这项研究得到了 nih/NIAID AI112423 和 nih/研究院 GM113885 给本杰明. 陈和 nih/NIAID K08-AI120806 的资助。我们想感谢在西奈山院长的流式细胞术核心的伊坎医学院。

1. 目标细胞线的生成
注意: 此步骤是可选的;如果使用现有的 RG 目标单元格行, 请从步骤2开始。
<ol><li>Cotransfect 70% 汇合 10 cm 板293T 细胞11 [在10毫升 Dulbecco 的被改进的老鹰的媒介 (DMEM) 与10% 胎牛血清 (血清)] 与 pMSCV-loxp-dsRed-loxp-eGFP-普洛 WPRE10和 pCL-10A112 (包装质粒) 在一个1:1 比例 (20 µg 总) 使用钙基转染13。</li>
	<li>收获10毫升的病毒上清 48 h 后转染通过吹打介质从板块成15毫升圆锥管和离心管在 3000 x g 5 摄氏度, 以颗粒任何细胞碎片。</li>
	<li>将0.45 µm 过滤器连接到10毫升注射器上。离心后, 取整个上清液并装入注射器。把样品通过进入一个干净的15毫升管。</li>
	<li>整除过滤的病毒上清到适当的大小 (通常, 0.5–1毫升整除数)。这些可以立即在下一步使用, 或可以冻结在-80 摄氏度和存储。</li>
	<li>使用50–100µL 的病毒上清液感染细胞线的选择, 在一个96井板。文化细胞在37°c 与 5% CO2。RFP 信号应在24小时内出现在荧光显微镜下 (40X 放大, 532 nm 激发, 588 nm 发射), 但可能需要多达 72 h。 注意: 在这些实验中, Jurkat 细胞被使用, 但其他类型也可以使用。</li>
	<li>通过设置一系列10口井并将嘌呤霉素添加到每个嘌呤霉素, 选择转基因细胞, 将至少1的未处理好的控制 (使用从0.5 µg/毫升到5µg/毫升的浓度范围); 这可能会因每个单元格类型而异)。监测细胞的生存能力 (细胞裂解将发生在没有嘌呤霉素抵抗的细胞中) 在数天的过程中与未经治疗的控制相比, 并使用嘌呤霉素集中, 只有 RFP 表达细胞生存。 注: 选择在转染细胞存活时 untransfected 细胞被杀死的浓度。</li>
	<li>使用单细胞流式细胞仪排序或限制稀释 (见步骤 1.8–1.10), 培养从单细胞1发展成克隆细胞线 (这可能需要几个星期的成长)。</li>
	<li>如果使用稀释方法, 用 hemocytometer 计数细胞, 并将样品稀释到大约500细胞/毫升。</li>
	<li>在一个96井板, 吸管50µL 细胞稀释成50µL 媒体 [罗斯威尔公园纪念研究所 (RPMI) 培养基与10% 血清和2% 青霉素-链霉素, 如果使用 Jurkat 细胞], 并执行1:1 串行稀释到11井, 包含50µL 的媒体。为达到最佳效果, 请至少执行10个复制操作。</li>
	<li>通过显微镜 (40X) 在组织培养孵化器 (37 °c 与 5% CO2) 或大约4周, 监测细胞生长。选择从最低的嘌呤霉素浓度与生长 (如显微镜, 40X) 的克隆文化的文化。</li>
	<li>冷冻整除数由离心20毫升的文化 (50万细胞/毫升, 计数与 hemocytometer) 在 800 x g 5 分钟在23°c 和 resuspending 它在500µL 的血清与10% 亚砜。把它放在-80 摄氏度使用一个细胞冻结容器, 然后储存在液氮。</li>
</ol>2. 细胞对细胞病毒的传播
<ol><li>
靶细胞的制备
	<ol>
<li>解冻一个500µL 瓶 Jurkat RG 记者细胞通过放置到一个37°c 水浴。将细胞从瓶子中注入10毫升的 RPMI 完全介质, 然后将混合物在 800 x g处离心, 在23摄氏度时为5分钟。并用重悬在一个 T-75 瓶中的20毫升 RPMI 完全培养基中的颗粒。隔夜孵化烧瓶 (37 °c 和 5% CO2)。 注: RPMI 完整培养基包含 RPMI 1640, 10% 血清, 2 毫米 l-谷氨酰胺, 100 单位/毫升青霉素, 100 毫克/毫升链霉素。</li>
		<li>第二天, 添加0.5 µg/毫升的嘌呤霉素 (1 µL 2 毫克/毫升的股票每8毫升的媒体)。培养细胞, 保持200,000–800,000 细胞/毫升的密度 (通过hemocytometer 计数)。</li>
		<li>在建立转移化验前的前一天, 将细胞分解为200,000–400,000 细胞/毫升, 在含有0.5 µg/毫升嘌呤霉素的新鲜 RPMI 培养基中。</li>
	</ol>
</li>
	<li>
供体细胞的制备
	<ol>
<li>解冻 untransduced Jurkat 细胞和培养他们在一个 T-75 瓶与20毫升 RPMI 完全培养基 (如步骤2.1 所述), 保持一个密度的200,000–800,000 细胞/毫升。</li>
		<li>离心750万细胞 (800 x g为5分钟) 和并用重悬他们在120µL 的 nucleofection 溶液 V 与补充 (参见材料表)。将细胞转移到电穿孔试管, 加入4.5 µg 地球复兴社区1的 DNA。染细胞 (通过以电穿孔为基础的方法, 看到材料表) 使用适当的程序 (如S-18), 然后立即转移到3毫升的 RPMI 培养基 (10% 血清)。</li>
		<li>允许细胞恢复通过孵化他们隔夜在37°c 与 5% CO2。</li>
		<li>第二天早晨, 离心细胞在 800 x g 5 分钟在23°c 和并用重悬他们在3毫升的 RPMI 完全培养基, 允许他们 2 h 恢复在37°c (5% CO2), 然后继续进行化验。</li>
	</ol>
</li>
	<li>
共同文化
	<ol>
<li>用 hemocytometer 计数细胞, 然后向下旋转5万个细胞 (800 x g 5 分钟, 23 °c), 每井 (包括捐献者和靶细胞) 进行测定。Resuspending 1 x 106细胞/毫升在 RPMI 完成没有嘌呤霉素。</li>
		<li>在一个板块, 增加25µL 的捐献细胞悬浮在每个井;在另一个板块中, 添加25µL 的目标细胞。</li>
		<li>对每个井, 添加25µL 的测试化合物稀释在 RPMI 完全培养基在适当的浓度。用该化合物孵育供体和靶细胞30分钟。 注意: 每一种药物的浓度都会有所不同。如果它有一个已知的 IC50, 使用它作为一个起点, 滴定上面和下面。如果 IC50是未知的, 准备一个200µM 库存的最后浓度为10µM。</li>
		<li>预处理后, 将供体细胞与靶细胞结合, 将一个板块的内容吹打到另一个盘子中, 在37摄氏度的组织培养孵化器中孵化出40小时的细胞, 5% CO2。</li>
	</ol>
</li>
	<li>
流式细胞仪
	<ol>
<li>将多聚甲醛 (粉煤灰) 添加到每个井中, 最终浓度为2%。 注: 对于16% 库存解决方案, 这是14µL 每100µL 井。</li>
		<li>注意: 粉煤灰对暴露的皮肤和眼睛有害。戴上实验室大衣、手套和安全护目镜, 并在安全罩中处理库存解决方案。</li>
		<li>阅读 mCherry 和 FITC 通道的流式细胞仪上的单元格。 注意: 有可能看到在感染细胞中表达 GFP 以上背景的细胞与5–20%。未感染的细胞。</li>
		<li>通过荧光显微镜 (40X) 在 gfp 特定通道上可视化 gfp 信号 (带有 400 nm 的励磁过滤器和 508 nm 的发射过滤器)。</li>
	</ol>
</li>
</ol>3. 无细胞病毒感染的替代议定书
<ol><li>
靶细胞的制备
	<ol>
<li>通过放入37°c 水浴, 解冻一个500µL 瓶 Jurkat RG 记者细胞。将细胞从瓶子中注入10毫升的 RPMI 完全培养基, 然后离心 RPMI 完全培养基在 800 x g 5 分钟在23摄氏度。并用重悬在一个 T-75 瓶中的20毫升 RPMI 完全培养基中的颗粒。隔夜孵化烧瓶 (37 °c 和 5% CO2)。</li>
		<li>第二天, 添加0.5 µg/毫升的嘌呤霉素 (1 µL 2 毫克/毫升的股票每8毫升的媒体)。培养细胞, 保持200,000–800,000 细胞/毫升的密度 (通过hemocytometer 计数)。</li>
		<li>在建立转移化验前的前一天, 将细胞分解为200,000–400,000 细胞/毫升, 在含有0.5 µg/毫升嘌呤霉素的新鲜 RPMI 培养基中。</li>
	</ol>
</li>
	<li>
无细胞病毒的生产
	<ol>
<li>染70% 汇合 10 cm 板293T 细胞11 (在10毫升 DMEM 与10% 血清) 与5µg 地球复兴社区1质粒使用钙基转染13。 注: 这一比例将产生10毫升的病毒, 这是足够的约四96井板, 根据效率的转染。</li>
		<li>通过将介质从盘子中吹打到15毫升圆锥管中, 并离心在 3000 x g处为5分钟, 将所有细胞碎片全部切片, 以48小时收割整个病毒上清液。</li>
		<li>将0.45 µm 过滤器连接到10毫升注射器上。离心后, 取整个上清液并装入注射器。把样品通过进入一个干净的15毫升管。</li>
		<li>使用病毒立即感染目标细胞 (步骤 3.3) 或冻结并将病毒存储在-80 摄氏度 (在使用前解冻样品)。</li>
	</ol>
</li>
	<li>
感染
	<ol>
<li>使用 hemocytometer 计算 RG Jurkat 目标单元格 (从步骤 3.1.3), 并在 800 x g处向下旋转5万个目标单元格5分钟, resuspending 1 x 106细胞/mL 中的细胞, RPMI 完全培养基中没有嘌呤霉素。</li>
		<li>在一个板块, 添加25µL 的 RG Jurkat 目标细胞的每一个井;在一个单独的板块, 添加25µL (2 ng) 病毒每一个井。</li>
		<li>对每一个井, 添加25µL 的测试化合物, 稀释在介质中适当的浓度。用化合物孵育细胞和病毒30分钟。 注: 测试浓度会因药物而异。如果它有一个已知的 IC50, 使用它作为一个起点, 滴定上面和下面。如果未知, 准备一个200µM 的股票培养基, 最终浓度为10µM。</li>
		<li>预处理后, 将病毒/复合物的全部内容 (50 µL) 从水井添加到目标细胞, 并在37摄氏度与 5% CO2的组织培养孵化器中孵化一切40小时。</li>
	</ol>
</li>
	<li>
流式细胞仪
	<ol>
<li>将粉煤灰添加到每个井中, 最终浓度为2%。 注: 对于16% 库存解决方案, 这是14µL 每100µL 井。</li>
		<li>阅读 mCherry 和 FITC 通道的流式细胞仪上的单元格。gfp 信号也可以通过荧光显微镜在 gfp 通道上进行可视化。 注: 一旦可以期望看到5–20% 的细胞表达 GFP 以上背景的感染细胞vs。未感染的细胞。</li>
	</ol>
</li>
</ol>

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</ol>

作者没有什么可透露的。

地球复兴社区的实验证明对筛选可能抑制病毒复制融合步骤的药物候选者非常有用。在执行此检测时, 获得良好信号的最重要步骤类似于大多数病毒感染化验。第一个关键步骤是产生高质量的病毒的高滴度。这一步要求293T 细胞频繁传代 (至少每48小时 1x), 这样它们就不会变成 overconfluent 并聚集在一起。此外, 它可能值得尝试不同的转染方法, 因为一些研究人员发现, 磷酸钙转染提供了更好的结果, 而...

新的抗逆转录病毒疗法的需要, 促使开发高通量的屏幕抑制剂的 HIV-1 进入。地球复兴社区报告的研究是为了识别细胞-细胞感染系统融合步骤中病毒进入的抑制剂, 具体测量与宿主细胞膜1的病毒膜融合。研究了一种新的抑制剂的筛选, 具体表现在 HIV-1 感染的早期阶段, 直至病毒膜融合点。测量细胞感染的一个挑战是最初的接种含有受感染的供体细胞和 ininfected 靶细胞, 因此测量新的感染很难从输入细胞中辨别出来。理想的系统将涉及一个外源基因标记, 可诱导的目标细胞感染的启动, 并没有出现在捐献细胞。一种流行的病毒含量混合检测基于病毒蛋白-酶融合, 砰-Vpr, 可以有效, 但有限制高通量, 细胞筛选2。这种检测包括一个β-酶 (砰) 报告基因, 融合到 HIV-1 Vpr, 包装成病毒, 并交付到靶细胞后, 病毒膜融合。基质 CCF2-AM 被装入靶细胞的细胞质中, 在卵裂时进行荧光转移。CCF2-AM 基板成本高昂, 可用于高吞吐量屏幕。为了区分供体和靶细胞, 有必要对目标种群进行染色标记。最后, 该协议需要几个洗涤和孵化步骤, 这可能是繁重和昂贵的测试大量的化合物。
以地球复兴社区为解决这些问题的方法, 开发了高通量的细胞间融合抑制剂筛选。此系统不需要将基板加载到单元格中。该检测也可用于无细胞研究, 并适用于其他病毒融合研究使用伪类型 HIV-1 地球复兴社区病毒粒子。艾滋病病毒的细胞融合检测可以测量病毒的 HIV-1 蛋白介导的细胞融合, 然而这些不使用实际的病毒粒子作为地球复兴社区的实验3,4,5。这种检测可以用流式细胞术或荧光显微镜阅读。它已成功地用于筛选 FDA 图书馆以及一个小图书馆的嘌呤抑制剂1,6。其他调查人员还通过一种经过改进和优化的高通量融合检测发现了 HIV-1 融合中的嘌呤抑制剂, 报告将病毒封装的β-酶转移到细胞质7、8.
地球复兴社区病毒的设计与 iGFP 病毒类似的方法, 插入 recombinase 之间的基质和衣壳, 两侧由 HIV-1 蛋白酶网站9。当 HIV-1 蛋白酶在新生的病毒微粒中激活时, 该酶是作为插入在 polyprotein 中的前驱体而制成的。因此,通过病毒膜融合过程将蛋白酶活化的 HIV-1 粒子的含量传递到目标细胞中, 该方法的交付是依赖的。这里提供了两个版本的协议。首先使用 HIV-1 感染的人群感染靶细胞, 研究 HIV 的直接细胞间传播。第二个版本使用无细胞病毒来研究无细胞病毒感染。细胞对细胞的透射检测需要7天的时间完成从细胞解冻的当天, 或5天, 如果细胞已经解冻和传代。如果细胞需要解冻, 如果细胞解冻和传代, 可以在5天内进行无细胞感染检测。还可以使用 Clevers 实验室10创建的质粒, 在所需的单元格类型中生成一个 rg (红到绿) 目标单元格线 (如果未使用现有的 rg 目标单元格线)。建议在本试验中采用病毒和病毒表达细胞进行适当的生物安全防护。我们在 BSL2+ 组织培养设施中进行这种检测的传染性部分。细胞固定后, 可在标准流式细胞仪和显微术中进行分析。
在这里, 我们描述了这种检测的应用, 以筛选新的化合物, 抑制 HIV-1 病毒膜融合 (图 1)。嘌呤受体是亲炎症介质。我们的实验室已经证明, 非选择性的嘌呤受体抑制剂作为 HIV-1 病毒膜融合的抑制剂6。我们报告说, 这种检测在高通量的利用可以识别新的 HIV-1 病毒膜融合抑制剂。我们证明, 嘌呤类受体抑制剂是一种新型的 HIV-1 病毒膜融合抑制剂。

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)</td><td>Sigma Aldrich</td><td>D5546</td><td>Media for 293 Cells</td></tr><tr><td>RPMI-1640</td><td>Sigma Aldrich</td><td>R0883</td><td>Media for Jurkats</td></tr><tr><td>Fetal Bovine Serum Albumin</td><td>Gibco</td><td>16140-071</td><td>Serum for 293 Cells</td></tr><tr><td>Cosmic Calf Serum</td><td>Hyclone</td><td>SH30087.03</td><td>Serum for Jurkat Cells</td></tr><tr><td>Hyclone Pennecillin Streptomycin solution</td><td>GE Healthcare Life sciences</td><td>SV30010</td><td>Penn/Strep used in both media</td></tr><tr><td>T75 Flasks</td><td>Corning</td><td>3073</td><td>Used for Culture of Jurkat Cells</td></tr><tr><td>10 cm Tissue Culture Plates</td><td>Corning</td><td>430167</td><td>Used for Culture of 293 Cells</td></tr><tr><td>96 Well Plates (tissue culture Treated)</td><td>Corning</td><td>3595</td><td>Used for fusion assay</td></tr><tr><td>Polyjet Transfection Reagent</td><td>Signagen</td><td>SL100688</td><td>Used to transfect 293 Cells</td></tr><tr><td>Dulbecco&#039;s Phosphate Buffered Saline</td><td>Sigma Aldrich</td><td>D8537-100ML</td><td>Used in wash steps</td></tr><tr><td>Hyclone Trypsin Protease</td><td>GE Healthcare Life sciences</td><td>SH30042.01</td><td>For Trypsinization of 293 cells</td></tr><tr><td>Amaxa Cell Line Nucleofector Kit V</td><td>Lonza</td><td>VACA-1003</td><td>For Nucleofection of Jurkats</td></tr><tr><td>Ficoll-Paque plus</td><td>GE Healthcare Life sciences</td><td>17144002</td><td>For Nucleofection of Jurkats</td></tr><tr><td>Serological Pipettes</td><td>Fisher Brand</td><td>13-678-11E</td><td>For all tissue culture</td></tr><tr><td>Pipettor Tips</td><td>Denville Scientific</td><td>P3020-CPS</td><td>For all tissue culture and liquid handling steps</td></tr><tr><td>Millex Syringe Filter (0.45 &amp;mu;m)</td><td>Millipore</td><td>SLHA033</td><td>For filtration of virus</td></tr><tr><td>BD Slip Tip Sterile syringes</td><td>BD Diagnostics</td><td>309656</td><td>For filtration of virus</td></tr><tr><td>Amaxa Nucleofector</td><td>Lonza</td><td>2b</td><td>for Nucleofection of Jurkats (various models available)</td></tr><tr><td>BD Fortessa Flow Cytometer</td><td>BD Biosciences</td><td></td><td>for flow cytometry analyss of samples</td></tr><tr><td>Tissue Culture Hood</td><td>Various models</td><td>Fortessa 2</td><td></td></tr><tr><td>pMSCV-loxp-dsRed-loxp-eGFP-Puro-W PRE</td><td>Addgene</td><td>32702</td><td>&lt;strong&gt;Koo BK&lt;em&gt; et al.&lt;/em&gt;&lt;/strong&gt; Controlled gene expression in primary Lgr5 organoid cultures. Nature Methods &lt;strong&gt;9:&lt;/strong&gt;81-83 (2011).</td></tr><tr><td>pCL10a1</td><td>Novus Bio</td><td>NBP2-29542</td><td>&lt;strong&gt;Naviaux, RK, Costanzi, E, Haas, M and Verma, I&lt;/strong&gt;. The pCL vector system: Rapid production of helper-free, high titer, recombinant retroviruses. Journal of Virology &lt;strong&gt;70&lt;/strong&gt;: 5701-5705 (1996)</td></tr><tr><td>Gag-iCre</td><td>Benjamin Chen Lab</td><td></td><td>&lt;strong&gt;Esposito AM&lt;em&gt; et al.&lt;/em&gt; &lt;/strong&gt;A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry. Virology &lt;strong&gt;490:&lt;/strong&gt;6-16 (2016).</td></tr></tbody></table>

a high-throughput cre-lox activated viral membrane fusion assay to identify inhibitors of hiv-1 viral membrane fusion

本试验旨在通过流式细胞术或荧光显微镜检测绿色荧光蛋白 (GFP) 的表达, 具体报告 HIV-1 融合。一个 HIV-1 报告病毒 (HIV-1 地球复兴社区) 是通过插入 recombinase 到 HIV-1 基因组之间的母体和外壳蛋白的 polyprotein。这导致了 recombinase 的包装成病毒微粒, 然后被释放入目标细胞线稳定地表达 recombinase 激活的红色荧光蛋白 (RFP) 到 GFP 交换盒。在基本状态下, 此卡带只表示 RFP。在 recombinase 进入目标细胞后, RFP 由 loxP 站点两侧切除, 导致 GFP 表达。该方法可用于检测无细胞和细胞对细胞感染系统中的任何病毒进入抑制剂 (特别是融合步骤), 并被用来鉴定一类嘌呤受体拮抗剂作为新的 HIV-1 病毒膜融合抑制剂。

未感染的 RG Jurkat 细胞展示了一个低级别的背景 GFP 信号 (0.3%) 与一个非常强的 RFP 信号 (图 2A, 未感染的专栏)。地球复兴社区的感染导致 GFP 信号 (24.9%) 的增加, 而 HIV-1 融合抑制剂 AMD3100 (20 µM) 的存在抑制了这一信号的发展, 使其下降到未感染的背景水平 (0.34%)。当使用反向转录抑制剂 AZT (2 µM) 的后融合事件抑制剂时, 信号不受显著影响 (23.5%), 表明该?...

Watch this Scientific Journal Video about A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion at JoVE.com

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

通过流式细胞术或荧光显微镜检测绿色荧光蛋白的表达, 我们描述了以细胞为基础的 HIV-1 融合报告。它可以用来测试病毒进入的抑制剂 (特别是在融合步骤) 在无细胞和细胞对细胞感染系统。

高通量的高效液氧活化病毒膜融合检测 HIV-1 病毒膜融合抑制剂的研究

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

a-high-throughput-cre-lox-activated-viral-membrane-fusion-assay-to

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

6.4K 次观看.  Icahn School of Medicine at Mount Sinai, Immunology Institute. 通过流式细胞术或荧光显微镜检测绿色荧光蛋白的表达, 我们描述了以细胞为基础的 HIV-1 融合报告。它可以用来测试病毒进入的抑制剂 (特别是在融合步骤) 在无细胞和细胞对细胞感染系统。

视频: A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes.
In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

重组融合蛋白在荧光蛋白酶检测平台中的应用及其凝胶内再生

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

科研

生物化学

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV逆转录酶和整合酶抑制剂的快速筛选

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

免疫与感染

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

限制性内切酶克隆方法，以评估<em&gt;在体外</em&gt; HIV-1 C亚型的Gag-MJ4嵌合病毒的复制能力

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

病毒融合动力学测量方法在单粒子能级

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

生物学

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

HIV-1的三聚体包膜糖蛋白的构象评估使用基于细胞的ELISA检测

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

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 ...

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune system and is not targeted by the current antiretroviral therapy (cART). Transcription and splicing have been shown to reinforce HIV-1 latency in resting CD4+ T cells. Reversal of latency by the use of latency reversal agents (LRAs) in the &#34;shock and kill&#34; approach has been studied extensively in an attempt to purge this reservoir but has thus far not shown any success in clinical trials due to the lack of development of adequate small molecules that can efficiently perturb this reservoir. The protocol presented here provides a method for reliably and efficiently assessing latency reversal agents (LRAs) on HIV transcription and splicing. This approach is based on the use of an LTR-driven dual color reporter that can simultaneously measure the effect of an LRA on transcription and splicing by flow cytometry. The protocol described here is adequate for adherent cells as well as the cells in suspension. It is useful for testing a large number of drugs in a high throughput system. The method is technically simple to implement and cost-effective. In addition, the use of flow cytometry allows the assessment of cell viability and thus drug toxicity at the same time.

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

hiv 转录和拼接中延迟逆转剂的高通量体外评价

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) formats. The flavivirus non-structural (NS) proteins are thought to co-translationally assemble on the endoplasmic reticulum (ER) membranes, forming the replication complex (RC). The NS3 and NS5 are the most studied enzymes of the RC and constitute the main targets for drug development due to their crucial roles in viral genome replication. NS3 protease domain, which requires NS2B as its cofactor, is responsible for the cleavage of the immature viral polyprotein into the mature NS proteins, whereas NS5 RdRp domain is responsible for the RNA replication. Herein, we describe in detail the protocols used in replicon-based screenings and enzymatic assays to test large compound libraries for inhibitors of the Zika virus (ZIKV) replication. Replicons are self-replicating subgenomic systems expressed in mammalian cells, in which the viral structural genes are replaced by a reporter gene. The inhibitory effects of compounds on viral RNA replication can be easily evaluated by measuring the reduction in the reporter protein activity. The replicon-based screenings were performed using a BHK-21 ZIKV replicon cell line expressing Renilla luciferase as a reporter gene. To characterize the specific targets of identified compounds, we established in-vitro fluorescence-based assays for recombinantly expressed NS3 protease and NS5 RdRp. The proteolytic activity of the viral protease was measured by using the fluorogenic peptide substrate Bz-nKRR-AMC, while the NS5 RdRp elongation activity was directly detected by the increase of the fluorescent signal of SYBR Green I during RNA elongation, using the synthetic biotinylated self-priming template 3&#8242;UTR-U30 (5&#39;-biotin-U30-ACUGGAGAUCGAUCUCCAGU-3&#39;).

In this work, we describe the protocols used in replicon-based and viral enzyme-based assays to screen for inhibitors of Zika virus replication in a high-throughput screening format.

高通量抗病毒检测筛查寨卡病毒复制抑制剂

Antiviral drug discovery requires the development of reliable biochemical and cellular assays that can be performed in high-throughput screening (HTS) ...

Single-Cell Characterization of Calcium Influx and HIV-1 Infection using a Multiparameter Optofluidic Platform

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience comorbidity related to chronic inflammation despite antiretroviral therapy. However, these inflammatory signaling has not been fully characterized. The role of early entry events on the activation of cellular signaling events and downstream gene expression has not been captured at the single-cell level. Here the authors describe a method that applies principles of live-cell fluorescence microscopy to an automated single-cell platform that cultures and images cells over user-customized time courses, allowing for high-throughput analysis of dynamic cellular processes. This assay can track single-cell live fluorescence microscopy of early events that immediately follow HIV-1 infection, notably the influx of calcium that accompanies exposure to the virus and the development of productive infection using a fluorescent reporter virus. MT-4 cells are loaded with a calcium-sensitive dye and cultured in isolated pens on a nanofluidic device. The cultured cells are infected with an HIV-1 reporter virus (HIV-1 NLCI). A fluorescence microscope positioned above the nanofluidic device measures calcium influx over an 8-min time course following acute HIV-1 exposure. HIV-1 productive infection is measured in those same cells over a 4-day interval. Imaging data from these time courses are analyzed to define virus-host receptor interactions and signaling pathway dynamics. The authors present an integrated, scalable alternative to traditional imaging methods using a novel optofluidic platform capable of single-cell sorting, culturing, imaging, and software automation. This assay can measure the kinetics of events under various conditions, including cell type, agonist, or antagonist effect, while measuring an array of parameters. This is the first established method for nanofluidic high-throughput longitudinal single-cell culture and imaging: This technique can be broadly adapted to study cellular signaling kinetics and dynamic molecular interactions.

Here, we present a protocol in which single cells are monitored for acute events and productive HIV-1 infection on a nanofluidic device. Imaging data define virus-host receptor interactions and signaling pathway dynamics. This is the first method for nanofluidic high-throughput longitudinal single-cell culture and imaging to study signaling kinetics and molecular interactions.

使用多参数光伏平台对钙流入和HIV-1感染进行单细胞特征描述

HIV-1 causes a chronic infection that affects more than 37 million people worldwide. People living with human immunodeficiency virus (HIV) experience ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

开源高内涵分析利用自动荧光寿命成像显微镜

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...