作者从Corus公司，MedSys进入细胞，CHAIN​​和血竭项目资助。

该协议被总结在图。 4。 1。采集血样<ol><li>在EDTA管收集至少3毫升的血液在临床的网站。 </li><li>可将样本存储在一个星期，在4℃下</li><li>只要可能的实验室通过普通邮件发送EDTA血液样本。 </li></ol> 2。艾滋病毒的RNA和/或DNA的分离<ol><li>获取从1000微升的全血，血清或血浆中的病毒RNA和/或DNA，使用在MagNA Pure契约核酸隔离I包我在MagNA Pure紧凑型系统。 </li><li>洗脱获得的RNA或DNA在50μlTE缓冲液中。或者，其他的核酸纯化程序可以被使用。 </li></ol> 3。 env区的扩增扩增的env无论从等离子体RNA或原病毒DNA的区域（图2和图4）。一个长1245 bp的产品，包括大多数的Env蛋白（新台币6556-7811 according至HXB2）被生成。 <ol><li> RNA样品的RT-PCR反应<ol><li>病毒RNA呈现一个非常复杂的二级结构（图4），可能会阻碍反转录（RT）反应。为了避免这个问题，在65℃下孵育10μlRNA提取10分钟（已经在PCR管中，并在PCR机器），然后将温度降低至50℃。 </li><li>加入40μl的PCR扩增预混试剂，包括引物env-F的和Env-R。 预混RT-PCR在内务系统： <table border="1"><tbody><tr><td></td><td> 体积/反应（微升） </td><td> 终浓度 </td></tr><tr><td>无RNA酶H 2 O </td><td> 14.4 </td><td></td></tr><tr><td> 5×缓冲液（Qiagen公司的一步法RT-PCR试剂盒） </td><td> 10 </td><td> 1个</td></tr><tr><td> 5倍Q-解决方案</td><td> 10 </td><td> 1个</td></tr><tr><td> dNTP混合物（10毫米） </td> &lt;TD&gt; 2 </td><td> 400μM的每种dNTP </td></tr><tr><td>体酶混合物混合（Qiagen公司的一步法RT-PCR试剂盒） </td><td> 2 </td><td></td></tr><tr><td>入门ENV-F（100μM） </td><td> 0.3 </td><td> 0.6μM </td></tr><tr><td>入门ENV-R（100μM） </td><td> 0.3 </td><td> 0.6μM </td></tr><tr><td> RNase抑制剂（RNA酶抑制剂） </td><td> 1 </td><td> 5单位/反应</td></tr></tbody></table> 的env-F：的5&#39;-CAAAGCCTAAAGCCATGTGTAAA -3&#39;（核苷酸6556→6586根据HXB2） ENV-R：的5&#39;-AGTGCTTCCTGCTGCTCCTAAGAACCC -3“（nt的7785←7811） </li><li>运行反转录（RT）反应在50℃下30分钟。 </li><li>运行第一PCR如下： <table border="1"><tbody><tr><td> 95°C，15分钟</td><td> 1个</td></tr><tr><td> 95°C，30秒 50℃，30秒 72°C，2分钟</tD&gt; <td> 1个</td></tr><tr><td> 95°C，30秒 56℃，30秒 72°C，2分钟</td><td> 38× </td></tr><tr><td> 72°C，10分钟 4°C∞ </td><td></td></tr></tbody></table></li></ol></li><li> DNA样品PC​​R反应 对于原病毒DNA样本，需要没有初始的反转录（RT）反应，PCR反应可以直接启动。 <ol><li>加入40μl的PCR主要混合液至10微升provial脱氧核糖核酸。 PCR反应混合液在内务系统： <table border="1"><tbody><tr><td></td><td> 体积/反应（微升） </td><td> 终浓度 </td></tr><tr><td>无RNA酶H 2 O </td><td> 15.4 </td><td></td></tr><tr><td> 5×缓冲液（HotStarTaq DNA聚合酶） </td><td> 10 </td><td> 1个</td></tr><tr><td> 5倍Q-解决方案</td><td> 10 </td><td> 1个</td></tr><tr><td> dNTP混合物（10毫米） </td><td> 2 </td><td> 400μM的每种dNTP </td></tr><tr><td>体酶混合物混合（HotStarTaq DNA聚合酶） </td><td> 2 </td><td></td></tr><tr><td>入门ENV-F（100μM） </td><td> 0.3 </td><td> 0.6μM </td></tr><tr><td>入门ENV-R（100μM） </td><td> 0.3 </td><td> 0.6μM </td></tr></tbody></table> ENV-F：的5&#39;-CAAAGCCTAAAGCCATGTGTAAA -3“（nt的6556→6586） ENV-R：的5&#39;-AGTGCTTCCTGCTGCTCCTAAGAACCC -3“（nt的7785←7811） </li><li>如先前所述的RNA样品（步骤3.1.4）运行的PCR条件。 </li></ol></li></ol> 4。巢式PCR扩增的V3区： <ol><li>巢式PCR，用5微升的第一次PCR反应（不包括前面的分析中琼脂糖凝胶电泳）为模板，加入95微升的PCR MAS后混合。 嵌套在内务PCR预混<table border="1"><tbody><tr><td></td><td> 体积/反应（微升） </td><td> 终浓度 </td></tr><tr><td> H 2 O </td><td> 61.9 </td><td></td></tr><tr><td> 10×PCR缓冲液</td><td> 10 </td><td> 1个</td></tr><tr><td> 5倍Q-解决方案</td><td> 20 </td><td> 1个</td></tr><tr><td> dNTP混合物（10毫米） </td><td> 2 </td><td> 200μM的每种dNTP </td></tr><tr><td>引物ENV-2F（100μM） </td><td> 0.3 </td><td> 0.6μM </td></tr><tr><td>引物ENV-2R（100μM） </td><td> 0.3 </td><td> 0.6μM </td></tr><tr><td> HotStarTaq DNA聚合酶</td><td> 0.5 </td><td> 2.5单位/反应</td></tr></tbody></table> ENV-2F：5&#39;-GTCCAAAGGTATCCTTTGAGCCAATTC的3&#39;（新台币6838→6864） <strong&gt;环境-2R：5&#39;-CACCACTCTTCTCTTTGCCTTGGTGGGTGC的的-3“（nt的7712←7742） </li><li>运行的PCR如下： <table border="0"><tbody><tr><td> 93°C，15分钟</td><td> 1个</td></tr><tr><td> 95°C，30秒 50℃，30秒 72°C，90秒</td><td> 1个</td></tr><tr><td> 95°C，30秒 56℃，30秒 72°C，90秒</td><td> 43× </td></tr><tr><td> 72°C，10分钟 4°C∞ </td><td></td></tr></tbody></table></li><li>分析的PCR产物在1％琼脂糖凝胶（图4）。预期的产品为902 bp的长。 </li><li>测序的样品纯化。 <ol><li>用Qiagen公司的PCR纯化试剂盒纯化PCR产物，使用的协议，由供应商提供的解决方案和微柱。在30-100微升PE缓冲液洗脱，根据带的强度。 </li><li>或者，可将样本用外切核酸酶我和快速AP（热碱性磷酸酶）。为了这个目的，添加6微升外AP混合到15微升的PCR产物，并孵育15分钟，在37℃。 <table border="1"><tbody><tr><td> 580微升H 2 O </td></tr><tr><td> 66微升的快速AP </td></tr><tr><td> 13.4微升埃克索我</td></tr></tbody></table></li></ol></li></ol> 5。测序反应的V3扩增子<ol><li>测序反应由仅使用其中一个下列引物的PCR反应： ENV-2：5&#39;-GTACAATGYACACATGGAATTAGGC的的-3（nt的6959→6980） ENV-5：5&#39;-AAAATTCCCCTCCACAATTA - 3&#39;（nt的7352←7371） ENV-6：5&#39;-GGCCAGTAGTATCAACTCAAC - 3&#39;（新台币6979→7000） ENV-7：5&#39;-TGTCCACTGATGGGAGGGGC - 3&#39;（nt的7530←7549） ENV-10：5 - GCAGAATAAAACAAATTATAAACATGTGGC - 3&#39;（nt的7478←7507） 信封-11：5&#39; - TACATTGCTTTTCCTACTTTCTGCCAC - 3&#39;（nt的7638←7664） 首选引物分别是：ENV-2，ENV-6或env-7，ENV-11 </li><li>加入1μl纯化的V3扩增产物为模板，并添加9μl的PCR主混合。 测序主混音： <table border="1"><tbody><tr><td> 4μL的顺序混合（艾滋病毒基因分型试剂盒） </td></tr><tr><td> 4微升H 2 O </td></tr><tr><td> 1μl引物（100皮摩尔/微升） </td></tr></tbody></table></li><li>运行序列反应如下： <table border="0"><tbody><tr><td> 96°C，持续10秒</td><td></td></tr><tr><td> 50℃，10秒</td><td> 36× </td></tr><tr><td> 60℃，45秒</td><td></td></tr><tr><td> 4°C∞ </td><td></td></tr></tbody></table></li></ol> 6。测序的样品纯化净化序列USI吴经Sephadex G-50超细。 <ol><li>在“列装载器”，用Sephadex G-50超细填写适当数量的井。转移交联葡聚糖滤板MAHVN4510的翻身。 </li><li>在过滤板，每孔加入300μl的Milli-Q H 2 O，下培养至少3小时，在2-8°C。 </li><li>离心5分钟的板在910Xg和4-15℃。丢弃流过。 </li><li>加入10μlH 2 O至序列的产品，然后移液管上的序列的溶胀的交联葡聚糖板。 </li><li>为了获得纯化的产物，离心5分钟的板。在910Xg和4-15°C，并收集流过。 </li></ol> 7。测序的测序仪ABI PRISM 3130 XL的纯化样品对<ol><li>每次运行之前，改变缓冲液和检查的聚合物（POP7）的体积。如果需要的话，由一个新的更换旧的聚合物烧瓶。 </li><li>使用所保存的运行条件，包括注射时间为18秒。 </li><li>在这台机器，采集的信号有16个序列样本数据的一个运行大约需要60分钟（36厘米毛细管）或150分钟（50厘米毛细管）。 </li></ol> 8。序列编辑<ol><li>使用该程序Lasergene（DNA明星）对齐和编辑的序列（图5）。可以使用其他序列编辑程序。使用一个“V3-共识”B“和作为参考的 env一致序列。 </li><li> Lasergene创建一个的共识序列分析样品所有的原始数据，并将其存储FASTA文件。的FASTA文件是一个文本文件，该文件包括一个标题用的样品和核苷酸序列的名称。 </li></ol> 9。测序数据的解释和取向预测<ol><li>测序数据解释执行的基于Web的解释系统geno2pheno 辅助受体（ <a href="http://coreceptor.bioinf.mpi-inf.mpg.de/index.php">http://coreceptor.bioinf.mpi-inf.mpg.de/</a>的index.php）。对于嗜性预测，可以选择不同的FPR设置。默认设置是根据德国和奥地利的治疗指引。对于FPR≥20％，该病毒被归类时，作为R5 FPR≥20％和X4为FPR &lt;12.5％。 </li><li> FASTA文件上传到服务器。此服务器的核苷酸序列翻译成氨基酸，它与V3-共识B序列对齐，并产生一个亚型分类。此外，它会产生一个预测的辅助受体的使用（图4）为假阳性率（FPR）表示。 </li></ol> 10。代表性的成果 geno2pheno 辅助受体输出显示了一个渐变的解释取向。根据使用的辅助受体的可能性，解释文本和背景颜色变化从绿色（FPR &lt;20％），安全管理提出了MVC，地黄，暗示可能，风险低，最后到红色。红色是颜色建议ING不规定MVC。此外，服务器生成一个PDF格式的报告可以打印，填写病人的样本数据，发送给医生。的geno2pheno [辅助受体]输出的实施例描绘在图6。 <img alt="图1" src="/files/ftp_upload/3264/3264fig1.jpg" /> 图。 1。原理的HIV的复制。病毒颗粒细胞CD4受体结合，无论是作为辅助受体CCR5或CXCR4。辅助受体CCR5基因可以阻止CCR5拮抗剂如Maraviroc（MVC，Celsentri，Selzentry）。病毒和细胞的膜融合后，病毒核衣壳在细胞质中被释放。核衣壳拆卸和病毒RNA复合物被释放到细胞质中。病毒的逆转录酶（RT）的基因组RNA转录成原病毒DNA，然后被输送到细胞核，并整合入宿主基因组中的HIV整合。细胞RNA聚合酶TRAnscribe的前病毒基因组中的病毒基因组和信使RNA。产生的病毒蛋白在细胞质中，并输送到细胞表面。不成熟的，非感染性的病毒颗粒从细胞中的病毒颗粒芽。 HIV蛋白酶切割传染性微粒的蛋白质的生产。抑制的步骤之一导致中断的复制周期。 抗逆转录病毒药物和复制的步骤，他们抑制被标记为红色。 病毒RNA和绿色标记的前病毒DNA（所用材料趋向性或耐药性分析）。 <img alt="图2" src="/files/ftp_upload/3264/3264fig2.jpg" /> 图2。建立HIV前病毒基因组的 HIV颗粒具有不同的结构蛋白和房屋的各种酶和蛋白质，病毒和细胞。下图展示S的病毒的基因组内的基因的位置。表面蛋白gp120和gp41的病毒粒子的表面上，构成尖峰人体细胞联系来执行膜融合。的PCR扩增产物和我们的协议中使用的引物，在该图的下部，示出。 <img alt="图3" src="/files/ftp_upload/3264/3264fig3.jpg" /> 图3。低病毒载量（VL）的患者比例测量分析研究所病毒学大学，科隆（德国）的耐药性或嗜性的判断。 <img alt="图4" src="/files/ftp_upload/3264/3264fig4.jpg" /> 图4。总体方案的实验 ..请<a href="https://www.jove.com/files/ftp_upload/3264/3264fig4_lg.jpg">点击这里</a>看到一个更大的版本，这个数字。 <img alt="图5" src="/files/ftp_upload/3264/3264fig5.jpg" /> 图5。序列编辑Lasergene V3的扩增子与至少一个正向和一个反向引物测序。 lasergene alignes“。阿比”的文件从音序获得与参考序列“V3-共识”B“和env。 ，Lasergene创建一个共识序列使用的所有行数据。可以被标记的参考序列（和然后以灰色显示），以便它们不有助于样品一致序列。 <img alt="图6" src="/files/ftp_upload/3264/3264fig6.jpg" /> 图6。归经预测报告所产生的geno2pheno [辅助受体工具。报告生成的PDF文件可以保存在电脑上完成。附加数据例如患者的姓名，血液提取，日期等，可手动包括使用的pdf作家程序。还可以添加具体意见。帖E数据完全是在用户的计算机和不上geno2pheno的[辅助受体]服务器。请<a href="https://www.jove.com/files/ftp_upload/3264/3264fig6_lg.jpg">点击这里</a>看到一个更大的版本，这个数字。

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<li>Toma, J., Frantzell, A., Hoh, R., Martin, J., Deeks, S., Petropoulos, C., Huang, W. Determining HIV-1 Co-receptor Tropism Using PBMC Proviral DNA Derived from Aviremic Blood Samples. 18th Conference on Retroviruses and Opportunistic Infections, San Francisco, USA, , (2010).</li>
</ol>

此的视频文章是由欢跃医疗保健和葛兰素史克公司。

的V3序列允许可靠的病毒嗜性预测，在临床研究中3-9所示。事实上，基因型确定拟在目前的欧洲，德国和奥地利的指引10。 表型检测相比，不仅是周转时间更短（相当于电阻测试），但也有成本。此外，基因型检测的一个主要优点是，结果评定为FPR和因此，可以适应患者的需求。另一方面，trofile结果，简单地R5或X4报告，还没有给定的原始数据的访问。 目前，欧洲指南建议FPR截止的20％10，而奥地利和德国的指引，允许适应 ​​的FPR截止专门的每个病人的需要11。在这条线，具有广泛的抗逆转录病毒药物的选择，更高的FPR开口销（&gt; 20％）的患者RECOMM结束。相反的，对于重预治疗的患者的治疗选择有限，低级的FPR切割冲销（&gt; 5％）可以被使用。这种治疗指导目前正在进行的NRTI类药物，NNRTI类药物，督察和INIS，其中局部活性药物可被包括在与减少的治疗选择的患者治疗的电阻测试。此外，基因型引导治疗的变化，越来越多的临床相关的FPR切的平衡不断被调整，由一组生物信息学家，病毒学家和临床医生。 的基因型的嗜性测试的另一个重要的优点是非常低，甚至检测不到病毒载量（VL）的临床样本分析的可能性。在这种情况下，当等离子体RNA是不扩增，原病毒DNA可以被测序，并用于可靠的预测9,12。值得注意的是，低或不可检测的病毒载量的患者的数量急剧增加在最近几年。至目前为止，在T从VL &lt;1000拷贝/ ml的患者，只允许rofile分析样品的分析。然而，初步研究表明，前病毒DNA，也可以由Trofile 13 adecuate要测试。

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>麦格纳的纯紧凑型核酸提取试剂盒我</td><td>罗氏诊断</td><td> 04 802 993 001 </td><td></td></tr><tr><td>的MagNA Pure Compact系统</td><td>罗氏诊断</td><td> 03731146001 </td><td></td></tr><tr><td> T3000热循环仪</td><td> Biometra </td><td> 050-723 </td><td></td></tr><tr><td>一步法RT-PCR试剂盒</td><td> Qiagen公司</td><td> 210215 </td><td></td></tr><tr><td> HotStarTaq预混试剂盒</td><td> Qiagen公司</td><td> 203445 </td><td></td></tr><tr><td> QIAquick PCR纯化试剂盒</td><td> Qiagen公司</td><td> 28106 </td><td></td></tr><tr><td>核酸外切酶I（埃克索我） </td><td> Fermentas公司</td><td> EN0582 </td><td></td></tr><tr><td>的Fastap热敏碱性磷酸酶</td><td> Fermentas公司</td><td> EF0651 </td> &lt;TD&gt; </td></tr><tr><td> BigDye Terminator v3.1循环测序试剂盒</td><td>美国应用生物系统公司</td><td> 4337457 </td><td></td></tr><tr><td>葡聚糖凝胶G-50超细</td><td> GE医疗集团</td><td> 17-0041-01 </td><td></td></tr><tr><td> ABI棱镜3130 XL毛细管测序</td><td>美国应用生物系统公司</td><td> 3130xl基因分析</td><td></td></tr></tbody></table>

prediction of hiv-1 coreceptor usage (tropism) by sequence analysis using a genotypic approach

Maraviroc（MVC）是第一家持牌的抗逆转录病毒药物的辅助受体拮抗剂类。它绑定到主机的辅助受体CCR5，它是由多数的艾滋病毒菌株感染人体免疫细胞（图1）。其他的艾滋病毒菌株使用不同的辅助受体，CXCR4。使用哪一个受体，被确定的Env蛋白（图2）中的病毒。根据所使用的辅助受体，这些病毒是分类为R5或X4分别。 MVC的绑定到CCR5受体抑制R5病毒进入靶细胞的条目。在疾病的过程中，X4病毒的出现和长大的R5病毒。测定的辅助受体的使用（也称为嗜性），因此前必须进行管理的MVC，EMA和FDA所要求的。 已进行的研究MVC效率的激励，MERIT和1029的Trofile分析会标，美国旧金山，这是一个高品质分析的基础上sophisticated重组的测试。接受本次测试的日常是相当低的，因为美国以外的欧洲的医生，而往往与分散的专家实验室，这也提供伴随性测试。这些实验室已经经历了几次的质量保证评估，最后在2011年1。 几年来，我们已进行趋向性测定HIV的env-V3（V3）2基因区域序列分析的基础上。这个地区进行足够的信息来进行可靠的预测。的辅助受体的基因型确定使用的优势，如：更短的周转时间（相当于电阻测试），更低的成本，可能适应的结果，患者的需求和临床样本进行分析的可能性非常低，甚至检测不到病毒载量（VL ），特别是因为与VL的分析样品的数量&lt;1000拷贝/μl大致在过去几年中增加（图3）。 嗜性测试的主要步骤（图4）在这个视频演示： 1。采集的血液样本 2。从血浆和/或从血液中单核细胞的HIV前病毒DNA的HIV RNA的分离 3。 env区的扩增 4。扩增的V3地区， 5。测序反应的V3扩增子 6。测序的样品纯化 7。测序的纯化样品 8。序列编辑 9。测序数据的解释和取向预测

Watch this Scientific Journal Video about Prediction of HIV-1 Coreceptor Usage Tropism by Sequence Analysis using a Genotypic Approach at JoVE.com

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

预测HIV-1的辅助受体的使用需要管理的一类新的抗逆转录病毒药物，即辅助受体拮抗剂。它可以通过序列分析<em&gt;环境</em基因和随后的解释，通过一个基于互联网的同声传译系统（geno2pheno<sub[辅助受体]</sub&gt;）。

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 ...

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prediction-hiv-1-coreceptor-usage-tropism-sequence-analysis-using

Universidad Científica del Sur

Research

JoVE Journal

Immunology and Infection

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

13.3K 次观看.  University of Cologne.  Español 

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

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 ...

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 ...

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 ...

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

用于下一代测序的近全长 HIV-1 Proviruses 的扩增

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

遗传学

Determining 3'-Termini and Sequences of Nascent Single-Stranded Viral DNA Molecules during HIV-1 Reverse Transcription in Infected Cells

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and host cell proteins on viral DNA synthesis. Here we address the lack of a cell-based, high-coverage, and high-resolution assay that is capable of defining retroviral reverse transcription intermediates within the physiological context of virus infection. The described method captures the 3'-termini of nascent complementary DNA (cDNA) molecules within HIV-1 infected cells at single nucleotide resolution. The protocol involves harvesting of whole cell DNA, targeted enrichment of viral DNA via hybrid capture, adaptor ligation, size fractionation by gel purification, PCR amplification, deep sequencing, and data analysis. A key step is the efficient and unbiased ligation of adaptor molecules to open 3'-DNA termini. Application of the described method determines the abundance of reverse transcripts of each particular length in a given sample. It also provides information about the (internal) sequence variation in reverse transcripts and thereby any potential mutations. In general, the assay is suitable for any questions relating to DNA 3'-extension, provided that the template sequence is known.

Here we present a deep sequencing approach that provides an unbiased determination of nascent 3&#39;-termini as well as mutational profiles of single-stranded DNA molecules. The main application is the characterization of nascent retroviral complementary DNAs (cDNAs), the intermediates generated during the process of retroviral reverse transcription.

hiv-1 反向转录过程中鼻部单链病毒 dna 分子的 3 '-特米尼和序列的测定

Monitoring of nucleic acid intermediates during virus replication provides insights into the effects and mechanisms of action of antiviral compounds and ...

Next-generation Viral RNA/DNA in situ Hybridization Applications in Human Immunodeficiency Virus/Simian Immunodeficiency Virus Research

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important insights into physiological processes and disease pathogenesis. In situ hybridization (ISH) has been used for many years to assess the location of cells infected by viruses, but recently a next-generation ISH approach was developed with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. This next-generation ISH is based on an approach like branched PCR, but performed in situ and is more facile, sensitive, and reproducible than classical ISH methods or in situ PCR approaches in routinely detecting RNA or DNA in formalin-fixed paraffin embedded (FFPE) tissues. For the last several years our laboratory has been applying this ISH platform for the detection of human immunodeficiency (HIV) and simian immunodeficiency (SIV) viral RNA (vRNA) and/or viral DNA (vDNA) positive cells within a multitude of FFPE tissues. With this detailed technical manuscript, we would like to share our knowledge and advice with all individuals interested in using next-generation ISH in their research.

<meta charset="utf8"></head><body>下一代病毒 RNA/DNA 原位杂交在人类免疫缺陷病毒/猿猴免疫缺陷病毒研究中的应用

Here we present a next-generation in situ hybridization assay to identify specific viral RNA or DNA sequences in formalin-fixed paraffin embedded (FFPE) tissues. This approach allows visualization of low copies of RNA and DNA in less than 24 h with very high sensitivity and specificity.

下一代病毒 RNA/DNA 原位杂交在人类免疫缺陷病毒/猿猴免疫缺陷病毒研究中的应用

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important ...

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 ...

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.

使用基因型的方法预测HIV-1辅助用途（归经）的序列分析

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