此意见得到了美国国立卫生赠款，AI074420 - 02，宝来惠康研究者奖，以BKC Hirschl职业科学家奖由美国国家科学基金会生物光子学科技中心，合作协议PHY012099，以及一个UCD卫生系统研究奖，以TH UCD CTSC NCRR ULRR024146。

使用这种方法是在报告的研究<a href="http://www.sciencemag.org/cgi/content/abstract/323/5922/1743">夜蛾等科学323：1743年至1747年（2009年）</a> 。 1。概述原是感染艾滋病毒的Jurkat细胞作为供体细胞和主要的CD4 + T淋巴细胞受体细胞1,2,3描述的细胞与细胞的艾滋病毒的蔓延。在这些研究中，使用抗体染色的固定细胞中检测到病毒。为了追踪病毒在活细胞中的细胞从细胞的转移，我们利用重组，传染病艾滋病毒的分子克隆称为艾滋病毒 GAG - iGFP 4。它携带绿色荧光蛋白（GFP）的内部插入MA和CA域之间的Gag蛋白。这对艾滋病毒的分子克隆保留而不需要辅助病毒的传染性。从这个克隆的病毒颗粒是化学计量装有绿色荧光蛋白，提供了较强的荧光信号来监测病毒的组装和细胞间的转移。当配合使用的惰性细胞跟踪荧光染料，受体细胞可以从输入供体细胞的歧视，并允许一个可视化的艾滋病毒从被感染的T细胞转移到未受感染的T细胞5的。病毒学突触的形成和病毒从一个细胞转移到另一个，然后可以观察到活细胞，而他们是在一个密封的，透气的成像室培养。 （第一天） 2。艾滋病毒GAG - iGFP转染的Jurkat T细胞的制备<ol><li>通过精心维护2 x 10 5和8 x 10 5细胞/毫升的Jurkat文化传媒（RPMI 1640培养液，10％胎牛血清的浓度细胞的人体的CD4 + T细胞线的Jurkat（ATCC，马纳萨斯，弗吉尼亚州） ， 100单位/ mL青霉素和100μg/ mL链霉素）。成功：文化浓度超过8 × 10 5细胞/ ml的Jurkat细胞的关键，将结果在转染效率的降低。 注：转染到T细胞的艾滋病毒GAG - iGFP前病毒DNA，如下所述，在一个复制能力的人类免疫缺陷病毒的生产的结果。因此，所有的程序都只能由受过训练的实验室工作人员进行认证的生物安全水平2 +或三级生物安全水平（BL2 + / BL3）组织培养室。在成像设施艾滋病毒具有传染性的工作必须由当地机构的生物安全办公室批准。 </li><li>转染质粒的病毒，艾滋病毒使用Amaxa nucleofection方法（龙沙，Walkersville，MD）GAG - iGFP的Jurkats。颗粒5 × 10 6细胞为10分钟，离心150小工吸上清，洗细胞，在无菌磷酸盐缓冲液（PBS）。离心机的细胞和悬浮颗粒97μL预热，nucleofector解决方案包含了制造商的补充V。三年内毒素免费艾滋病毒GAG - iGFP质粒（1微克/μL）μL细胞悬液轻轻混匀。使用程序的S - 18细胞悬液转移到比色皿和nucleofect。立即转移的细胞，以3毫升不含抗生素的培养基预热的Jurkat。 </li><li>为了准备病毒学突触的CD4 +靶细胞​​，我们取得巴菲大衣的人外周血单核细胞，通过使用标准的聚蔗糖 - 帕确（通用电气医疗集团，瑞典乌普萨拉）协议。 CD4 + T细胞，然后产生负面选择使用磁珠分离试剂盒（美天旎生物技术，赤褐色，CA）。这些可能会在5 × 10 6 cells/500冻结媒体μL（90％胎牛血清serum/10％DMSO）等分液氮低温存储。解冻的主要CD4 + T细胞培养在RPMI 10％胎牛血清辅以10单位/ mL的IL - 2，。 </li></ol> （2天） 3。回收的生活与艾滋病毒GAG - iGFP和靶细胞与荧光染料染色的细胞从Jurkats <ol><li>允许Jurkat细胞转染在一夜之间恢复。然后取出离心多聚蔗糖hypaque密度梯度材料的细胞碎片。分配1.5毫升的聚蔗糖帕确15 mL锥形管的底部。轻轻覆盖在5 mL体积的RPMI转Jurkats梯度材料。在室温为20分钟关闭制动离心400 XG的细胞。小心取出的细胞用吸管从媒体/聚蔗糖接口。它们转移到一个新的15 mL锥形管。与RMPI稀释的细胞在10分钟的300 XG总量的15毫升和离心机。重悬浮颗粒在2厘米（6孔板）3毫升对Jurkat文化传媒和返回的细胞组织培养的孵化器。 </li><li>为了区分在细胞与细胞转移实验的供体细胞的靶细胞，我们prelabel的CD4 +与荧光染料的靶细胞。甚至小学CD4 + T细胞被标记ING之前​​使用。 CellTracker和CellTrace染料（Invitrogen公司，卡尔斯巴德，加利福尼亚州），我们有优秀的成功标签的T细胞。虽然染料浓度和孵育时间取决于特定的染料和所使用的细胞类型，必须优化，代表协议如下。佩莱4 × 10 6小学的CD4 + T细胞在10分钟的400 XG纺。 5毫升的PBS重悬在2毫升的PBS清洗细胞。添加CellTracker橙（CMTMR，Invitrogen公司，卡尔斯巴德，加利福尼亚州）至终浓度为1.5微米和孵育在37 ° C加热20分钟。加入8毫升的完整的​​Jurkat媒体和离心机为10分钟400 XG。重悬细胞在3毫升10单位/ mL的IL - 2和在组织培养孵化器放置过夜为辅的完整的媒体。 </li></ol> （3天） 4。监测艾滋病毒的细胞与细胞转移GAG - iGFP使用活细胞成像我们经常使用艾滋病毒GAG - iGFP转染Jurkat细胞转染后48小时。不过，我们已经成功地用作早在24小时细胞，转染后72小时才。 <ol><li>转染后大约48小时，艾滋病毒GAG - iGFP表达Jurkats都计算在内，与CO 洗 2独立媒体（Invitrogen公司，卡尔斯巴德，CA），并悬浮在活细胞成像介质（CO 2个独立的媒体，10％胎牛血清， 100 U / mL青霉素和100μg/ mL链霉素，10个单位/ mL的IL - 2）1-3 × 10 7细胞/ ml的浓度。 </li><li>以类似的方式，清洗和重新悬浮标记的初级CD4 + T细胞在浓度为1-3 × 10 7细胞/毫升在活细胞成像介质。 </li><li>混合艾滋病毒GAG - iGFP转Jurkats主要的CD4 + T细胞在一个1:2或1:3的比例和成一个组织处理文化，透气，microchamber（Ibidi，维罗纳，WI）的负载。例如，20μL艾滋病毒GAG - iGFP转Jurkats，40μL标记的初级CD4 + T细胞混合。装入Ibidi室50μL这种混合物和带插头的密封腔。安全包装封口膜插件/ Ibidi室界面的插头。 </li></ol> 5。显微镜平台的注意事项：旋转盘共焦成像加载Ibidi成像腔与细胞后，我们立即安装到一个倒置光学显微镜（奥林巴斯IX71，谷中心，PA）的设备。 Ibidi室应转移到显微镜，只能由BL2 +训练有素的实验室人员穿着适当的安全服装。虽然活细胞成像通常使用昂贵的孵化室要保持一个稳定的37 ° C的环境，这是一个简单和经济的恒温加热器（400路ASI，Nevtek，威廉斯维尔，弗吉尼亚州）与T型热电偶尖登上旁边适当的温度反馈的样本。我们发现使用CO 2独立的媒体使我们能够保持高的细胞活力，同时避免使用的 CO 2室。以缓冲对温度波动的平台，并在房间里来阻挡背景室内光线，一个黑色的大导流罩披在整个显微镜/加热器设置，创建一个系统周围的绝缘空气口袋。这大大降低了温度变化和相关的温度相关的样本漂移，但需要预热30分钟前安装样品。 6。数据采集​​，传输和图像处理图片是一个60X 1.42 NA油浸目标（UPlanApo氮，奥林巴斯）使用高数值孔径（NA），大大提高了分辨率。要创建三维共聚焦图像，扫描是由一个旋转盘共聚焦单元（CSU - 10，Yokagawa，日本），同时使用800焦点，大大提高扫描速度。赞扬它的速度，CSU - 10是一个高度敏感的电子倍增电荷耦合器件（EMCCD，IXON + 897，安道尔，爱尔兰）相机，以便快速，低成本的光成像，减少了漂白和光毒性的配对。荧光光源，多波长ArKr离子气体激光器（伊诺70C，连贯，圣克拉拉市）前所需的波长（488纳米）和激光功率测量显微镜物镜（&lt;1毫瓦），是由一个选定声光可调谐滤波器（AOTF），（安道尔）。为了进一步降低漂白和延长总成像时间，AOTF的快速（微秒）关闭激光束每次相机处于非活动状态（10-20毫秒），而新收购的图像从CCD（15-30毫秒暴露）到计算机。激光光耦合到旋转盘系统使用一个405/488/568/647四频分色镜（Semrock，罗切斯特，纽约州）。荧光发射过滤内使用一个过滤器轮（Ludl电子产品，霍桑，NY）一个50分之525的带通滤波器（Semrock）装between EMCCD相机和共聚焦旋转盘单位。所有的硬件​​和收购，包括Z -阶段（疯狂的城市实验室，麦迪逊，威斯康星州），安道尔IQ V1.8软件控制。为了最大限度地提高采集速度约为1.2-1.9秒的三维图像栈，我们作物的EMCCD的相机记录区域细胞对邻近地区。此外，在牺牲在Z的一些信息，0.45-0.75微米之间的一个大Z -步可用于加快收购。在这些条件下的成像可以持续长达6个小时 - 使用连续20-60分钟的环节 - 以最小的漂白。总之，每个数据段通常占5-20 GB的空间，是数以万计的图像。为了便于运输和分析大量的数据文件，每次采集设置需要被分解和1 ​​GB TIFF图像文件段使用安道尔IQ V1.8软件出口。从这里，许多优秀的软件程序包可用于图像的分析，如Metamorph，Volocity和ImageJ（我们建议其变化斐济），。 当两个荧光标记被跟踪，我们同时记录他们都没有放慢收购使用“画面分割模式。”由两个不同的ArKr一次激光线（通常为488和568纳米）两项指标很高兴。直接插入前EMCCD相机是分隔两个不同的图像，图像的分离器（OptoSplit二，山，肯特，英国）。每幅图像，然后独立使用相机上曾经创造了“分屏”效应的荧光过滤器（Semrock）和预计端侧的过滤。这将需要人工裁剪和调整后的图像在图像处理。 7。图像处理和数据分析的注意事项我们通常执行与图像分析Volocity（珀金埃尔默，马萨诸塞州沃尔瑟姆，在Macintosh计算机上）（苹果公司）。旋转盘激光共聚焦显微镜图像先调整正确Volocity deconvolved然后漂白，在它们的强度。加格puncta强度测量和跟踪执行Volocity定量模块。对于预先形成的突触相对运动需要计算的图像集，采用自动跟踪​​算法跟踪整个序列的突触按钮。由自动跟踪软件定义的利息地区的人工检验，必须执行，以确认该软件已经正确地跟踪期望的对象。手动跟踪对象与周围物体的对比度太弱，可能是一帧帧的基础上进行。 Volocity软件包允许出口X​​YZ的位置，数量和范围内所需的对象集成的信号。正常化运动突触按钮的中心，突触和速度的跟踪对象的距离计算。 8。代表性的成果在10至15分钟，搅拌，应该能够看到艾滋病毒GAG - iGFP表达Jurkat细胞坚持初级CD4 + T细胞。成像这些结合物，可以评估在受感染的细胞，以及在靶细胞后synaspe加格puncta变动。人们可以观察到靶细胞的GAG - iGFP puncta运动后不久，突触形成。

<ol>
	<li>Blanco, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+level+of+coreceptor-independent+HIV+transfer+induced+by+contacts+between+primary+CD4+T+cells.">High level of coreceptor-independent HIV transfer induced by contacts between primary CD4 T cells.</a> J Biol Chem. 279, 51305-51314 (2004).</li><li>Sourisseau, M., Sol-Foulon, N., Porrot, F., Blanchet, F., Schwartz, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inefficient+human+immunodeficiency+virus+replication+in+mobile+lymphocytes.">Inefficient human immunodeficiency virus replication in mobile lymphocytes.</a> J Virol. 81, 1000-1012 (2007).</li><li>Jolly, C., Kashefi, K., Hollinshead, M., Sattentau, Q. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+cell+to+cell+transfer+across+an+Env-induced,+actin-dependent+synapse.">HIV-1 cell to cell transfer across an Env-induced, actin-dependent synapse.</a> J Exp Med. 199, 283-293 (2004).</li><li>Hubner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+of+human+immunodeficiency+virus+type+1+(HIV-1)+Gag+localization+and+oligomerization+monitored+with+live+confocal+imaging+of+a+replication-competent,+fluorescently+tagged+HIV-1.">Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1.</a> J Virol. 81, 12596-12607 (2007).</li><li>Hubner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+3D+video+microscopy+of+HIV+transfer+across+T+cell+virological+synapses.">Quantitative 3D video microscopy of HIV transfer across T cell virological synapses.</a> Science. 323, 1743-1747 (2009).</li></ol>

我们在这里介绍一个简单的方法，以可视化的艾滋病毒细胞与细胞间的转移。从我们的实验室和其他人最近的研究表明，这可能是T细胞之间传播艾滋病毒的主要模式。这里介绍的方法采用了艾滋病毒的重组形式，被称为艾滋病毒GAG - iGFP，它带有一种基因编码的绿色荧光蛋白标记为核心的结构蛋白，GAG的。由于每个病毒粒子产生的绿色荧光蛋白的水平高，这对艾滋病毒的复制能力的克隆使研究人员能够实时跟踪单个病毒颗粒。关于艾滋病毒的组装和细胞与细胞转移的研究，这种病毒应该特别有用。 艾滋病毒的细胞与细胞转移的转移，是高效的。在三个小时的合作文化，我们日常看到病毒转移到20-30％的原发性T细胞流式细胞仪评估。在活细胞成像定性看到类似的效率。开始后几乎立即开始联合培养的T细胞与未感染的主要病毒感染的T细胞形成突触。有趣的，并非所有的主要的T细胞相互作用与激光镊子（未发表意见，GM）被迫与艾滋病毒感染的Jurkat细胞相互作用，尽管的能力。确定T细胞亚群细胞-细胞在体外和体内的转移，是最容易受到艾滋病毒GAG - iGFP无疑将是有益的。

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Jurkat T Cells</td>
<td>&nbsp;</td>
<td>ATCC</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Leukocyte buffy coats from whole blood</td>
<td>&nbsp;</td>
<td>New York Blood Center</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>RPMI</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Bovine Serum</td>
<td>&nbsp;</td>
<td>Hyclone</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin/Streptomycin</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Nucleofector System and Solution V</td>
<td>&nbsp;</td>
<td>Lonza</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>EndoFree Maxi Prep Kits</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>HIV Gag-iGFP plasmid</td>
<td>&nbsp;</td>
<td>Chen Laboratory</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CD4+ T Cell Isolation Kit</td>
<td>&nbsp;</td>
<td>Miltenyi Biotec</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Ficoll-Paque</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Celltracker/CellTrace Dyes</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CO2-Independent Media</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Imaging Chambers</td>
<td>&nbsp;</td>
<td>Ibidi</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Olympus IX-71 Inverted Microscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>PlanApo N 1.42NA 60X oil objective</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Innova 70C ArKr ion gas laser</td>
<td>&nbsp;</td>
<td>Coherent </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>CSU-10 Nipkow-type spinning disk confocal unit</td>
<td>&nbsp;</td>
<td>Yokogawa</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Di01-T405/488/568/647-13x15x0.5 dichroic beamsplitter</td>
<td>&nbsp;</td>
<td>Semrock</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>525/50 bandpass filter</td>
<td>&nbsp;</td>
<td>Semrock</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>iXon+ 897 electron-multiplied charged coupled devise camera</td>
<td>&nbsp;</td>
<td>Andor</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ASI 400 Thermostatic Heater</td>
<td>&nbsp;</td>
<td>Nevtek</td>
<td>&nbsp;</td>
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visualizing cell-to-cell transfer of hiv using fluorescent clones of hiv and live confocal microscopy

他们最喜爱的蛋白质的绿色荧光蛋白融合，生物学家现在有学习能力，生活复杂的细胞过程中使用荧光视频显微镜。要跟踪人类免疫缺陷病毒核心蛋白在细胞与细胞的人类免疫缺陷病毒的传播运动，我们有GFP标记的Gag蛋白中的一种感染艾滋病毒的分子克隆的情况下，被称为艾滋病毒GAG - iGFP。我们研究这种病毒的克隆，使用视频共聚焦显微镜。我们在下面的可视化实验，转染人类与艾滋病毒GAG - iGFP的T细胞系，我们用荧光标记的未受感染的CD4 + T细胞作为靶细胞病毒。使用不同的荧光标记，我们可以很容易地按照病毒的生产和运输跨间结构称为病毒学突触。简单透氧成像商会让我们从几分钟到几天的现场共聚焦显微镜观察的突触。这些方法可用于跟踪病毒的蛋白质，因为他们移动从一个细胞到下一个。

Watch this Scientific Journal Video about Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy at JoVE.com

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

这种可视化的实验是利用荧光艾滋病毒的分子克隆，现场共聚焦成像实验的指导。

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 ...

visualizing-cell-to-cell-transfer-hiv-using-fluorescent-clones-hiv

Research

JoVE Journal

Immunology and Infection

16.3K 次观看.  Mount Sinai School of Medicine. 这种可视化的实验是利用荧光艾滋病毒的分子克隆，现场共聚焦成像实验的指导。

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

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

HIV - 1使用转速依赖的慢病毒表达载体的绿色荧光蛋白阳性细胞的特定标记

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

科研

免疫与感染

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

使用低温电子断层扫描和自动子断层扫描平均HIV包膜糖蛋白分子结构的测定

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Prediction of HIV-1 Coreceptor Usage (Tropism) by Sequence Analysis using a Genotypic Approach

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. 
The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 20111.
For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3)2. This region carries enough information to perform a reliable prediction. 
The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL&lt;1000 copies/&mu;l roughly increased in the last years (Fig. 3).
The main steps for tropism testing (Fig. 4) demonstrated in this video: 
1. Collection of a blood sample 
 2. Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells 
 3. Amplification of the env region 
 4. Amplification of the V3 region 
 5. Sequence reaction of the V3 amplicon 
 6. Purification of the sequencing samples 
 7. Sequencing the purified samples 
 8. Sequence editing 
 9. Sequencing data interpretation and tropism prediction

Prediction of HIV-1 Coreceptor Usage Tropism by Sequence Analysis using a Genotypic Approach

The prediction of the coreceptor usage of HIV-1 is required for the administration of a new class of antiretroviral drugs, i.e. coreceptor antagonists. It can be performed by sequence analysis of the env gene and subsequent interpretation through an internet based interpretation system (geno2pheno[coreceptor]).

使用基因型的方法预测HIV-1辅助用途（归经）的序列分析

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by ...

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

在体外对HIV - 1核心脱壳

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

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 ...

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 ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

从HIV-1阳性者的血浆分离外来体的

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.

吞噬体偏移和速度与HIV-1感染的活小学的人巨噬细胞测量

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

测量<em&gt;体外</em&gt; HIV-1 Preintegration配合物的整合活动

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...