这项工作是支持的比尔和梅林达盖茨基金会（＃38631）和美国国立卫生研究院的资助，其中包括DP2 OD004631和R01 AI084119。

1。方法<ol><li>该协议的第一步是选择你想在硅片倍V3冠序列。对于R2菌株，该片段的序列是KSIPMGPGRAFYT。 </li><li>这个序列的肽相应的三维原子结构应建立在计算机的虚拟空间。 ICM这个命令是： buildpep“KSIPMGPGRAFYT” 或文件：新功能：可以选择下拉菜单下的肽</li><li>然后设置几个参数的过程，包括搜索步骤（搜索的长度），模拟温度，搜索策略参数，包括变量的约束，以实现合理的地区，能源条件，选择不同的能量计算方法偏见的搜索，应记录和参数说明如何搜索将被记录，例如是否记录电影，有多少中间得分的构象。所有这些参数进行了优化，由以前的出版物（见www.molsoft.com）。 </li><li>折叠，然后用下面的命令启动： 蒙特卡洛 或文件：新的肽，可以选择下拉下拉菜单中的分子力学：最小化：全球 在前一种情况下，从第3步中的参数应设置在命令行之一。在后一种情况下，选择最常用的参数复选框窗格中提供的命令被执行之前。为方便起见，本实验利用的实验，并察看相同的折叠ICM的脚本如下所示： <img src="/files/ftp_upload/2118/2118output.jpg" alt="输出" /> 这个脚本可以被保存在一个文本文件，并运行的计算机操作系统的命令行快速使用的命令（通常是LINUX的）： ICM _foldingscript </li></ol> 2。成功的秘诀<ol><li> V3环是一个常数35个氨基酸长度在几乎每一个著名的应变，所以氨基酸的位置编号从1到35，保税胱氨酸的原产二硫化碳1开始，并与相应的二硫化物保税在35胱氨酸终止。既然是一个循环的一部分，应慎重选择冠V3的界限。如果选择过大的一个片段，它是不太可能表现为一个自由灵活的部分，就好像它是一个自由肽，使折叠模拟，将无法正确评估的结构。如果选择太小的一个片段，翔实的三级结构可能不会在模拟的形式。我们以前的研究表明，从第10位22位的片段抗体绑定的晶体构象密切相关，所以应该选择折叠，这是任何V3环的片段。 </li><li> ，虽然使用的图形用户界面和下拉菜单是用户友好的，事先一般肽折叠的成功的工作，特别是使用ICM和V3环冠折叠使用上面的脚本，简单修改buildpep线的顺序和运行从上面的命令行脚本建议。 </li></ol> 3。代表性的成果 R2的折叠的结果是代表任何V3环结果。要评估的结果，应打开和项目文件（它将被命名为“newProject1.icb”从上面的脚本）“分子力学，堆栈，查看”选择。会出现一个堆栈的构象表。栈构象可以通过点击“打印/直方图图标图形化显示。 “分子力学，栈，播放”将使堆栈的电影视觉欣赏发现折叠的构象喜好。对于R2的序列，构象是β-发夹状预计为V3的循环2 -尤其是在位置的片段12日至14一个明确的β链偏好是整个堆栈，和很少的α-螺旋构象被视为。此外，近3个单位的能源缺口之间的能量最低的构象和第二个最低能量构象。从一个充满活力的观点来看，这意味着只有闪烁的结构的最低能量构象不到一％的时间：折叠的结果，因此建议在R2 V3冠是一个刚性的，而不是灵活，结构。可能有其他重要的构象合奏结构特点，但它们很难系统地欣赏。 <table border="1"><tbody><tr><td></td><td> JRFL </td><td> SF162_V3JRFL </td><td> SF162_V3R2 </td></tr><tr><td> 447 - 52D格林尼治标准时间50 </td><td> 15 </td><td> 0.00061 </td><td> 0.00078 </td></tr></tbody></table> 表1：JRFL和R2 V3相对中立蒙面东窗事发设置循环。数据曾报道在卡多佐T.， 等。ARHR（2008）和品特，A.， 等。人研究Virol（2004年）。简单地说，中和活性测定一个单周期的传染性，使用的env缺陷的荧光素酶表达质粒JRFL环境保护或上文所述的SF162 V3的变种假型pNL4 - 3.Luc.RE产生psVs分析：SF162_V3JRFL包含JRFL V3 SF162 V3环序列进行循环序列。 SF162_V3R2包含R2 V3环序列在SF162 V3环序列的地方。 psVs孵育447 - 52D单克隆抗体系列稀释为1.5 h，在37 ° C，然后添加对CD4 + CCR5 + U87目标细胞在96孔板接种于聚凝胺（10毫克= ML）。 24小时后，细胞refed用RPMI含有10％FBS和10毫克= ML聚凝胺，一个额外的24-48 h孵化中期。测定荧光素酶的活性测定48-72 h，用一个微孔板光度计（HARTA公司）使用Promega公司，公司的几何平均滴度为50％的中（GMT50）检测试剂447 - 52D postinfection的中和曲线插值至少有三个独立实验的平均值。

<ol>
	<li>Almond, D., Kimura, T., Kong, X., Zolla-Pazner, S. &. a. m. p. ;. a. m. p., Cardozo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+characterization+of+the+V3+loop+crown.">Dynamic characterization of the V3 loop crown.</a> Antiviral Therapy. 12, 13-31 (2007).</li><li>Almond, D., Kimura, T., Kong, X., Swetnam, J., Zolla-Pazner, S., Cardozo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+conservation+predominates+over+sequence+variability+in+the+crown+of+HIV+type+1's+V3+loop.">Structural conservation predominates over sequence variability in the crown of HIV type 1's V3 loop.</a> AIDS Res Hum Retroviruses. 26, (2010).</li><li>Abagyan, R., Totrov, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biased+probability+Monte+Carlo+conformational+searches+and+electrostatic+calculations+for+peptides+and+proteins.">Biased probability Monte Carlo conformational searches and electrostatic calculations for peptides and proteins.</a> J Mol Biol. 235, 983-1002 (1994).</li><li>Quinnan, G. V., Zhang, P. F., Fu, D. W., Dong, M., Alter, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+characterization+of+HIV+type+1+envelope+protein+associated+with+a+broadly+reactive+neutralizing+antibody+response.">Expression and characterization of HIV type 1 envelope protein associated with a broadly reactive neutralizing antibody response.</a> AIDS Res Hum Retroviruses. 15, 561-5670 (1999).</li><li>Zhang, P. F., Bouma, P., Park, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variable+region+3+(V3)+mutation+determines+a+global+neutralization+phenotype+and+CD4-independent+infectivity+of+a+human+immunodeficiency+virus+type+1+envelope+associated+with+a+broadly+cross-reactive,+primary+virus-neutralizing+antibody+response.">A variable region 3 (V3) mutation determines a global neutralization phenotype and CD4-independent infectivity of a human immunodeficiency virus type 1 envelope associated with a broadly cross-reactive, primary virus-neutralizing antibody response.</a> J Virol. 76, 644-655 (2002).</li></ol>

从头计算折叠揭示了高等教育的结构特性，这可能不是研究单个氨基酸顺序明显的影响。 R2 V3环冠几个先前晦涩的结构特性，揭示折叠模拟。这些观察到的结构的偏好可能关联与观察到的功能特性R2的应变，即灵敏度，以中和hyperinfectivity 5。 首先，一个清晰的β链N -末端的立场12-14 R2 V3的冠一半的趋势是观察。这是R2的应变不寻常的异亮氨酸，脯氨酸-蛋氨酸（IP...

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assessment of immunologically relevant dynamic tertiary structural features of the hiv-1 v3 loop crown r2 sequence by ab initio folding

的HIV - 1的抗原多样性，长期以来一直是疫苗设计的障碍，而这种变化是，尤其是在病毒表面的包膜糖蛋白V3环明显。我们以前提出的V3环的冠，虽然动态和序列变量，是整个人口的HIV - 1病毒免疫相关的β-发夹高等教育结构的制约。重要的是，HIV - 1病毒在流传有成千上万不同的V3环冠序列，使得整个晶体或核磁共振困难或不可能的病毒多样性的趋势的三维结构表征。我们以前的1，2冠的V3折叠成功研究用从头算法3的ICM - Pro的分子建模软件包（Molsoft有限责任公司，拉霍亚，CA）的访问和建议，V3环的冠，专门从位置10至22，足够的灵活性和其侧翼茎长度的行为在很大程度上就好像它是一个不受约束的肽溶液中的折叠自由的好处。作为等，迅速​​AB只是这部分的任何个人的60000株的V3 环的从头折叠+循环HIV - 1毒株，可以翔实的。在这里，我们折叠的V3环的R2菌株，争取到其独特的性能结构基础的洞察力。 R2蕴藏着难得的V3环序列被认为是负责这株精湛的灵敏度，以中和病人血清和单克隆抗体，4， 5。应变介导CD4独立的感染和出现，引起广泛中和抗体。我们将演示如何评价结果的折叠可以在折叠与R2的观察免疫活动相关联的观察结构信息。

Watch this Scientific Journal Video about Assessment of Immunologically Relevant Dynamic Tertiary Structural Features of the HIV-1 V3 Loop Crown R2 Sequence by ab initio Folding at JoVE.com

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

冠表面的HIV - 1包膜糖蛋白（gp120的）不同的V3的循环序列区域的结构特点，在许多情况下，在硅片的立场折叠10日至22日对一个国家的最先进的循环使用<em&gt;从头</em&gt;折叠算法。在这里，我们展示了这一地区从R2的HIV - 1菌株，一个独特的令人费解的功能特性的敏感株中和与V3环的折叠和评价。

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' ...

assessment-immunologically-relevant-dynamic-tertiary-structural

Research

JoVE Journal

Immunology and Infection

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

9.5K 次观看.  School of Medicine, New York University. 冠表面的HIV - 1包膜糖蛋白（gp120的）不同的V3的循环序列区域的结构特点，在许多情况下，在硅片的立场折叠10日至22日对一个国家的最先进的循环使用从头折叠算法。在这里，我们展示了这一地区从R2的HIV - 1菌株，一个独特的令人费解的功能特性的敏感株中和与V3环的折叠和评价。

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

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 ...

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

在艾滋病毒感染者病毒载量低于检测限制标准的临床实验放大和量化HIV - 1 RNA

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

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 ...

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 ...

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 ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

核衣壳退火介导电泳（NAME）分析允许HIV-1核衣壳抑制剂的快速鉴定

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

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. ...