Name: purification of viral dna for the identification of associated viral and cellular proteins
Uploaded: 2017-08-31T15:00:00.000+00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins at JoVE.com

我们感谢汉娜在准备这份手稿方面的帮助。这项工作得到了 NIH 补助金 R01AI030612 的支持。

1. 细胞培养、病毒感染和标记 ( 图 1 ) 以下协议涉及使用病毒。有关安全处理病毒和其他生物制剂的问题, 请参阅您的机构和 #39 的生物安全协议。该议定书得到了匹兹堡大学机构审查委员会的批准. 
<ol> <li> 处理一个汇合的 150 cm 2 组织培养 MRC-5 细胞的烧瓶, 并将细胞转移到 600 cm 2 组织培养板中, 其中含有100毫升 DMEM 加 10% FBS。孵育在37和 #176; C 在 5% CO 2 的存在下, 3-4 天到达70-100 和固定相。汇合的 600 cm 2 盘包含 ~ 7 x 10 7 单元格。为每个条件准备1板和1板为每个对应的阴性控制. 
	注意: 细胞必须到达固定阶段, 以抑制细胞 DNA 复制, 以确保只有 vDNA 在后续步骤中标记和纯化. 
	注: 每个样品都需要使用相同的感染条件和时间点, 而无需添加附加的无标记阴性对照. 
	注: 本实验的缩小版应串联进行, 以通过可比细胞生长、感染、标记条件和时间点的成像来测试细胞 DNA 的标记 (参见步骤 2). </li>
	<li> 稀释 7 x 10 8 PFU HSV-1 在7毫升冷三缓冲盐水 (TBS)。37、#176 中移除培养基及贮存;为了感染, 在室温下将7毫升稀释的病毒添加到 MRC-5 细胞和岩石中1小时。吸附后, 去除接种, 用50毫升室温 TBS 冲洗, 取代原生长培养基。在 5% CO 2 的存在下孵育37和 #176; C。这一步应进行的每一个实验条件和负控制. 
	注: 使用 HSV-1 突变体的病毒 dUTPase (UL50 基因) 和/或嘧啶 glycosylase (UL2 基因) 的表达有缺陷, 可获得增加的氯乙烷标记。HSV-1 dUTPase 具有较低的基底特异性, 可减少几种核苷类似物的活化 25 , 27 。
	<li> 标签的病毒基因组。按照指示来标记传入的基因组 (1.3.1), 复制基因组 (1.3.2), 或病毒复制叉 (1.3.3). 
	注意: 根据传入的基因组、复制的基因组或复制叉是否要分别在后续步骤中进行分析, 只能执行步骤1.3.1、1.3.2 或 1.3.3. 
	注: 可替代教育和教育。核苷类似物的毒性应加以监测和尽量减少。
	<ol> <li> 使用 prelabeled 库存在步骤1.2 中进行感染, 并在 4 h 后感染 (hpi) 内进行步骤2或 3, 以调查复制 vDNA ( 图 1A ). 
		注意: 使用前面描述的协议 25 准备 prelabeled 病毒储存。病毒储存必须通过一个 G-25 的专栏, 以消除任何残余的氯乙烷. </li>
		<li> 标签复制 vDNA 通过添加 5-25 和 #181; M 对细胞培养培养基的感染细胞为 2-4 h ( 图 1B )。加入前, 稀释1毫升的生长培养基. </li>
		<li> 对病毒复制叉进行标记, 在 vDNA 复制 (#8805; 4 hpi) 后, 用 5-25 和 #181 复制 vDNA 的脉冲标签; M. 5-20 分钟追逐, 冲洗3x 与追逐媒介包含 25-100 和 #181; M 2 和 #180;d eoxycytidine (deoxyC), 然后孵育在追逐媒介面前为另外 20-40 min ( 图 1C ). 
		注: 脉冲和大通介质应平衡到37和 #176; C 和 5% CO 2 , 然后使用. 
		注意: 对于脉冲追逐实验, 重要的是要考虑到苷进入细胞和磷酸化前所需的时间, 才能将其纳入复制 DNA。这必须是经验主义地确定的. 
		注: 要确定脉冲追逐实验的分辨率, 在实验条件下计算病毒复制率。这可以通过定量 PCR 的病毒基因组在单步生长时间课程 26 . 
		注: 对于影像病毒基因组进行步骤2和纯化病毒基因组进行步骤 3. </li>
	</ol> </li> </ol> 2。标记 DNA 的图像 ( 图 2A ) 注意: 在与病毒基因组纯化方法同时进行成像, 以验证细胞 DNA 在实验中没有标记是有用的。成像也可以用来可视化的性质, 标记 vDNA 和验证蛋白质组数据。细胞和病毒 DNA 染色模式的例子如 图 3 所示. 
<ol> <li> 执行步骤1中所示的感染和片标记, 但在包含1毫升生长培养基的12良好组织培养皿中使用缩放条件时除外. </li>
	<li> 在 RT 1x PBS 中, 用1毫升3.7% 甲醛固定电池15分钟。在固定后, 冲洗细胞2x 与1毫升 3% BSA 在 PBS. 
	警告: 甲醛可能导致严重或永久性的伤害。遵循安全防范措施. 
	注意: 协议可以在这里暂停, 片可以存储在4和 #176; C 在第二次洗涤3天. </li>
	<li> 带 1 mL 性缓冲区的 Permeabilize 单元格 (请参见 材料表 ] 在摇晃时室温下为20分钟. </li>
	<li> 用1毫升 3% bsa 冲洗, 然后在 PBS 中用1毫升 3% bsa 在室温下以30分钟的温度进行清洗. </li>
	<li> 添加1毫升的点击反应鸡尾酒 [见 材料的表 ] 片到共价键共轭 Alexa 的488叠氮化物的标记 DNA。在室温下孵育30分钟的摇摆。用1毫升的 PBS 吸气和冲洗两次. </li>
	<li> 染色细胞 DNA 1 毫升 1:2, 000 稀释赫斯特在 PBS 为30分钟的室温, 而摇摆。用1毫升的 PBS 吸气和冲洗两次. </li>
	<li> 根据标准的间接免疫荧光协议对主要和次要抗体进行染色 25 . 
	注: 标记 vDNA colocalizes 与 ICP8, ICP4, 或 UL42 和标记细胞 DNA colocalizes 与赫斯特染色. </li>
	<li> 使用荧光显微镜在幻灯片和图像单元上安装片. 
	注: 幻灯片可以存储在4和 #176; C 几个星期. </li>
</ol> 3。vDNA 及其相关蛋白的纯化 注意: 此协议的几个方面已从梁 et al. 中进行了改编 22 注意: 所有的缓冲器和试剂在使用前应冷藏在冰上, 除非另有说明, 所有步骤应在冰上进行. 
<ol> <li> 收获核. 
	注: 在细胞核分离之前, 甲醛可用于将蛋白质与 DNA 交联。更严格的洗涤条件, 然后可以使用在 DNA 纯化的亲和涂层珠。crosslinking 和洗涤条件请参见 Sirbu et al. 21 . <ol> <li> 用20毫升核提取缓冲剂 (纳布) 取代生长培养基, 在4和 #176 中孵育20分钟; C. 偶尔摇摆。在显微镜下, 原子核会变得可见. </li>
		<li> 用刮板机将细胞核从板材上刮起, 转移到50毫升锥形管上。离心机10分钟在 2500 x g 在4和 #176; C 对颗粒核, 丢弃上清. 
		注意: 台盼蓝染色可用于验证细胞核与细胞的分离. </li>
		<li> 在10毫升 pbs 中轻轻地将核小球取出, 转移到15毫升的锥形管, 并在4和 #176 上用离心10分钟, 在 2500 x g 处进行颗粒的移动; c. 完全删除 pbs. 
		注意: 原子核可以被冻结, 在这一点上可以暂停协议。要做到这一点, 轻轻地重500和 #181 的核颗粒; 冻结缓冲液, 在乙醇/干冰浴中孵育试管。将冷冻核储存在-80 和 #176;在使用前, 在室温下解冻细胞核, 迅速转移到冰上, 加入10毫升 pbs, 轻轻地混合, 在4和 #176 ° C 的 2500 x g 处进行离心10分钟的颗粒, 并完全去除 pbs。冷冻核可能导致产量下降. </li>
	</ol> </li> <li> 共价共轭生物素-叠氮化为标记为 vDNA. 
	<ol> <li> 重在10毫升单击反应混合中的核颗粒 (参见 材料表 ), 用10毫升吸管轻轻移上下5次. 
		注意: 重要的是, 包括在点击反应混合的试剂被添加在指定的顺序. </li>
		<li> 4 #176 旋转1h ° C。当旋转准备和冷却缓冲 B1, B2 和 B3 [请参阅 材料表 ]. </li>
		<li> 球团核由离心为10分钟, 在 2500 x g 在4和 #176; c. 完全删除单击反应混合, 并通过轻轻溶液10毫升 PBS 和离心10分钟 2500 x g 在4和 #176, 清洗颗粒; c. </li>
		<li> 轻轻地重1毫升 PBS 中的核颗粒, 并使用大口径吸管尖端转移到1.5 毫升离心管。球团核由离心10分钟在 2500 x g 在4和 #176; c. 完全去除液态氮中的 PBS 和闪光冷冻. 
		注意: 该协议可以在这一点上暂停, 冻结的原子核可以储存在-80 和 #176; c. 冻结解冻有助于随后的裂解, 因此建议, 即使在同一天进行步骤 3.3, 核也被冻结. </li>
	</ol> </li> <li> 溶解核和片段 DNA. 
	<ol> <li> 将500和 #181 的冰和重中的原子核解冻; 移向上和向下缓冲 B1。在冰上孵育45分钟. </li>
		<li> 几种样本的6倍, 三十年代每40% 振幅使用3毫米 mircotip 探头。在冰上放置样品三十年代在脉冲之间。超声后, 样品应清晰显示, 不浑浊. 
		注: 超声条件应针对个人 sonicators 进行优化。有关如何优化超声条件的详细说明, 请参阅梁 et al. 22 . </li> <li> 颗粒细胞碎片的离心在 1.4万 x g 处为10分钟, 在4和 #176;颗粒大小应大幅度降低。过滤上清, 通过100和 #181; M 细胞过滤器, 并保持流经. </li>
		<li> 添加500和 #181; 对过滤后的上清液进行缓冲 B2. 900 和 #181; 此示例中的 l 将在步骤3.4.2 中使用. </li>
		<li> 分对每个条件进行 DNA 隔离 (50 和 #181; l 或 1/第二十卷) 并添加50和 #181; l 2x SDS-盐解决方案 [请参见 材料表 ]。继续执行 DNA 隔离协议 (步骤 3.6). </li>
		<li> 对输入蛋白质 (50 和 #181; l 或 1/第二十卷) 的每个条件进行分, 并添加50和 #181; l 2x Laemmli 样品缓冲液, 在液氮中冷冻, 在-80 和 #176 中贮存; c. 在步骤3.5.6 中使用. </li>
	</ol> </li> <li> 将化的 DNA 绑定到亲和涂层的珠子. 
	<ol> <li> 通过转移300和 #181 的亲和 T1 磁珠; 在1.5 毫升离心管上进行珠浆的 L。每样都准备一管珠子。洗涤珠3x 与1毫升缓冲 B2 由涡流到重, 应用磁铁分离的珠子, 并吸取洗涤缓冲区. 
		注意: 优化的绑定条件导致最大收益率和最小背景绑定。实验证明, 亲和 T1 磁珠具有比琼脂糖珠更大的结合能力, 以及其他亲和珠, 比亲和 C1 珠更少的背景绑定 ( 图4A).</li>
		<li> 添加900和 #181; 从步骤3.3.4 中抽取样品 L. 到被洗涤的珠子和隔夜在4和 #176 转动; C. 
		注: 在添加样品后不要涡流珠. 
		注意: 如果在未标记的负控制中分离出大量的 DNA 或蛋白质, 这一步可以从一夜之间减少到 4 h, 以减少背景绑定. </li>
	</ol> </li> <li> 洗涤珠子和洗 vDNA 及相关蛋白. 
	注意: 建议对其余步骤使用筛选提示。
	<ol> <li> 将样品放入磁性离心管机架中, 取出上清, 在1毫升缓冲 B2 中轻轻重, 在4和 #176 旋转; C 为 5 min. </li>
		<li> 重复步骤3.5.1 三次. </li>
		<li> 将样品放入磁性离心管机架中, 取出上清, 在1毫升缓冲 B3 中轻轻重, 并在4和 #176 旋转; C 5 min. </li>
		<li> 传输100和 #181; L 珠混合物 (1/10 th 容量) 到一个新的管子为束缚的脱氧核糖核酸隔离。将此管应用于磁体, 取出上清, 重100和 #181 中的珠子; L 1x SDS-盐溶液, 并进行 DNA 隔离协议 (步骤 3.6). </li>
		<li> 应用包含剩余900和 #181 的管状物; 从台阶3.5.3 的珠混合物. 到磁铁, 去除上清, 和重珠在50和 #181; l 2x Laemmli 样品缓冲液洗蛋白和 DNA 蛋白复合物. </li>
		<li> 在95和 #176 中煮沸样品; C 为15分钟, 涡流, 快速旋转在离心, 并应用磁铁。将洗转换为新的管, 闪存冻结, 并存储在-80 和 #176; C. 
		注: 使用瓶盖锁, 以确保管不弹出开放, 而沸腾. 
		注意: 此时可以暂停协议, 并且可以将示例存储在-80 和 #176; C 数周. </li>
		<li> 通过标准程序对马斯亮蓝染色、西方印迹或质谱法分析蛋白质样品 25 。代表性结果如 图 4 和 图 5 所示。
		注: 为了确定蛋白质的产量是否足够的质谱, 7.5 和 #181; 每个样本的 l 用于分析马斯亮蓝染色和7.5 和 #181; l 为西方印迹的一个代表性的病毒基因组相关蛋白 (ICP4, ICP8, 或 UL42)。同一体积的裂解从步骤3.3.6。在控制输入蛋白质水平的同时运行。剩下的样品 (35 和 #181; L) 然后通过质谱法分析, 如前面所描述的 25 . </li>
	</ol> </li> <li> DNA 隔离 <ol> <li> 将示例从步骤3.3.5 和3.5.4 中孵化. 在65和 #176; C 为 4-16 h. 从磁性珠子中取出样品, 如有必要. </li>
		<li> 用150和 #181 提取样品; L phenol:chloroform:isoamyl 酒精 (PCI), 并将水相转移到一个新的管. </li>
		<li> 用100和 #181 提取剩余的 PCI; 三 EDTA (TE), 并将水相添加到新的管中. </li>
		<li> 抽取200和 #181 的样品; L 氯仿: 异戊醇 (24:1). </li>
		<li> 根据制造商和 #39 的协议, 使用 PCR 纯化试剂盒净化水相. 
		注: 当添加到样品中时, 缓冲铅中的 pH 值会变紫。添加 10 & #181;L 3M NaOAc ph 值 5.0, 以降低样品的 ph 值. </li>
		<li> 使用荧光确定 DNA 浓度. 
		注: 本协议通常产生 100-400 ng 珠绑定的 DNA。在1.3 步中未添加洗的负控制中, 不应检测到任何 DNA. </li>
		<li> dna 可以储存在低 dna 结合管4或-20 和 #176; C, 可用于 real-time PCR 或高通量测序分析. </li>
	</ol>
	</li>
</ol>

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L., McAnany, P. K., Bloom, 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=A+chromatin+insulator-like+element+in+the+herpes+simplex+virus+type+1+latency-associated+transcript+region+binds+CCCTC-binding+factor+and+displays+enhancer-blocking+and+silencing+activities.">A chromatin insulator-like element in the herpes simplex virus type 1 latency-associated transcript region binds CCCTC-binding factor and displays enhancer-blocking and silencing activities.</a> J Virol. 80, 2358-2368 (2006).</li><li>Cliffe, A. R., Knipe, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+ICP0+promotes+both+histone+removal+and+acetylation+on+viral+DNA+during+lytic+infection.">Herpes simplex virus ICP0 promotes both histone removal and acetylation on viral DNA during lytic infection.</a> J Virol. 82, 12030-12038 (2008).</li><li>Herrera, F. J., Triezenberg, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VP16-dependent+association+of+chromatin-modifying+coactivators+and+underrepresentation+of+histones+at+immediate-early+gene+promoters+during+herpes+simplex+virus+infection.">VP16-dependent association of chromatin-modifying coactivators and underrepresentation of histones at immediate-early gene promoters during herpes simplex virus infection.</a> J Virol. 78, 9689-9696 (2004).</li><li>Kent, J. R., 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=During+lytic+infection+herpes+simplex+virus+type+1+is+associated+with+histones+bearing+modifications+that+correlate+with+active+transcription.">During lytic infection herpes simplex virus type 1 is associated with histones bearing modifications that correlate with active transcription.</a> J Virol. 78, 10178-10186 (2004).</li><li>Oh, J., Fraser, N. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+association+of+the+herpes+simplex+virus+genome+with+histone+proteins+during+a+lytic+infection.">Temporal association of the herpes simplex virus genome with histone proteins during a lytic infection.</a> J Virol. 82, 3530-3537 (2008).</li><li>Catez, F., 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=HSV-1+genome+subnuclear+positioning+and+associations+with+host-cell+PML-NBs+and+centromeres+regulate+LAT+locus+transcription+during+latency+in+neurons.">HSV-1 genome subnuclear positioning and associations with host-cell PML-NBs and centromeres regulate LAT locus transcription during latency in neurons.</a> PLoS Pathog. 8, e1002852 (2012).</li><li>Everett, R. D., Murray, J., Orr, A., Preston, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+type+1+genomes+are+associated+with+ND10+nuclear+substructures+in+quiescently+infected+human+fibroblasts.">Herpes simplex virus type 1 genomes are associated with ND10 nuclear substructures in quiescently infected human fibroblasts.</a> J Virol. 81, 10991-11004 (2007).</li><li>Tang, Q., 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=Determination+of+minimum+herpes+simplex+virus+type+1+components+necessary+to+localize+transcriptionally+active+DNA+to+ND10.">Determination of minimum herpes simplex virus type 1 components necessary to localize transcriptionally active DNA to ND10.</a> J Virol. 77, 5821-5828 (2003).</li><li>Ishov, A. M., Maul, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+periphery+of+nuclear+domain+10+(ND10)+as+site+of+DNA+virus+deposition.">The periphery of nuclear domain 10 (ND10) as site of DNA virus deposition.</a> J Cell Biol. 134, 815-826 (1996).</li><li>Maroui, M. 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=Latency+Entry+of+Herpes+Simplex+Virus+1+Is+Determined+by+the+Interaction+of+Its+Genome+with+the+Nuclear+Environment.">Latency Entry of Herpes Simplex Virus 1 Is Determined by the Interaction of Its Genome with the Nuclear Environment.</a> PLoS Pathog. 12, e1005834 (2016).</li><li>Sirbu, B. M., Couch, F. B., Cortez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+the+spatiotemporal+dynamics+of+proteins+at+replication+forks+and+in+assembled+chromatin+using+isolation+of+proteins+on+nascent+DNA.">Monitoring the spatiotemporal dynamics of proteins at replication forks and in assembled chromatin using isolation of proteins on nascent DNA.</a> Nat Protoc. 7, 594-605 (2012).</li><li>Leung, K. H., Abou El Hassan, M., Bremner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+efficient+method+to+purify+proteins+at+replication+forks+under+native+conditions.">A rapid and efficient method to purify proteins at replication forks under native conditions.</a> BioTechniques. 55, 204-206 (2013).</li><li>Hobbs, W. E., DeLuca, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perturbation+of+cell+cycle+progression+and+cellular+gene+expression+as+a+function+of+herpes+simplex+virus+ICP0.">Perturbation of cell cycle progression and cellular gene expression as a function of herpes simplex virus ICP0.</a> J Virol. 73, 8245-8255 (1999).</li><li>Lomonte, P., Everett, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Herpes+simplex+virus+type+1+immediate-early+protein+Vmw110+inhibits+progression+of+cells+through+mitosis+and+from+G(1)+into+S+phase+of+the+cell+cycle.">Herpes simplex virus type 1 immediate-early protein Vmw110 inhibits progression of cells through mitosis and from G(1) into S phase of the cell cycle.</a> J Virol. 73, 9456-9467 (1999).</li><li>Dembowski, J. A., DeLuca, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+recruitment+of+nuclear+factors+to+productively+replicating+herpes+simplex+virus+genomes.">Selective recruitment of nuclear factors to productively replicating herpes simplex virus genomes.</a> PLoS Pathog. 11, e1004939 (2015).</li><li>Dembowski, J. A., Dremel, S. E., DeLuca, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication-Coupled+Recruitment+of+Viral+and+Cellular+Factors+to+Herpes+Simplex+Virus+Type+1+Replication+Forks+for+the+Maintenance+and+Expression+of+Viral+Genomes.">Replication-Coupled Recruitment of Viral and Cellular Factors to Herpes Simplex Virus Type 1 Replication Forks for the Maintenance and Expression of Viral Genomes.</a> PLoS Pathog. 13, e1006166 (2017).</li><li>Bjornberg, O., Nyman, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dUTPases+from+herpes+simplex+virus+type+1+and+mouse+mammary+tumour+virus+are+less+specific+than+the+Escherichia+coli+enzyme.">The dUTPases from herpes simplex virus type 1 and mouse mammary tumour virus are less specific than the Escherichia coli enzyme.</a> J Gen Virol. 77 (Pt 12), 3107-3111 (1996).</li><li>Chiou, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-+and+protein-scission+activities+of+ascorbate+in+the+presence+of+copper+ion+and+a+copper-peptide+complex.">DNA- and protein-scission activities of ascorbate in the presence of copper ion and a copper-peptide complex.</a> J Biochem. 94, 1259-1267 (1983).</li><li>Kennedy, D. C., 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=Cellular+consequences+of+copper+complexes+used+to+catalyze+bioorthogonal+click+reactions.">Cellular consequences of copper complexes used to catalyze bioorthogonal click reactions.</a> J Am Chem Soc. 133, 17993-18001 (2011).</li><li>Snel, B., Lehmann, G., Bork, P., Huynen, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STRING:+a+web-server+to+retrieve+and+display+the+repeatedly+occurring+neighbourhood+of+a+gene.">STRING: a web-server to retrieve and display the repeatedly occurring neighbourhood of a gene.</a> Nucleic Acids Res. 28, 3442-3444 (2000).</li></ol>

作者没有什么可透露的。

该协议包括多个步骤, 如果不小心, 可能会导致蛋白质的产量和细胞 DNA 的污染显著降低。至关重要的是, 固定细胞用于所有的实验, 以确保细胞 DNA 没有标记和纯化。这可以证实, 由于缺乏细胞 dna 聚合在蛋白质样本, 因为 HSV-1 不利用细胞 dna 聚合酶的基因组合成。在标记和核收获步骤期间, 样品应错开, 以确保每一个处理平等。在纯化步骤中, 应注意不要过度移地操纵细胞核。这可能导致过早的原子?...

病毒具有执行基本功能的能力有限, 因此取决于宿主因素, 以促进感染的关键方面, 包括病毒基因表达、复制、修复、重组和运输。这些寄主因素的活动通常由病毒编码的蛋白质增强。此外, 病毒必须避免对病毒感染的细胞反应进行检测和干扰。因此, 病毒宿主交互决定感染的结果。最重要的是了解病毒如何改变细胞环境, 以适应细胞的机器, 以促进病毒的过程。特别感兴趣的是确定哪些因素和过程对病毒基因组在整个感染周期中的作用。
单纯疱疹病毒1型 (HSV-1) 是一种双链 DNA 病毒, 感染了相当一部分人的人口。在感染的第一个小时内, 病毒基因组进入细胞核, 病毒基因表达的有序级联随后与病毒 DNA (vDNA) 复制1进行协调。在细胞核中, 基因组受后生调节, 经过修复和重组, 并被包装成衣, 这样, 第一个子代病毒在不到六小时内生产。在整个感染过程中, 对病毒基因组相关蛋白的综合评价将为研究病毒基因组过程的分子细节打下基础, 并将提供对病毒和细胞因子的洞察参与感染的各个阶段。
以前的方法调查病毒感染的寄主因素包括亲和纯化病毒蛋白的相关细胞蛋白的分析2,3,4,5,6,7,8,9. 这些检测方法有助于识别宿主抗病毒应答中涉及的细胞因子, 以及病毒染色质修饰、基因表达和 DNA 修复。然而, 很难确定相互作用是否依赖于与 vDNA 的关联, 蛋白质组学只提供对作为特定病毒因素的功能而发生的相互作用的洞察。染色质沉淀 (芯片) 已被用来确定特定的病毒和细胞蛋白绑定到病毒基因组10,11,12,13,14, 15和荧光原位杂交 (fish) 结合免疫细胞化学技术, 使 colocalize 与 vDNA16,17,18的单元格因子的可视化19,20. 这些化验结果可以进行时空分析。然而, 限制包括对高度特异的抗体的需要, 有限的灵敏度, 以及对病毒宿主相互作用之前的洞察力的需要。因此, 我们开发了一种基于 iPOND (在新生的 DNA 上分离蛋白质)21和 aniPOND (加速本机 iPOND)22的方法, 以有选择地标记和纯化感染细胞的 vDNA, 以无偏见地识别病毒基因组。相关蛋白的质谱。iPOND 对细胞复制叉动力学的研究起到了很重要的作用。
对于被感染细胞的病毒基因组的选择性纯化, 复制 vDNA 被标记为乙炔修饰的苷、5-乙炔-2´-脱氧 (教育) 或 5-乙炔-2´-脱氧 () (图 1), 其次是共价键结合到生物素叠氮化通过点击化学促进单步纯化病毒基因组和相关蛋白的亲和涂层珠子 (图 2B)。重要的是, 感染是在固定细胞中进行的, 它们不从事细胞 DNA 复制, 从而能够对 vDNA 进行特定的标记。此外, HSV-1 感染导致细胞周期阻滞和抑制细胞 DNA 复制23,24。病毒可在感染前 prelabeled, 用于分析与传入病毒基因组相关的蛋白质 (图 1A), 或在 DNA 复制过程中标记, 用于分析与新合成的 vDNA 相关的蛋白质 (图 1B)25. 此外, 脉冲追踪分析可用于研究与病毒复制叉 (图 1C)26相关的蛋白质的性质。此外, 乙炔修饰的 vDNA 可以共价键共轭到一个荧光的空间研究蛋白质动力学 (图 2A和图 3)。成像允许直接可视化的 vDNA, 是一个免费的方法来验证 vDNA-蛋白质相互作用, 并可以适应跟踪病毒基因组在整个感染。我们预计, 这些方法可以进一步修改, 以研究任何方面的 herpesviral 感染, 包括潜伏期和重新激活, 并研究其他 DNA 病毒。此外, 用 5-乙炔苷 (EU) 进行标记可以分析 RNA 病毒基因组。

<table><tbody><tr><td>MRC-5 cells</td><td>ATCC</td><td>CCL-171</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>26140-179</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>12800-082</td><td>Substituted with 10% FBS, 2 mM L-glutamine, 12 mM&amp;nbsp; (for growth in flasks) or 30 mM (for growth in dishes) sodium-bicarbinate</td></tr><tr><td>600 cm&lt;sup&gt;2&lt;/sup&gt; tissue culture dish</td><td>Thermo Fisher Scientific</td><td>166508</td><td></td></tr><tr><td>Tris Buffered Saline (TBS), pH 7.4</td><td></td><td></td><td>137 mM NaCl, 5 mM KCl, 491 mM MgCl, 680 mM CaCl, 25.1 mM Tricine</td></tr><tr><td>HSV-1 stock</td><td></td><td></td><td>Stocks with titers greater than 1x10&lt;sup&gt;9&lt;/sup&gt; PFU/mL work best</td></tr><tr><td>Sephadex G-25&amp;nbsp; column (PD-10 Desalting Column)</td><td>GE Healthcare</td><td>17085101</td><td></td></tr><tr><td>Dimethyl sulfoxide (DMSO)</td><td>Fisher Scientific</td><td>D128-1</td><td></td></tr><tr><td>5&amp;acute;-Ethynyl-2&amp;acute;-deoxycytidine (EdC)</td><td>Sigma-Aldrich</td><td>T511307</td><td>Dissolve in DMSO to prepare 40 mM stock, aliquot, and store at -20 &amp;deg;C&amp;nbsp;</td></tr><tr><td>2&amp;acute;-deoxycytidine (deoxyC)</td><td>Sigma-Aldrich</td><td>D3897</td><td>Dissolve in water to prepare 40 mM stock, aliquot, and store at -20 &amp;deg;C&amp;nbsp;</td></tr><tr><td>Nuclear Extraction Buffer (NEB)</td><td></td><td></td><td>Prepare fresh (20 mM Hepes pH 7.2, 50 mM NaCl, 3 mM MgCl&lt;sub&gt;2&lt;/sub&gt;, 300 mM Sucrose, 0.5% Igepal)</td></tr><tr><td>Cell scraper</td><td>Bellco glass</td><td>7731-22000</td><td>Autoclave before use</td></tr><tr><td>Trypan blue solution</td><td>Sigma-Aldrich</td><td>T8154</td><td></td></tr><tr><td>PBS, pH 7.2 (10x)</td><td></td><td></td><td>1.37 M NaCl, 27 mM KCl, 100 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 18 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt; (dilute to 1x in sterile water before use)</td></tr><tr><td>Copper (II) sulfate pentahydrate (CuSO4-5H2O)</td><td>Fisher Scientific</td><td>C489</td><td>Prepare 100 mM stock and store at 4 &amp;deg;C for up to 1 month</td></tr><tr><td>(+) Sodium L-ascorbate</td><td>Sigma-Aldrich</td><td>A4034</td><td>Freshly prepare 100 mM stock and store on ice until use</td></tr><tr><td>Biotin azide</td><td>Invitrogen</td><td>B10184</td><td>Prepare 10 mM stock in DMSO, aliquot, and store at -20 &amp;deg;C for up to 1 year&amp;nbsp;</td></tr><tr><td>Click Reaction Mix</td><td></td><td></td><td>Prepare immediately before use by adding reagents in the indicated order (10 mL: 8.8 mL 1x PBS, 200 mL 100 mM CuSO&lt;sub&gt;4&lt;/sub&gt;, 25mL 10 mM Biotin Azide, 1 mL 100 mM sodium ascorbate)&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Complete Protease Inhibitor Cocktail</td><td>Roche</td><td>11697498001</td><td>Dissolve in 1 mL water to prepare 50x stock, can store at 4 &amp;deg;C for up to 1 week, or directly add 1 pill to 50 mL buffer</td></tr><tr><td>Freezing buffer</td><td></td><td></td><td>Prepare fresh (7 mL 100% glycerol, 3 mL NEB, 200 &amp;mu;L 50x protease inhibitor)</td></tr><tr><td>Buffer B1</td><td></td><td></td><td>Prepare fresh (25 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl pH 8, 1% Igepal, 1x protease inhibitor)</td></tr><tr><td>Buffer B2</td><td></td><td></td><td>Prepare fresh (150 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl pH 8, 0.5% Igepal, 1x protease inhibitor)</td></tr><tr><td>Buffer B3</td><td></td><td></td><td>Prepare fresh (150 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl pH 8, 1x protease inhibitor)</td></tr><tr><td>Vibra Cell Ultra Sonic Processer equipped with a 3 mm microtip probe</td><td>Sonics</td><td>VCX 130</td><td></td></tr><tr><td>Cell strainer</td><td>Falcon</td><td>352360</td><td></td></tr><tr><td>Dynabeads MyOne Streptavidin T1</td><td>Life Technologies</td><td>65601</td><td></td></tr><tr><td>DynaMag-2 Magnet</td><td>Life Technologies</td><td>12321D</td><td></td></tr><tr><td>Mini-Tube Rotator</td><td>Fisher Scientific</td><td>260750F</td><td></td></tr><tr><td>2x Laemmli sample buffer</td><td></td><td></td><td>Mix 400 mg of SDS, 2 mL 100% glycerol, 1.25 mL of 1 M Tris (pH 6.8), and 10 mg of bromophenol blue in 8 mL water. Store at 4&amp;nbsp; &amp;deg;C for up to 6 months. Before use add 1 M DTT to a final concentration of 200 mM.</td></tr><tr><td>Cap locks for 1.5 mL tube</td><td>Fisher Scientific</td><td>NC9679153</td><td></td></tr><tr><td>Standard western blotting reagents</td><td></td><td></td><td></td></tr><tr><td>NOVEX Colloidal Blue Staining Kit</td><td>Invitrogen</td><td>LC6025</td><td></td></tr><tr><td>12-well tissue culture dish</td><td>Corning</td><td>3513</td><td></td></tr><tr><td>Coverslips</td><td>Fisher Scientific</td><td>12-545-100</td><td>Autoclave before use</td></tr><tr><td>Microscope slides</td><td>Fisher Scientific</td><td>12-552-5</td><td></td></tr><tr><td>16% paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>15710</td><td>Dilute to 3.7% in 1x PBS before use</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Fisher Scientific</td><td>BP1605</td><td>Prepare 3% in 1x PBS</td></tr><tr><td>Permeabilization buffer (0.5% Triton-X 100)</td><td>Sigma-Aldrich</td><td>T8787</td><td>Prepare 0.5% Triton-X 100 in 1x PBS</td></tr><tr><td>Click reaction cocktail - Click-iT EdU Alexa Fluor 488 Imaging Kit</td><td>Molecular Probes</td><td>C10337</td><td>Prepare according to manufactorer&#039;s protocol</td></tr><tr><td>Hoechst 33342</td><td>Life Technologies</td><td>H1399</td><td>Prepare 10 mg/mL in water, can store at 4 &amp;deg;C for up to 1 year</td></tr><tr><td>Mouse anti-ICP8 primary antibody</td><td>Abcam</td><td>ab20194</td><td>Use a 1:200 dilution in 1x PBS</td></tr><tr><td>Mouse anti-UL42 primary antibody (2H4)</td><td>Abcam</td><td>ab19311</td><td>Use a 1:200 dilution in 1x PBS</td></tr><tr><td>Goat anti-mouse 594-conjugated secondary antibody</td><td>Life Technologies</td><td>a11005</td><td>Use a 1:500 dilution in 1x PBS</td></tr><tr><td>Immu-mount</td><td>Thermo Fisher Scientific</td><td>9990402</td><td></td></tr><tr><td>2x SDS-bicarb solution</td><td></td><td></td><td>2% SDS, 200 mM NaHCO3</td></tr><tr><td>Phenol:chloroform:isoamyl alcohol</td><td></td><td></td><td>Mix 25:24:1 at least 1 day before use, store at 4 &amp;deg;C in the dark</td></tr><tr><td>chloroform:isoamyl alcohol</td><td></td><td></td><td>Mix 24:1 at least 1 day before use, store at room temperature in the dark</td></tr><tr><td>10x Tris EDTA (TE), pH 8.0</td><td></td><td></td><td>100 mM Tris, 10 mM EDTA (dilute to 1x before use)</td></tr><tr><td>Qiaquick PCR purification kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>3 M sodium acetate pH 5.2</td><td></td><td></td><td></td></tr><tr><td>Qubit 2.0 Fluorometer</td><td>Invitrogen</td><td>Q32866</td><td></td></tr><tr><td>Qubit dsDNA HS Assay Kit</td><td>Invitrogen</td><td>Q32851</td><td></td></tr><tr><td>Qubit assay tubes</td><td>Invitrogen</td><td>Q32856</td><td></td></tr><tr><td>Fluorescence microscope equipped with imaging software</td><td></td><td></td><td></td></tr><tr><td>Microcentrifuge for 1.5 mL tubes</td><td></td><td></td><td></td></tr><tr><td>Tabletop centrifuge for 15 and 50 mL tubes</td><td></td><td></td><td></td></tr><tr><td>Cell culture incubator</td><td></td><td></td><td></td></tr><tr><td>Biosafety cabinet</td><td></td><td></td><td></td></tr><tr><td>Heat blocks</td><td></td><td></td><td>65 &amp;deg;C and 95 &amp;deg;C</td></tr></tbody></table>

purification of viral dna for the identification of associated viral and cellular proteins

该协议的目的是分离单纯疱疹病毒1型 (HSV-1) DNA 从感染的细胞, 以确定相关的病毒和细胞蛋白的质谱。尽管与病毒基因组相互作用的蛋白质在决定感染的结果方面起着重要作用, 但对病毒基因组相关蛋白的综合分析在以前是不可行的。在这里, 我们演示了一种方法, 能够直接纯化 HSV-1 基因组从受感染的细胞。复制病毒 DNA 有选择性地用含有炔烃功能基团的修饰核苷酸标记。然后通过铜 (I)-催化叠氮化-炔烃加或单击反应, 通过生物素叠氮化物的共价键, 对标记的 DNA 进行具体和不可逆转的标记。生物素标记的 DNA 是纯化亲和包覆的珠子和相关的蛋白质是洗和鉴定的质谱。这种方法使 HSV-1 复制叉或整个基因组在复杂的生物环境中有选择的目标和隔离。此外, 这种方法的适应将允许调查 herpesviral 感染的各个方面, 以及检查其他 DNA 病毒的基因组。

使用单击化学从细胞纯化 DNA 首先是通过 iPOND 方法21完成的。iPOND 的目的是纯化细胞复制叉, 用于鉴定相关的蛋白质。我们已经适应了这一技术, 专门研究 vDNA 蛋白相互作用期间感染。使用 vDNA 标记病毒基因组的方法 (图 1) 和同步感染的操作, 允许选择性地隔离和调查不同的种群。HSV-1 DNA 被贴上 (图 1), 以促进荧...

Watch this Scientific Journal Video about Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins at JoVE.com

Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

该协议的目的是专门标记和有选择地分离病毒 DNA 从受感染的细胞, 以表征病毒基因组相关的蛋白质。

病毒 DNA 的纯化及相关病毒和细胞蛋白的鉴定

The goal of this protocol is to isolate herpes simplex virus type 1 (HSV-1) DNA from infected cells for the identification of associated viral and ...

purification-viral-dna-for-identification-associated-viral-cellular

Research

JoVE Journal

Immunology and Infection

13.4K 次观看.  University of Pittsburgh School of Medicine. 该协议的目的是专门标记和有选择地分离病毒 DNA 从受感染的细胞, 以表征病毒基因组相关的蛋白质。

视频: Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins

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

Isolation and Genome Analysis of Single Virions using 'Single Virus Genomics'

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which are ubiquitous and the most numerous entities on our planet 4 and important in all environments 5, have yet to be revealed via similar approaches. Here we describe an approach for isolating and characterizing the genomes of single virions called 'Single Virus Genomics' (SVG). SVG utilizes flow cytometry to isolate individual viruses and whole genome amplification to obtain high molecular weight genomic DNA (gDNA) that can be used in subsequent sequencing reactions.

Single Virus Genomics (SVG) is a method to isolate and amplify the genomes of single virons. Viral suspensions of a mixed assemblage are sorted using flow cytometry onto a microscope slide with discrete wells containing agarose, thereby capturing the virion and reducing genome shearing during downstream processing. Whole genome amplification is achieved using multiple displacement amplification (MDA) resulting in genomic material that is suitable for sequencing.

使用“单个病毒基因组的单病毒颗粒的分离和基因组分析&#39;

Whole genome amplification and sequencing of single microbial cells enables genomic characterization without the need of cultivation 1-3. Viruses, which ...

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

Isolation of Viral Replication Compartment-enriched Sub-nuclear Fractions from Adenovirus-infected Normal Human Cells

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.

We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.

由腺病毒感染正常细胞分离病毒复制车厢富集亚核分数

During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments ...

Combining Analysis of DNA in a Crude Virion Extraction with the Analysis of RNA from Infected Leaves to Discover New Virus Genomes

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low titers in their host or cannot be mechanically inoculated to another host are difficult to propagate to achieve a greater titer of infectious material. Infected leaves are ground in a mild buffer with optimal pH and ionic composition recommended for purifying most bacilliform Para retroviruses. Urea is used to break up inclusion bodies that trap virions and to dissolve cellular components. Differential centrifugation provides further separation of virions from plant contaminants. Then proteinase K treatment removes the capsids. Then the viral DNA is concentrated and used for next-generation sequencing (NGS). The NGS data are used to assemble contigs which are submitted to NCBI-BLASTn to identify a subset of virus sequences in the generated dataset. In a parallel pipeline, RNA is isolated from infected leaves using a standard column-based RNA extraction method. Then ribosome depletion is carried out to enrich for a subset of mRNA and virus transcripts. Assembled sequences derived from RNA sequencing (RNA-seq) were submitted to NCBI-BLASTn to identify a subset of virus sequences in this dataset. In our study, we identified two related full-length badnavirus genomes in the two datasets. This method is preferred to another common approach which extracts the aggregate population of small RNA sequences to reconstitute plant virus genomic sequences. This latter metagenomic pipeline recovers virus related sequences that are retro-transcribing elements inserted into the plant genome. This is coupled to biochemical or molecular assays to further discern the actively infectious agents. The approach documented in this study, recovers sequences representative of replicating viruses that likely indicate active virus infection.

Here we present a new approach to identify plant viruses with double-strand DNA genomes. We use standard methods to extract DNA and RNA from infected leaves and carry out next-generation sequencing. Bioinformatic tools assemble sequences into contigs, identify contigs representing virus genomes and assign genomes to taxonomic groups.

粗病毒提取中 DNA 的组合分析及对感染叶 RNA 的分析发现新的病毒基因组

This metagenome approach is used to identify plant viruses with circular DNA genomes and their transcripts. Often plant DNA viruses that occur in low ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

In Vitro Selection of Aptamers to Differentiate Infectious from Non-Infectious Viruses

Virus infections have a major impact on society; most methods of detection have difficulties in determining whether a detected virus is infectious, causing delays in treatment and further spread of the virus. Developing new sensors that can inform on the infectability of clinical or environmental samples will meet this unmet challenge. However, very few methods can obtain sensing molecules that can recognize an intact infectious virus and differentiate it from the same virus that has been rendered non-infectious by disinfection methods. Here, we describe a protocol to select aptamers that can distinguish infectious viruses vs non-infectious viruses using systematic evolution of ligands by exponential enrichment (SELEX). We take advantage of two features of SELEX. First, SELEX can be tailor-made to remove competing targets, such as non-infectious viruses or other similar viruses, using counter selection. Additionally, the whole virus can be used as the target for SELEX, instead of, for example, a viral surface protein. Whole virus SELEX allows for the selection of aptamers that bind specifically to the native state of the virus, without the need to disrupt of the virus. This method thus allows recognition agents to be obtained based on functional differences in the surface of pathogens, which do not need to be known in advance.

We provide a protocol that can be generally applied to select aptamers that bind to infectious viruses only and not to viruses that have been rendered non-infectious by a disinfection method or to any other similar viruses. This opens the possibility of determining infectivity status in portable and rapid tests.

体外选择核酸适配体以区分感染性和非感染性病毒

Virus infections have a major impact on society; most methods of detection have difficulties in determining whether a detected virus is infectious, ...

生物工程

<meta charset="utf8"></head><body>使用双报告基因和多重技术分析 SARS-CoV-2

Profiling of Surface Protein Epitopes on Viral Particles by Multiplex Dual-Reporter Strategy

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of viral particles to host cells, host-virus interactions, and disease pathogenesis. Furthermore, viral membrane proteins on virus particles and presented on host cell surfaces have proven to be excellent targets for antivirals and vaccines. Here, we describe a protocol to investigate surface proteins on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) particles using the dual-reporter flow cytometric system. The assay exploits multiplex technology to obtain a triple detection of viral particles by three independent affinity reactions. Magnetic beads conjugated to recombinant human angiotensin-converting enzyme-2 (ACE2) were used to capture viral particles from the supernatant of cells infected with SARS-CoV-2. Then, two detection reagents labeled with R-phycoerythrin (PE) or Brilliant Violet 421 (BV421) were applied simultaneously. As a proof-of-concept, antibody fragments targeting different epitopes of the SARS-CoV-2 surface protein Spike (S1) were used. The detection of viral particles by three independent affinity reactions provides strong specificity and confirms the capture of intact virus particles. Dose-dependency curves of SARS-CoV-2 infected cell supernatant were generated with replicate coefficient variances (mean/SD) &#706;14%. Good assay performance in both channels confirmed that two virus surface target protein epitopes are detectable in parallel. The protocol described here could be applied for (i) high-multiplex, high-throughput profiling of surface proteins expressed on enveloped viruses; ii) detection of active intact viral particles; and (iii) assessment of specificity and affinity of antibodies and antiviral drugs for surface epitopes of viral antigens.The application can be potentially extended to any type of extracellular vesicles and bioparticles, exposing surface antigens in body fluids or other liquid matrices.

<meta charset="utf8"></head><body>作者聚焦:通过新型免疫捕获和质谱技术推进抗病毒策略

Here, we describe a newly developed multiplex fluorescent immunoassay that uses a dual-reporter flow cytometric system to concurrently detect two unique spike protein epitopes on intact severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral particles that had been captured by angiotensin-converting enzyme-2-coupled magnetic microspheres.

通过多重双报告基因策略分析病毒颗粒上的表面蛋白表位

Membrane proteins on enveloped viruses play an important role in many biological functions involving virus attachment to target cell receptors, fusion of ...

病毒 DNA 的纯化及相关病毒和细胞蛋白的鉴定

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从核衣壳的病毒DNA的制备

使用“单个病毒基因组的单病毒颗粒的分离和基因组分析'

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

A型流感病毒的核蛋白复合物的分离纯化及可视化

由腺病毒感染正常细胞分离病毒复制车厢富集亚核分数

粗病毒提取中 DNA 的组合分析及对感染叶 RNA 的分析发现新的病毒基因组

鉴定蛋白质相互作用的合作伙伴使用串联亲和纯化

体外选择核酸适配体以区分感染性和非感染性病毒

通过多重双报告基因策略分析病毒颗粒上的表面蛋白表位

通过多重双报告基因策略分析病毒颗粒上的表面蛋白表位