这项研究得到了国家卫生研究院 (R01-DE025985) 和美国癌症协会研究学者奖 (RSG-13-383-MPC) 的支持。这项工作还得到了美国农业部 (2015-67015-23268 和 2014-67015-21787) 的赠款和加利福尼亚大学戴维斯分校的新的研究计划赠款的支持。

注意: 此过程中使用的细胞系含有传染性病毒, 要小心, 只在2级 BSL 设施或更高的地方进行。
1. 细胞的制备和维护
<ol><li>培养龙 BCBL-1 细胞 (或其他 KSHV 感染的 PEL 细胞系) 在 RPMI1640 培养基中补充15% 胎牛血清 (FBS) 和1% 青霉素链霉素谷氨酰胺溶液。</li>
	<li>生长在37° c 的细胞与5% 的二氧化碳 (CO2), 确保不断增长和分裂细胞的比率为1:4 每2-4 天。</li>
	<li>每天用光显微镜监测细胞培养, 以确认良好的细胞生长和形态学, 否则建议重新启动细胞培养或相应调整细胞培养条件。</li>
</ol>2. 胸苷同步和溶解诱导
<ol><li>在 10 cm 培养皿中, 平板龙-K-BCBL-1 细胞在新制备的培养基中 (1 x 106 cells/2 介质的 mL)</li>
	<li>加入 200 mm 胸苷 (最终浓度: 2 毫米) 的菜, 混合和孵化为18小时。</li>
	<li>离心机的细胞为5分钟, 在 500 x g. 用无菌1x 磷酸缓冲盐 (PBS) 洗涤细胞, 再次离心, 和重细胞在新准备的媒体。重复共3洗。重在新鲜的准备的媒介。</li>
	<li>允许单元格退出 S 阶段8小时。</li>
	<li>添加胸苷 (最终浓度: 2 毫米), 混合和孵育16小时。</li>
	<li>离心机的细胞为5分钟, 在 500 x g. 用无菌1x 磷酸缓冲盐 (PBS) 洗涤细胞, 再次离心, 和重细胞在新准备的媒体。重复共3洗。重在新鲜的准备的媒介。</li>
	<li>为了诱导病毒活化, 添加12邻十四-phrobol-13-乙酸 (TPA, 最终浓度 20 ng/毫升) 和强力霉素 (DOX, 最终浓度 100 ng/毫升) 的细胞培养, 混合和孵化为 4 h。对于没有 K-的诱导盒的细胞系, 使用 TPA 刺激活化 (最终浓度 20 ng/毫升和丁酸钠 (1 mM))。</li>
	<li>将细胞离心5分钟, 在 500 x g. 用无菌1x 磷酸缓冲盐 (PBS) 冲洗细胞, 再次离心, 并在完整培养基中重细胞。重复共3洗。重在完整的文化媒体。</li>
	<li>允许细胞生长24小时。</li>
</ol>3. 固定和性
注: 在进行前, 确保固定解决方案 (3.7% 甲醛在 DEPC 处理 pbs), 二乙基焦处理的磷酸盐缓冲盐水 (DEPC-pbs), 甘氨酸 DEPC pbs (甘氨酸的最终浓度: 100 毫米) 和 permeabilizating 溶液 (50%丙酮, 50% 甲醇) 准备。通常情况下, 每张幻灯片至少需要50万单元格, 如果需要, 可以进行乘法运算, 特别是在单元格不健康时, 还要包括额外的单元格。
<ol><li>将细胞收集到1.5 毫升的离心管。</li>
	<li>将细胞离心2分钟, 200 x g. 用1毫升无菌 DEPC PBS 冲洗细胞3次。重在1.2 毫升的 DEPC PBS。</li>
	<li>将适当数量的片 (由实验装置确定) 放入6井板的底部。</li>
	<li>吸管200µL 的细胞 DEPC PBS 混合物到每个片, 确保每张幻灯片上至少有50万单元格。允许细胞沉淀2分钟, 并抽出多余的 DEPC PBS 只留下一层薄薄的细胞。在此过程中移除单元格是正常的。 注意: 粉煤灰是有毒的, 穿戴适当的保护。</li>
	<li>小心地添加1毫升的固定解决方案 (3.7% 甲醛在 DEPC PBS), 以每个盖子滑动。允许单元格修复10分钟。</li>
	<li>用 DEPC PBS 清洗片3次。</li>
	<li>小心地添加1毫升的甘氨酸 DEPC PBS (最后浓度的甘氨酸: 100 毫米) 到每个盖子滑动。允许细胞淬火5分钟。</li>
	<li>添加1.5 毫升的 DEPC pbs 和任何地方在一个振动筛5分钟或动摇, 但轻轻地在手1分钟, 吸入过量的 DEPC pbs。注意不要破坏细胞。重复此步骤共3洗。</li>
	<li>小心地将1毫升的 permeabilizing 溶液 (50% 丙酮, 50% 甲醇) 添加到每个盖板上。允许细胞 permeabilize 15 分钟。 注意: 在这一点上, 片可以储存在一个-20 ° c 的冰箱不超过一周的时间。避免冻结或减少在冷冻库中的时间, 因为图像质量会迅速降低。确保在冷冻室中, 细胞保持湿润并覆盖在甲醇/丙酮中, 并确保它们在从冷冻库中取出后, 与 DEPC PBS 适当水化。</li>
	<li>添加1.5 毫升的 DEPC pbs 和任何地方在一个振动筛5分钟或动摇, 但在手轻轻地在1分钟. 吸入过量的 DEPC pbs, 小心不要扰乱细胞。重复此步骤共3洗。</li>
</ol>4. IFA 和 RNA-鱼
注: 关于 RNA 鱼探针标记和准备: 对于959碱基对的 K-含子内含子, 生成31不同的探针分别为20碱基对, 其中含有大约50% 的 GC 含量。探头的工作和储存溶液分别 aliquoted 为100µM 和2.5 µM 浓度。
<ol><li>标签显微镜幻灯片与必要的实验信息或某种可区分的标签。</li>
	<li>用湿纸巾把密封塑料容器的底部排成一行。</li>
	<li>
准备主要抗体溶液 (1.5 µL 的拉娜抗体, 0.75 µL 的 RNA II 抗体, 3 µL 贝克酵母 tRNA, DEPC PBS 30 µL), 并为每片目前的食谱。将30µL 的主抗体溶液放在每个有标记的显微镜幻灯片上。
	<ol>
<li>首先滴定的主要抗体, 并使用最佳的数量。一定要使用纯化的 IgG, 因为血清 (腹水液) 可能含有大量的核糖核酸。</li>
	</ol>
</li>
	<li>将片 (单元格朝向解决方案) 放置在有标记的显微镜幻灯片上, 并将滑块移到容器中, 用湿纸巾。注意不要引入气泡。用塑料包盖住容器的外部。将容器移动到37° c 的恒温箱中1小时。</li>
	<li>准备鱼洗涤缓冲液 (2x 盐水柠檬酸钠缓冲液 (SSC) (最后), 10% 甲酰胺, 90% DEPC dH2O)。</li>
	<li>准备2x 杂交缓冲 (4x SSC, 20% 葡聚糖硫酸盐)。</li>
	<li>准备二次抗体和鱼内含子探针缓冲 (20 µL 2x 杂交缓冲 (最后的 1x Hyb 缓冲), 4 µL 贝克的酵母 tRNA (最后 10%), 4 µL 甲酰胺 (最后 10%), 内含子 K-贸易量探针 (最后 125 nM), 0.8 µg 二级抗体, DEPC dH2高达40µL)。将每张幻灯片所需的食谱相乘。</li>
	<li>在孵育后, 洗涤细胞3次与 DEPC PBS 在一个6井板5分钟每。</li>
	<li>洗涤用鱼洗涤缓冲3次为5分钟每。保持片在洗鱼缓冲液中, 直到完成第二抗体和内含子探针的混合 (步骤 4.7)。</li>
	<li>用于杂交的初级抗体孵化用清洁玻片。</li>
	<li>放置40µL 的二次抗体和鱼内含物探针, 包含溶液在每个玻片上。</li>
	<li>将片与单元格放在幻灯片上, 并将单元侧朝下放在缓冲区顶部, 注意不要引入气泡。</li>
	<li>将幻灯片放入塑料容器中, 底部有一条湿润的纸巾。将塑料容器包装在第一个塑料包装上, 然后用铝箔。</li>
	<li>在37° c 的16-24 小时内用幻灯片孵化容器。</li>
	<li>在孵化后, 小心地从玻璃的边缘滑动片, 并将其放回6个正面朝上的板块, 并在6井盘中洗涤3次的清洗细胞, 每次5分钟。</li>
	<li>用 2x SSC 冲洗细胞2次。</li>
	<li>添加 DAPI (1:1, 000) 到 2x SSC 和允许坐5分钟的室温。</li>
	<li>用 2x SSC 冲洗细胞2次。</li>
	<li>清洁用于杂交的玻片, 并在玻璃片上添加10µL 的安装液。</li>
	<li>将片面向下放置到安装解决方案中。注意不要引入气泡。</li>
	<li>用实验室组织, 轻轻地移除多余的安装解决方案, 注意不要挤压细胞。</li>
	<li>使用指甲油, 应用在片边缘的一个充足的层, 并允许干燥。</li>
	<li>进行荧光显微镜检查。</li>
</ol>5. 3D 荧光显微镜
注意: 虽然协议将随所使用的荧光显微镜系统的类型而异, 但以下步骤将有助于确保获得最佳图像数据以进行定量分析。
<ol><li>预处理固定样品和贴装在防褪色介质中延迟荧光漂白在成像过程中。</li>
	<li>使用高品质的目标 (如 60X 1.42 N. 油浸透镜), 可以捕获整个细胞 (或细胞核) 的视野。</li>
	<li>为每个荧光色通道设置曝光参数 (例如、激发功率、曝光时间), 使用正负控制样本。获取荧光图像。 注: 只包含一个荧光标签的阳性对照样品也应用于确定颜色通道之间的 "串扰" 级别。对于成像固定细胞标本, 荧光激发功率可以减少 (与稍长的曝光时间), 以尽量减少漂白。</li>
	<li>一旦建立了成像协议, 保持相同的曝光参数一致的所有样本的研究, 使图像数据可以得到准确的分析和比较。</li>
	<li>执行采集后图像处理和3D 重建。用 RNA-鱼信号 (红色)、拉娜分子 (绿色) 和核染色质 (适用的地方) 染色 DAPI (蓝色) 识别 K。 注: 细胞细胞核的区域, 在其中 K 和拉娜是共聚的, 因此被认为与病毒转录工厂和复制复合体有关。</li>
</ol>

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本文作者没有金融联系或利益冲突。

该议定书的某些方面可以改变, 以适应不寻常的情况。固定和性缓冲的选择也可以改变。对于固定缓冲器, 甲醛也是有效的, 乙醇可用于性。
推荐用甘氨酸 pbs 猝灭甲醛, 因为它能防止甲醛在没有牛血清白蛋白 (BSA) 的情况下禁用抗体, 但如果片洗得足够彻底, 可以跳过甘氨酸 pbs 的步骤。在协议中, 避免使用牛血清白蛋白作为阻断溶液。在 Izumiya 实验室进行的试验研究显示, rna-鱼?...

越来越清楚的是, 原子核的时空组织在真核生物中调节微调基因表达中起着重要的作用。大多数组织特异性基因分布在许多染色体上, 需要同步调节, 以协调的方式响应特定的刺激1。细胞构建活性染色质中枢 (乙酰胆碱) 是一种将基因及其顺式调节成分汇集到特定核空间的方法1。
各种研究也表明, 尽管细胞核看起来非常致密和粘性, 但生物活性分子可以通过扩散2快速地穿过细胞核。由于这种短暂的性质, 大多数 DNA 结合蛋白 ' 跳跃 ' 从绑定站点到绑定站点, 感觉他们的方式周围的核空间, 这使得一个高度适应和多才多艺的核2。
尽管细胞核内的生物分子有这种动态行为, 但没有膜的核组织 (但不限于) 核仁、卡哈尔体和早幼粒细胞白血病核体 (PML) 仍然存在。它是通过各种机制, 如串联 DNA 重复 (核仁), rRNA (核仁) 和结构蛋白, 如 coilin (卡哈尔体) 或 pml 蛋白 (pml-NB), 持有这些构造一起3,4,5.这些结构和其他会众, 如转录工厂, 作为脚手架, 不仅增加了所需成分的局部浓度, 而且还调节了蛋白质和核酸的组成, 最终形成有效蜂窝功能的中心站点6。
对研究表观遗传学的研究人员来说, 核结构的形成提供了大量的信息。从病毒学的角度来看, 重新激活潜在感染病毒, 如 KSHV, 极大地改变了细胞核的景观和核酶的分布, 将转录主要从细胞基因转移到病毒基因, 最终生成功能完备的病毒子代7,8。KSHV 如何操纵细胞基因表达机制来促进病毒基因表达？这些信息也可以揭示颞细胞基因调控机制。
和所有其他疱疹一样, KSHV 有两个生命周期称为分解复制和延迟。KSHV 主要驻留在潜伏期阶段, 其中的大多数病毒基因表达是沉默的, 除了潜伏期相关基因9,10。在潜伏期 KSHV 产生潜伏期相关的核抗原 (拉娜), 其中组成结合病毒基因组和系病毒染色质的人类染色体11。由于拉娜与病毒基因组的密切关系, 有可能使用 IFA 和 DAPI 染色和定位的病毒 episomes 与寄主染色质。
为了研究 KSHV 在单个 episome 水平上的重新激活以及与感染细胞中其他病毒 episomes 的联系, 已经建立了一种在原位转录病毒染色质的有效方法。因此, 拉娜和 RNAPII IFA 与内含子 rna-鱼结合, 通过生成 rna 鱼探针, 绑定到内含子区域 (确切的探针序列可以发现在 Izumiya 实验室的最新出版物8) 的 KSHV K-贸易协定-关键的病毒蛋白, 是必要和足够的 KSHV 重新激活-它是可能的, 以确定何处转录实际上发生8,11,12,13,14,15.这种内含子 RNA-鱼技术使研究人员能够直观地看到 mRNA 在被剪接之前被转录到细胞质中, 然后被输出到胞浆16,17。
KSHV 可以重新激活的各种化学刺激, 包括佛波酯类, 如 12 O-Tetradeconoyl-佛波-13 醋酸盐 (TPA) 和组蛋白乙酰抑制剂, 如丁酸钠, 另外 KSHV 可以诱发活化的过度表达病毒转录因子, K-贸易协定19。研究人员成功地提高了 KSHV 激活的效率, 在诱导重新激活18之前同步细胞周期。因此, 对于这些特殊的研究, 细胞是同步使用双胸苷块 (协议描述), 并与 TPA 和强力霉素 (Dox) 孵育短时间。Doxycyline 的使用是因为这些实验中所用的细胞系有一种强力霉素诱导的 k-区域化盒, 它是从 cDNA 中克隆出来的, 不包括 k 区域的内含子区。虽然可以使用仅诱导的 k-KSHV 表达式来重新激活它, 但其他研究人员已经证明, 由于各种不同的生物化学因素, k-贸易协定的表达仅仅证明是一个微弱的活化刺激20。通过结合所有这些, 并通过限制药物孵化到短时间内, 一个强大的, 但不是过度人工 KSHV 的重新激活是实现了成像。
在对拉娜、RNAPII、K-的内含子和 DNA 进行标记后, 采用 widefield 反褶积显微镜进行3D 荧光成像。利用成像软件进行处理后, 可以正确评价活性病毒 episomes 的空间分布。利用这一技术, 可以研究关于形成活性染色质中枢和其他核结构的基本性质的核心问题。具有相同的病毒 episomes 在一个单一的细胞, 处理相同的调控元素可能代表一个独特的研究工具, 以加深对时空基因调控机制的理解。
在不同时间点固定的成像多细胞标本的局限性, 以表征一个固有的动态分子过程是, 微妙或小规模的变化, 荧光分布是不被发现或认为微不足道。这是事实, 除非在罕见的情况下, 每个细胞观察显示同样微妙的变化。因此, 有效的病毒转录和其他核结构的完全时空关系不能用固定成像进行严格的评估。为了解决这些技术难题, 最好的方法是对有标记的病毒 episomes 的活细胞进行图像处理, 并随着时间的推移, 跟踪关键细胞酶的位置。

<table><tbody><tr><td>RPMI medium 1650 GlutaMAX</td><td>Gibco by Life Technologies</td><td>61870-036</td><td>Needs to be warmed to 37 &amp;deg;C</td></tr><tr><td>Fetal Bovine Serum</td><td>Omega Scientific Inc.</td><td>FB-11</td><td></td></tr><tr><td>Pen Strep Glutamine (100x)</td><td>Gibco by Life Technologies</td><td>10378-016</td><td></td></tr><tr><td>Thymidine&amp;nbsp;</td><td>Sigma Aldrich</td><td>T9250</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Solution</td><td>Gibco by Life Technologies</td><td>14190-144</td><td></td></tr><tr><td>TPA</td><td>Sigma Aldrich</td><td>P1585</td><td></td></tr><tr><td>Sodium Butyrate&amp;nbsp;</td><td>Sigma Aldrich</td><td>B5887</td><td></td></tr><tr><td>Formaldehyde Solution&amp;nbsp;</td><td>Sigma Aldrich</td><td>252549</td><td>Corrosive, toxic, health hazard</td></tr><tr><td>Diethyl Pyrocarbonate</td><td>Sigma Aldrich</td><td>D-5758</td><td>Acute toxicity&amp;nbsp;</td></tr><tr><td>Glycine</td><td>Sigma Aldrich</td><td>410225</td><td></td></tr><tr><td>Acetone</td><td>Sigma Aldrich</td><td>179124</td><td>Flammable, toxic</td></tr><tr><td>Methanol</td><td>Fisher Chemical&amp;nbsp;</td><td>67-56-1</td><td>Flammable, toxic, health hazard</td></tr><tr><td>Rat monoclonal antibody to KSHV/HHV-8 ORF73</td><td>Advanced Biotechnologies</td><td>13-210-100</td><td></td></tr><tr><td>Anti-RNA Polymerase II Antibody clone CTD4H8</td><td>EMD Milipore</td><td>05-623</td><td></td></tr><tr><td>Ribonucleic acid, transfer from baker&amp;rsquo;s yeast (S. cerevisiae)&amp;nbsp;</td><td>Sigma Aldrich</td><td>9014-25-9</td><td></td></tr><tr><td>Saline sodium citrate buffer 20x (SSC)</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>15557044</td><td></td></tr><tr><td>Formamide&amp;nbsp;</td><td>Sigma Aldrich</td><td>295876</td><td></td></tr><tr><td>Omnipur Dextran Sulfate, 50% solution</td><td>EMD Milipore</td><td>1916C093</td><td></td></tr><tr><td>DAPI (4&amp;rsquo;,6-diamindino-2-phenylindole, Dihydrochloride)</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>D1306</td><td></td></tr><tr><td>slowFade Gold antifade reagent</td><td>Life Technologies</td><td>S36936</td><td></td></tr><tr><td>Micro cover glass 22x22</td><td>Matsunami</td><td>C022221</td><td></td></tr><tr><td>VoloCITY</td><td></td><td>3D imaging software</td><td></td></tr><tr><td>DeltaVision microscope</td><td></td><td></td><td></td></tr><tr><td>Superfrost/Plus microscope slides</td><td>Fisher Scientific</td><td>12-550-15</td><td></td></tr></tbody></table>

functional imaging of viral transcription factories using 3d fluorescence microscopy

众所周知, 基因的时空调控是控制适当基因表达的一个组成部分。因此, 了解在核空间内发生转录的位置和时间, 以及在同一细胞核内感染 episomes 之间的关系是非常宝贵的。在这里, 免疫荧光 (IFA) 和 RNA 鱼已经 combinedto 识别积极转录卡波肉瘤相关的疱疹病毒 (KSHV) episomes。通过染色 KSHV 潜伏期相关的核抗原 (拉娜), 可以找到在细胞核内存在病毒 episomes 的地方。此外, 通过设计 rna 鱼探针来瞄准病毒基因的内含子区域, 这只在生产感染期间表达, 可以找到新生的 rna 转录。利用这种分子探针的组合, 可以可视化大型病毒转录工厂的组装, 分析 KSHV 活化过程中病毒基因表达的空间调节。通过包括抗 rna 聚合酶 ii 抗体染色, 你也可以可视化 rna 聚合酶 ii (RNAPII) 聚集和 KSHV 转录之间的关联在重新激活。

在使用 BCBL-1 细胞的地方, 只有拉娜和 K-区域内 RNA 被染色 (图 1), 才执行了略微删节的协议。这项实验使我们可以检查在哪里积极转录病毒 episomes 是和异质性的反应, KSHV 活化刺激在一个群体的细胞。在图 1中箭头指示的单元格中, 与病毒 episomes 的分布紧密匹配的区域 (与 LANA 标记的) 中的明显的 K-位置荧光, 是接近 KSHV 基因组的?...

Watch this Scientific Journal Video about Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy at JoVE.com

Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

病毒转录工厂是分散的结构, 富含细胞 RNA 聚合酶 II, 以增加病毒基因转录在重新激活。本文介绍了一种通过免疫荧光染色和原位RNA 杂交技术在3D 核空间中主动转录病毒染色质位置的方法。

基于3D 荧光显微镜的病毒转录工厂功能成像

It is well known that spatial and temporal regulation of genes is an integral part of governing proper gene expression. Consequently, it is invaluable to ...

functional-imaging-viral-transcription-factories-using-3d

Research

JoVE Journal

Biology

7.0K 次观看.  University of California, Davis. 病毒转录工厂是分散的结构, 富含细胞 RNA 聚合酶 II, 以增加病毒基因转录在重新激活。本文介绍了一种通过免疫荧光染色和原位RNA 杂交技术在3D 核空间中主动转录病毒染色质位置的方法。

视频: Functional Imaging of Viral Transcription Factories Using 3D Fluorescence Microscopy

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

可视化的细胞与细胞转移的艾滋病毒，艾滋病毒和现场共聚焦显微镜的荧光克隆

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

科研

免疫与感染

Live Cell Imaging of Alphaherpes Virus Anterograde Transport and Spread

Advances in live cell fluorescence microscopy techniques, as well as the construction of recombinant viral strains that express fluorescent fusion proteins have enabled real-time visualization of transport and spread of alphaherpes virus infection of neurons. The utility of novel fluorescent fusion proteins to viral membrane, tegument, and capsids, in conjunction with live cell imaging, identified viral particle assemblies undergoing transport within axons. Similar tools have been successfully employed for analyses of cell-cell spread of viral particles to quantify the number and diversity of virions transmitted between cells. Importantly, the techniques of live cell imaging of anterograde transport and spread produce a wealth of information including particle transport velocities, distributions of particles, and temporal analyses of protein localization. Alongside classical viral genetic techniques, these methodologies have provided critical insights into important mechanistic questions. In this article we describe in detail the imaging methods that were developed to answer basic questions of alphaherpes virus transport and spread.

Live cell imaging of alphaherpes virus infections enables analysis of the dynamic events of directed transport and intercellular spread. Here, we present methodologies that utilize recombinant viral strains expressing fluorescent fusion proteins to facilitate visualization of viral assemblies during infection of primary neurons.

顺行运输Alphaherpes病毒传播的活细胞成像

Advances in live cell fluorescence microscopy  techniques, as well as the construction of recombinant viral strains that  express fluorescent fusion ...

Methodology for the Efficient Generation of Fluorescently Tagged Vaccinia Virus Proteins

Tagging of viral proteins with fluorescent proteins has proven an indispensable approach to furthering our understanding of virus-host interactions. Vaccinia virus (VACV), the live vaccine used in the eradication of smallpox, is particularly amenable to fluorescent live-cell microscopy owing to its large virion size and the ease with which it can be engineered at the genome level. We report here an optimized protocol for generating recombinant viruses. The minimal requirements for targeted homologous recombination during vaccinia replication were determined, which allows the simplification of construct generation. This enabled the alliance of transient dominant selection (TDS) with a fluorescent reporter and metabolic selection to provide a rapid and modular approach to fluorescently label viral proteins. By streamlining the generation of fluorescent recombinant viruses, we are able to facilitate downstream applications such as advanced imaging analysis of many aspects of the virus-host interplay that occurs during virus replication.

A rapid and modular protocol for the generation of recombinant vaccinia viruses expressing fluorescently&#160;tagged proteins simultaneously using the method of transient dominant selection is described here.

有效生成荧光标记疫苗病毒蛋白的方法

Tagging of viral proteins with fluorescent  proteins has proven an indispensable approach to furthering our understanding  of virus-host interactions. ...

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

牛痘病毒记者量化病毒功能在基因表达的各个阶段

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

Temporal Analysis of the Nuclear-to-cytoplasmic Translocation of a Herpes Simplex Virus 1 Protein by Immunofluorescent Confocal Microscopy

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is responsible for the proteasomal degradation of host restrictive factors and the subsequent viral gene activation. ICP0 contains a canonical nuclear localization sequence (NLS). It enters the nucleus immediately after de novo synthesis and executes its anti-host defense functions mainly in the nucleus. However, later in infection, ICP0 is found solely in the cytoplasm, suggesting the occurrence of a nuclear-to-cytoplasmic translocation during HSV-1 infection. Presumably ICP0 translocation enables ICP0 to modulate its functions according to its subcellular locations at different infection phases. In order to delineate the biological function and regulatory mechanism of ICP0 nuclear-to-cytoplasmic translocation, we modified an immunofluorescent microscopy method to monitor ICP0 trafficking during HSV-1 infection. This protocol involves immunofluorescent staining, confocal microscope imaging, and nuclear vs. cytoplasmic distribution analysis. The goal of this protocol is to adapt the steady state confocal images taken in a time course into a quantitative documentation of ICP0 movement throughout the lytic infection. We propose that this method can be generalized to quantitatively analyze nuclear vs. cytoplasmic localization of other viral or cellular proteins without involving live imaging technology.

ICP0 undergoes nuclear-to-cytoplasmic translocation during HSV-1 infection. The molecular mechanism of this event is not known. Here we describe the use of confocal microscope as a tool to quantify ICP0 movement in HSV-1 infection, which lays the groundwork for quantitatively analyzing ICP0 translocation in future mechanistic studies.

免疫荧光共聚焦显微镜对单纯疱疹病毒1蛋白核细胞质转移的时间分析

Infected cell protein 0 (ICP0) of herpes simplex virus 1 (HSV-1) is an immediate early protein containing a RING-type E3 ubiquitin ligase. It is ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

荧光检测病毒RNA<em&gt;原位</em&gt;杂交（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

生物学

A Luciferase-fluorescent Reporter Influenza Virus for Live Imaging and Quantification of Viral Infection

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, studying IAV requires the use of laborious secondary approaches to detect the presence of the virus in infected cells and/or in animal models of infection. This limitation has been recently circumvented with the generation of recombinant IAVs expressing easily traceable fluorescent or bioluminescent (luciferase) reporter proteins. However, researchers have been forced to select fluorescent or luciferase reporter genes due to the restricted capacity of the IAV genome for including foreign sequences. To overcome this limitation, we have generated a recombinant replication-competent bi-reporter IAV (BIRFLU) stably expressing both a fluorescent and a luciferase reporter gene to easily track IAV infections in vitro and in vivo. To this end, the viral non-structural (NS) and hemagglutinin (HA) viral segments of influenza A/Puerto Rico/8/34 H1N1 (PR8) were modified to encode the fluorescent Venus and the bioluminescent Nanoluc luciferase proteins, respectively. Here, we describe the use of BIRFLU in a mouse model of IAV infection and the detection of both reporter genes using an in vivo imaging system. Notably, we have observed a good correlation between the expressions of both reporters and viral replication. The combination of cutting-edge techniques in molecular biology, animal research and imaging technologies, provides researchers the unique opportunity to use this tool for influenza research, including the study of virus-host interactions and dynamics of viral infections. Importantly, the feasibility to genetically alter the viral genome to express two foreign genes from different viral segments opens up opportunities to use this approach for: (i) the development of novel IAV vaccines, (ii) the generation of recombinant IAVs that can be used as vaccine vectors for the treatment of other human pathogen infections.

Influenza A viruses (IAVs) are contagious respiratory pathogens that cause annual epidemics and occasional pandemics. Here, we describe a protocol to track viral infections in vivo using a novel recombinant luciferase and fluorescence-expressing bi-reporter IAV (BIRFLU). This approach provides researchers with an excellent tool to study IAV in vivo.

用于病毒感染活成像和定量的路西酶-荧光报告流感病毒

Influenza A viruses (IAVs) cause human respiratory disease that is associated with significant health and economic consequences. As with other viruses, ...

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

单细胞多重荧光成像，可可视化病毒核酸和蛋白质，并监测 HIV、HTLV、HBV、HCV、寨卡病毒和流感感染

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...

Live-Cell Imaging of Transcriptional Activity at DNA Double-Strand Breaks

DNA double-strand breaks (DSB) are the most severe type of DNA damage. Despite the catastrophic consequences on genome integrity, it remains so far elusive how DSBs affect transcription. A reason for this was the lack of suitable tools to simultaneously monitor transcription and the induction of a genic DSB with sufficient temporal and spatial resolution. This work describes a set of new reporters that directly visualize transcription in live cells immediately after the induction of a DSB in the DNA template. Bacteriophage RNA stem-loops are employed to monitor the transcription with single-molecule sensitivity. For targetting the DSB to a specific gene region, the reporter genes are engineered to contain a single recognition sequence of the homing endonuclease I-SceI, otherwise absent from the human genome. A single copy of each reporter gene was integrated into the genome of human cell lines. This experimental system allows the detection of single RNA molecules generated by the canonical gene transcription or by DNA break-induced transcription initiation. These reporters provide an unprecedented opportunity for interpreting the reciprocal interactions between transcription and DNA damage and to disclose hitherto unappreciated aspects of DNA break-induced transcription.

This protocol presents a new reporter gene system and the experimental setup to detect transcription at DNA double-strand breaks with single-molecule sensitivity.

DNA双链断裂时转录活性的活细胞成像

DNA double-strand breaks (DSB) are the most severe type of DNA damage. Despite the catastrophic consequences on genome integrity, it remains so far ...