我们感谢艾滋病和癌症的病毒程序，上汽冯检基，公司的生物制品部分，提供纯化，AT - 2治疗的HIV - 1病毒，史蒂芬费里尼和他的同事们利用高性能计算能力的Biowulf的援助Linux集群在国立卫生研究院，马里兰州贝塞斯达<a href="http://biowulf.nih.gov">（http://biowulf.nih.gov） ，</a>飞电子显微镜的帮助公司和Ethan泰勒专家援助与数字。这项工作是由国立卫生研究院，马里兰州贝塞斯达的国家癌症研究所，癌症研究中心的资金支持。

<html>这里所描述的方法被开发使用的仪器，工具和软件的具体设置。因为所有的实验室将不会使用这一相同的实验装置，努力作了概括的方法，并在可能的情况下提供替代品，这是不可能的。 1。准备玻璃化冷冻的病毒样本在下面的部分玻化电网使用费Vitrobot Mark III的一个印迹和暴跌冻结机器人准备。 Iancu， 等上使用这个系统7的详细协议。作为替代机器人柱塞，柱塞断头台式或重力是完全足够6。 在第1.2。和1.3。一个加坦Solarus 950辉光放电单元使用干净的样品网格和提高其亲水性，虽然任何辉光放电装置设计与使用电子显微镜网格可以取代。 内容“&gt;虽然应用于印迹和投身冻结前网格的样本量应在2μL范围内，病毒和混合样品中蛋白- A金胶体量会有所不同，取决于源。因此，很可能是病毒和黄金稀释或比率的范围将进行测试，并在显微镜成像，凭经验确定一个理想的混合物。注意，当图像数据是在第3节获得10和20之间，金基准标记应目前适当倾斜系列对齐。 注意：本节介绍的是高度易燃气体乙烷，氢气和氧气的使用。使用这些气体时，应采取适当的谨慎。此外，应小心处理病毒样本，按照生物安全建议。最后，总是穿防护眼镜和衣物时，液态氮（N 2）和乙烷。 <ol><li>准备飞Vitrobot马克。第三，通过打开其N 2气体供应，安装加湿器的墨盒，然后填充墨盒过滤的去离子水。 Vitrobot和电源设置其气候室温度22℃，湿度为100％的目标，杂交时间为6秒，和印迹抵销-2毫米。 </li><li>加坦Solarus 950辉光放电单元和开放的H 2和O 2罐，供应单位的电源。运行辉光放电为50瓦1分钟的预清洗标本室。 </li><li>排列的玻璃盖玻片，碳的一面朝上，和辉光放电，通过样品室顶部装载门里面地点上的多孔碳电网所需的数字。清洁电网运行辉光放电6-10秒，在25瓦。 （请注意，在本议定书中使用的Quantifoil品牌电网的碳端是在外观，磨砂，这会不会是所有商业电网的真实，所以最好是从制造商获得此信息。） </li><li>内首席运营官位发车框（ES）lant容器碗，和中央杯内冷却液碗坐在的热扩散主轴。慢慢倒入碗里的液体氮，直到轰轰烈烈的冒泡消退，表明碗和液体之间的平衡。保持液体N 2平整个过程的其余部分，照顾，以保持中央杯。 </li><li>将塑料软管的一端乙烷气体罐，和一个玻璃吸管的另一端。保持玻璃枪头中央杯的底部，建立一个缓慢的，稳定的乙烷流，乙烷将开始凝结。继续填充，直到中央杯满。从中央杯主轴。 </li><li>准备加入2μL蛋白黄金胶体基准，10μL，AT - 2灭活HIV - 1病毒悬液样品混合物。轻轻吸液管的混合物，使解决方案基准。置于冰上。 </li><li>抓住一个专门Vitrobot镊子的网格的外缘。应用碳小号2μL样品混合物IDE电网。立即指示机器人在液态乙烷的样品装入湿润的气候室，杂交和投身冻结。 </li><li>玻化电网传输到电网中。重复步骤1.7。生产所需数量的电网。当完成后，拧紧螺丝电网盒盖（S），然后快速交接箱（ES）小液氮运输杜瓦。 </li></ol> 2。样品载入到透射电子显微镜在本节中，装载室的液态氮的2级应高度警觉，监测，和液体按需补充，以确保该网格框仍然沉浸。一个工具，使电网接触之前，冷却浸在液态氮 ，直到它与液体达到平衡。每次使用后，工具应使用热风枪解冻。 在本节费Tecnai G2 Polara透射电子显微镜（TEM）一起使用加坦低温工作站。虽然装载样品液态氮条件下的原则是适用于所有的冷冻电子显微镜的工作，本节中的步骤是在实验中使用的设备。针对不同的设备所使用的步骤会有所不同制造商。 注意：在本节中，始终处理液态氮时戴防护眼镜和服装。 <ol><li>确保，透射电镜Compustage不包含标本盒，标本选择杆和棒插入低于110 K。 </li><li>冷却和低温工作站准备装入标本存储块，这是样本选择杆的一部分电网。 </li><li>从显微镜的冷却（&lt;110 K）选择杆转移低温工作站。 </li><li>幻灯片选择杆插入氮浴低温工作站选择块。使用专门的彗星从存储块中删除所有标本墨盒artridge处理镊子。位发车盒（ES）装载工作站，用螺丝刀松开盖子（S）。 </li><li>使用镊子取出墨盒网格和地方电网。使用C型夹专门放置工具，沉积在网格上方的一个C夹，确保电网在墨盒。 </li><li>重复步骤2.5。直到所需的格数是在墨盒。标本选择块传输墨盒。 </li><li>准备切除标本选择杆冷冻工作站。将选择杆，以去除氮浴选择块。取出冷冻工作站选择杆和附加到的TEM。 </li></ol> 3。收购低温电子显微镜数据在本节中，飞批次断层扫描软件接口是用来建立一个批处理文件，存储所有感兴趣的发车位置的坐标。当用户的指示，将重新批断层扫描软件的每个岗位，并会协调通过数码显微倾斜的一系列收购。如果这个软件无法使用，Leginon软件包是可行的免费，开源替代品13。成像在200​​千电子伏，一个加坦成像过滤器（GIF）使用一个后的GIF 2K x 2K分辨率的CCD相机（加坦）。 <ol><li>的位置Compustage，这样电子束穿过真空。切换到高放大倍率（34kX）曝光模式，并进行直接的路线。 </li><li>将零能源滤波器的损耗峰。 （这提供与电子零能量损失的参考能源过滤器）。 </li><li>移动的Compustage一个网格区域特色的碳。在低倍率（4.5kX）搜寻模式，使用摇摆功能，来回倾斜+ / -15 °之间的阶段。同时调整，直到最小的阶段转变是观察Z轴的舞台上的地位。 （这个职位eucentric高度约阶段）。禁用摇摆时没有舞台的转变是观察。 </li><li>使用自动Eucentric高度F<html>油膏完善eucentric高度。 （此例程使用交叉相关，以获得更精确​​的eucentric高度比在步骤3.3）。 Eucentric高度自动存储批量层析成像软件以后参考。 </li><li>查找区，具有约三至六个月的病毒颗粒的利益和不低于10的基准指标。这个发车位置添加到批处理断层扫描文件。 </li><li>重复步骤3.4。和3.5。直到所需的位置数指定。定义批处理参数（± 60 ° 2 °倾斜的增量，1-2 E - / 2 /倾斜视图）和运行批处理。 </li></ol> 4。重建的冷冻电子tomograms <ol><li>猛禽1使用自动基准基于对齐，对齐倾斜系列。 </li><li>重建tomograms使用的R -加权反投影的IMOD 8。产生的数据量的标准文件格式是湄公河委员会的文件，文件扩展名。MRC。 </li></ol>ve_title“&gt; 5。分割病毒颗粒子卷，并确定尖峰本节使用一个定制的IMOD的扩展，允许用户指定范围内的断层扫描病毒颗粒子卷。在第6条，用户定义的病毒粒子的边界是用来作为沿尖峰自动选择表面。 <ol><li>打开的IMOD，负载与病毒粒子填充断层扫描。 </li><li>沿Z轴的断层扫描，直到通过中央切片的病毒粒子之一是位于导航。确认，已发现的病毒粒子的质心。这样做之后，存入一个病毒粒子标记。此标记将被用于自动化的病毒粒子在第6分割工具。 </li><li>继续质心的称号，直到所有的病毒粒子卷确定。 </li><li>关闭断层扫描并保存标记集，确定病毒颗粒的重心。重复，直到已在所有tomograms指定的病毒粒子。 </li><li>使用由四个因素的IMOD，羽绒样品病毒粒子subtomograms。 </li><li>使用边缘增强的各向异性扩散降噪病毒颗粒作为实施的IMOD。 </li><li>主题的病毒颗粒无监督膜分割使用能源为基础的三维方法（详见Bartesaghi等，2005）3 。 </li><li>尖峰确定分段病毒颗粒表面的特定位置和合成对称穗状卷之间的交叉相关的局部最大值对应的位置。指定的阈值以上这些位置添加到公认的穗子卷的坐标列表。 </li></ol> 6。分类和平均粒子<ol><li>计算提取断层扫描子卷（100 × 100 × 100体素），在每个步骤5.8中确定的的位置。 （这是没有去噪或分级）。结果集合的子卷包含在下面的步骤将分类和平均穗蛋白质。 </li><li> ðetermine穗长，正常使用每穗的位置自动分段膜轴的方向。这种方法提供了两个三个欧拉角的初步估计。 </li><li>随机其余就地旋转，以防止任何可能在随后的路线的偏见。 </li><li>应用欧拉角子卷，然后翻译对齐子卷到他们的圆柱形平均的全球平均水平，以确保它们都共享同一中心的质量。 </li><li>删除的子卷的最微弱关联与更新的全球平均水平的10％。这样做是为了清除被错误地确定峰值的风险最大密度。 </li><li>对齐和分类 ​​，而无需使用外部引用和扣球缺少楔适当的会计（详细Bartesaghi 等人，2008年）4卷。逐步完善子卷的路线，并在每一次迭代分类秒杀卷。 </li><li>应清楚地观察到三倍对称，在分类的早期阶段，并在第四迭代，对称的3倍征收。在每一轮中，最良好定义的类平均，并为下一轮的参考使用。 </li><li>通常〜4000尖峰应选择为每个数据集。 〜5-12细化轮后获得最后的密度图将包括从〜50％，分卷，每个数据集的贡献。 </li></ol> 7。坐标拟合<ol><li>步骤6.8密度图是开在加州大学旧金山分校的奇美12。 </li><li>产生一个模拟的20地图上的X射线坐标使用奇美拉或货物舱单10。 </li><li>基座坐标为随机取向。使用奇美拉的建在陡峭的上升局部优化，适合执行100陡峭的上升步骤的倍数，直到获得收敛的坐标。 </li></ol>8。代表性的成果使用3D平均和协调装修，各种HIV和SIV环境保护尖峰结构已得到解决。图3A是信封尖峰结构的HIV - 1株，BAL（紫色）。秒杀的3倍〜120尺寸测量尖峰尖顶膜的对称性，并锥〜35穗基地的一个最大宽度约150。图3B，三聚环境保护相结合的密度图，确定使用低温电子断层扫描单体gp120的晶体结构（红色，从PDB ID，2NY7派生）确定，我们能够获得一个工作的分子模型三聚体的包膜糖蛋白复合物（PDB ID，3DNN）9 。 我们的方法还可以用于环境保护之间的三聚体和各种gp120的特定蛋白质复合物的结构形成分析片段和抗体。这方面的例子，其中环境保护是具有广谱中和B12的Fab复合物显示在图4A（整复是在紫色所示）。在这个数字中，额外的密度对应B12是从穗向外投射，和平行膜。这种复合物结构的解释，可以延长到分子水平，通过拟合穗的部分，必然要相应的配体，与原子坐标的密度图。图4B，当这个密度图是装有B12晶圆厂（白），三个gp120的核心相对方向的约束坐标gp120的刺突蛋白（红色，从PDB ID，2NY7获得）中可以看出，就可以看出端倪（PDB编号，3DNL）。这些构象关系的理解等配体的相互作用与尖峰和干扰病毒活性的启发。 unliganded和liganded环境保护的一些其他结构已解决方法，包括在这里提出乌兰乌德这些HIV - 1的R3A，SIV病毒的CP - MAC，SIVmneE11S和SIVmac239，HIV - 1巴尔环境保护，sCD4及17B厂之间的三元复合，CP - Mac的环境保护和SIV复合7D3 抗体 9,14 。 <img src="/files/ftp_upload/2770/2770fig1.jpg" align="Figure 1" /> 图1病毒悬浮的三维结构：在低温电子显微镜和三维重建的概念步骤。 <img src="/files/ftp_upload/2770/2770fig2.jpg" align="Figure 2" /> 图2（a）从代表的HIV - 1病毒颗粒断层扫描片。 （二）一个单一的HIV - 1病毒颗粒的核心和表面尖峰图示。 <img src="/files/ftp_upload/2770/2770fig3.jpg" align="Figure 3" /> 图3（A）的HIV - 1 BAL病毒膜表面上显示的包膜糖蛋白（面尖峰）的三维结构。 （二）分子模型的TRIMERIC尖峰由配售单体gp120的X射线晶体学所得密度图9（红色）三种结构的副本。 <img src="/files/ftp_upload/2770/2770fig4.jpg" align="Figure 4" /> 图4：（一）在复杂的一个广泛的中和抗体Fab片段，B12的HIV - 1 BAL的包膜糖蛋白的三维结构。 （二）为复杂的分子模型，由配售单体gp120的维生素B12厂成密度图9 X射线晶体学派生的复杂结构的三个副本。

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Chem. 25, 1605-1612 (2004).</li><li>Suloway, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+molecular+microscopy:+the+new+Leginon+system.">Automated molecular microscopy: the new Leginon system.</a> J. Struct. Biol. 151, 41-60 (2005).</li><li>White, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+architectures+of+trimeric+SIV+and+HIV-1+envelope+glycoproteins+on+intact+viruses:+strain-dependent+variation+in+quaternary+structure.">Molecular architectures of trimeric SIV and HIV-1 envelope glycoproteins on intact viruses: strain-dependent variation in quaternary structure.</a> PLoS. Pathog. 6, e1001249-e1001249 (2010).</li></ol>

没有利益冲突的声明。

这里介绍的低温电子显微镜和立体的分类和平均方法允许〜20 Å分辨率的包膜糖蛋白复合物的三维结构的测定。 X射线拟合坐标密度图单体组成部分，允许在分子水平上的结构解释。与在开放源码的科学工具和网络基础设施的扩散的发展相结合的低成本计算设备的不断改进使以前难以想象的科学的协作努力。我们采取的这些发展优势，并表明可以在许多年龄的学生达到带来了先进的科学技术。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>多一个多孔碳网格</td><td> Quantifoil </td><td> - </td><td> 200目</td></tr><tr><td>蛋白黄金胶体</td><td> Utrech大学 - 荷兰</td><td> - </td><td>直径为10纳米</td></tr><tr><td> HIV - 1 BAL </td><td>国立卫生研究院，国家癌症研究所，冯检基，MD </td><td> - </td><td> 〜10月11日的病毒颗粒/毫升</td></tr><tr><td>冷冻电镜储物盒</td><td>太平洋电网技术</td><td> GB - 4R </td><td> 4孔，圆</td></tr><tr><td> Vitrobot马克III </td><td>费</td><td> - </td><td> 6秒的印迹时间，抵消-2毫米，湿度为100％ </td></tr><tr><td> Tecnai G2 Polara透射电子显微镜</td><td>费</td><td> - </td><td> 200千电子伏</td></tr><tr><td>加坦成像<html>过滤器</td><td>加坦</td><td> - </td><td> - </td></tr><tr><td>加坦后的GIF CCD </td><td>加坦</td><td> - </td><td> 2K x 2K分辨率像素</td></tr><tr><td>加坦低温工作站</td><td>加坦</td><td> - </td><td> - </td></tr><tr><td>加坦Solarus 950等离子清洗系统</td><td>加坦</td><td> - </td><td>氧和氢等离子体</td></tr><tr><td> - 剪辑</td><td>费</td><td> ZIT0634 </td><td> - </td></tr><tr><td> Biowulf计算星团</td><td>国家卫生研究院</td><td> - </td><td> <a href="http://biowulf.nih.gov/">http://biowulf.nih.gov/</a> </td></tr><tr><td>加州大学旧金山分校的奇美拉</td><td>美国加州大学 - 旧金山</td><td> - </td><td> <a href="http://www.cgl.ucsf.edu/chimera/">http://www.cgl.ucsf.edu/chimera/</a> </td></tr><tr><td> IMOD </td><td>科罗拉多大学博尔德分校 - </td><td> - </td><td>/ IMOD /“&gt; http://bio3d.colorado.edu/imod/ </td></tr><tr><td>电子舱单</td><td>贝勒医学院</td><td> - </td><td> <a href="http://blake.bcm.tmc.edu/eman/">http://blake.bcm.tmc.edu/eman/</a> </td></tr><tr><td>猛禽</td><td>斯坦福大学</td><td> - </td><td> <a href="http://www-vlsi.stanford.edu/TEM/index.htm">http://www-vlsi.stanford.edu/TEM/index.htm</a> </td></tr></tbody></table>

determination of molecular structures of hiv envelope glycoproteins using cryo-electron tomography and automated sub-tomogram averaging

自从近30年前它的发现，超过60万人已经感染了人类免疫缺陷病毒（HIV） <a href="http://www.usaid.gov">（www.usaid.gov） 。</a>该病毒感染和破坏的CD4 + T细胞，从而削弱免疫系统，并造成获得性免疫缺陷综合症（艾滋病）2。感染开​​始时，艾滋病病毒包膜糖蛋白“秒杀”接触的CD4 + T细胞表面的CD4受体。这种相互作用引起的构象变化，促进互动与细胞表面受体5,9在穗。在艾滋病毒感染途径的这些蛋白质相互作用的意义，使他们从根本上艾滋病毒研究的深刻重要性，并在追求一种艾滋病疫苗。 需要更好地了解艾滋病毒的细胞接触和中分子尺度的相互作用的动机技术的开发，以确定结构与细胞表面受体的蛋白质和分子，阻止感染艾滋病毒的相互作用穗。使用低温电子断层扫描和三维图像处理，我们最近表现出的能力，以确定对本土病毒的表面，在20〜Å分辨率9,14，这样的结构。这种方法不仅限于解决HIV包膜结构，并可以扩展到其他病毒的膜蛋白和脂质体重组蛋白。在这个协议中，我们描述了如何获取从纯化艾滋病毒的病毒颗粒，进而逐步通过玻璃化冷冻样品的准备，收集，低温电子显微镜数据，重组和3D数据处理量，平均和分类3D蛋白子卷的HIV包膜糖蛋白的结构，并解释结果产生一种蛋白质模型。我们的方法计算方面改编成模块可以访问和远程执行使用Biowulf GNU / Linux的平行p在美国<a href="http://biowulf.nih.gov">国立卫生研究院</a> （http://biowulf.nih.gov ） rocessing集群。这种远程访问，与低成本的计算机硬件和高速网络接入相结合，取得了有可能从学校或家中工作的研究人员和学生的参与。

Watch this Scientific Journal Video about Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging at JoVE.com

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

协议描述了高通量的方法，以确定使用低温电子断层扫描和三维图像处理的膜蛋白的结构。它涵盖了样品制备的详细信息，数据采集，数据处理和解释，最后一个有代表性的目标的方法，HIV - 1包膜糖蛋白的生产。这些计算程序设计的方式，使研究人员和学生远程工作和作出贡献的数据处理和结构分析。

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 ...

determination-molecular-structures-hiv-envelope-glycoproteins-using

Research

JoVE Journal

Immunology and Infection

41.3K 次观看.  National Institutes of Health. 协议描述了高通量的方法，以确定使用低温电子断层扫描和三维图像处理的膜蛋白的结构。它涵盖了样品制备的详细信息，数据采集，数据处理和解释，最后一个有代表性的目标的方法，HIV - 1包膜糖蛋白的生产。这些计算程序设计的方式，使研究人员和学生远程工作和作出贡献的数据处理和结构分析。

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

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

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, ...

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 ...

Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein complexes and assemblies. In fact, single particle electron microscopy1 and two-dimensional (2D) electron crystallography2 have become relatively routine methodologies and a large number of structures have been solved using these methods. At the same time, image processing and three-dimensional (3D) reconstruction of helical objects has rapidly developed, especially, the iterative helical real-space reconstruction (IHRSR) method3, which uses single particle analysis tools in conjunction with helical symmetry. Many biological entities function in filamentous or helical forms, including actin filaments4, microtubules5, amyloid fibers6, tobacco mosaic viruses7, and bacteria flagella8, and, because a 3D density map of a helical entity can be attained from a single projection image, compared to the many images required for 3D reconstruction of a non-helical object, with the IHRSR method, structural analysis of such flexible and disordered helical assemblies is now attainable.
In this video article, we provide detailed protocols for obtaining a 3D density map of a helical protein assembly (HIV-1 capsid9 is our example), including protocols for cryo-EM specimen preparation, low dose data collection by cryo-EM, indexing of helical diffraction patterns, and image processing and 3D reconstruction using IHRSR. Compared to other techniques, cryo-EM offers optimal specimen preservation under near native conditions. Samples are embedded in a thin layer of vitreous ice, by rapid freezing, and imaged in electron microscopes at liquid nitrogen temperature, under low dose conditions to minimize the radiation damage. Sample images are obtained under near native conditions at the expense of low signal and low contrast in the recorded micrographs. Fortunately, the process of helical reconstruction has largely been automated, with the exception of indexing the helical diffraction pattern. Here, we describe an approach to index helical structure and determine helical symmetries (helical parameters) from digitized micrographs, an essential step for 3D helical reconstruction. Briefly, we obtain an initial 3D density map by applying the IHRSR method. This initial map is then iteratively refined by introducing constraints for the alignment parameters of each segment, thus controlling their degrees of freedom. Further improvement is achieved by correcting for the contrast transfer function (CTF) of the electron microscope (amplitude and phase correction) and by optimizing the helical symmetry of the assembly.

This article describes a method to obtain a three-dimensional (3D) structure of helically assembled molecules using cryo-electron microscopy. In this protocol, we use HIV-1 capsid assemblies to illustrate the detailed 3D reconstruction procedure for achieving a density map by the iterative helical real-space reconstruction method.

HIV - 1衣壳大会通过低温电子显微镜的结构和迭代螺旋实时空间重构

Cryo-electron microscopy (cryo-EM), combined with image processing, is an increasingly powerful tool for structure determination of macromolecular protein ...

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not representative of the behavior, status or phenotype of single cells. Due to this new insight the number of single cell studies rises continuously (for recent reviews see 1,2,3). However, many of the single cell techniques applied do not allow monitoring the development and behavior of one specific single cell in time (e.g. flow cytometry or standard microscopy).
Here, we provide a detailed description of a microscopy method used in several recent studies 4, 5, 6, 7, which allows following and recording (fluorescence of) individual bacterial cells of Bacillus subtilis and Streptococcus pneumoniae through growth and division for many generations. The resulting movies can be used to construct phylogenetic lineage trees by tracing back the history of a single cell within a population that originated from one common ancestor. This time-lapse fluorescence microscopy method cannot only be used to investigate growth, division and differentiation of individual cells, but also to analyze the effect of cell history and ancestry on specific cellular behavior. Furthermore, time-lapse microscopy is ideally suited to examine gene expression dynamics and protein localization during the bacterial cell cycle. The method explains how to prepare the bacterial cells and construct the microscope slide to enable the outgrowth of single cells into a microcolony. In short, single cells are spotted on a semi-solid surface consisting of growth medium supplemented with agarose on which they grow and divide under a fluorescence microscope within a temperature controlled environmental chamber. Images are captured at specific intervals and are later analyzed using the open source software ImageJ.

Live Cell Imaging of Bacillus subtilis and Streptococcus pneumoniae using Automated Time-lapse Microscopy

This protocol provides a step-by-step procedure to monitor single cell behavior of different bacteria in time using automated fluorescence time-lapse microscopy. Furthermore, we provide guidelines how to analyze the microscopy images.

活细胞成像枯草芽孢杆菌肺炎链球菌使用自动定时显微镜

During the last few years scientists became increasingly aware that average data obtained from microbial population based experiments are not ...

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 ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

艾滋病毒的成像1信封引起的病毒学突触信号合成脂双层

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

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 ...

Averaging of Viral Envelope Glycoprotein Spikes from Electron Cryotomography Reconstructions using Jsubtomo

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these glycoprotein &#8216;spikes&#8217; is often technically challenging but important for understanding viral pathogenesis and in drug design. Here, a protocol is presented for viral spike structure determination through computational averaging of electron cryo-tomography data. Electron cryo-tomography is a technique in electron microscopy used to derive three-dimensional tomographic volume reconstructions, or tomograms, of pleomorphic biological specimens such as membrane viruses in a near-native, frozen-hydrated state. These tomograms reveal structures of interest in three dimensions, albeit at low resolution. Computational averaging of sub-volumes, or sub-tomograms, is necessary to obtain higher resolution detail of repeating structural motifs, such as viral glycoprotein spikes. A detailed computational approach for aligning and averaging sub-tomograms using the Jsubtomo software package is outlined. This approach enables visualization of the structure of viral glycoprotein spikes to a resolution in the range of 20-40 &#197; and study of the study of higher order spike-to-spike interactions on the virion membrane. Typical results are presented for Bunyamwera virus, an enveloped virus from the family Bunyaviridae. This family is a structurally diverse group of pathogens posing a threat to human and animal health.

An approach is presented for determining structures of viral membrane glycoprotein complexes using a combination of electron cryo-tomography and sub-tomogram averaging with the computational package Jsubtomo.

使用Jsubtomo从电子Cryotomography重构平均病毒包膜糖蛋白穗

Enveloped viruses utilize membrane glycoproteins on their surface to mediate entry into host cells. Three-dimensional structural analysis of these ...

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 ...