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 border="0" cellpadding="0" cellspacing="0" width="1930">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">MRC-5 cells</td>
			<td style="width:197px;">ATCC</td>
			<td style="width:101px;">CCL-171</td>
			<td style="width:1215px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:197px;">Gibco</td>
			<td style="width:101px;">26140-179</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:197px;">Gibco</td>
			<td style="width:101px;">12800-082</td>
			<td>substituted with 10% FBS, 2 mM L-glutamine, 12 mM&#160; (for growth in flasks) or 30 mM (for growth in dishes) sodium-bicarbinate</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:419px;">600 cm2 tissue culture dish</td>
			<td style="width:197px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:101px;">166508</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Tris Buffered Saline (TBS), pH 7.4</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>137 mM NaCl, 5 mM KCl, 491 mM MgCl, 680 mM CaCl, 25.1 mM Tricine</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:419px;">HSV-1 stock</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>stocks with titers greater than 1x109 PFU/mL work best</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Sephadex G-25&#160; column (PD-10 Desalting Column)</td>
			<td style="width:197px;">GE Healthcare</td>
			<td align="right" style="width:101px;">17085101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Dimethyl sulfoxide (DMSO)</td>
			<td style="width:197px;">Fisher Scientific</td>
			<td style="width:101px;">D128-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">5&#180;-Ethynyl-2&#180;-deoxycytidine (EdC)</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:101px;">T511307</td>
			<td>Dissolve in DMSO to prepare 40 mM stock, aliquot, and store at -20 &#176;C&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">2&#180;-deoxycytidine (deoxyC)</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:101px;">D3897</td>
			<td>Dissolve in water to prepare 40 mM stock, aliquot, and store at -20 &#176;C&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:419px;">Nuclear Extraction Buffer (NEB)</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>prepare fresh (20 mM Hepes pH 7.2, 50 mM NaCl, 3 mM MgCl2, 300 mM Sucrose, 0.5% Igepal)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Cell scraper</td>
			<td style="width:197px;">Bellco glass</td>
			<td style="width:101px;">7731-22000</td>
			<td>Autoclave before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Trypan blue solution</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:101px;">T8154</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:419px;">PBS, pH 7.2 (10x)</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, 18 mM KH2PO4 (dilute to 1x in sterile water before use)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Copper (II) sulfate pentahydrate (CuSO4-5H2O)</td>
			<td style="width:197px;">Fisher Scientific</td>
			<td style="width:101px;">C489</td>
			<td>Prepare 100 mM stock and store at 4 &#176;C for up to 1 month</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">(+) Sodium L-ascorbate</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:101px;">A4034</td>
			<td>Freshly prepare 100 mM stock and store on ice until use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Biotin azide</td>
			<td style="width:197px;">Invitrogen</td>
			<td style="width:101px;">B10184</td>
			<td>Prepare 10 mM stock in DMSO, aliquot, and store at -20 &#176;C for up to 1 year&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:419px;">Click Reaction Mix</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>prepare immediately before use by adding reagents in the indicated order (10 mL: 8.8 mL 1x PBS, 200 mL 100 mM CuSO4, 25mL 10 mM Biotin Azide, 1 mL 100 mM sodium ascorbate)&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">cOmplete Protease Inhibitor Cocktail</td>
			<td style="width:197px;">Roche</td>
			<td align="right" style="width:101px;">11697498001</td>
			<td>Dissolve in 1 mL water to prepare 50x stock, can store at 4 &#176;C for up to 1 week, or directly add 1 pill to 50 mL buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">freezing buffer</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>prepare fresh (7 mL 100% glycerol, 3 mL NEB, 200 &#956;L 50x protease inhibitor)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Buffer B1</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>prepare fresh (25 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl pH 8, 1% Igepal, 1x protease inhibitor)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Buffer B2</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></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 height="21">
			<td height="21" style="height:21px;width:419px;">Buffer B3</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>prepare fresh (150 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl pH 8, 1x protease inhibitor)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibra Cell Ultra Sonic Processer equipped with a 3 mm microtip probe</td>
			<td style="width:197px;">Sonics</td>
			<td style="width:101px;">VCX 130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Cell strainer</td>
			<td style="width:197px;">Falcon</td>
			<td align="right" style="width:101px;">352360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Dynabeads MyOne Streptavidin T1</td>
			<td style="width:197px;">Life Technologies</td>
			<td align="right" style="width:101px;">65601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">DynaMag-2 Magnet</td>
			<td style="width:197px;">Life Technologies</td>
			<td style="width:101px;">12321D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Mini-Tube Rotator</td>
			<td style="width:197px;">Fisher Scientific</td>
			<td style="width:101px;">260750F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">2X Laemmli sample buffer</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></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&#160; &#176;C for up to 6 months. Before use add 1 M DTT to a final concentration of 200 mM.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">cap locks for 1.5 mL tube</td>
			<td style="width:197px;">Fisher Scientific</td>
			<td style="width:101px;">NC9679153</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Standard western blotting reagents</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">NOVEX Colloidal Blue Staining Kit</td>
			<td style="width:197px;">Invitrogen</td>
			<td style="width:101px;">LC6025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">12-well tissue culture dish</td>
			<td style="width:197px;">Corning</td>
			<td align="right" style="width:101px;">3513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Coverslips</td>
			<td style="width:197px;">Fisher Scientific</td>
			<td style="width:101px;">12-545-100</td>
			<td>Autoclave before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Microscope slides</td>
			<td style="width:197px;">Fisher Scientific</td>
			<td style="width:101px;">12-552-5</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">16% paraformaldehyde</td>
			<td style="width:197px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:101px;">15710</td>
			<td>dilute to 3.7% in 1x PBS before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Bovine serum albumin (BSA)</td>
			<td style="width:197px;">Fisher Scientific</td>
			<td style="width:101px;">BP1605</td>
			<td>prepare 3% in 1x PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Permeabilization buffer (0.5% Triton-X 100)</td>
			<td style="width:197px;">Sigma-Aldrich</td>
			<td style="width:101px;">T8787</td>
			<td>prepare 0.5% Triton-X 100 in 1x PBS</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:419px;">Click reaction cocktail - Click-iT EdU Alexa Fluor 488 Imaging Kit</td>
			<td style="width:197px;">Molecular Probes</td>
			<td style="width:101px;">C10337</td>
			<td>prepare according to manufactorer&#39;s protocol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Hoechst 33342</td>
			<td style="width:197px;">Life Technologies</td>
			<td style="width:101px;">H1399</td>
			<td>prepare 10 mg/mL in water, can store at 4 &#176;C for up to 1 year</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">mouse anti-ICP8 primary antibody</td>
			<td style="width:197px;">Abcam</td>
			<td style="width:101px;">ab20194</td>
			<td>Use a 1:200 dilution in 1x PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">mouse anti-UL42 primary antibody (2H4)</td>
			<td style="width:197px;">Abcam</td>
			<td style="width:101px;">ab19311</td>
			<td>Use a 1:200 dilution in 1x PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Goat anti-mouse 594-conjugated secondary antibody</td>
			<td style="width:197px;">Life Technologies</td>
			<td style="width:101px;">a11005</td>
			<td>Use a 1:500 dilution in 1x PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Immu-mount</td>
			<td style="width:197px;">Thermo Fisher Scientific</td>
			<td align="right" style="width:101px;">9990402</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">2x SDS-bicarb solution</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>2% SDS, 200 mM NaHCO3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Phenol:chloroform:isoamyl alcohol</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>mix 25:24:1 at least 1 day before use, store at 4 &#176;C in the dark</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">chloroform:isoamyl alcohol</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>mix 24:1 at least 1 day before use, store at room temperature in the dark</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">10x Tris EDTA (TE), pH 8.0</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>100 mM Tris, 10 mM EDTA (dilute to 1x before use)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Qiaquick PCR purification kit</td>
			<td style="width:197px;">Qiagen</td>
			<td align="right" style="width:101px;">28104</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">3 M sodium acetate pH 5.2</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Qubit 2.0 Fluorometer</td>
			<td style="width:197px;">Invitrogen</td>
			<td style="width:101px;">Q32866</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Qubit dsDNA HS Assay Kit</td>
			<td style="width:197px;">Invitrogen</td>
			<td style="width:101px;">Q32851</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Qubit assay tubes</td>
			<td style="width:197px;">Invitrogen</td>
			<td style="width:101px;">Q32856</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Fluorescence microscope equipped with imaging software</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Microcentrifuge for 1.5 mL tubes</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Tabletop centrifuge for 15 and 50 mL tubes</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Cell culture incubator</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Biosafety cabinet</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:419px;">Heat blocks</td>
			<td style="width:197px;"></td>
			<td style="width:101px;"></td>
			<td>65 &#176;C and 95 &#176;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

Using a Pan-Viral Microarray Assay (Virochip) to Screen Clinical Samples for Viral Pathogens

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence of novel viral pathogens, such as SARS coronavirus and 2009 pandemic H1N1 influenza virus, that are not detectable by traditional methods. To address these challenges, we have previously developed and validated a pan-viral microarray platform called the Virochip with the capacity to detect all known viruses as well as novel variants on the basis of conserved sequence homology1. Using the Virochip, we have identified the full spectrum of viruses associated with respiratory infections, including cases of unexplained critical illness in hospitalized patients, with a sensitivity equivalent to or superior to conventional clinical testing2-5. The Virochip has also been used to identify novel viruses, including the SARS coronavirus6,7, a novel rhinovirus clade5, XMRV (a retrovirus linked to prostate cancer)8, avian bornavirus (the cause of a wasting disease in parrots)9, and a novel cardiovirus in children with respiratory and diarrheal illness10. The current version of the Virochip has been ported to an Agilent microarray platform and consists of ~36,000 probes derived from over ~1,500 viruses in GenBank as of December of 2009. Here we demonstrate the steps involved in processing a Virochip assay from start to finish (~24 hour turnaround time), including sample nucleic acid extraction, PCR amplification using random primers, fluorescent dye incorporation, and microarray hybridization, scanning, and analysis.

Using a Pan-Viral Microarray Assay Virochip to Screen Clinical Samples for Viral Pathogens

The Virochip is a pan-viral microarray designed to simultaneously detect all known viruses as well as novel viruses on the basis of conserved sequence homology. Here we demonstrate how to run a Virochip assay to analyze clinical samples for the presence of both known and unknown viruses.

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

The diagnosis of viral causes of many infectious diseases is difficult due to the inherent sequence diversity of viruses as well as the ongoing emergence ...

科研

免疫与感染

Separation of Single-stranded DNA, Double-stranded DNA and RNA from an Environmental Viral Community Using Hydroxyapatite Chromatography

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2. Viruses modulate host cell abundance and diversity, contribute to the cycling of nutrients, alter host cell phenotype, and influence the evolution of both host cell and viral communities through the lateral transfer of genes 3. Numerous studies have highlighted the staggering genetic diversity of viruses and their functional potential in a variety of natural environments. 
Metagenomic techniques have been used to study the taxonomic diversity and functional potential of complex viral assemblages whose members contain single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and RNA genotypes 4-9. Current library construction protocols used to study environmental DNA-containing or RNA-containing viruses require an initial nuclease treatment in order to remove nontargeted templates 10. However, a comprehensive understanding of the collective gene complement of the virus community and virus diversity requires knowledge of all members regardless of genome composition. Fractionation of purified nucleic acid subtypes provides an effective mechanism by which to study viral assemblages without sacrificing a subset of the community&#x2019;s genetic signature. 
Hydroxyapatite, a crystalline form of calcium phosphate, has been employed in the separation of nucleic acids, as well as proteins and microbes, since the 1960s11. By exploiting the charge interaction between the positively-charged Ca2+ ions of the hydroxyapatite and the negatively charged phosphate backbone of the nucleic acid subtypes, it is possible to preferentially elute each nucleic acid subtype independent of the others. We recently employed this strategy to independently fractionate the genomes of ssDNA, dsDNA and RNA-containing viruses in preparation of DNA sequencing 12. Here, we present a method for the fractionation and recovery of ssDNA, dsDNA and RNA viral nucleic acids from mixed viral assemblages using hydroxyapatite chromotography.

We describe an efficient method to separate single-stranded DNA, double-stranded DNA and RNA molecules from environmental viral communities. Nucleic acids are fractionated using hydroxyapatite chromatography with increasing concentrations of phosphate-containing buffers. This method permits the isolation of all viral nucleic acid types from environmental samples.

分离单链DNA，双链DNA和RNA病毒使用羟基磷灰石色谱从环境社区

Viruses, particularly bacteriophages (phages), are the most numerous biological entities on Earth1,2.  Viruses modulate host cell abundance and diversity, ...

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. ...

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

重组腺相关病毒载体的生产和滴定

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex (pMHC) proteins expressed on the surface of antigen presenting cells (APCs). The TCR complex is composed of the ligand binding TCR&alpha;&beta; heterodimer that associates non-covalently with CD3 dimers (the &#949;&delta; and &#949;&gamma; heterodimers and the &zeta;&zeta; homodimer)1. Upon engagement of the receptor, the CD3 &zeta; chains are phosphorylated by the Src family kinase, Lck. This leads to the recruitment of the Syk family kinase, Zap70, which is then phosphorylated and activated by Lck. After that, Zap70 phosphorylates the adapter proteins LAT and SLP76, initiating the formation of the proximal signaling complex containing a large number of different signaling molecules2. 
The formation of this complex eventually results in calcium and Ras-dependent transcription factor activation and the consequent initiation of a complex series of gene expression programs that give rise to T cell differentiation2. TCR signals (and the resulting state of differentiation) are modulated by many other factors, including antigen potency and crosstalk with co-stimulatory/co-inhibitory, chemokine, and cytokine receptors 3-4. Studying the spatial and temporal organization of the proximal signaling complex under various stimulation conditions is, therefore, key to understanding the TCR signaling pathway as well as its regulation by other signaling pathways.
One very useful model system to study signaling initiated by the TCR at the plasma membrane in T cells is glass-supported lipid bilayers, as described previously5-6. They can be utilized to present antigenic pMHC complexes, adhesion, and co-stimulatory molecules to T cells-serving as artificial APCs. By imaging the T cells interacting with the lipid bilayer using total internal reflection fluorescence microscopy (TIRFM), we can restrict the excitation to within 100 nm of the space between the glass and the cell surface 7-8. This allows us to image primarily the signaling events occurring at the plasma membrane. As we are interested in imaging the recruitment of signaling proteins to the TCR complex, we describe a two-camera TIRF imaging system wherein the TCR, labeled with fluorescent Fab (fragment antigen binding) fragments of the H57 antibody (purified from hybridoma H57-597, ATCC, ATCC Number:HB-218) which is specific for TCR&beta;, and signaling proteins, tagged with GFP, may be imaged simultaneously and in real time. This strategy is necessary due to the highly dynamic nature of both the T cells and of the signaling events that are occurring at the TCR. This imaging modality has allowed researchers to image single ligands 9-11 as well as recruitment of signaling molecules to activated receptors and is an excellent system to study biochemistry in-situ12-16.

The compartmentalization of proteins either within the plasma membrane or into intracellular locations is one regulatory mechanism that can greatly influence signaling outcomes; hence, to understand signaling it is important to study the spatial and temporal behavior of the proteins involved. We describe here a TIRF microscopy based system to study signal transduction in T cells, but is broadly applicable.

一个实时，同步成像的TCR和其相关的信号转导蛋白的T​​IRF显微镜技术

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

Phage Phenomics: Physiological Approaches to Characterize Novel Viral Proteins

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage sequences share no homology to current annotated proteins. As a result, a large proportion of phage genes are annotated as hypothetical. This reality heavily affects the annotation of both structural and auxiliary metabolic genes. Here we present phenomic methods designed to capture the physiological response(s) of a selected host during expression of one of these unknown phage genes. Multi-phenotype Assay Plates (MAPs) are used to monitor the diversity of host substrate utilization and subsequent biomass formation, while metabolomics provides bi-product analysis by monitoring metabolite abundance and diversity. Both tools are used simultaneously to provide a phenotypic profile associated with expression of a single putative phage open reading frame (ORF). Representative results for both methods are compared, highlighting the phenotypic profile differences of a host carrying either putative structural or metabolic phage genes. In addition, the visualization techniques and high throughput computational pipelines that facilitated experimental analysis are presented.

Here, we present phenomic approaches for the functional characterization of putative phage genes. Techniques include a developed assay capable of monitoring host anabolic metabolism, the Multi-phenotype Assay Plates (MAPs), in addition to the established method of metabolomics, capable of measuring effects to catabolic metabolism.

噬菌体型组学:生理途径来表征新型病毒蛋白

Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

Early Viral Entry Assays for the Identification and Evaluation of Antiviral Compounds

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic approach to identify and evaluate antiviral agents that target specific steps of the viral entry. In this report, the methods of examining viral inactivation, viral attachment, and viral entry/fusion as antiviral assays for such purposes are described, using hepatitis C virus as a model. These assays should be useful for discovering novel antagonists/inhibitors to early viral entry and help expand the scope of candidate antiviral agents for further drug development.

Here, we present a protocol that examines specific steps of the viral entry to identify and evaluate novel antiviral agents.

早期的病毒进入测定的鉴定和抗病毒化合物的评价

Cell-based systems are useful for discovering antiviral agents. Dissecting the viral life cycle, particularly the early entry stages, allows a mechanistic ...

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. burnetii DNA in cell cultures and clinical samples. PCR requires specialized equipment and extensive end user training, and therefore, it is not suitable for routine work especially in a resource-constrained area. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect the presence of C. burnetii in patient samples. This method is performed at a single temperature around 60 &#176;C in a water bath or heating block. The sensitivity of this LAMP assay is very similar to PCR with a detection limit of about 25 copies per reaction. This report describes the preparation of the reaction using lyophilized reagents and visualization of results using hydroxynaphthol blue (HNB) or a UV lamp with fluorescent intercalating dye in the reaction. The LAMP reagents were lyophilized and stored at room temperature (RT) for one month without loss of detection sensitivity. This LAMP assay is particularly robust because the reaction mixture preparation does not involve complex steps. This method is ideal for use in resource-limited settings where Q fever is endemic.

The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

We described here a simple loop-mediated isothermal amplification (LAMP) method using lyophilized reagents for the detection of C. burnetii in patient samples.

冻干环介导等温扩增试剂的检测中的发展<em&gt;贝氏柯克斯体</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional methods to measure NA inhibiting (NI) antibody titers are not practical for routine serology. This protocol describes the enzyme-linked lectin assay (ELLA), a practical alternative method to measure NI titers that is performed in 96 well plates coated with a large glycoprotein substrate, fetuin. NA cleaves terminal sialic acids from fetuin, exposing the penultimate sugar, galactose. Peanut agglutinin (PNA) is a lectin with specificity for galactose and therefore the extent of desialylation can be quantified using a PNA-horseradish peroxidase conjugate, followed by addition of a chromogenic peroxidase substrate. The optical density that is measured is proportional to NA activity. To measure NI antibody titers, serial dilutions of sera are incubated at 37 &#176;C O/N on fetuin-coated plates with a fixed amount of NA. The reciprocal of the highest serum dilution that results in &#8805;50% inhibition of NA activity is designated as the NI antibody titer. The ELLA provides a practical format for routine evaluation of human antibody responses following influenza infection or vaccination.

We describe the enzyme-linked lectin assay (ELLA) for measuring influenza neuraminidase (NA)-inhibition antibody titers in sera. The assay uses peanut agglutinin to quantify galactose residues that become accessible when NA removes sialic acid from fetuin-coated, 96-well plates.

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

病毒 DNA 的纯化及相关病毒和细胞蛋白的鉴定

摘要

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此视频中的章节

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

分离单链DNA，双链DNA和RNA病毒使用羟基磷灰石色谱从环境社区

从核衣壳的病毒DNA的制备

重组腺相关病毒载体的生产和滴定

一个实时，同步成像的TCR和其相关的信号转导蛋白的TIRF显微镜技术

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应

噬菌体型组学:生理途径来表征新型病毒蛋白

早期的病毒进入测定的鉴定和抗病毒化合物的评价

冻干环介导等温扩增试剂的检测中的发展

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

病毒 DNA 的纯化及相关病毒和细胞蛋白的鉴定

摘要

探索更多视频

此视频中的章节

使用泛病毒基因芯片检测（Virochip）屏幕病毒病原体的临床标本

分离单链DNA，双链DNA和RNA病毒使用羟基磷灰石色谱从环境社区

从核衣壳的病毒DNA的制备

重组腺相关病毒载体的生产和滴定

一个实时，同步成像的TCR和其相关的信号转导蛋白的T​​IRF显微镜技术

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应

噬菌体型组学:生理途径来表征新型病毒蛋白

早期的病毒进入测定的鉴定和抗病毒化合物的评价

冻干环介导等温扩增试剂的检测中的发展

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

一个实时，同步成像的TCR和其相关的信号转导蛋白的TIRF显微镜技术