HIV - 1脱壳在艾肯实验室的研究是由NIH的拨款AI40364，AI50423和AI076121支持。几个关键材料，包括在P24 ELISA试剂，由美国国立卫生研究院艾滋病研究和参考程序，艾滋病司，NIAID的，国立卫生研究院提供。

1。生产的HIV - 1颗粒在继续之前，必须获得许可，并按照您的机构的生物安全办公室的工作在艾滋病毒传染性批准了一项生物安全设施的指引。您必须确保适当的照顾，并在生物安全实验室工作期间的安全防范措施。 HIV - 1颗粒的生产通常是由瞬时转染293T细胞与HIV - 1前病毒DNA使用一个磷酸钙转染方法7，8 。也适用于高滴度病毒感染T细胞的文化成长股票。 <ol><li>文化293T细胞贝科的修改鹰中等（DMEM）中含有10％胎牛血清（FBS），并用抗生素为辅[青霉素（100国际单位/毫升）和链霉素（100微克/毫升）]在37 °彗星，5％的CO 2 。 </li><li>从近融合的菜肴分离细胞，用0.25％胰蛋白酶- EDTA和种子 2X10 6 </s9毫升每100毫米培养皿&gt;细胞转染前一天。种子六个100毫米的培养皿中，病毒颗粒的生产。如果核心的集中准备是必需的，更多的菜，可用于转染种子。 </li><li>第二天，细胞单层应该是25％左右相连，并准备转染。 6转染细胞的菜肴，带来120微克的前病毒DNA 2.7毫升无菌水的体积。这种混合物添加0.3毫升2.5米氯化钙2和3毫升2XBBS，混合移液器上下几次正确。让混合物在室温下静置10-20分钟。 </li><li>加入1毫升的混合物滴加，每道菜的中心，而纷飞轻轻混合。放置在一个孵化器设定在35 ° C和3％的CO 2的菜过夜（〜16小时）。 </li><li>吸出培养基，轻轻地添加5毫升PBS。 </li><li>吸PBS和添加5毫升新鲜培养基。文化的细胞培养箱中培养37及度; C和5％CO 2额外的24-48小时。 </li><li>收集含有病毒颗粒的培养上清，转移至50 ml离心管。从所有菜肴相结合的上清，离心5分钟沉淀细胞和碎片在1500 XG。使用0.45微米孔径的针头式过滤器，过滤上清液。应采取的预防措施，以尽量减少气溶胶的形成，而与活病毒的工作。这包括使用锥形管，用O型环，或在转子水桶盖的离心步骤。如果这是不可能的，管的螺丝帽应包装封口膜。 </li></ol> 2。浓度的HIV - 1病毒颗粒通过超速离心<ol><li>放置在一个38.5毫升polyallomer的离心管（贝克曼SW32Ti转子或同等学历）的培养上清（30毫升）。轻轻衬底5毫升20％的PBS中，用5毫升吸管蔗糖溶液含有病毒上清。 </li><li> 3小时离心32000 RPM在4 ° C（175,000 r 最高 XG）沉淀病毒颗粒。 </li><li>取出吸上清，照顾不打扰沉淀在试管底部。加入0.5毫升1XSTE缓冲区（10毫米的Tris - HCl [pH值7.4，100 mM氯化钠，1 mM EDTA的）管的地方，并在4 ° C为1小时放松沉淀。使用一个大口径的1毫升吸管尖轻轻吸管上下几次从管底沉淀分离。接下来，转让松动颗粒到1.5 ml离心管，照顾，以避免起泡。孵育另一个1-3个小时，让病毒的小团块分散在4 ° C。在此期间，准备在步骤3.1中所述的蔗糖梯度。 </li><li>轻轻地将吸管上下数次，并在8000转（6000 XG）在4℃冷藏在1分钟离心彗星离心机的病毒悬液，以去除残留的团块。在此过程中保持样品冷。 </li></ol> 3。蔗糖梯度离心隔离内核 HIV - 1核心是孤立的“自旋式”方法9，这是修改先前所描述的方法纯化HIV - 2 核心 10 。 <ol><li>准备14.5毫升polyallomer SW32.1Ti转子的离心管12毫升1XSTE缓冲区的线性30％至70％蔗糖密度梯度。要做好准备梯度，使用前20毫升的梯度;附近放置6毫升70％的蔗糖溶液（靠近出气口）和远方30％的蔗糖溶液6毫升。确保气泡没有被困在通道连接两院。使用前从底部到顶部包含底部85％的蔗糖溶液1毫升的管泵的梯度自动Densi流梯度。梯度轻轻将在4 ° C，直到冷却（2-4小时）。 </li><li>使用一个大口径的1毫升吸管尖轻轻地覆盖在科技教育的梯度含有1％的Triton X - 100的0.25毫升15％的蔗糖溶液。 </李&gt; <li>轻轻覆盖，用0.25毫升7.5％蔗糖科技教育，用一个大口径的1毫升吸管尖端的解决方案。一定要小心，不要混合层。 </li><li>从步骤2.4与0.5毫升病毒悬液轻轻叠加。这一步应该认真执行，以避免在渐变中的任何干扰和混合underlaid蔗糖层。 </li><li>管放置在预冷SW32.1Ti桶和离心32,000 RPM（r 最高 XG 187000）过夜（16-20小时）在4 ° C。为了避免暂停离心机，不启动离心真空离心机，直到达到250微米</li><li>收集1毫升分数从顶部底部使用自动Densi流量梯度分馏梯度。管收集后立即置于冰上。反相几次混合管的内容，并撤回50μL每个ELISA或逆转录酶活性检测的量化分数。 </li></ol> 4。 HIV - 1核心的本地化和存储的核心<ol><li>执行脱壳分析之前，重要的是要确定哪些分数包含完整的HIV - 1核心。这可以由P24 ELISA或逆转录酶的活性测定步骤3.6使用50μL等份。 </li><li>与核相关的CA的复苏往往涉及到稳定的核心。 9 P24 ELISA法使用样品从每一个分数衡量的核心相关的CA 。另外，衡量每个分数的逆转录酶活性。使用这两种方法的峰值应该围绕分数10。 </li><li>池的分数包含核心。如果不脱壳立即检测，到0.2至0.3毫升分装，液氮和存储的闪存冻结在-80 ° C等分汇集核心的核心是以这种方式冻结的核心是适合脱壳检测。核心相关的CA的收益率通常是1-2微克每毫升P24或约15％的总的病毒粒子相关P24。 </li></ol> 5。 HIV - 1脱壳动力学检测<ol><li>每脱壳反应所需的HIV - 1核心的金额约为50纳克。要执行在体外脱壳检测，predilute核心等分等体积冷1XSTE缓冲区，以减少悬浮液的粘度，并尽量减少移液错误。 </li><li>进一步稀释到0.15毫升1.5毫升的离心管中含有10微克/ ml的BSA的缓冲冷1XSTE 100μL的核心。我们补充与牛血清白蛋白（10微克/毫升）的STE缓冲，以尽量减少CA的吸附管的墙壁。混合反相管的几倍。不要涡旋样品。水浴管在37 ° C的时间间隔（15，30，60和120分钟）。管应沉浸在水浴中至少内部采样水平，以确保即使变暖。 </li><li>孵化过程中，轻轻混匀管的内容定期（一般为5 -10分钟）弹管。对于零分钟控制，稀释成0.15毫升冷1XSTE缓冲液100μL的核心，并在冰上孵育实验（120分钟）的整个长度。零分钟控制提供了基础脱壳值，用于确定在37 ° C下孵育不同的时间间隔的核心脱壳增加。 </li><li>在潜伏期结束，停止脱壳过程中，在冰中放置10分钟的管。 </li><li>离心管，45,000 RPM（TLA - 55转子; 125,000 r最高XG）20分钟，在4 ° C 。这将颗粒完整的核心和自由为核心脱壳的CA发布将继续留在上清。预冷的转子应在4 ° C之前加载样品。 </li><li>将上清转移至新预冷的离心管，最好使用在室温下储存的凝胶加载提示。请注意，颗粒将肉眼不可见，所以采取极端的照顾瓦特hile吹打出上清液。 ELISA样品稀释液250μL（0.5％的Triton X - 100和5％的捐助者的小牛血清的PBS）重悬的颗粒。为了确保颗粒溶解后加入ELISA样品稀释液的高效，涡。使用P24 ELISA法如以下部分所述，量化的沉淀和上清的CA内容。 </li><li>脱壳程度获得通过计算上清液中的CA的一小部分。 </li></ol> 6。为CA P24酶联免疫吸附测定<ol><li>许多商业ELISA试剂盒，可用于量化CA。使用任何商业或“内部”ELISA法，包括P24的标准来确定检测的线性。稀释的样品应当检测，以确保准确性。 </li><li>我们已经开发出一个自制的ELISA我们经常使用的CA检测。的过程，是适应从以前的报告第11和使用捕获和检测抗体可从NIH援助的研究和参考试剂的计划。 </li></ol> 7。代表性的成果： 从ELISA结果确定分数包含核心的一个例子是如图1所示。后收集的分数（每1 mL），50μL的样品从每个分数是用来做系列稀释，并用ELISA分析。 P24增加12分数值总额为14.5微克。正如图中的分数显示8日至10所载的核心。 P24价值为核心含分数为2.17微克。核心相关的P24的比例（14.97％）是由P24价值的核心成分的比例（2.17微克）的P24值总额（14.5微克）。分数1或2将始终包含最高的P24数额;这免费的CA，这是不符合相关的核心。其次是每个后续的分数与P24值逐渐减少，然后急剧增加，终于在锐减的P24值。如果采取适当的照顾，同时加载的梯度，然后核心相关的CA峰附近发现的一小部分10。 化验HIV - 1芯脱壳动力学得到一个典型的结果如图2所示。图表显示在野生型HIV - 1核心脱壳的时间依赖性增加。 ％脱壳值计算上清液中的CA的一小部分。通过练习，检测是高度重复性，并确定在体外的病毒核心的稳定非常有用。请注意，检测的可重复性是高度依赖的样品在操作过程中妥善处理。由于内核是热不稳定，样品应保持在4 ° C整个检测，除在潜伏期。 <img alt="图1" src="/files/ftp_upload/3384/3384fig1.jpg" /> 图1平衡密度梯度离心HIV - 1核心。浓缩病毒悬液被应用到了30％至70％的线性蔗糖梯度覆盖1％TRITON - X - 100的顶部。以下过夜离心187000 XG和4 ° C，1毫升的分数（分数1）从顶部的底部（分数12）收集和P24用ELISA量化。 <img alt="图2" src="/files/ftp_upload/3384/3384fig2.jpg" /> 图2 在体外对HIV - 1脱壳的时间当然。纯化的HIV - 1核心是稀释和培养在37℃，在指定的时间间隔。零分钟样品孵育实验的整个长度的冰。经过孵化，20分钟样品ultracentrifuged 125000 XG在4 ° C。上清和沉淀分离和P24酶联免疫吸附分析。脱壳程度被认定为在协议文本描述。每个脱壳反应是一式两份;误差线跨越两个值的范围。

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没有利益冲突的声明。

这里描述的方法来净化HIV - 1核心，研究在体外脱壳研究这种HIV - 1生命周期的阶段，特别是由于没有一个有效的方法来分析靶细胞的HIV - 1脱壳。虽然这种方法使用平衡离心净化核心，其他人使用简要裂解后直接制粒，1％的Triton - X - 100 12 13或通过造粒蔗糖垫覆盖0.1％的Triton X - 100 的14，15 。 通过这种方法获得的内核的复苏是受实验误差。一个变量是在这个协议...

<table border="1"><tbody><tr><td width="190"> 试剂名称 </td><td width="197"> 公司 </td><td width="114"> 目录编号 </td><td width="161"> 评论（可选） </td></tr><tr><td> DMEM培养液</td><td> Cellgro </td><td> 10 - 013 - CV </td><td></td></tr><tr><td> PBS </td><td> Cellgro </td><td> 21 - 031 - CV </td><td></td></tr><tr><td> TRITON - X - 100 </td><td> Mallinckrodt贝克公司</td><td> 9002-93-1 </td><td></td></tr><tr><td>吐温20 </td><td>阿库罗斯福冈</td><td> 9005-64-5 </td><td></td></tr><tr><td> 0.25％胰蛋白酶EDTA </td><td> Cellgro </td><td> 25 - 053 - CI </td><td></td></tr><tr><td> 2XBBS </td><td></td><td></td><td>组成：50MM百世H 6.95 [P]，280毫米NaCl和1.5毫米的Na 2 HPO 4。 pH值调整到6.95在室温下。过滤消毒及分装储存在-20 ° C </td></tr><tr><td>涂料抗体：单克隆抗体P24（183 - H12 - 5C） </td><td>美国国立卫生研究院艾滋病研究和参考试剂</td><td> 3537 </td><td></td></tr><tr><td>一抗HIV - IG（高免人类患者血清） </td><td>国立卫生研究院艾滋病研究并参考试剂计划</td><td> 3957 </td><td></td></tr><tr><td>二抗： 山羊抗人IgG，过氧化物酶标记</td><td>刺穿</td><td> 31130 </td><td></td></tr><tr><td> HRP的底物</td><td>嘉里建设有限公司</td><td> 50-76-11 </td><td></td></tr><tr><td> Immulon 2HB 96孔板</td><td> Thermo Scientific的</td><td> 3455 </td><td></td></tr><tr><td> SW32Ti和SW32.1 TI转子和兼容超速离心机</td> &lt;TD&gt;贝克曼</td><td></td><td></td></tr><tr><td> TLA - 55转子和台式超速离心机</td><td> Beckman Coulter公司</td><td></td><td></td></tr><tr><td>高速离心管</td><td> Beckman Coulter公司</td><td> 326823，358123，357448 </td><td></td></tr><tr><td>自动Densi流密度梯度分馏</td><td> Labconco公司</td><td> 4517000 </td><td></td></tr><tr><td>渐变前</td><td> CBS科技有限责任公司</td><td> GM - 20 </td><td></td></tr></tbody></table>

in vitro uncoating of hiv-1 cores

的逆转录病毒的基因组是由脂质包膜包围一个衣壳，包裹。慢病毒，如HIV - 1，锥形衣壳外壳组成的六角晶格排列的CA蛋白。衣壳是关闭的7 pentamers广泛年底，在窄的一端的锥1，2 5。在这衣壳外壳外壳是病毒的核蛋白复杂，和他们一起组成的核心。 HIV - 1病毒膜与靶细胞膜的融合，是释放到细胞质。衣壳，然后拆卸 3中的可溶性形式释放游离Ca简称为脱壳的过程。细胞内的位置和HIV - 1脱壳的时间是知之甚少。在CA的单一氨基酸替换改变衣壳的稳定性也削弱的HIV - 1感染细胞的能力4。这表明衣壳的稳定性是HIV - 1感染的关键。 </p&gt; HIV - 1脱壳已经很难研究由于缺乏这个过程中的敏感和可靠的检测。在这里，我们描述了一个定量方法研究使用体外传染性的HIV - 1颗粒分离的核心脱壳。该方法涉及了一层洗涤剂，并通过集中的病毒颗粒的沉淀核心的隔离成线性蔗糖梯度，在寒冷的。要量化脱壳，孤立的核心是在37 ° C的各种时间间隔，随后离心沉淀。脱壳程度上清液中的CA的一小部分通过量化分析。已采用这种方法分析HIV - 1衣壳的稳定性4，5，6的病毒突变的影响。研究HIV - 1脱壳的细胞因子的作用，还应该有用。

Watch this Scientific Journal Video about In vitro Uncoating of HIV-1 Cores at JoVE.com

In vitro Uncoating of HIV-1 Cores

脱壳是在HIV - 1生命周期的早期阶段的重要一步，并定义为衣壳外壳的拆卸和释放病毒核蛋白复合物（vRNP）。在这里，我们展示了隔离HIV - 1病毒颗粒的完整的核心和量化其脱壳技术<em&gt;在体外</em&gt;。

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 ...

Research

JoVE Journal

Immunology and Infection

In vitro Uncoating of HIV-1 Cores

15.6K 次观看.  Vanderbilt University School of Medicine. 脱壳是在HIV - 1生命周期的早期阶段的重要一步，并定义为衣壳外壳的拆卸和释放病毒核蛋白复合物（vRNP）。在这里，我们展示了隔离HIV - 1病毒颗粒的完整的核心和量化其脱壳技术在体外。

视频: In vitro Uncoating of HIV-1 Cores

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 ...

科研

免疫与感染

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 ...

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 ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

一个<em&gt;在体外</em&gt;共感染模型研究<em&gt;恶性疟原虫</em人原发性单核细胞源性的免疫细胞-HIV-1相互作用

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

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

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

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

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

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

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

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. ...

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an essential step for virus particle formation, since a defect in Gag membrane binding results in severe impairment of viral particle production. To gain mechanistic details of Gag-lipid membrane interactions, in vitro methods based on NMR, protein footprinting, surface plasmon resonance, liposome flotation centrifugation, or fluorescence lipid bead binding have been developed thus far. However, each of these in vitro methods has its limitations. To overcome some of these limitations and provide a complementary approach to the previously established methods, we developed an in vitro assay in which interactions between HIV-1 Gag and lipid membranes take place in a &#34;cell-like&#34; environment. In this assay, Gag binding to lipid membranes is visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique. Here we describe the background and the protocols to obtain myristoylated full-length Gag proteins and GUV membranes necessary for the assay and to detect Gag-GUV binding by microscopy.

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle GUV Membranes

We illustrate here an in vitro membrane binding assay in which interactions between HIV-1 Gag and lipid membranes are visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique.

HIV-1 Gag的可视化绑定到巨人的单片囊泡（GUV）膜

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an ...

Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential to be used as drug therapies for HIV-1 infection. Post-integration inhibitors include ribozymes, short hairpin (sh) RNAs, small interfering (si) RNAs, U1 interference (U1i) RNAs and RNA aptamers. Many of these have been identified using transient co-transfection assays with an HIV-1 expression plasmid and some have advanced to clinical trials. In addition to measures of efficacy, small RNAs have been evaluated for their potential to affect the expression of human RNAs, alter cell growth and/or differentiation, and elicit innate immune responses. In the protocols described here, a set of transient transfection assays designed to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps in the HIV-1 replication cycle are described. We have used these assays to identify new ribozymes and optimize the format of shRNAs and siRNAs targeting HIV-1 RNA. The methods provide a quick set of assays that are useful for screening new anti-HIV-1 RNAs and could be adapted to screen other post-integration inhibitors of HIV-1 replication.

Methods to evaluate the efficacy and toxicity of RNA molecules targeting post-integration steps of the HIV-1 replication cycle are described. These methods are useful for screening new molecules and optimizing the format of existing ones.

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

Small RNA therapies targeting post-integration steps in the HIV-1 replication cycle are among the top candidates for gene therapy and have the potential ...

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 ...