作者要感谢技术支持公仆赵博士和丹霞柯。我们感谢博士。爱德华Egelman和尼科Grigorieff分享他们的图像处理软件。我们也承认的工作人员，支持冷冻电镜设施，并在美国匹兹堡大学医学院的Beowulf集群和电网结构生物学。这项工作是支持由GM082251和GM085043。

1。冷冻水合EM样品制备由于HIV - 1衣壳蛋白（CA）的集会9只在高盐（1M氯化钠）的缓冲区，这有助于在冷冻电镜图像的强大的背景噪声的稳定，我们使用了快速稀释和背面印迹方法瞬时减少盐浓度在编制冻结水合EM网格。 <ol><li>辉光放电R2 200目/ 1 Quantifoil铜电网下25毫安的碳端为25秒。 </li><li>使用雾化器带来的湿度环境试验箱，一个自制的手册重力活塞，80％。 </li><li>在费Vitrobot柱塞杜瓦，用液氮冷却液态乙烷。装载到人工重力活塞的暴跌冻结杜瓦。 </li><li>应用到碳端钳上安装的网格，这是预装的CA解决方案的2.5μL，并加载到柱塞与电网面临远离你的碳端的钳。 </li><li> 3低盐缓冲液稀释（100毫米氯化钠）液添加到电网的背面，并立即印迹对电网的滤纸背面。整个电网的背面应密切联系，与滤纸约6秒钟，然后取出滤纸。取出滤纸后，立即投身到液态乙烷的网格。 </li><li>从柱塞取出镊子，并迅速转移到一个网格储物盒电网。 </li></ol> 2。 CA管状组件的低温电子显微镜<ol><li>费Polara G2电子显微镜操作装入冷冻水合电网200KV及装备与加坦4Kx4K CCD相机。 </li><li>在低剂量的搜索模式，在〜200倍的放大倍率，并具有剂量&lt;0.001e - / 2，屏幕上的整个电网合适的冰，并保存在一个阶段文件这些领域的岗位领域。 </li><li>记得，在放大3900 x在低剂量搜索模式保存的位置，并进一步屏幕上这些地区。选择一个统一的，薄薄的冰含有的过孔层为数据收集，分离良好，长管领域。第二阶段的文件保存在这些地区的位置。 </li><li>切换到放大5.9 X，插图100微米的目标光圈曝光模式，客观stigmatism和束流强度，并调整剂量〜15 E - / 2％曝光。 </li><li>返回到低剂量的搜索模式，移动到保存的位置，并确定和良好的管使用的CCD摄像头的中心。切换对焦模式，调整的重点，并设置一个散焦值，通常在0.5到2.5微米。切换曝光模式，设置为0.3-0.5秒的曝光时间，剂量的15项电子 - / 2，并收集图像。在盘子上的摄像头，图像采集的电影应该被允许定居前10秒曝光。 </li><li>移动到下一个保存的位置，并重复步骤5，以收集更多的图像。 </li><li>电影发展充满力量D19尼康SUPER COOLSCAN 9000 ED扫描仪采用了6.35微米的像素尺寸为12分钟和数字化。图像文件的格式为TIFF。 </li></ol> 3。螺旋索引螺旋对象可以被索引由两个参数：贝塞尔秩序，N，和层的行号，L。每个特点（N，L），傅立叶变换层线，线表面晶格的螺旋对象表示指数（H，K），使用一个二维符号。对于任何（H，K），层线（N 港元 ， 升 香港 ）是两个基本向量（N 10，L 10）和（N 01，L 01），这是N和L值的线性组合可以得到两个主要的层线（1，0）（0，1），L线沿着Z轴的傅立叶变换测量高度层。 n 的值可使用下列公式10估计 πRr≈J ñ≈1.1 | N | -0.9 .....................( 1） 其中J n是贝塞尔函数，这就决定第n层线的强度，R为半径的螺旋对象，R为半径层线的最大振幅。层行号，L，是关系到 n选择第11条 L = TN + UM ....................( 2） 其中T和 U螺旋常数。对于任何给定的的螺旋结构，有可能完全 ü单位完全相同（或非常密切）T Complete打开。 <ol><li>开箱与统一使用电子舱单的方案12 helixboxer在湄公河格式保存的图像的直径比较直，长管。 </li><li>确定螺旋重复距离，C，如“imgccf&#39;使用基于互相关方案，在MRC包13，。 </li><li>计算傅立叶变换一个新的框的长度，是一个不可分割的重复距离，C 。 </li><li>选择两个主要的层线定义了两个基本的表面晶格向量（1,0）和（0,1）。 </li><li>测量管的半径，R，和R 10，L 10，傅立叶变换， 升 01 ř 01，（图1）的两个主要的层线的高度和半径。 </li><li>计算N 10 和 N 01根据公式（1）。 </li><li>由于n值只能估算，N 10 和 N 01几个组合进行测试（见步骤4.2），在以后的步骤来找到正确的螺旋对称使用的程序包IHRSR 。 </li><li>计算两个实数所描述的：亚基（Δφ）和轴向一星级螺旋上升（ΔZ）（N = 1）之间的旋转螺丝对称。鉴于01 L 10，L，N 10 和 N 01的值，U和 T可通过测试的M值的范围内（例如，-50 &lt;M &lt;50）按照选拔规则。最后，Δφ和ΔZ计算Δφ= 360吨/ u和ΔZ= C / U。 </li></ol> 4。三维重建<ol><li>粒子分割<ol><li>打开一个包含螺旋颗粒的形貌，使用图形化的方案义和团，这是一个在电子舱单包方案。 </li><li>剪切成螺旋颗粒重叠段。在义和团的控制面板，选择“螺旋模式”，并设置拳击参数：框的大小应大于粒子的直径和“OLAP”应框的大小〜90％的价值。 </li><li>螺旋颗粒的两端左击后，义和团会自动生成一个粒子箱沿耳轮长度的系列。 </li><li>保存盒装领域以及它们的坐标。 </li></ol></li><li>初始三维重建，使用IHRSR方案<ol><li>反转冷冻电镜图像的对比度，适用于低通滤波前 14（可选）处理与迭代螺旋真正的空间重构法（IHRSR） 。 </li><li>打开IHRSR程序的图形界面，键入“发电机”。提供盒装颗粒堆栈的所有信息，包括堆栈，堆栈中的图像的名称和路径的图形界面，为对称参数的值，等点击“完成”按钮创建重建脚本， b25.spi。 </li><li>作为一个初始参考使用固体或空心圆筒，并允许循环的过程，直到有没有定义的螺丝对称的变化，这通常发生后几个周期。一个正确的螺旋对称，应该提供一个稳定的融合重建。在过去的周期中所产生的重建将被用来作为一个初始参考进一步细化。 IHRSR进行三维重建，使用天基信息平台方案。 </li></ol></li><li> IHRSR生成三维重建，重建迭代细化 15现在用来作为一个额外细化的初始参考。在细化，螺旋对称固定在Δφ和ΔZ，这是从IHRSR程序确定。 <ol><li>确定在显微镜的使用方案CTFFIND3和CTFTILT 16的散焦和散光目前。 </li><li>乘以使用天基信息平台方案17周大福的粒子段。金融时报“称为天基信息平台套件的操作是用于计算二维图像傅立叶变换（FFT）。此后，FFT乘以由周大福值，在前面的步骤确定，使用操作称为亩的天基信息平台套件内。然后逆变换，形成一个新的形象，在现实空间（CTF -校正），使用天基信息平台操作，金融时报再次得到的值。 </li><li>执行周大福校正后的图像进行比较，参考量的预测，采用多参考对齐投影匹配。在平面倾斜角度的变化是有限的+ / -10 °和1 °取样。引进的限制，如高的相关系数，面角度0 °或180 °，和有限的X -转移附近，为每个细分市场的定位参数。包括在重建只有那些细分，满足约束条件。 </li><li>在每个迭代的细化周期，使用背投影生成一个三维重建和除以总和超过周大福2。 </li><li>对螺旋对称生成一个对称的音量。迭代的细化是终止时，没有发生进一步的改善，在解决新的三维重建。 </li><li>图像处理包蜘蛛是用于最细化的步骤。一系列的操作控制蜘蛛的脚本，这是用户创建的批处理控制文件中包含的操作和参数值序列。最终的重建是计算蜘蛛，浴室中使用的方案。 </li></ol></li></ol> 5。代表性的成果： 一个单一的HIV - 1 CA A92E管（图1A）盒装出螺旋索引计算其傅立叶变换（图1b）。层线（1，0）（0，1），L 10 = 28，L 01 = 37，R 10 = 55，R 01 = 44。鉴于一个211.57Å管半径，我们近似N 10 = 12，N 01 = 11（这里，预定的霸道）。的5195.48Å重复距离，管的螺丝对称被确定为​​ΔZ= 6.8093Å，Δφ= 328.88 °。 ΔZ和Δφ进行了细化7.1321Å和328.86 ° IHRSR（图2a）和图的初步重建。 2B。最终的重建（图3），迭代的细化后，提高密度图明显IHRSR（图2b）计算初始模型。 <img src="/files/ftp_upload/3041/3041fig1.jpg" alt="图1"/> 图1。HIV - 1 CA斜管的索引。 （一）一个单一的HIV - 1 CA A92E管形象。比例尺，30纳米。 （二）在（a）所示的管傅里叶变换。螺旋指数（N 10 = 12，N 01 = 11）表示。在第23A条的决议层线的箭头指向。 <img src="/files/ftp_upload/3041/3041fig2.jpg" alt="图2"/> 图2。使用IHRSR初步重建。 （一）为每个迭代周期的螺丝对称的决心。 Δφ和ΔZ，从初始值开始，10后收敛到稳定值迭代的细化周期。 （二）10后最初的三维密度图迭代周期。 <img src="/files/ftp_upload/3041/3041fig3.jpg" alt="图3"/> 图3。迭代细化后的三维密度图。 （AC）的CA管密度图显示三个正交切片：管轴平行，接近表面（A），垂直于管轴线（二），和并行，并通过管轴（C） 。比例尺，10纳米。 （四）表面三维密度图呈现封闭100％的体积轮廓在1.8s。

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没有利益冲突的声明。

我们目前的协议来提供一个简单的方法来获得螺旋物体的三维结构。使用所描述的程序，我们收购了从单管（176段）的HIV - 1衣壳大会的三维结构。包括更多的图像数据结构，可以实现更高的分辨率。 有最佳的数据收集和分析的几个关键点：第一，在一个冷冻电镜标本的制备，样品溶液应被涂抹了，留下一个统一的，略高于样本大小厚的一层薄薄的解决方案。有几种不同的方式印迹样品。对于细菌的细胞和管状的标本，如HIV - 1 CA大会，印迹，特别是从背面，从一个侧，是最合适的。 二，螺旋霸道的需要确定，因为这不能由螺旋索引或重建。一个常见的做法是用冷冻蚀刻，旋转阴影18，以确定霸道。偏手也可以被确定后重建时的密度图的分辨率足够高，各个组件的三维原子模型应符合密度图时假设是正确的霸道。否则，应假设相反的霸道。 第三，应该是一个维纳滤波器在图像处理，相位和幅度校正，以减少噪声放大。由于从一个单一的形象CTF，总是有过零丢失，在倒易空间的信息的一部分。因此，有必要拥有多个投影数据集为每个影像三维重建，包括在不同的散焦值。

<table border="1"><tbody><tr><td> 名称 </td><td> 来源 </td><td> 评论 </td></tr><tr><td>辉光放电设备100X </td><td>辉光放电设备100X </td><td></td></tr><tr><td> Tecnai Polara F30显微镜，场发射枪</td><td>费，希尔斯伯勒，或</td><td></td></tr><tr><td>加坦的4K x 4K CCD相机</td><td>加坦，普莱森顿，CA </td><td></td></tr><tr><td>切入式冻结装置</td><td></td><td>自制手册重力活塞</td></tr><tr><td> Quantifoil R2 / 1 200目holely碳铜网</td><td> Quantifoil微型工具，德国耶拿</td><td></td></tr><tr><td> EM软件电子舱单</td><td> <a href="http://blake.bcm.edu/EMAN/">http://blake.bcm.edu/EMAN/</a> </td><td></td></tr><tr><td> EM软件IHRSR </td><td> <a href="http://people.virginia.edu/~ehe2n/">http://people.virginia.edu/〜ehe2n /</a> </td><td>与爱德华H Egelman </td></tr><tr><td> EM软件蜘蛛</td><td> <a href="http://www.wadsworth.org/spider_doc/spider/docs/spider.html">http://www.wadsworth.org/spider_doc/spider/docs/spider.html</a> </td><td></td></tr><tr><td> MRC基于螺旋处理软件</td><td> <a href="http://www.riken.jp/biostrmech/index.html">http://www.riken.jp/biostrmech/index.html</a> </td><td>程序从弘治米仓</td></tr><tr><td> CTFFIND3/CTFTILT和空间螺旋细化软件</td><td> <a href="http://emlab.rose2.brandeis.edu/software">http://emlab.rose2.brandeis.edu/software</a> </td><td></td></tr></tbody></table>

structure of hiv-1 capsid assemblies by cryo-electron microscopy and iterative helical real-space reconstruction

低温电子显微镜（冷冻电镜），与图像处理相结合是一个日益强大的工具结构的大分子蛋白复合物和集会的决心。事实上，单粒子电子显微镜1和二维（2D）电子晶体已成为相对常规的方法和大量的结构已经解决了使用这些方法。同时，图像处理和三维（3D）重建螺旋对象迅速的发展，特别是迭代螺旋真正空间重建（IHRSR） 方法 3，它采用单粒子分析工具，结合螺旋对称。许多生物实体的功能丝状或螺旋形式，包括微丝，微管 5，淀粉样纤维 6，烟草花叶病毒 7，细菌鞭毛 8，因为可以从一个单一的投影实现三维密度图的螺旋实体现在达到的形象，相比非螺旋对象的三维重建所需的许多图像，使用的IHRSR方法，结构分析等灵活和无序的螺旋集会。 在这个视频文章中，我们获得的螺旋蛋白大会的三维密度图（HIV - 1衣壳是我们的榜样），包括冷冻电镜冷冻电镜样品制备，低剂量的数据收集协议，索引提供了详细的协议螺旋衍射图案，图像处理和三维重建，使用IHRSR。与其他技术相比，冷冻电镜接近原生的条件下提供最佳的标本保存。样品嵌在一层薄薄的玻璃体冰，快速冷冻，在电子显微镜和影像在低剂量条件下，在液氮温度下，以尽量减少辐射的伤害。牺牲在低信号和低对比度的记录显微接近原生的条件下获得的样品图像。幸运的是，螺旋重建的过程中已经在很大程度上实现了自动化，索引螺旋衍射图样的异常。在这里，我们描述了一个方法，指数螺旋结构，并确定螺旋对称性，从数字化显微，三维螺旋重建的重要一步（螺旋参数）。简单地说，我们得到一个初始的3D密度地图应用IHRSR方法。这最初的地图，然后反复提炼为每个细分市场的定位参数推出的约束，从而控制他们的自由程度。进一步改善，取得了纠正的对比传递函数（CTF）的电子显微镜（振幅和相位校正），并通过优化大会的螺旋对称性。

Watch this Scientific Journal Video about Structure of HIV-1 Capsid Assemblies by Cryo-electron Microscopy and Iterative Helical Real-space Reconstruction at JoVE.com

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

本文介绍了一种方法来获得一个三维（3D）螺旋组装使用低温电子显微镜的分子结构。在这个协议中，我们使用的HIV - 1衣壳集会，说明详细的三维重建过程，实现了由迭代螺旋空间重构方法的密度图。

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 ...

structure-hiv-1-capsid-assemblies-cryo-electron-microscopy-iterative

Research

JoVE Journal

Immunology and Infection

17.3K 次观看.  University of Pittsburgh School of Medicine. 本文介绍了一种方法来获得一个三维（3D）螺旋组装使用低温电子显微镜的分子结构。在这个协议中，我们使用的HIV - 1衣壳集会，说明详细的三维重建过程，实现了由迭代螺旋空间重构方法的密度图。

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

Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' surface envelope glycoprotein. We previously proposed that the crown of the V3 loop, although dynamic and sequence variable, is constrained throughout the population of HIV-1 viruses to an immunologically relevant &beta;-hairpin tertiary structure. Importantly, there are thousands of different V3 loop crown sequences in circulating HIV-1 viruses, making 3D structural characterization of trends across the diversity of viruses difficult or impossible by crystallography or NMR. Our previous successful studies with folding of the V3 crown1, 2 used the ab initio algorithm 3 accessible in the ICM-Pro molecular modeling software package (Molsoft LLC, La Jolla, CA) and suggested that the crown of the V3 loop, specifically from positions 10 to 22, benefits sufficiently from the flexibility and length of its flanking stems to behave to a large degree as if it were an unconstrained peptide freely folding in solution. As such, rapid ab initio folding of just this portion of the V3 loop of any individual strain of the 60,000+ circulating HIV-1 strains can be informative. Here, we folded the V3 loop of the R2 strain to gain insight into the structural basis of its unique properties. R2 bears a rare V3 loop sequence thought to be responsible for the exquisite sensitivity of this strain to neutralization by patient sera and monoclonal antibodies4, 5. The strain mediates CD4-independent infection and appears to elicit broadly neutralizing antibodies. We demonstrate how evaluation of the results of the folding can be informative for associating observed structures in the folding with the immunological activities observed for R2.

Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding

The crown region of different V3 loop sequences of the surface envelope glycoprotein (gp120) of HIV-1 can be structurally characterized in many cases by in silico folding of positions 10 to 22 of the loop using a state-of-the-art ab initio folding algorithm. Here we demonstrate the folding and evaluation of this region of the V3 loop from the R2 strain of HIV-1, a uniquely neutralization sensitive strain with puzzling functional properties.

免疫相关动态三级的HIV - 1 V3环冠R2的序列结构特点的评估从头折叠

The antigenic diversity of HIV-1 has long been an obstacle to vaccine design, and this variability is especially pronounced in the V3 loop of the virus' ...

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 ...

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample preparation for transmission electron microscopy (TEM) such investigations can take several days1,2 and are therefore not suited for a rapid diagnosis of plant virus diseases. Microwave fixation can be used to drastically reduce sample preparation time for TEM investigations with similar ultrastructural results as observed after conventionally sample preparation3-5. Many different custom made microwave devices are currently available which can be used for the successful fixation and embedding of biological samples for TEM investigations5-8. In this study we demonstrate on Tobacco Mosaic Virus (TMV) infected Nicotiana tabacum plants that it is possible to diagnose ultrastructural alterations in leaves in about half a day by using microwave assisted sample preparation for TEM. We have chosen to perform this study with a commercially available microwave device as it performs sample preparation almost fully automatically5 in contrast to the other available devices where many steps still have to be performed manually6-8 and are therefore more time and labor consuming. As sample preparation is performed fully automatically negative staining of viral particles in the sap of the remaining TMV-infected leaves and the following examination of ultrastructure and size can be performed during fixation and embedding.

This study describes a method that allows the rapid and clear diagnosis of plant virus diseases in about half a day by using a combination of microwave assisted plant sample preparation for transmission electron microscopy and negative staining methods.

微波辅助植物病毒病的快速诊断用透射电子显微镜

Investigations of ultrastructural changes induced by viruses are often necessary to clearly identify viral diseases in plants. With conventional sample ...

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 ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

新工具展开调节性T细胞的HIV-1感染的个体

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the protocols described here use freshly isolated human peripheral blood mononuclear cells (PBMCs) or plasmacytoid dendritic cells (pDCs) to sense infections in either T cell line (MT4) or heterologous primary CD4+ T cells. In order to ensure proper sensing, it is essential that PBMC are isolated immediately after blood collection and that optimal percentage of infected T cells are used. Furthermore, multi-parametric flow cytometric staining can be used to confirm that PBMC samples contain the different cell lineages at physiological ratios. A number of controls can also be included to evaluate viability and functionality of pDCs. These include, the presence of specific surface markers, assessing cellular responses to known agonist of Toll-Like Receptors (TLR) pathways, and confirming a lack of spontaneous type-I interferon (IFN) production. In this system, freshly isolated PBMCs or pDCs are co-cultured with HIV-1 infected cells in 96 well plates for 18-22 hr. Supernatants from these co-cultures are then used to determine the levels of bioactive type-I IFNs by monitoring the activation of the ISGF3 pathway in HEK-Blue IFN-&#945;/&#946; cells. Prior and during co-culture conditions, target cells can be subjected to flow cytometric analysis to determine a number of parameters, including the percentage of infected cells, levels of specific surface markers, and differential killing of infected cells. Although, these protocols were initially developed to follow type-I IFN production, they could potentially be used to study other imuno-modulatory molecules released from pDCs and to gain further insight into the molecular mechanisms governing HIV-1 innate sensing.

Assessing the Innate Sensing of HIV-1 Infected CD4+ T Cells by Plasmacytoid Dendritic Cells Using an Ex vivo Co-culture System.

Unlike cell-free HIV-1 particles, infected CD4+ T cells are effectively sensed by plasmacytoid dendritic cells (pDCs). This manuscript describes a method where peripheral blood mononuclear cells (PBMCs) or isolated pDCs are co-cultured with HIV-1 infected T cells to evaluate innate sensing by pDCs as assessed by release of type-I IFN.

评估的HIV-1感染的CD4的先天传感<sup&gt; +</sup&gt;性T细胞浆细胞样树突细胞使用<em&gt;体外</em&gt;共培养体系。

HIV-1 innate sensing requires direct contact of infected CD4+ T cells with plasmacytoid dendritic cells (pDCs). In order to study this process, the ...

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. ...

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 ...