这个实验的发展是支持欢跃医疗保健和卫生研究所（CIHR）加拿大研究所和葛兰素史克公司在临床病毒学博士哈里根/ CIHR主席通过。 由辉瑞和Viiv医疗提供支持。

程序： 1。提取： 此基因型的艾滋病毒嗜测试样本输入所需的至少500μL血浆。 <ol><li> easyMAG（生物梅里埃）自动提取，或提取您的实验室的首选方法500μL血浆中提取的HIV RNA。 成60微升等分洗脱提取的病毒RNA。贮存在-20摄氏度或反向转录- PCR解压后立即开始。 </li></ol> 2。 RT - PCR检测： 样品提取液4μL将被放大，用巢式RT - PCR方法一式三份。必须建立的RT - PCR反应在PCR洁净室。 <ol><li>计算被放大的样本数量所需的试剂用量。按照下面的说明1反应试剂卷表。 <table border="1"><tbody><tr><td> 试剂 </td><td> 量（μl） （1X反应） </td></tr><tr><td> DEPC处理水</td><td> 10.56 </td></tr><tr><td> 2倍的反应缓冲液</td><td> 20 </td></tr><tr><td> 50％的蔗糖和0.04％溴酚蓝混合</td><td> 4 </td></tr><tr><td>针对艾滋病毒V3区的上游引物</td><td> 0.32 </td></tr><tr><td>反向引物</td><td> 0.32 </td></tr><tr><td>酶 - 标​​第三步白金Taq DNA聚合酶RT - PCR系统</td><td> 0.8 </td></tr><tr><td> 共有 </td><td> 36 </td></tr></tbody></table> 表1 1单步RT - PCR反应所需的试剂卷。 每一个步骤RT - PCR反应需要以下几招： <ul><li> 10.56μL，DEPC处理水</li><li> 20μL的反应缓冲液2倍</li><li> 4μL，50％的蔗糖和0.04％溴酚蓝混合</li><li> 0.32μL的远期目标艾滋病毒V3区引物</li><li> 0.32μL的反向引物</li><li> 0.8μL的酶</li></ul>对于一个总的36μL每反应。 </li><li>打成线了旁边一个空行中的样品提取液管，96孔PCR板。 </li><li>使用一个中继器移液器，加36μL样本混合，以每孔PCR板，有3口井是每个样本提取物准备。 *注意：此过程必须一式三份，以最低的，以确保有足够的采样病毒的人口。 </li><li>使用8通道多道移液器，将其3井的样品提取液4μL。更改彼此之间另外的提示。 </li><li>用PCR地带帽盖井。 </li><li>当转移是comlete，放置在一个热循环PCR板。设置热循环与下面的程序运行：30分钟52℃2分钟94 ° C，40个周期（15秒94℃，在5530秒° C和1.5分钟在68℃）5分钟68 ° C </li><li>删除从热循环PCR板。板可以在室温下保存。 </li></ol>继续进行第二轮PCR步骤 3。第二轮PCR： <ol><li>计算被放大的样本数量所需的试剂用量。按照下面的说明1反应试剂卷表。 <table border="1"><tbody><tr><td> 试剂 </td><td> 量（μl） （1X反应） </td></tr><tr><td> DEPC处理水</td><td> 13.45 </td></tr><tr><td> 60％的蔗糖，0.08％甲酚红混合</td><td> 2 </td></tr><tr><td>罗氏公司的HiFi系统缓冲区2 </td><td> 2 </td></tr><tr><td> MgCl 2的 </td><td> 0.8 </td></tr><tr><td>的dNTP </td><td> 0.16 </td></tr><tr><td>正向PCR引物</td><td> 0.15 </td></tr><tr><td>反向PCR引物</td><td> 0.15 </td></tr><tr><td>展开高保真酶</td><td> 0.29 </td></tr><tr><td> 共有 </td><td> 19 </td></tr></tbody></table> 表2：1 的第二轮PCR反应所需试剂卷。 每个第二轮PCR反应需要以下几招： <ul><li> 13.45μL，DEPC处理水</li><li> 2μL60％蔗糖，0.08％甲酚红混合</li><li> 2μL罗氏高保真系统缓冲区2 </li><li> 0.8μLMgCl 2的 25毫米</li><li> 0.16μL，25毫米的dNTP </li><li> 0.15μL，两者的正向和反向PCR引物</li><li> 0.29微升展开高保真酶</li></ul>总额的19％μL反应。 </li><li在PCR的洁净室，使用中继器的吸管，添加到一个干净的PCR板，每孔19μL反应缓冲液。 </li><li>在后放大室，采用8通道多道移液器，转移到第二轮PCR缓冲液1μLRT - PCR的模板。更改彼此之间另外的提示。 </li><li> PCR地带帽盖井和第二轮PCR板转移到热循环。 </li><li>设置热循环与下面的程序运行：2分钟94 ° C 35个周期（15秒94℃，30秒，在55℃，1分钟72 ° C）7分钟，在72 ° C </li><li>删除板从热循环后的温度已恢复到25 ° C。 </li></ol> 4。凝胶电泳： 为了确认，V3区已成功地扩增，凝胶电泳的步骤执行。 <ol><li>准备琼脂糖凝胶琼脂糖粉0.8克，50毫升的1X TAE缓冲。搅拌融化〜1分钟或微波，直到琼脂糖完全溶解。 </li><li>加入6μL安全的SYBR凝胶染色和漩涡混合。 </li><li>倒入凝胶托盘的解决方案，并补充足够的凝胶梳子，以适应PCR检测井的数量。 </li><li>一旦凝胶干燥，取出梳子和地方凝胶，凝胶仪器，以确保它是完全淹没在1X TAE缓冲。 </li><li>使用10μL多道移液器，加其在自己的凝胶以及每个PCR样品8μL。盖PCR板，扩增后的凝胶已被添加到。 </li><li>加入6μL的DNA阶梯，每行至少有一个。 </li><li>运行〜100V〜10分钟的凝胶仪器，确保，带不跑凝胶。 </li><li>到紫外线的反式照明灯凝胶和凝胶的图片。 PCR扩增的DNA井会出现亮，说明一个成功的放大。 </li><li>请注意：所有样品的三个扩增成功。如有必要，重复这些样本少于3扩增RT - PCR检测。 </li></ol>进行测序 5。测序： 病毒基因扩增后，样品可以准备进行测序。测序反应，应在一个放大后室地方。 <ol><li>从冰箱和测序引物，从冰箱中取出BigDye。让BigDye在室温下解冻，不超过1小时。 </li><li>计算运行的样本数量所需的试剂用量。按照下面的说明1反应试剂卷表。 <table border="1"><tbody><tr><td> 试剂 </td><td> 量（μl） （1X反应） </td></tr><tr><td> BigDye </td><td> 0.3 </td></tr><tr><td> BigDye缓冲区</td><td> 2.1 </td></tr><tr><td>底漆</td><td> 2.6 </td></tr><tr><td> 共有 </td><td> 5.0 </td></tr></tbody></table> 表3。试剂卷1测序反应。 *注：准备为每个​​引物单独组合。 **注：BigDye缓冲区用于测序反应，可作如下准备：混合17.5毫升Trizma盐酸缓冲溶液（pH9.0）（1米）和氯化镁0.125毫升（1米）。把100毫升的试剂级水终体积。这应该是存储在2-8 ° C。 </li><li>从3.3中使用的计算，准备为每个​​引物和不超过5秒的旋涡混合的测序反应。分装成悬液0.2ml地带管。 </li><li>分装5μL测序反应混合到适当的板采用多道移液器井。罩板（S），其中包含一个Kimwipe测序反应混合，待用。 </li><li>准备加入180μL无菌水巴克斯特，其余的PCR产物的扩增PCR产物1:15稀释。 </li><li>加入1μL稀释后的样品，以适当的板井底部，改变枪头。 </li><li>当完成转移样品，密封带帽子和涡板1秒</li><li>放置在离心机板。 145克，当旋转速度达到145克设置速度，停止旋转。 </li><li>板放置在下面的程序设置为一个热循环仪（S）：25个循环（10秒@ 96 ° C，5秒，50℃，55秒@ 60 ° C。）量应至少6μL 。这个周期应该采取大约1.2小时。 </li></ol>继续降水 6。降水： “清理”病毒的基因测序反应，进行乙醇沉淀，用95％乙醇。 <ol><li>从热循环检索板块，并带来一个后放大器fication房间。 </li><li>删除地带的上限和地点，以便他们在一个干净的纸巾或Kimwipe。 </li><li>移液器4μL工作EDTA的醋酸钠溶液每个样品的一面。塔盘轻轻EDTA钠溶液下降到井底。可以使用同样的技巧，只要不接触样品在井底。 *注：工作EDTA的醋酸钠溶液可作如下准备： <ol><li>准备246.1克醋酸钠溶解于1 L试剂级水3M醋酸钠。 0.45微过滤器的过滤解决方案，并准备50毫升分装。 </li><li>准备库存EDTA -乙酸钠溶液（1.5米NaOAc，250毫米EDTA）混合等量的醋酸钠（3米）和EDTA溶液（500毫米），pH值8.0。 </li><li>准备工作EDTA - 醋酸钠溶液混合EDTA - 醋酸钠溶液与试剂级水（10毫升+ 30毫升）三部分组成的一个部分股票。 </li></ol></li><li>移取40μL到对面的样品井，并冷冻95％的乙醇取代地带帽。 </li><li>密封帽和旋涡10秒板。点触板，驱逐任何气泡。 </li><li>放置至少30分钟，最多2小时冷冻在-20 ° C的板块。 </li><li><ol class="lalpha"><li>一旦发生降水，从冰箱和一个20分钟3700转离心中删除的板块。 </li><li>从冰箱中取出适量的高迪甲酰胺（美国应用生物系统公司），使其能够解冻之前变性。 </li></ol> *注：一个完​​整​​的96孔板变性要求960μL希迪甲酰胺，因此它有利于事先准备〜1.1mL等分。 </li><li>在纺织板块中，删除和丢弃的条形帽。反转盘在废物容器（如垃圾箱），倒出上清液。任何剩余的上清摆脱印迹纸巾倒板。 </li><li>折板面2张纸巾和地点。在离心机和旋转板面朝下放置在145克，停止旋转，当它到达145克。 </li><li>从离心机中取出的钢板和检查，以确保井空。移液器155μL冷冻入井95％的乙醇。 </li><li>丢弃到废物的容器和纸巾的污点上清，重复离心4.10。 </li><li>从离心机中取出盘子后，丢弃用过的纸巾，让板的立场面对2-5分钟，以允许任何剩余的乙醇蒸发。 </li></ol>继续以变性 7。变性<ol><li>检索到一个容器足够大的多道移液器解冻希迪甲酰胺和倒出， *注：如果使用预分装的措施为6.7b指出，一管是要运行的每个96孔板需要。 </li><li>使用多道移液器，分发到所有的孔10μL希迪甲酰胺盘上，包括那些都是空的。 </li><li>与隔垫覆盖板，形成一个密封。 </li><li> 2分钟，放置于90 ° C的板在热循环。 </li><li>变性后，放置在一个盘子持有的板块，然后加载到音序器。上传一个准备测序布局。板可保持在-20 ° C，最多一个星期前执行的测序运行。 </li></ol> 8。使用基本电话软件产生的数据分析。 <ol><li>使用召回执行调用，无需人工干预的自动基地，单引物覆盖是可以接受的的。召回是一个自定义的基本电话软件，可以在以下网址找到：http://pssm.cfenet.ubc.ca/ *注：需要登录的帐户。要建立一个帐户，点击“注册”按钮，在登录页面。一旦帐户已经创建，您可以访问上传页面。 </li><li>登录后，您可以选择上传样本文件。使用“浏览”选项，您可以找到您的原始序列数据的上传与最大的20MB上传。 *注：。。网站召回将只接受在一个zip ABI和SCF文件中的数据文件，tar或tar.gz文件</li><li>一旦上载的文件已被选中，选择您的参考序列。在这种情况下，可以肯定的参考序列设置为“V3”。现在您可以点击“过程数据。” </li><li>处理过的数据会出现“标题下的旧的样本。”页面右侧每个运行或样本集处理，将被放置在标有日期的文件夹。这些可以被重新标记，点击“重命名”按钮。 </li><li>点击文件夹，将弹出的样本清单。是红色的那些失败，那些都是绿色的已经过去了。通过点击一个特定的样本和“查看”按钮，你可以审查序列。按照页面右侧的指示导航THROUGH序列。 *注：对于这种方法，是可以接受的出口通过召回（以绿色显示），无需人工干预，自动序列的。 </li><li>如果您对结果感到满意，你可以选择点击下载通过序列“下载”。一个压缩文件夹中的文件将被下载。 *注：根据“设置”，您可以选择下载和电子邮件选项，包括文件类型。 </li><li>未能出示清洁一式三份序列的样品应reamplified摘录一式三份。如果RePCR未能提供一个清洁的顺序，最小的2个序列向性推断是可以接受的。 </li></ol> 9。使用Geno2pheno合作受体算法人口为基础的测序序列推断病毒嗜性。 <ol><li>序列可以在以下网址：http://coreceptor.bioinf.mpi-inf.mpg.de/index.php geno2pheno辅助受体服务贯穿。 </li><li>要上传的样品可以被标注为挺直腰杆。从降下来的意​​义设置菜单，选择“从临床数据分析为基础的优化截断激励（2％和5.75％，FPR）” </li><li>选择上传或粘贴序列进行分析，FASTA文件是可以接受的的。 </li><li> geno2pheno FPR可以作如下解释：G2P FPR&gt; 5.75代表一个R5的使用病毒的人口可能回应CCR5的拮抗剂G2P FPR &lt;5.75代表非R5病毒的人口;不大可能回应CCR5的拮抗剂。 G2P &lt;2是不太可能有任何CCR5拮抗剂的反应。如果从样品中的三个序列中的任何一个推断非R5，病人是没有可能回应到CCR5的拮抗剂。 </li></ol> 10。代表性的成果正确执行协议时，不要指望成功序列2个或3个，每个样品重复。样品一起运行的底片应该没有任何指示的R​​NA。大多数的序列的“通行证”召回basecalling。 基于R5和非R5社区内的病毒人口的分布，大多数的随机选择的病人样本，预计将有R5，使用病毒。因此，除非该项目的设计建议，否则，不要指望有超过非R5样品R5。 表1：A）1反应的一步法RT - PCR和B）的热循环程序要进行RT - PCR反应所需的试剂卷。 一） <table border="1"><tbody><tr><td> 试剂 </td><td> 量（μl） （1X反应） </td></tr><tr><td> DEPC处理水</td><td> 10.56 </td></tr><tr><td> 2倍的反应缓冲液</td><td> 20 </td></tr><tr><td> 50％的蔗糖和0.04％溴酚蓝混合</td><td> 4 </td></tr><tr><td>正向引物SQV3F1 </td><td> 0.32 </td></tr><tr><td>反向引物CO602 </td><td> 0.32 </td></tr><tr><td>酶 - 标​​第三步白金Taq DNA聚合酶RT - PCR系统 </td><td> 0.8 </td></tr><tr><td> 共有 </td><td> 36 </td></tr></tbody></table> *注意： SQV3F1引物序列：5&#39;GAG文建会ATT CCC认证的ATA CAT达TGT的3&#39; CO602引物序列：5&#39;GCC CAT AGT GCT TCC TGC TGC TCC民航局GAA CC 3“ 二） <table border="1"><tbody><tr><td>循环号</td><td>时间</td><td>温度</td></tr><tr><td> 1 </td><td> 30分钟</td><td> 52℃ </td></tr><tr><td> 1 </td><td> 2分钟</td><td> 94℃ </td></tr><tr><td rowspan="3"> 40 </td><td> 15秒</td><td> 94℃ </td></tr><tr><td> 30秒</td><td> 55℃ </td></tr><tr><td> 1.5分钟</td><td> 68℃ </td></tr><tr><td> 1 </td><td> 5分钟</td><td> 68℃ </td></tr></tbody></table> 表2 A）第二轮PCR反应1和B）的热循环程序要进行第二轮PCR反应所需的试剂卷。 一） <table border="1"><tbody><tr><td> 试剂 </td><td> 量（μl） （1X反应） </td></tr><tr><td> DEPC处理水</td><td> 13.45 </td></tr><tr><td> 60％的蔗糖，0.08％甲酚红混合</td><td> 2 </td></tr><tr><td>罗氏公司的HiFi系统缓冲区2 </td><td> 2 </td></tr><tr><td>氯化镁<suB&gt; 2 </td><td> 0.8 </td></tr><tr><td>的dNTP </td><td> 0.16 </td></tr><tr><td>正向引物SQV3F2 </td><td> 0.15 </td></tr><tr><td>反向引物CD4R </td><td> 0.15 </td></tr><tr><td>展开高保真酶</td><td> 0.29 </td></tr><tr><td> 共有 </td><td> 19 </td></tr></tbody></table> *注意： SQV3F2引物序列：5&#39;TGT的海湾合作​​委员会共同国家评估GCT GGT TTT GCG在3&#39; CD4R引物序列：5&#39;达亚洲空运中心TCA铁通反恐委员会民航局TTG TCC的3&#39; 二） <table border="1"><tbody><tr><td> 循环号</td><td>时间</td><td>温度</td></tr><tr><td> 1 </td><td> 2分钟</td><td> 94℃ </td></tr><tr><td> 35 </td><td> 15秒</td><td> 94℃ </td></tr><tr><td> 30秒</td><td> 55℃ </td></tr><tr><td> 1分钟</td><td> 72℃ </td></tr><tr><td> 1 </td><td> 7分钟</td><td> 72℃ </td></tr></tbody></table> 表3 A）1测序反应和二）热循环程序要进行测序反应所需的试剂卷。 一） <table border="1"><tbody><tr><td> 试剂 </td><td> 量（μl） （1X反应） </td></tr><tr><td> BigDye </td><td> 0.3 </td></tr><tr><td> BigDye缓冲区</td><td> 2.1 </td></tr><tr><td>底漆 远期V3FO2F 反向SQV3R1 </td><td> 2.6 </td></tr><tr><td> 共有 </td><td> 5.0 </td></tr></tbody></table> *注意：不要将引物，引物各准备一个单独的组合应 V3O2F引物序列：5&#39;亚洲空运中心GTC的AGY ACA的多伦多民航局的TGT ACA的C 3“ SQV3R1引物序列：5&#39;GAA TTC AAA级CCT TCC ACA的AT＆T AAA级3“ 二） <table border="1"><tbody><tr><td> 循环号</td><td>时间</td><td>温度</td></tr><tr><td> 25 </td><td> 10秒</td><td> 96℃ </td></tr><tr><td> 5秒</td><td> 50℃ </td></tr><tr><td> 55秒</td><td> 60℃ </td></tr></tbody></table>

<ol>
	<li>Feng, Y., Broder, C. C., Kennedy, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+entry+cofactor:+functional+cDNA+cloning+of+a+seven+transmembrane,+G+protein-coupled+receptor.">HIV-1 entry cofactor: functional cDNA cloning of a seven transmembrane, G protein-coupled receptor.</a> Science. 272, 872-877 (1996).</li><li>Dragic, T., Litwin, V., Allaway, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HIV-1+entry+into+CD4++cells+is+mediated+by+the+chemokine+receptor+CC-CKR-5.">HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5.</a> Nature. 381, 667-673 (1996).</li><li>Dorr, P., Westby, M., Dobbs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maraviroc+(UK-427,857),+a+potent,+orally+bioavailable,+and+selective+small-molecule+inhibitor+of+chemokine+receptor+CCR5+with+broad-spectrum+anti-human+immunodeficiency+virus+type+1+activity.">Maraviroc (UK-427,857), a potent, orally bioavailable, and selective small-molecule inhibitor of chemokine receptor CCR5 with broad-spectrum anti-human immunodeficiency virus type 1 activity.</a> Antimicrob. Agents Chemother. 49, 4721-4732 (2005).</li><li>Gulick, R. M., Lalezari, J., Goodrich, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maraviroc+for+previously+treated+patients+with+R5+HIV-1+infection.">Maraviroc for previously treated patients with R5 HIV-1 infection.</a> N. Engl. J. Med. 359, 1429-1441 (2008).</li><li>Cooper, D. A., Heera, J., Goodrich, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maraviroc+versus+efavirenz,+both+in+combination+with+zidovudine/lamivudine,+for+the+treatment+of+antiretroviral-na&#237;ve+subjects+with+CCR5-tropic+HIV-1.">Maraviroc versus efavirenz, both in combination with zidovudine/lamivudine, for the treatment of antiretroviral-na&#237;ve subjects with CCR5-tropic HIV-1.</a> J. Infect. Dis. 201, 803-813 (2010).</li><li>Low, A. J., McGovern, R. A., Harrigan, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trofile+HIV+co-receptor+usage+assay.">Trofile HIV co-receptor usage assay.</a> Expert Opin. Med. Diagnostics. 3, 181-191 (2000).</li><li>Sing, T., Low, A. J., Beerenwinkel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+HIV+co-receptor+usage+based+on+genetic+and+clinical+covariates.">Predicting HIV co-receptor usage based on genetic and clinical covariates.</a> Antiviral Ther. 12, 1097-1106 (2007).</li><li>Harrigan, P. R., McGovern, R., Dong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+for+HIV+tropism+using+population-based+V3+genotypic+analysis:+a+retrospective+virological+outcome+analysis+using+stored+plasma+screening+samples+from+MOTIVATE-1.">Screening for HIV tropism using population-based V3 genotypic analysis: a retrospective virological outcome analysis using stored plasma screening samples from MOTIVATE-1.</a> , (2009).</li><li>Harrigan, P. R., McGovern, R., Dong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+clinically+relevant+cut-points+for+the+determination+of+HIV+co-receptor+usage+to+predict+maraviroc+responses+in+treatment+experienced+(TE)+patients+using+population+V3+genotyping.">Optimization of clinically relevant cut-points for the determination of HIV co-receptor usage to predict maraviroc responses in treatment experienced (TE) patients using population V3 genotyping.</a> , (2009).</li><li>Swenson, L. C., McGovern, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+clinically+relevant+cutoffs+for+determining+HIV+co-receptor+use+by+population+and+"deep"+sequencing+methods.">Optimization of clinically relevant cutoffs for determining HIV co-receptor use by population and "deep" sequencing methods.</a> , (2009).</li><li>Swenson, L., McGovern, R., Dong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+screening+for+HIV+tropism+versus+both+population-based+and+"deep"+sequencing.">Phenotypic screening for HIV tropism versus both population-based and "deep" sequencing.</a> , (2009).</li><li>Swenson, L. C., Moores, A., Low, A. J., Thielen, A., Dong, W., Woods, C., Jensen, M. A., Wynhoven, B., Chan, D., Glascock, C., Harrigan, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Detection+of+CXCR4-Using+HIV+by+V3+Genotyping:+Application+of+Population-based+and+'Deep'+Sequencing+to+Plasma+RNA+and+Proviral+DNA.">Improved Detection of CXCR4-Using HIV by V3 Genotyping: Application of Population-based and 'Deep' Sequencing to Plasma RNA and Proviral DNA.</a> Journal of Acquired Immune Deficiency Syndromes. 54, 506-510 (2010).</li><li>Swenson, L. C., Boehme, R., Thielen, A., McGovern, R. A., Harrigan, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genotypic+determination+of+HIV-1+tropism+in+the+clinical+setting.">Genotypic determination of HIV-1 tropism in the clinical setting.</a> HIV Therapy. 4, 293-303 (2010).</li></ol>

P.理查德哈里根欢跃医疗保健，Virco，默克，雅培，和Quest的利益冲突。此视频文章是由欢跃医疗赞助。

这里介绍的方法是一个标准的测序方法应用于向性测试。 HIV包膜V3循环测序预测病毒嗜性的临床应用，直到深夜被限制。 maraviroc（欢跃医疗）临床试验的回顾性分析，此方法已被证明至少同样能够预测病毒嗜性，在临床上常用的其他验证分析比较。 有许多好处执行向性预测V3环的基因型分析。首先，这个程序可以在任何设施进行经营的测序设备，大大增加了获取和减少周转时间向性测试。相比较而言，表型增强敏感性Trofile含量（ESTA）（的Monogram Biosciences公司），其中有向性测试的黄金标准，是在南加州的一个中心。其他好处包括要求的起始原料和最低要求的血浆病毒载量的500copies/mL。同时，运行的基因型检测的营运成本相对较低的表型检测的比较。召回的软件的使用，特别是消除了手动顺序审查的要求，这往往是一个劳动力密集的基因型分析。 这里介绍的方法非常简单，没有特别的又一步骤抵销的重要性。我们建议运行PCR和测序，一式三份，以增加病毒样本人口内捕捉少数物种的可能性。可进行复制大于3，但是我们已经发现一式三份，高效，可靠的。我们也建议采用一步到位逆转录PCR试剂盒（QIAGEN）执行同样的反应，同时保持高灵敏度和特异性都RT - PCR和第一轮PCR。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>RNA extraction using NucliSens easyMAG version 1.0</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NucliSens easyMAG Magnetic Silica</td>
<td>&nbsp;</td>
<td>bioMerieux</td>
<td>cat. # 280133</td>
<td>(48 x 0.6 ml)</td>
</tr>
<tr>
<td>NucliSens easyMAG Lysis Buffer</td>
<td>&nbsp;</td>
<td>bioMerieux</td>
<td>cat. # 280134</td>
<td>(4 x 1000 ml)</td>
</tr>
<tr>
<td>NucliSens easyMAG Extraction Buffer 1</td>
<td>&nbsp;</td>
<td>bioMerieux</td>
<td>cat. # 280130</td>
<td>(4 x 1000 ml)</td>
</tr>
<tr>
<td>NucliSens easyMAG Extraction Buffer 2</td>
<td>&nbsp;</td>
<td>bioMerieux</td>
<td>cat. # 280131</td>
<td>(4 x 1000 ml)</td>
</tr>
<tr>
<td>NucliSens easyMAG Extraction Buffer 3</td>
<td>&nbsp;</td>
<td>bioMerieux</td>
<td>cat. # 280132</td>
<td>(4 x 1000 ml)</td>
</tr>
<tr>
<td>NucliSens easyMAG version 1.0 </td>
<td>&nbsp;</td>
<td>bioMerieux</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Class II A2 biological safety cabinet</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>One-Step Reverse Transcriptase PCR and Second Round PCR</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>DEPC-treated water</td>
<td>&nbsp;</td>
<td>Ambion Corp.</td>
<td>cat. # 9922</td>
<td>&nbsp;</td>
<tr>
<td>SuperScrip III One-Step RT-PCR System with Platinum Taq High Fidelty</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>cat#12574-035</td>
<td>Kit components used: 
- SuperScript III RT/ Platinum Taq High Fidelty Enzyme Mix 
- 2X Reaction Mix (a buffer containing 0.4mM of each dNTP, 2.4mM MgSO4)</td>
</tr>
<tr>
<td>Expand High Fidelity PCR System</td>
<td>&nbsp;</td>
<td>Roche Diagnostics</td>
<td>cat. # 1 759 078</td>
<td>Kit components used: 
- Expand High Fidelity Buffer 10X conc. with 15 mM MgCl2 (lids labelled 2) 
- Expand HF PCR Enzyme Mix (3.5U/&mu;l) 
- MgCl2 Stock Solution (25mM) (lids labelled 4)</td>
</tr>
<tr>
<td>dNTPs: dATP, dGTP, dCTP, dTTP</td>
<td>&nbsp;</td>
<td>Roche Diagnostics</td>
<td>cat. # 11969064001</td>
<td>(100 mM)</td>
</tr>
<tr>
<td>Sucrose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>cat. # S0389-500G</td>
<td>&nbsp;</td>
<tr>
<td>Bromophenol blue sodium salt</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>cat.# B5525</td>
<td>&nbsp;</td>
<tr>
<td>Cresol Red</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>cat.# 114472-5G</td>
<td>indicator grade</td>
</tr>
<tr>
<td>Thermocycler </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gel Electrophoresis</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50X TAE buffer</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>cat. # 24710030</td>
<td>(1000 ml) </td>
</tr>
<tr>
<td>SYBR Safe DNA gel stain</td>
<td>&nbsp;</td>
<td>Invitrogen Life Technologies</td>
<td>cat. # S33102</td>
<td>&nbsp;</td>
<tr>
<td>Agarose (ultra pure)</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>cat. # 15510-027</td>
<td>&nbsp;</td>
<tr>
<td>E-Gel Low Range Quantitative DNA Ladder</td>
<td>&nbsp;</td>
<td>Invitrogen Life Technologies</td>
<td>cat. # 12373-031</td>
<td>&nbsp;</td>
<tr>
<td>Reagent Grade Type II water</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Gel apparatus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Sequencing</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ABI PRISM BigDye Terminator Cycle Sequencing Kit</td>
<td>&nbsp;</td>
<td>Applied Biosciences</td>
<td>cat. # 4337458</td>
<td>(25000 reactions)</td>
</tr>
<tr>
<td>Hi-Di Formamide</td>
<td>&nbsp;</td>
<td>Applied Biosciences</td>
<td>cat. # 4311320</td>
<td>&nbsp;</td>
<tr>
<td>Sodium Acetate (NaOAc)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>cat. # S-2889</td>
<td>&nbsp;</td>
<tr>
<td>EDTA</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>Cat.# 03690-100ML</td>
<td>0.5M, pH=8.0</td>
</tr>
<tr>
<td>TRIZMA Hydrochloride Buffer Solution</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>cat. # T-2819</td>
<td>1 M, pH 9.0</td>
</tr>
<tr>
<td>Magnesium Chloride (MgCl2)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>cat. # M-1028</td>
<td>1 M</td>
</tr>
<tr>
<td>95% Ethanol</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Running Buffer (10X) with EDTA</td>
<td>&nbsp;</td>
<td>Applied Biosystems</td>
<td>cat. # 4335613</td>
<td>500 mL</td>
</tr>
<tr>
<td>Polymer Pop-7 (25mL) Or Polymer Pop-7 (10mL)</td>
<td>&nbsp;</td>
<td>Applied Biosystems</td>
<td>cat. # 44363929 or cat. # 4352759</td>
<td>&nbsp;</td>
<tr>
<td>3700/3730 BigDye Terminator v3.1 Sequencing Standard Kit</td>
<td>&nbsp;</td>
<td>Applied Biosciences</td>
<td>Cat# 4336943</td>
<td>&nbsp;</td>
<tr>
<td>Thermocycler </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Centrifuge with plate holders</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>3730xl DNA Analyzer</td>
<td>&nbsp;</td>
<td>Applied Biosciences</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

genotypic inference of hiv-1 tropism using population-based sequencing of v3

背景：收到CCR5拮抗剂类艾滋病毒治疗的一个药物之前，患者必须接受艾滋病毒嗜性测试，以确认他或她的病毒的人口使用蜂窝进入了CCR5辅助受体，而不是替代辅助受体。向性测试的方法之一，是研究HIV包膜，与辅助受体相互作用的V3区序列。 方法：从血浆中提取病毒RNA。 V3区是一式三份嵌套逆转录PCR扩增。的扩增，测序和分析软件，RE_Call。如geno2pheno生物​​信息学算法推断病毒嗜从V3区序列，然后提交。推断序列被非R5，如果他们geno2pheno的假阳性率低于5.75％。如果从样品中的三个序列中的任何一个推断非R5，病人是不会回应一个CCR5拮抗剂。

Watch this Scientific Journal Video about Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3 at JoVE.com

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

可以推断出艾滋病毒嗜性病毒包膜V3区。 V3是一式三份，采用巢式RT - PCR扩增，测序，PCR扩增，并解释使用生物信息学软件。被列为非R5病毒与G2P分数低1个或多个序列（S）的样品。

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 ...

<a class="grey_button" href="/video/3538">Espa&ntilde;ol </a>

genotypic-inference-hiv-1-tropism-using-population-based-sequencing

Research

JoVE Journal

Immunology and Infection

12.1K 次观看.  BC Centre for Excellence in HIV/AIDS.  Español 

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

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, ...

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

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by the majority of HIV strains in order to infect the human immune cells (Fig. 1). Other HIV isolates use a different coreceptor, the CXCR4. Which receptor is used, is determined in the virus by the Env protein (Fig. 2). Depending on the coreceptor used, the viruses are classified as R5 or X4, respectively. MVC binds to the CCR5 receptor inhibiting the entry of R5 viruses into the target cell. During the course of disease, X4 viruses may emerge and outgrow the R5 viruses. Determination of coreceptor usage (also called tropism) is therefore mandatory prior to administration of MVC, as demanded by EMA and FDA. 
The studies for MVC efficiency MOTIVATE, MERIT and 1029 have been performed with the Trofile assay from Monogram, San Francisco, U.S.A. This is a high quality assay based on sophisticated recombinant tests. The acceptance for this test for daily routine is rather low outside of the U.S.A., since the European physicians rather tend to work with decentralized expert laboratories, which also provide concomitant resistance testing. These laboratories have undergone several quality assurance evaluations, the last one being presented in 20111.
For several years now, we have performed tropism determinations based on sequence analysis from the HIV env-V3 gene region (V3)2. This region carries enough information to perform a reliable prediction. 
The genotypic determination of coreceptor usage presents advantages such as: shorter turnover time (equivalent to resistance testing), lower costs, possibility to adapt the results to the patients' needs and possibility of analysing clinical samples with very low or even undetectable viral load (VL), particularly since the number of samples analysed with VL&lt;1000 copies/&mu;l roughly increased in the last years (Fig. 3).
The main steps for tropism testing (Fig. 4) demonstrated in this video: 
1. Collection of a blood sample 
 2. Isolation of the HIV RNA from the plasma and/or HIV proviral DNA from blood mononuclear cells 
 3. Amplification of the env region 
 4. Amplification of the V3 region 
 5. Sequence reaction of the V3 amplicon 
 6. Purification of the sequencing samples 
 7. Sequencing the purified samples 
 8. Sequence editing 
 9. Sequencing data interpretation and tropism prediction

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

The prediction of the coreceptor usage of HIV-1 is required for the administration of a new class of antiretroviral drugs, i.e. coreceptor antagonists. It can be performed by sequence analysis of the env gene and subsequent interpretation through an internet based interpretation system (geno2pheno[coreceptor]).

使用基因型的方法预测HIV-1辅助用途（归经）的序列分析

Maraviroc (MVC) is the first licensed antiretroviral drug from the class of coreceptor antagonists. It binds to the host coreceptor CCR5, which is used by ...

In vitro Uncoating of HIV-1 Cores

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

在体外对HIV - 1核心脱壳

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

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

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

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

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

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

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

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

HIV-1的三聚体包膜糖蛋白的构象评估使用基于细胞的ELISA检测

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

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 ...

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.