol density gradient centrifugation purifies full AAV particles based on particle density. Additionally, three steps are included in this now-ubiquitous methodology in order to increase total virus yield, decrease the risk of precipitation due to contaminating proteins, and further concentrate the final virus product, respectively: precipitation of viral particles from cell media using a solution of polyethylene glycol (PEG) and sodium chloride, the introduction of a second round of iodixanol density gradient centrifugation, and buffer exchange via a centrifugal filter. Using this method, it is possible to consistently achieve titers in the range of 1012 viral particles/mL of exceptional purity for in vivo use.

作者聚焦：用于临床前应用的高效腺相关病毒分离

Adeno-associated virus is produced in suspension cell culture and purified by double iodixanol density gradient centrifugation. Steps are included to increase total virus yield, decrease the risk of virus precipitation, and further concentrate the final virus product. Expected final titers reach 1012 viral particles/mL and are suitable for pre-clinical in vivo use.