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="http://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="http://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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P., Wensing, A. M., Kaiser, R., Brun-Vezinet, F., Clotet, B., De Luca, A., Dressler, S., Garcia, F., Geretti, A. M., Klimkait, T., Korn, K., Masquelier, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=European+guidelines+on+the+clinical+management+of+HIV-1+tropism+testing.">European guidelines on the clinical management of HIV-1 tropism testing.</a> Lancet Infect Dis. 11 (5), 394-407 (2011).</li><li>Walter, H., Eberle, J., M&#246;ller, H., Noah, C., Wolf, E., St rmer, M., Braun, P., Krn, K., D umer, M., Berg, T., Obermeier, M., Thielen, A., Kaiser, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Empfehlung+zur+Bestimmung+des+HIV-1-Korezeptor-Gebrauchs+(DAIG+Recommendations+for+the+HIV-1+tropism+testing).">Empfehlung zur Bestimmung des HIV-1-Korezeptor-Gebrauchs (DAIG Recommendations for the HIV-1 tropism testing).</a> , (2011).</li><li>Obermeier, M. J., Carganico, A., Dupke, S., Schranz, D., Cordes, C., Freiwald, M., Klausen, G., K&#246;ppe, S., Schuler, C., Mayr, C., Schleehauf, D., Berg, T., Krznaric, I., Baumgarten, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+application+of+genotypic+co-receptor+tropism+testing+from+viral+RNA+and+proviral+DNA:+week+24+analysis+of+the+Berlin+maraviroc+cohort.">Clinical application of genotypic co-receptor tropism testing from viral RNA and proviral DNA: week 24 analysis of the Berlin maraviroc cohort.</a> Journal of the International AIDS Society. 13, 129-129 (2010).</li><li>Toma, J., Frantzell, A., Hoh, R., Martin, J., Deeks, S., Petropoulos, C., Huang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+HIV-1+Co-receptor+Tropism+Using+PBMC+Proviral+DNA+Derived+from+Aviremic+Blood+Samples.">Determining HIV-1 Co-receptor Tropism Using PBMC Proviral DNA Derived from Aviremic Blood Samples.</a> , (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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Research

JoVE Journal

Immunology and Infection

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

13.1K 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

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, the LTR is also responsive to cellular activation states and to the local chromatin activity where the integration has occurred. This can result in high HIV-independent activity, and has restricted the usefulness of LTR-based reporter to mark HIV positive cells 1,2,3. Here, we constructed an expression lentiviral vector that possesses, in addition to the Tat-responsive LTR, numerous HIV DNA sequences that include the Rev-response element and HIV splicing sites 4,5,6. The vector was incorporated into a lentiviral reporter virus, permitting highly specific detection of replicating HIV in living cell populations. The activity of the vector was measured by expression of the green fluorescence protein (GFP). The application of this vector as reported here offers a novel alternative approach to existing methods, such as in situ PCR or HIV antigen staining, to identify HIV-positive cells. The vector can also express therapeutic genes for basic or clinical experimentation to target HIV-positive cells.

We have developed a lentiviral vector that possesses, in addition to the Tat-responsive LTR, the Rev-response element (RRE) that can regulate reporter gene expression in an HIV-1 Tat- and Rev-dependent fashion. The vector permits the specific detection of replicating HIV in living cells via the expression of GFP.

HIV - 1使用转速依赖的慢病毒表达载体的绿色荧光蛋白阳性细胞的特定标记

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

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

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

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

HIV - 1衣壳大会通过低温电子显微镜的结构和迭代螺旋实时空间重构

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

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 ...

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 ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.