Name: generation and assembly of virus-specific nucleocapsids of the respiratory syncytial virus
Uploaded: 2021-07-27T14:00:00
Description: Watch this Scientific Journal Video about Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus at JoVE.com

埃默里梁实验室的研究项目由美国国家普通医学科学研究院 （NIGMS）、国家卫生研究院 （NIH） 提供，奖励编号为 R01GM130950，以及埃默里大学医学院的研究启动基金。作者感谢梁实验室的成员给予的帮助和支持和批判性讨论。

1. 分子克隆
注：利气独立克隆（LIC）用于制造RSV双语气联表达构造质粒。LIC是一种在20世纪90年代初开发的方法，它利用T4DNA聚合酶的3'-5'Exo活性来制造载体和DNA插入21，22之间的互补性悬垂。构造使用 2BT-10 矢量 DNA 进行，该 DNA 由他在开放读取帧 （ORF） （图 1）N 端的标记组成。
<ol><li>使用 SSPI ?...

<ol>
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已知的核胶囊状粒子（NCLP）结构的非隔离负感（NNS）RNA病毒表明，组装的NCLP是复杂的N与宿主细胞RNA时，过度表达在细菌或真核表达系统15，16，17，18，19。先前的研究已经尝试通过各种方法获得RNA免费N，如RNA酶A消化，高盐洗涤，或调整不同的pH缓冲器，以去除非特异性细胞...

非隔离性负感（NNS）RNA病毒包括许多重要的人类病原体，如狂犬病、埃博拉和呼吸道同步病毒（RSV）1、2。RSV是导致全球幼儿和老年人患支气管炎和肺炎等呼吸系统疾病的主要原因。目前，没有有效的疫苗或抗病毒疗法可用于预防或治疗RSV4。作为生命周期的一部分，RSV基因组作为RSV RNA依赖RNA聚合酶复制的模板，以产生抗原体，而抗基因组又充当生成后代基因组的模板。基因组和抗基因组RNA都完全被核蛋白（N）封装，形成核胶囊（NCs）3。由于NC是RSV聚合酶复制和转录的模板，因此适当的NC组装对于聚合酶获得RNA合成5的模板至关重要。有趣的是，根据NNS病毒聚合酶的结构分析，假设几个N蛋白暂时脱离NC，允许聚合酶进入，并在RNA合成6、7、8、9、10、11、12后重新绑定RNA。
目前，RSV RNA聚合检测已建立使用纯化RSV聚合酶短裸RNA模板13，14。然而，RSV聚合酶的活动没有达到最佳水平，正如RSV聚合酶在使用裸RNA模板时产生的非加工和流产产品中观察到的。缺乏带有病毒特异性RNA的NC是进一步机械化地理解RSV RNA合成的主要障碍。因此，使用正宗的RNA模板成为推进RSV RNA合成基础知识的关键需要。来自RSV和其他NNS RNA病毒的核胶囊状粒子（NCLPs）的已知结构显示，NCLP中的RNA要么是随机细胞RNA，要么是平均病毒基因组RNA15、16、17、18、19。总之，主要障碍是，当N在主机单元中表达过度时，N与细胞RNA结合，形成NCLP。
为了克服这一障碍，我们建立了一个协议，首先获得无RNA（N0），并组装N0 与正宗的病毒基因组RNA到NCLP20。该协议的原则是获得大量的重组RNA无N（N0）通过共同表达N与伴郎，RSV磷蛋白（PNTD）的N终端域。纯化的 N0P 可以通过添加 RSV 特异性 RNA 寡头来刺激并组装成 NCLP，在组装过程中，伴郎 PNTD 在添加 RNA 寡头后会被移位。
在这里，我们详细说明了 RSV 特定 RNA 特定 NC 的生成和组装协议。在此协议中，我们描述了分子克隆、蛋白质制备、 体外 组装和复杂组件的验证。我们强调克隆策略，为分子克隆的蛋白质共表达生成双柠檬结构。在蛋白质制备方面，我们描述了细胞培养、蛋白质提取和蛋白质复合物的纯化过程。然后，我们讨论RSV RNA特定NC的 体外 组装方法。最后，我们使用大小排除色谱 （SEC） 和负污渍电子显微镜 （EM） 来描述和可视化组装的 NCLP。

<table border="0" cellpadding="0" cellspacing="0" style="width:1293px;" width="1293">
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	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Agarose</td>
			<td style="width:302px;">SIgma</td>
			<td style="width:301px;">A9539-500G</td>
			<td style="width:319px;">making construct using LIC method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amicon Ultra-15 Centrifugal Filter Unit</td>
			<td>Millipore</td>
			<td>UFC901024</td>
			<td>concentrate the protein sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Ampicillin sodium</td>
			<td style="width:302px;">GOLD BIOTECHNOLOGY</td>
			<td style="width:301px;">5118.111317A</td>
			<td>antibiotic for cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">AseI</td>
			<td style="width:302px;">NEB</td>
			<td style="width:301px;">R0526S</td>
			<td>making construct using LIC method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cobalt (High Density) Agarose Beads</td>
			<td style="width:302px;">Gold Bio</td>
			<td>H-310-500</td>
			<td>For purification of His-tag protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Corning LSE Digital Dry Bath Heater</td>
			<td style="width:302px;">CORNING</td>
			<td style="width:301px;">6885-DB</td>
			<td>Heate the sample</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">dCTP</td>
			<td style="width:302px;">Invitrogen</td>
			<td style="width:301px;">10217016</td>
			<td>making construct using LIC method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">dGTP</td>
			<td style="width:302px;">Invitrogen</td>
			<td style="width:301px;">10218014</td>
			<td>making construct using LIC method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Glycerol</td>
			<td style="width:302px;">Sigma</td>
			<td style="width:301px;">G5516-4L</td>
			<td>making solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">HEPES</td>
			<td style="width:302px;">Sigma</td>
			<td style="width:301px;">H3375-100G</td>
			<td>making solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">HiTrap Q HP</td>
			<td style="width:302px;">Sigma</td>
			<td style="width:301px;">GE29-0513-25</td>
			<td>Protein purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Imidazole</td>
			<td style="width:302px;">Sigma</td>
			<td style="width:301px;">I5513-100G</td>
			<td>making solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">IPTG (Isopropyl-beta-D-thiogalactopyranoside)</td>
			<td style="width:302px;">GOLD BIOTECHNOLOGY</td>
			<td style="width:301px;">1116.071717A</td>
			<td>induce the expression of protein</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Microcentrifuge Tubes</td>
			<td style="width:302px;">VWR</td>
			<td style="width:301px;">47730-598</td>
			<td>for PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Misonix Sonicator XL2020 Ultrasonic Liquid Processor</td>
			<td style="width:302px;">SpectraLab</td>
			<td style="width:301px;">MSX-XL-2020</td>
			<td>sonicator for lysing cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Negative stain grids</td>
			<td style="width:302px;">Electron Microscopy Sciences</td>
			<td style="width:301px;">CF400-Cu-TH</td>
			<td>For making negative stain grids</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">New Brunswick Innova 44/44R</td>
			<td style="width:302px;">eppendorf</td>
			<td style="width:301px;">M1282-0000</td>
			<td>Shaker for culturing the cell</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Nonidet P 40 Substitute</td>
			<td style="width:302px;">Sigma</td>
			<td style="width:301px;">74385-1L</td>
			<td>making solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">OneTaq DNA Polymerase</td>
			<td style="width:302px;">NEB</td>
			<td style="width:301px;">M0480L</td>
			<td>PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">QIAquick Gel Extraction Kit</td>
			<td style="width:302px;">QIAGEN</td>
			<td style="width:301px;">28706</td>
			<td>Purify DNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">SSPI-HF</td>
			<td style="width:302px;">NEB</td>
			<td style="width:301px;">R3132S</td>
			<td>making construct using LIC method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Superose 6 Increase 10/300 GL</td>
			<td style="width:302px;">Sigma</td>
			<td>GE29-0915-96</td>
			<td>Protein purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">T4 DNA polymerase</td>
			<td style="width:302px;">Sigma</td>
			<td>70099-3</td>
			<td>making construct using LIC method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Thermo Scientific Sorvall RC 6 Plus Centrifuge</td>
			<td style="width:302px;">Fisher Scientific</td>
			<td style="width:301px;">36-101-0816</td>
			<td>Centrifuge, highest speed 20,000 rpm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Trizma hydrochloride</td>
			<td style="width:302px;">Sigma</td>
			<td style="width:301px;">T3253-250G</td>
			<td>making solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:371px;">Uranyl Formate</td>
			<td style="width:302px;">Electron Microscopy Sciences</td>
			<td style="width:301px;">22451</td>
			<td>making negative stain solution</td>
		</tr>
	</tbody>
</table>

generation and assembly of virus-specific nucleocapsids of the respiratory syncytial virus

使用正宗的RNA模板对于推进病毒RNA合成的基本知识至关重要，该知识可以指导机械发现和病毒学的检测发展。非受教负感 （NNS） RNA 病毒（如呼吸道同步病毒 （RSV））的 RNA 模板不是单单 RNA 分子，而是核蛋白 （N） 封装核糖核蛋白复合物。尽管正宗RNA模板很重要，但这种核糖核蛋白复合物的生成和组装仍然复杂，需要深入阐明。主要的挑战是，过度表达的RSV N非具体地与细胞RNA结合，形成随机核胶囊状粒子（NCLPs）。在这里，我们建立了一个协议，通过与伴郎磷蛋白 （P） 共同表达 N，然后用 RNA 寡头与 RSV 特异性 RNA 序列组装 N0来获得无RNA N （N0），以获得病毒特异性核胶囊 （NCs）。该协议展示了如何克服在准备这个传统上具有挑战性的病毒核糖蛋白复合物的困难。

无RNA N0P蛋白的纯化 通过此协议，可以获得大规模可溶性异质 RSV N0P 复合体。P蛋白的N和N终端部分的全长与大肠杆菌中的N蛋白的10倍共同表达。N0P 使用钴柱、离子交换和大小排除色谱进行纯化。N0P 包含全长 N 端和 N 端 P，但不包含基于紫外线吸收 A260 /A 280比率20 （图4）的蜂窝RNA。
<p class="jove_...

Watch this Scientific Journal Video about Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus at JoVE.com

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

为了深入分析呼吸道同步病毒 （RSV） RNA 合成，我们报告了利用伴生磷蛋白 （P） 共同表达无RNA核蛋白 （N0）的协议，用于病毒特定核胶囊 （NCs） 的后续 体外 组装。

呼吸系统同步病毒病毒特定核胶囊的生成和组装

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and ...

这种方法利用各种方法解决重组病毒蛋白表达挑战使用一种病毒蛋白P'作为伴随另一个病毒蛋白N'获得无RNAN蛋白质。无RNA的RSV N蛋白是在将病毒特定RNA组装成体外核胶囊之前获得的。该方法还可用于其他非细分负感RNA病毒。结对独立克隆法是获取共同表达结构的快速技术。为什么或选修课污渍电子显微镜是检查生细胞中大组装的快速方法。我们想给你两个提示。首先，在进入下一步之前，每一步都获得高收益和扎实的结果。第二，建立适当的色谱程序，并练习用钳子拾取网格。对于N和P共同表达的双语制结构，在37摄氏度的LB介质中生长四个一升培养大肠杆菌BL21DE3应变细胞。当 600 纳米的光学密度达到 0.6 时，将温度降低到 16 摄氏度。一小时后，通过治疗诱导蛋白质表达与0.5毫升IPTG过夜。第二天早上，通过离心收集细胞，并在200毫升的裂解缓冲器中补充颗粒。通过声波解化细胞15分钟，3秒开，3秒关闭脉冲。并通过离心收集裂解物。将超自然物装载到 2.5 乘 10 厘米的钴重力柱中，并带有大约 10 毫升的珠子。预平衡 5 到 10 列的裂解缓冲器。并用五列缓冲器 B 和五卷缓冲 C 清洗柱子。使用两卷缓冲 D 从珠子中去除蛋白质。并平衡Q列与五个一毫升的Q缓冲器体积。使用围射泵将稀释的样品加载到柱子上。将加载的 Q 列与新鲜的 QA 和 QB 缓冲器一起熔化到 HPLC 机器上。运行泵清洗，用一到两卷 QB 和 QA 缓冲器成功清洗机器。最后一次洗涤后，将流速设定为每分钟一毫升，并设置紫外线1至280纳米和紫外线2至260纳米，使用96深井板收集分数。然后使用每浓度的三到四卷洗脱剂的分步梯度应用来阐明蛋白质，从0%的QB浓度开始，每次增加5%的百分比。N-0 P 蛋白复合物将在 15% QB 缓冲浓度下稀释。通过凝胶过滤分离蛋白质，在一个30厘米的小规模纯化柱中，用缓冲器E等于，然后通过SDS页面分析含有分数的蛋白质。对于病毒特定的核胶囊组装，混合和孵育纯净的N-0 P复合物与RNA寡头在室温下1：1.5分子比一小时。用缓冲器 E 预平衡小规模纯化凝胶过滤柱，并通过离心去除任何沉淀物。加载超自然，到大小排除色谱列。并将蛋白质的大小排除色谱图像与RNA组装进行比较，并单独对照蛋白质控制样品，结合核酸纯度比，确定哪些峰值是组装的N RNA、N-0 P和自由RNA。要组装 N RNA 复合物，收集所有 N RNA 峰值分数，执行 RNA 提取，并运行尿素页凝胶以仔细检查特定 RNA 的长度。要为负污渍电子显微镜准备负污渍网格，使用热板加热 5 mL 的双蒸馏水煮沸，并在水中加入 37.5 毫克尿素，以获得 0.75% 的尿素供友染色溶液。将溶液转移到铝箔覆盖烧嘴上。并加入4微升的10摩尔氢氧化钠。搅拌15分钟后，通过0.22微米滤芯将溶液过滤到试管中。要使连续的碳涂层电子显微镜网格具有亲水性，请将电网放置在连接到电源的腔室中，并将带负电荷的离子应用于电网。剪切并折叠两英寸的胶片条。将两滴 40 微升蒸馏水添加到条带的一端。和两个 40 微升下降 0.75% 尿素为队友染色溶液到另一端。在每个网格中加入三微升蛋白质样本。一分钟后，将网格阻塞在印迹纸上，并将网格浸入蒸馏水滴中两次。一旦在第一个染色溶液液滴。然后将网格浸入第二个染色溶液液滴中 30 秒，然后将网格与印迹纸擦黑，以去除任何多余的污渍溶液，并允许网格在成像前风干。利用本协议，可获得大规模可溶性异地呼吸道同步病毒N0 P复合物。在大肠杆菌中，P蛋白的N和N-终端部分的全长与N蛋白上的10 X标签共同表达。根据紫外线吸收核酸纯度比，N0 P 包含全长 N 和 N 端 P，但不包含细胞RNA。然后，纯化的N0 P可以通过特定RNA寡头的孵化，像电子显微镜网格上的粒子一样被刺激并组装成核胶囊。当净化蛋白质时，在柱子纯化过程中避免气泡，用 QA 缓冲器稀释样品，以调整 pH 和盐浓度，然后将样品加载到柱子上。小心将电子显微镜网格保持在外缘，以免对网格进行污染物处理。这将有助于我们确定非阳性RSV基因组包装问题，无论是左手还是右手的单体结构。按照这些程序，可以执行RSV聚合酶活性检测的RNA模板。而这种RSV病毒特异性核胶囊可用于高分辨率低温EM分析。

Research

JoVE Journal

Biochemistry

Examining Proteasome Assembly with Recombinant Archaeal Proteasomes and Nondenaturing PAGE:  The Case for a Combined Approach

检查蛋白酶体大会与重组古菌蛋白酶体和非变性PAGE：该案例相结合的办法

Proteasomes are found in all domains of life. They provide the major route of intracellular protein degradation in eukaryotes, though their assembly is ...

科研

生物化学

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

拟南芥线粒体的分离和呼吸测量

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Generation of Native, Untagged Huntingtin Exon1 Monomer and Fibrils Using a SUMO Fusion Strategy

利用相扑融合策略生成国产、未加标签的杭丁顿蛋白 Exon1 单体和纤维

Huntington&#39;s Disease (HD) is an inherited fatal neurodegenerative disease caused by a CAG expansion (&#8805;36) in the first exon of the HD gene, ...

Manipulating Living Cells to Construct Stable 3D Cellular Assembly Without Artificial Scaffold

操纵活细胞构建稳定的3D 蜂窝组件, 无需人工支架

Regenerative medicine and tissue engineering offer several advantages for the treatment of intractable diseases, and several studies have demonstrated the ...

How to Stabilize Protein: Stability Screens for Thermal Shift Assays and Nano Differential Scanning Fluorimetry in the Virus-X Project

如何稳定蛋白质: 病毒 x 项目中热移检测和纳米差动扫描荧光测量的稳定性筛选

The Horizon2020 Virus-X project was established in 2015 to explore the virosphere of selected extreme biotopes and discover novel viral proteins. To ...

Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes

混合光/蓝色原生电泳在线粒体呼吸链超综合体分离与分析中的作用

Complexes of the oxidative phosphorylation machinery form supramolecular protein arrangements named supercomplexes (SCs), which are believed to confer ...

Interfacial Molecular-level Structures of Polymers and Biomacromolecules Revealed via Sum Frequency Generation Vibrational Spectroscopy

通过相频生成振动光谱揭示聚合物和生物大分子的界面分子级结构

As a second-order nonlinear optical spectroscopy, sum frequency generation (SFG) vibrational spectroscopy has widely been used in investigating various ...

Real-Time, Semi-Automated Fluorescent Measurement of the Airway Surface Liquid pH of Primary Human Airway Epithelial Cells

一次人气道上皮细胞气道表面液体 pH 值的实时、半自动荧光测量

In recent years, the importance of mucosal surface pH in the airways has been highlighted by its ability to regulate airway surface liquid (ASL) ...

Deciphering Molecular Mechanism of Histone Assembly by DNA Curtain Technique

Chromatin is a higher-order structure that packages eukaryotic DNA. Chromatin undergoes dynamic alterations according to the cell cycle phase and in ...

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice

推进液体和冰中病毒组件的高分辨率成像

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It ...