这项工作是由ARRA补充，寿命/塔夫茨/布朗恒虚警，P30AI042853-13S1，NIH P20GM103421，P01AA019072，R01HD072693和K24HD080539为BR支持。这项工作也是由寿命试验研究基金（＃701- 5857），罗得岛基金会医学研究资助（＃20133969），和NIH COBRE URI / RIH试点研究基金（P20GM104317）到ML支持。我们感谢詹姆斯的美荷和Vy党与手稿图制作提供帮助。

1.细胞培养和HIV-1感染注:在开始实验之前，建议通过MTT法12以检查细胞的通过台盼蓝染色11和生存能力它们的增殖。它也使用新制备SILAC介质的关键。各种细胞系都可以使用，只要它们是在一个主动增殖阶段，很容易受到HIV-1感染，或所选择的测试条件。在这个协议中，使用的H9细胞系作为例子。 <ol><li>种子2×10 6个 H9细胞到每个细胞培养瓶。生长一组在标有含有10％透析的胎牛血清（FBS），100毫克/升13 C 6 -L-赖氨酸和100毫克/升13 C- 6 15 N 4 L-精氨酸的RPMI 1640培养基中10毫升生长另一组在10毫升的未标记的RPMI 1640培养基中，用透析的FBS 10％，100毫克/升的L-赖氨酸和100mg / L的L-精氨酸。 </li><li>生长的细胞与重氨基酸在该点在标记的培养基中的细胞的蛋白质实际上完全标记6倍增（&gt; 99％）。 （取决于细胞类型每1-3天。对于H9细胞，改变每3天培养基）添加新鲜的培养基或定期更换介质。在标记结束时，增加培养物体积（ 例如约30毫升），以容纳更多的细胞的生长。 注:确定确切细胞在通过台盼蓝染色和细胞计数实验开始倍增时间。 </li><li>感染标记细胞HIV-1 NL4-3使用标准的HIV-1感染的协议13 48小时用。孵育适当量的病毒与感染的周围0.3复数（MOI）将细胞。通过使用HIV-1 p24抗原ELISA试剂盒培养上清液的P24定量检查HIV-1感染。执行免疫荧光法14来确认细胞的成功感染。记p在不断增加中未标记的未标记的细胞无HIV-1感染。 </li><li>在感染结束时（步骤2.1）收获两组上清液。 </li></ol> 2.外来体隔离注:通过一系列的步骤，超速离心从培养上清液外体组分富集15。执行在4℃以下所有步骤，用超速离心机转子，可以达到至少100,000×g的速度。 <ol><li>收集培养物上清液在50ml锥形管中，避免了细胞。离心他们在300 xg离心10分钟以除去剩余的细胞。 </li><li>收集在新50ml锥形管中的上清液并在2000 xg离心离心它们10分钟，以去除死细胞。转让所得上清液商业转子兼容管是能够承受超离心。 </li><li>一定要平衡超速离心管。离心30分钟试管以10,000 xg离心以除去细胞碎片。 </li><li>收集70分钟的超速离心管上清和离心机在100,000×g的。弃去上清液。 </li><li>悬浮丰富的外来体颗粒在5毫升新鲜的PBS。在100,000×g的重新传送解决方案，以新鲜的超速离心管，离心70分钟。 </li><li>丢弃上清液。来存储外来体长期，悬浮颗粒在50μlPBS中并存储在-80℃。或者，如下所示直接进行蛋白质提取。 </li></ol> 3.蛋白提取和准备<ol><li>溶解在添加了蛋白酶抑制剂鸡尾酒100-200微升的RIPA裂解和提取缓冲液分离外来体粒料。缓冲区是由25毫摩尔Tris-盐酸盐，150mM氯化钠，1％NP-40，1％脱氧胆酸钠和0.1％SDS（十二烷基硫酸钠）的，在pH 7.6。蛋白酶抑制剂鸡尾酒应含有多种蛋白酶抑制剂，如抑肽酶，苯丁抑制素，升eupeptin，胃蛋白酶抑制素A和EDTA（乙二胺四乙酸），能够抑制全方位的蛋白酶。 </li><li>以13,000 xg离心（4℃）离心10分钟溶解的解决方案，以及清除上清液转移至新的1.5 ml微量离心管。 </li><li>量化使用二辛可宁酸（BCA）或Bradford测定16的外来体的各样品的蛋白质浓度。 </li><li>混合蛋白质从标记和未标记的样品等量（2微克），并在120毫安/运行在4-20％SDS-PAGE凝胶的等量混合物200伏30分钟。 </li><li>随后脱色17用考马斯蓝染色凝胶。 </li><li>用刀片从凝胶上切样品车道。然后切泳道成10-15等份。把每一块到一个新的1.5 ml离心管，总共10-15管。 </li><li>沉浸在立方体25毫米NH 4 HCO 3在50％乙腈，旋涡，并丢弃上清液。重复两次。干立方体在真空浓缩器18，19。 </li><li>再水合用10mM二硫苏糖醇（DTT），涡旋，并短暂离心立方体。孵育在56℃进行1小时。弃上清。 </li><li>沉浸凝胶立方体55毫米碘乙酰胺，漩涡和旋转。在室温下孵育在黑暗中45分钟。弃上清。 </li><li>浸入25毫米NH 4 HCO 3，旋涡，旋转立方体，弃上清。 </li><li>沉浸在立方体25毫米NH 4 HCO 3在50％乙腈，涡旋和旋转。重复3.9和3.10。 </li><li>干燥，在真空浓缩的立方体。添加25μl的测序级改良的胰蛋白酶在25mM的NH 4 HCO 3，孵育在4℃下进行30分钟，并弃去过量的溶液。浸入25毫米NH 4 HCO 3立方体没有胰蛋白酶，和孵育过夜在37℃。 </li><li>旋转简要立方体下来，转移所产生的肽的额外克拉到新管中。在50％乙腈中添加30微升的5％的甲酸的立方体，涡旋30分钟，和自旋。结合上清液用提取物。干燥用真空浓缩所述肽萃取到小于5微升。 </li><li>提交样品质谱核心设施用于LC-MS / MS分析。该MS分析数据，其可以从开放源码软件来产生，典型地包括用于标识的每个蛋白的登录号，标记/未标记的比率，并确定了独特肽的数量。 </li></ol> 4.免疫印迹验证注:Western印迹，建议以验证质谱结果。 <ol><li>遵循标准Western印迹协议和保证蛋白的从各组等量装载。使用由MS来识别针对目标蛋白的抗体（ 例如膜联蛋白A5，乳酸脱氢酶B链）。免疫印迹检测，符合密度analysi一起S，可以重申，MS蛋白质鉴定和定量是正确的。 </li></ol> 5.蛋白质组数据分析注:每个MS复制显著蛋白质候选人的数据质量评估，数据预处理，计算和确定单独完成。一旦上述分析完成后，从重复分析的数据进行比较和组合6，20，21。 <ol><li>评估MS数据的质量。 <ol><li>首先，LOG2改造SILAC-MS标记/定量蛋白质未标记的比例在电子表格程序。 </li><li>在科学绘图和统计软件，组的比例为40-100比箱并绘制每仓比的数目，以产生一个直方图。直方图的正态分布表示MS数据6的优良品质。做到这一步对所有MS复制。 </li></ol></li><li>为了提高MS肽比率的信心和准确性，考虑取消有不到两个量化肽蛋白。 </li><li>要确定显著上调和下调的蛋白质的考生，请使用以下步骤计算阈值的意义22。 <ol><li>在科学绘图和统计软件，生成一个非线性回归或曲线拟合到频率分布数据。此步骤产生可用于计算截止值的值。计算的截止值作为平均±1.96σ为95％置信限或2.56σ为99％置信限。 注:计算临界值的具体情况可以在艾默特等人发现。 22。 </li><li>选择的候选蛋白质的比例，其要么是大于1.96（或2.56）的标准偏差的中位数（显著过表达）高于或低于1.96（或2.56）标准差低于中位数（显著下表达）。 </li></ol></li><li>比较上述标题显著候选人的重复来实现的一致性。确保它们符合以下条件，通过这最后的选择步骤:考生必须在所有重复被一致确定;和候选的重复必须在其调节的方向是一致的（优选地，至少2选自候选的3个重复的要么是上调或下调）。 </li><li>最终确定由合并他们重复&#39;数据符合上述标准的候选人的数据。 </li></ol> 6.生物信息学检验和检定注:现有基因组和生物信息学的信息提供了丰富的信息，几乎所有的蛋白质。数据挖掘和生物信息学分析这些信息可以深入了解大量进入显著候选人的财产和功能的帮助。这PROCESS通常是必要的，以设计恰当下游湿实验室实验。 <ol><li>使用他们的GenBank登记号或的UniProt的ID，搜索针对当前外来体数据库的候选人（Exocarta 23 Evpedia 24），以验证该候选蛋白已在外来体先前已经找到。这一步增加了信心层的考生确实在外来体。 <ol><li>访问http://www.exocarta.org/，点击"查询"输入无论是基因或蛋白质名称/登录号。摘要页面将出现，并在外来体的证据将显示，提供，如果有任何。 </li></ol></li><li>搜索针对HIV-1和人类蛋白质相互作用数据库25的候选者。可替代地，搜索特定数据库能够提供关于所述候选蛋白质的相互作用和测试条件的信息。 <ol><li>在www.ncbi.nlm.nih.gov/RefSeq/HIV访问数据库互动。在"蛋白质域名"条目，输入候选人的名称或登录号，然后点击搜索。搜索结果可以提供深入的HIV-1与蛋白质候选人之间的相互作用，并提出它的候选人可能是真正的HIV-1相关联。 </li></ol></li><li>进口GenBank登记号或考生进入GO分析软件（如FunRich，功能丰富的分析工具26）的UniProt ID和运行该软件。 <ol><li>下载在http://www.funrich.org/软件。安装完成后，打开软件，在富集分析，单击"添加数据集"，并上传的蛋白质/基因名单。接下来，选择图表类型的可视化分析GO。 注:GO的分析结果将在饼图的形式显示。此步骤有助于我们获得与生物过程（BP），蜂窝组件等领域的考生有关环球透视（CC）和分子功能（MF）。 </li></ol></li><li>访问DAVID在http://david.ncifcrf.gov/ 27，点击其网站上的"功能性注释工具"。进入候选列表中，选择基因"标识"，如"的UniProt ID"，选择"基因名单"和搜索。富集的GO术语，p值等参数可以通过点击在"Gene_Ontology"类别中的"注释摘要结果"页面中找到。 </li><li>调查与其他蛋白质的候选潜在的相互作用，可以使用公开地可访问STRING数据库28阐明潜在的蛋白-蛋白相互作用和可能的生化途径。 注:GO分析和功能注释（6.3-6.4节），也可以使用最新的软件STRING执行。 <ol><li>在http://string-db.org/访问数据库。输入的蛋白质ID或子序列插入为S操作搜索框，然后选择分析正确的物种。点击"搜索"。结果会就直接（物理）和间接（功能）协会的信息。对于外来体的候选人的十大知名匹配结果将显示，并应考虑显著候选人的选择。 注:与考生相关的大量信息可以使用上面的步骤进行分析。最显著候选可以被选择用于进一步下游分子和生化分析。 </li></ol></li></ol>

<ol>
	<li>Kowal, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+comparison+defines+novel+markers+to+characterize+heterogeneous+populations+of+extracellular+vesicle+subtypes.">Proteomic comparison defines novel markers to characterize heterogeneous populations of extracellular vesicle subtypes.</a> Proc Natl Acad Sci U S A. 113 (8), 968-977 (2016).</li><li>Schorey, J. S., Bhatnagar, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosome+function:+from+tumor+immunology+to+pathogen+biology.">Exosome function: from tumor immunology to pathogen biology.</a> Traffic. 9 (6), 871-881 (2008).</li><li>Thery, C., Ostrowski, M., Segura, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+vesicles+as+conveyors+of+immune+responses.">Membrane vesicles as conveyors of immune responses.</a> Nat Rev Immunol. 9 (8), 581-593 (2009).</li><li>Booth, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+and+HIV+Gag+bud+from+endosome-like+domains+of+the+T+cell+plasma+membrane.">Exosomes and HIV Gag bud from endosome-like domains of the T cell plasma membrane.</a> J Cell Biol. 172 (6), 923-935 (2006).</li><li>Van Engelenburg, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+ESCRT+machinery+at+HIV+assembly+sites+reveals+virus+scaffolding+of+ESCRT+subunits.">Distribution of ESCRT machinery at HIV assembly sites reveals virus scaffolding of ESCRT subunits.</a> Science. 343 (6171), 653-656 (2014).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomic+analysis+of+exosomes+from+HIV-1-infected+lymphocytic+cells.">Quantitative proteomic analysis of exosomes from HIV-1-infected lymphocytic cells.</a> Proteomics. 12 (13), 2203-2211 (2012).</li><li>Ong, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+isotope+labeling+by+amino+acids+in+cell+culture,+SILAC,+as+a+simple+and+accurate+approach+to+expression+proteomics.">Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.</a> Mol Cell Proteomics. 1 (5), 376-386 (2002).</li><li>Ross, P. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+protein+quantitation+in+Saccharomyces+cerevisiae+using+amine-reactive+isobaric+tagging+reagents.">Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents.</a> Mol Cell Proteomics. 3 (12), 1154-1169 (2004).</li><li>Anderson, L., Hunter, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+mass+spectrometric+multiple+reaction+monitoring+assays+for+major+plasma+proteins.">Quantitative mass spectrometric multiple reaction monitoring assays for major plasma proteins.</a> Mol Cell Proteomics. 5 (4), 573-588 (2006).</li><li>Ong, S. E., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+isotope+labeling+by+amino+acids+in+cell+culture+for+quantitative+proteomics.">Stable isotope labeling by amino acids in cell culture for quantitative proteomics.</a> Methods Mol Biol. 359, 37-52 (2007).</li><li>Strober, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Immunology. , (2001).</li><li>Verma, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluation+of+the+MTT+lymphocyte+proliferation+assay+for+the+diagnosis+of+neurocysticercosis.">Evaluation of the MTT lymphocyte proliferation assay for the diagnosis of neurocysticercosis.</a> J Microbiol Meth. 81 (2), 175-178 (2010).</li><li>Cepko, C., Pear, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retrovirus+infection+of+cells+in+vitro+and+in+vivo.">Retrovirus infection of cells in vitro and in vivo.</a> Curr Protoc Mol Biol. , (2001).</li><li>Lennette, E. T., Karpatkin, S., Levy, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indirect+immunofluorescence+assay+for+antibodies+to+human+immunodeficiency+virus.">Indirect immunofluorescence assay for antibodies to human immunodeficiency virus.</a> J Clin Microbiol. 25 (2), 199-202 (1987).</li><li>Thery, C., Amigorena, S., Raposo, G., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+exosomes+from+cell+culture+supernatants+and+biological+fluids.">Isolation and characterization of exosomes from cell culture supernatants and biological fluids.</a> Curr Protoc Cell Biol. , 22 (2006).</li><li>Olson, B. J. S. C., Markwell, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Protein Science. , (2001).</li><li>Gauci, V. J., Padula, M. P., Coorssen, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coomassie+blue+staining+for+high+sensitivity+gel-based+proteomics.">Coomassie blue staining for high sensitivity gel-based proteomics.</a> J Proteom. 90, 96-106 (2013).</li><li>Soldi, M., Bonaldi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ChroP+approach+combines+ChIP+and+mass+spectrometry+to+dissect+locus-specific+proteomic+landscapes+of+chromatin.">The ChroP approach combines ChIP and mass spectrometry to dissect locus-specific proteomic landscapes of chromatin.</a> J Vis Exp. (86), (2014).</li><li>Trompelt, K., Steinbeck, J., Terashima, M., Hippler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+approach+for+the+comparative+analysis+of+multiprotein+complexes+based+on+15N+metabolic+labeling+and+quantitative+mass+spectrometry.">A new approach for the comparative analysis of multiprotein complexes based on 15N metabolic labeling and quantitative mass spectrometry.</a> J Vis Exp. (85), (2014).</li><li>Li, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem-loop+binding+protein+is+a+multifaceted+cellular+regulator+of+HIV-1+replication.">Stem-loop binding protein is a multifaceted cellular regulator of HIV-1 replication.</a> J Clin Invest. 126 (8), 3117-3129 (2016).</li><li>Li, M., Ramratnam, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+Characterization+of+Exosomes+from+HIV-1-Infected+Cells.">Proteomic Characterization of Exosomes from HIV-1-Infected Cells.</a> Methods Mol Biol. 1354, 311-326 (2016).</li><li>Emmott, E., Goodfellow, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+protein+interaction+partners+in+mammalian+cells+using+SILAC-immunoprecipitation+quantitative+proteomics.">Identification of protein interaction partners in mammalian cells using SILAC-immunoprecipitation quantitative proteomics.</a> J Vis Exp. (89), (2014).</li><li>Keerthikumar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ExoCarta:+A+Web-Based+Compendium+of+Exosomal+Cargo.">ExoCarta: A Web-Based Compendium of Exosomal Cargo.</a> J Mol Biol. 428 (4), 688-692 (2016).</li><li>Kim, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EVpedia:+a+community+web+portal+for+extracellular+vesicles+research.">EVpedia: a community web portal for extracellular vesicles research.</a> Bioinformatics. 31 (6), 933-939 (2015).</li><li>Fu, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunodeficiency+virus+type+1,+human+protein+interaction+database+at+NCBI.">Human immunodeficiency virus type 1, human protein interaction database at NCBI.</a> Nucleic Acids Res. 37, 417-422 (2009).</li><li>Pathan, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FunRich:+An+open+access+standalone+functional+enrichment+and+interaction+network+analysis+tool.">FunRich: An open access standalone functional enrichment and interaction network analysis tool.</a> Proteomics. 15 (15), 2597-2601 (2015).</li><li>Huang da, W., Sherman, B. T., Lempicki, 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=Systematic+and+integrative+analysis+of+large+gene+lists+using+DAVID+bioinformatics+resources.">Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.</a> Nat Protoc. 4 (1), 44-57 (2009).</li><li>Szklarczyk, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+STRING+database+in+2011:+functional+interaction+networks+of+proteins,+globally+integrated+and+scored.">The STRING database in 2011: functional interaction networks of proteins, globally integrated and scored.</a> Nucleic Acids Res. 39, 561-568 (2011).</li><li>Thery, C., Amigorena, S., Raposo, G., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+exosomes+from+cell+culture+supernatants+and+biological+fluids.">Isolation and characterization of exosomes from cell culture supernatants and biological fluids.</a> Curr Protoc Cell Biol. , (2006).</li><li>Lobb, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+exosome+isolation+protocol+for+cell+culture+supernatant+and+human+plasma.">Optimized exosome isolation protocol for cell culture supernatant and human plasma.</a> J Extracell Vesicles. 4, 27031 (2015).</li><li>Webber, J., Clayton, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+pure+are+your+vesicles.">How pure are your vesicles.</a> J Extracell Vesicles. 2, (2013).</li><li>Cantin, R., Diou, J., Belanger, D., Tremblay, A. M., Gilbert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrimination+between+exosomes+and+HIV-1:+purification+of+both+vesicles+from+cell-free+supernatants.">Discrimination between exosomes and HIV-1: purification of both vesicles from cell-free supernatants.</a> J Immunol Methods. 338 (1-2), 21-30 (2008).</li><li>Chertova, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+and+biochemical+analysis+of+purified+human+immunodeficiency+virus+type+1+produced+from+infected+monocyte-derived+macrophages.">Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages.</a> J Virol. 80 (18), 9039-9052 (2006).</li><li>Nikolov, M., Schmidt, C., Urlaub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+mass+spectrometry-based+proteomics:+an+overview.">Quantitative mass spectrometry-based proteomics: an overview.</a> Methods Mol Biol. 893, 85-100 (2012).</li></ol>

The authors have declared no conflict of interest.

在本文中描述的方法，我们证明了SILAC技术的应用来调查HIV-1感染的主机上的外来体蛋白质组的效果。最初，未感染和HIV-1感染的细胞中差异同位素标记。然后差异标记外泌体进行蛋白质提取前进行纯化。接着，液相色谱 - 串联质谱法被用来分析外来体的蛋白质组。最后，将得到的质谱数据以及潜在的候选蛋白质进​​行统计和生物信息学的前下游生化解剖分析。 整个协议的?...

许多人类疾病，包括病毒感染，通常与所发生和周围的影响的细胞独特的细胞过程有关。蛋白质，经常充当最终细胞效应，调解这些进程。蛋白质的分析往往可以提供宝贵的资料，受影响细胞的局部环境，并帮助我们理解疾病的发病机制的基本机制。在各种蛋白质分析技术，蛋白质组学拥有特别大的承诺。作为一个强大的，大规模的工具，蛋白质组学可以提供细胞过程的全身性的了解，特别是在功能和蛋白质相互作用的区域。判断特定蛋白质是通过标记技术，它允许研究者监测细胞成分，特别是蛋白质的表达的发展作出了简单，在调查的部位。虽然许多蛋白质组学分析都在细胞被执行蛋白质组的规模，对亚细胞区室蛋白质组学表征已被证明是特别信息1。这是在HIV-1感染的研究很好的例子。 外来体，通过广泛的细胞类型2，3，分泌30-100纳米膜囊泡间通讯以及分子运输的关键组成部分。他们先前发现的HIV-1出芽过程4,5发挥重要作用。通过蛋白质组学分析与解剖功能结合起来，我们发现，从HIV-1感染的细胞释放的外来体是由一个独特的和定量不同的蛋白质的签名和港口监管分子在邻国接受细胞，包括细胞凋亡与增殖6影响细胞特性的。该方法在此协议中描述，即SILAC从HIV-1感染细胞的外来体7基于蛋白质组学表征（在细胞培养物中的氨基酸稳定同位素标记）。类似的方法可以适用于通过调整试验应力的特定隔室或感兴趣的级分，使对所描述的程序进行必要的更改更好地理解发病期间其他亚细胞区室。 鉴于最近的定量蛋白质组学方法的发展，有许多从选择最有效的方法为一个特定的实验时选择。其中包括基于化学的iTRAQ（用于相对和绝对定量的等压标记）8和无标记MRM（多反应监测）9技术。这两种方法是强大的工具，并且是特定的设置很好的选择。对于一个典型的实验室主要与细胞系的工作，然而，这两种方法具有relativelÿ较高的成本，而且更费时相比SILAC基础的方法时。 SILAC是一个基于代谢标记技术并入的氨基酸从培养基进入细胞蛋白质的非放射性的同位素形式。通常情况下，SILAC实验开始与两种细胞群体，例如，感染和未感染的。每个不同标记在其特定的同位素环境满为止标注的实现。然后这些细胞的标记的外来体经受蛋白提取。一旦萃取，将标记的外来体的蛋白质是使用液相色谱串联质谱法10进行分析。最后，质谱结果和显著标记的蛋白质经受统计和生物信息学分析以及严格生物化学验证。我们以前的研究报告表明，SILAC /外来体程序更适合的细胞系比初级细胞，如细胞系通常是在一个活跃的增殖状态高效同位素标记，

<table border="0" cellpadding="0" cellspacing="0" width="625">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">H9 cell line</td>
			<td style="width:121px;">ATCC</td>
			<td style="width:111px;">HTB-176</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Trypan Blue</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">15250061</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">MTT assay kit</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">V13154</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:26px;width:227px;">Dialyzed fetal bovine serum (FBS)</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">26400044</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">SILAC Protein Quantitation Kit &#8211; RPMI 1640</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">89982</td>
			<td style="width:168px;">DMEM version (89983)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">L-Arginine-HCl, 13C6, 15N4 for SILAC</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">88434</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">L-Lysine-2HCl, 13C6 for SILAC</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">88431</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">HIV-1NL4-3&#160;&#160;</td>
			<td style="width:121px;">NIH AIDS Reagent Program</td>
			<td style="width:111px;">2480</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Alliance HIV-1 p24 Antigen ELISA kit</td>
			<td style="width:121px;">PerkinElmer</td>
			<td style="width:111px;">NEK050001KT</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Refrigerated super-speed centrifuge</td>
			<td style="width:121px;">Eppendorf</td>
			<td style="width:111px;">22628045</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Refrigerated ultracentrifuge</td>
			<td style="width:121px;">Beckman Coulter</td>
			<td style="width:111px;">363118</td>
			<td style="width:168px;">Should be able to reach 100,000g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">50mL Conical Centrifuge Tubes</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">14-432-22</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Ultracentrifuge Tubes</td>
			<td style="width:121px;">Beckman Coulter</td>
			<td style="width:111px;">326823</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">SW 32 Ti Rotor</td>
			<td style="width:121px;">Beckman Coulter</td>
			<td style="width:111px;">369694</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">RIPA buffer</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">89900</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Protease Inhibitor Cocktails</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">&#160;78430</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">ThermoMixer&#160;</td>
			<td style="width:121px;">Eppendorf</td>
			<td style="width:111px;">5384000020</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">BCA Protein Assay Kit&#160;</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">23250</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Spectrophotometer</td>
			<td style="width:121px;">Biorad</td>
			<td style="width:111px;">1702525</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">SDS PAGE Gel apparatus</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">EI0001</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Novex 4-20% Tris-Glycine Mini Gels</td>
			<td style="width:121px;">Novex</td>
			<td style="width:111px;">XV04200PK20</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Gel staining reagent</td>
			<td style="width:121px;">Sigma Aldrich</td>
			<td style="width:111px;">G1041</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Sequencing Grade Modified Trypsin</td>
			<td style="width:121px;">Promega</td>
			<td style="width:111px;">V5111</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">SpeedVac Concentrator</td>
			<td style="width:121px;">Thermo Fisher</td>
			<td style="width:111px;">SPD131DDA</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:227px;">Antibody to human annexin A5</td>
			<td style="width:121px;">Abcam</td>
			<td style="width:111px;">ab14196</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Antibody to human lactate dehydrogenase B chain</td>
			<td style="width:121px;">Abcam</td>
			<td style="width:111px;">ab53292</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="28">
			<td height="28" style="height:28px;width:227px;">Graphing and Statistical Software</td>
			<td style="width:121px;">Systat&#160;</td>
			<td style="width:111px;">SigmaPlot&#160;</td>
			<td style="width:168px;">Or GraphPad Prism</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:227px;">Quantitative proteomics software suite</td>
			<td style="width:121px;">Max Planck Institue of Biochemistry</td>
			<td style="width:111px;">Maxquant&#160;</td>
			<td style="width:168px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:227px;">Software and databases</td>
			<td style="width:121px;">Various vendors</td>
			<td style="width:111px;"></td>
			<td style="width:168px;">Refer to main text for details</td>
		</tr>
	</tbody>
</table>

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.

图1A是概述SILAC标记程序21的流程图。为了净化外来体，样品必须通过离心纺丝下来。 图1B示出了通过串行超速离心21外来体纯化的步骤。一旦纯化，如在程序中概述的外来体受实验蛋白质组学分析。 图2A是用于从蛋白质组数据21确定显...

Watch this Scientific Journal Video about SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells at JoVE.com

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

在这里，我们描述了使用通过在细胞培养物（SILAC）氨基酸稳定同位素标记的技术来分析HIV-1感染的主机外来体蛋白质组的作用的定量蛋白质组学方法。该协议可以很容易地适应于不同的压力或感染的条件下的细胞。

从HIV-1感染细胞的外来体基于SILAC蛋白质组学表征

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

silac-based-proteomic-characterization-exosomes-from-hiv-1-infected

Research

JoVE Journal

Immunology and Infection

10.6K 次观看.  Brown University. 在这里，我们描述了使用通过在细胞培养物（SILAC）氨基酸稳定同位素标记的技术来分析HIV-1感染的主机外来体蛋白质组的作用的定量蛋白质组学方法。该协议可以很容易地适应于不同的压力或感染的条件下的细胞。 

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

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

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

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

V3的HIV - 1取向的基因型推理使用人口为基础的测序

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

科研

免疫与感染

Preparation and Use of HIV-1 Infected Primary CD4+ T-Cells as Target Cells in Natural Killer Cell Cytotoxic Assays

Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK cells to recognize and lyse HIV-1 infected cells because identifying any aberrancy in NK cell function against HIV-infected cells could potentially lead to therapies that would enhance their cytolytic activity. There is a need to use HIV-infected primary T-cell blasts as target cells rather then infected-T-cell lines in the cytotoxicity assays. T-cell lines, even without infection, are quite susceptible to NK cell lysis. Furthermore, it is necessary to use autologous primary cells to prevent major histocompatibility complex class I mismatches between the target and effector cell that will result in lysis. Early studies evaluating NK cell cytolytic responses to primary HIV-infected cells failed to show significant killing of the infected cells 1,2. However, using HIV-1 infected primary T-cells as target cells in NK cell functional assays has been difficult due the presence of contaminating uninfected cells 3. This inconsistent infected cell to uninfected cell ratio will result in variation in NK cell killing between samples that may not be due to variability in donor NK cell function. Thus, it would be beneficial to work with a purified infected cell population in order to standardize the effector to target cell ratios between experiments 3,4. Here we demonstrate the isolation of a highly purified population of HIV-1 infected cells by taking advantage of HIV-1's ability to down-modulate CD4 on infected cells and the availability of commercial kits to remove dead or dying cells 3-6. The purified infected primary T-cell blasts can then be used as targets in either a degranulation or cytotoxic assay with purified NK cells as the effector population 5-7. Use of NK cells as effectors in a degranulation assay evaluates the ability of an NK cell to release the lytic contents of specialized lysosomes 8 called "cytolytic granules". By staining with a fluorochrome conjugated antibody against CD107a, a lysosomal membrane protein that becomes expressed on the NK cell surface when the cytolytic granules fuse to the plasma membrane, we can determine what percentage of NK cells degranulate in response to target cell recognition. Alternatively, NK cell lytic activity can be evaluated in a cytotoxic assay that allows for the determination of the percentage of target cells lysed by release of 51Cr from within the target cell in the presence of NK cells.

Cytotoxicity assays to measure natural killer cell lytic responses to HIV-infected cells is limited by the purity of the target cells. We demonstrate here the isolation of a highly purified population of HIV-1 infected primary T-cell blasts by taking advantage of HIV-1 s ability to down-modulate CD4.

制备和使用艾滋病毒感染主的CD4 + T细胞作为靶细胞，自然杀伤细胞的细胞毒性检测

 Natural killer (NK) cells are a vital component of the innate immune response to virus-infected cells. It is important to understand the ability of NK ...

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 ...

New Tools to Expand Regulatory T Cells from HIV-1-infected Individuals

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to measurable increases in antigen-specific T cell responses in vaccine settings for cancer and infectious pathogens. However, their role in HIV-1 immuno-pathogenesis remains controversial, as they could either serve to suppress deleterious HIV-1-associated immune activation and thus slow HIV-1 disease progression or alternatively suppress HIV-1-specific immunity and thereby promote virus spread. Understanding and modulating Treg function in the context of HIV-1 could lead to potential new strategies for immunotherapy or HIV vaccines. However, important open questions remain on their role in the context of HIV-1 infection, which needs to be carefully studied.
Representing roughly 5% of human CD4+ T cells in the peripheral blood, studying the Treg population has proven to be difficult, especially in HIV-1 infected individuals where HIV-1-associated CD4 T cell and with that Treg depletion occurs. The characterization of regulatory T cells in individuals with advanced HIV-1 disease or tissue samples, for which only very small biological samples can be obtained, is therefore extremely challenging. We propose a technical solution to overcome these limitations using isolation and expansion of Tregs from HIV-1-positive individuals.
Here we describe an easy and robust method to successfully expand Tregs isolated from HIV-1-infected individuals in vitro. Flow-sorted CD3+CD4+CD25+CD127low Tregs were stimulated with anti-CD3/anti-CD28 coated beads and cultured in the presence of IL-2. The expanded Tregs expressed high levels of FOXP3, CTLA4 and HELIOS compared to conventional T cells and were shown to be highly suppressive. Easier access to large numbers of Tregs will allow researchers to address important questions concerning their role in HIV-1 immunopathogenesis. We believe answering these questions may provide useful insight for the development of an effective HIV-1 vaccine.

CD4+ Regulatory T cells are potent immune-modulators and serve important functions in immune homeostasis. The paucity of these cells in peripheral blood makes functional studies challenging, specifically in the context of HIV-1-infection. We here describe a method to isolate and expand functional CD4+ Tregs from peripheral blood from HIV-1-infected individuals.

新工具展开调节性T细胞的HIV-1感染的个体

CD4+ Regulatory T cells (Tregs) are potent immune modulators and serve an important function in human immune homeostasis. Depletion of Tregs has led to ...

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 ...

Phagosome Migration and Velocity Measured in Live Primary Human Macrophages Infected with HIV-1

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human immunodeficiency virus (HIV)-1 and because of their resistance to its cytopathic effects they can be considered to be persistent viral reservoirs. In addition, HIV-infected macrophages exhibit defective functions that contribute to the development of opportunistic diseases.
The exact mechanism by which HIV-1 impairs the phagocytic response of macrophages was unknown. We had previously shown that the uptake of various particulate material by macrophages was inhibited when they were infected with HIV-1. This inhibition was only partial and phagosomes did form within HIV-infected macrophages. Therefore, we focused on analyzing the fate of these phagosomes. Phagosome maturation is accompanied by migration of these compartments towards the cell center, where they fuse with lysosomes, generating phagolysosomes, responsible for degradation of the ingested material. We used IgG-opsonized Sheep Red Blood Cells as a target for phagocytosis. To measure the speed of centripetal movement of phagosomes in individual HIV-infected macrophages, we used a combination of bright field and fluorescence confocal microscopy. We established a method to calculate the distance of phagosomes towards the nucleus, and then to calculate the velocity of the phagosomes. HIV-infected cells were identified thanks to a GFP-expressing virus, but the method is applicable to non-infected cells or any type of infection or treatment.

We describe a method to measure the velocity of phagosomes moving towards the cell center in living cells infected with or without the human immunodeficiency virus (HIV) type 1, using spinning disk confocal fluorescence microscopy to identify fluorescent infected cells and bright field microscopy to detect phagosomes.