这项工作部分由拨款DA024558，DA30896，DA033892和DA021471从NIDA / NIH到CD的支持。我们也承认RCMI批G12MD007586，范德比尔特CTSA授予UL1RR024975的梅哈里转化研究中心从NCRR / NIH，从NIMHD / NIH的U54补助MD007593，和田纳西CFAR补助P30 AI110527（MeTRC）CTSA补助U54 RR026140。

1.病毒生产注意:为了产生感染性的HIV-1的高滴度，使用活化基于树枝状大分子转染试剂转染与感染HIV-1的分子克隆的人类胚胎肾（HEK）细胞系293T。已经观察到，磷酸钙转染方法产生可比较的结果。 <ol><li>在生物安全等级2（BSL2）实验室中，种子3×10 6个 293T细胞每10cm培养皿中8的Dulbecco补充有10％胎牛血清（FBS），青霉素和链霉素改良的Eagle培养基（DMEM）中的溶液。培养细胞在设置在37℃的培养箱中5％的CO 2 O / N。 </li><li>第二天，在每个10cm皿转染细胞，稀释8微克pNL4-3质粒DNA（见材料数据表 ）的对的DMEM 300微升的缺乏血清和抗生素的最终体积。加入80微升转染试剂，吹打拌匀，并在室温孵育5 - 10分钟的转染复合物的形成。 </li><li>吸取下菜中，每含有血清和抗生素的DMEM新鲜菜加7毫升。 </li><li>添加1毫升含DMEM血清和抗生素的对转染复合物，通过移液混合，并将该混合物添加到细胞中。轻轻地摇晃菜方便，甚至转染复合物的分布。 </li><li>立即将细胞转移到BSL3实验室地点中的CO 2设定在37℃和5％的培养箱，并培养所述细胞48小时。 </li><li>收集用吸管含有病毒的组织培养上清液中，然后将其转移到一个离心管中。离心机在300×g离心5分钟以沉淀细胞和碎片，然后用0.45μm孔径的注射器过滤器过滤上清液。 </li><li>以消除在此无细胞病毒制备任何夹带的质粒DNA，在37℃下处理用DNA酶1（20微克/毫升）1小时。 </li><li>量化HIV-1的效价，可使用特定的p24-酶联免疫吸附测定（ELISA），因为它是常规使用22迅速并且易于方法。 注意:应该指出的是，p24的ELISA测量物理滴度（病毒颗粒的数量）。另外，测量病毒相关的RT活性（外源性RT法）或病毒感染（TZM-BL-基于线指示细胞感染性测定）将提供感染性病毒颗粒的独家读数。 </li></ol> 2. SUP-T1细胞病毒感染（BSL3实验室） <ol><li>在一个BSL2实验室中，保持SUP-T1细胞1细胞1.0和2.0之间×10 6×10 6 / mL的罗斯韦尔园区纪念研究所（RPMI）补充有10％热灭活的FBS，青霉素培养基，链霉素（分流比2，每2 - 3天）。汇集SUP-T1细胞从培养瓶中，吹打混匀，并通过使用血细胞计数器和台盼蓝排除ST确定活菌数癌宁方法。 <ol><li>制备所需数目的50毫升锥形离心管中80×10 6个细胞（每病毒感染）的等分试样。 </li><li>离心在吊桶式转子的细胞以500 xg离心和25℃下5分钟。不去除细胞培养基，含有沉淀的细胞在管转移到BSL3实验室用于进一步处理。小心吸从管细胞培养基而不干扰细胞沉淀。 </li></ol></li><li> DNA酶I处理过的病毒接种物的适量添加到管中的细胞，并通过温和移液拌匀。为80×10 6个细胞，使用30毫升DNA酶I处理过的病毒（p24的相当于病毒的50微克）的。 注:太平洋岛国功能的分离需要与感染非常高的多重性（MOI）感染靶细胞。 </li><li>均匀分布两个6孔组织培养板（ 即 12×2.5毫升等分试样的混合物;使用每个侵染2板离子）。 </li><li>为spinoculation，放置用在480 xg离心吊桶式转子和25℃2小时的板载体和离心机的培养板中。 </li><li>孵育spinoculated细胞在37℃和5％的CO 2 5小时。 </li></ol> 3.收获和裂解感染的细胞（BSL3实验室） <ol><li>池对应于从培养板放入50毫升锥形离心管中的每个感染（总共30毫升〜）细胞。 </li><li>收获离心细胞在300 xg离心和25℃下进行10分钟。小心吸从管的上清液（可替代地，可使用移液管）。 </li><li>以除去任何残留的病毒颗粒，通过加入缓冲的K 2毫升洗涤各细胞沉淀- / - （150毫米氯化钾，20mM的HEPES，pH值7.6，5mM的MgCl 2的），并离心分离，在300 xg离心样品和25℃下10分钟。然后，在不干扰颗粒，小心地取出通过抽吸或通过使用移液管的上清液。 </li><li> RepeaT高低3.3。 </li><li>小心通过使用微量除去任何残余缓冲液。重悬每个细胞沉淀在2ml冰冷的裂解缓冲液的K + / +（缓冲的 K - / -补充有0.025％毛地黄皂苷，1毫摩尔DTT，20微克/毫升抑肽酶），混合通过温和移液的内容，并转移到2毫升离心管中。 </li></ol> 4.准备细胞质的PIC（BSL3实验室） <ol><li>岩石上在RT旋转振荡器10分钟的样品。 </li><li>离心样品，用预冷却至4℃的转子，在1500 xg离心和4℃下进行4分钟。 </li><li>不干扰沉淀，小心地将上清液转移到新的微量离心管中并离心以16,000 xg离心和4℃1分钟。 </li><li>小心地将上清液转移到新的微量离心管中。添加RNA酶A（20毫克/毫升），以20微克/毫升（2微升2毫升的PIC）的终浓度和孵育在室温的内容为30分钟（无摇摆）。&lt;/ LI&gt; <li> （ - / -在缓冲ķ60％重量/体积）加入蔗糖至7％的终浓度，并通过温和移液拌匀。制备的PIC的等分试样（ 例如，200微升）在微量离心管，闪光冷冻在液氮中，并发生在-80℃冷冻用于长期存储。 </li></ol> 5. 在体外集成（IVI）分析使用HIV-1图片<ol><li>准备用特制的引物，phiX174-F和phiX174-R从PCR的phiX174质粒DNA扩增2.0 kb的区域在IVI试验使用的目标DNA。 <ol><li>组装如表1中描述的PCR混合物中。使用表2中建议的热循环条件在热循环仪进行PCR。凝胶纯化用凝胶纯化试剂盒（按照制造商推荐的协议）的PCR产物，并在-80℃下将纯化的靶DNA作为等份储存直至使用。 </li></ol></li><li>加入靶DNA的600毫微克至200 - ＆＃181;太平洋岛国L的等份，混合均匀，在37℃孵育45分钟。 注:IVI反应省略目标DNA或包括整合酶抑制剂（如雷特格韦）推荐的控制反应。 </li><li>通过添加SDS，EDTA和蛋白酶K至0.5％，8毫米，和0.5毫克/毫升，终浓度分别停止IVI反应。充分混合反应的内容和孵育过夜在56℃。 </li><li>第二天，净化从通过苯酚/氯仿萃取和乙醇沉淀的IVI反应的总DNA。开展16000 XG和RT所有离心步骤。 </li><li>酚等体积添加到脱蛋白质样品，通过涡旋剧烈混合，并离心2分钟。小心取出上层水相，并将其转移至新管。 </li><li>重复步骤5.5。上层水相转移至新管。 </li><li>加1的等体积:1的苯酚:氯仿混合物中，通过涡旋剧烈混合，并离心FOR 2分钟。上层水相转移至新管。 </li><li>添加氯仿等体积的，通过涡旋剧烈混合，并离心2分钟。上层水相转移至新管。 </li><li>沉淀的DNA，0.1微克/微升，醋酸钠3 M的最终浓度添加糖原到0.3M的最终浓度，和2.5体积的100％乙醇（冰冷）的。涡旋混合，短暂离心，收集的内容，并将该管在-80℃CO / N。 </li><li>第二天，离心样品30分钟，以16,000 xg离心4℃30分钟。小心取出上清液，而不会干扰颗粒。 </li><li>添加80％乙醇（冰冷），以沉淀和离心的500μL的16000 xg离心4℃10分钟。在RT风干的DNA沉淀，并在不含核酸酶的水加入50μL彻底重悬。在-20℃保存DNA样本。 </li></ol> 6. PCR方法量化PIC活动<ol><li>使用引物（分别为LTR和phiX174引物）靶向病毒LTR和phiX174的DNA扩增整合的HIV-1的DNA的结执行第一轮常规PCR。 <ol><li>组装如表3中描述的第一轮PCR混合物。组装不含模板DNA所有组件的主结构，取45微升等分到单独的PCR管，并添加5μL模板DNA或无核酸酶水（对照）。 </li><li>使用表4中建议的热循环条件在热循环仪进行PCR。 </li></ol></li><li>执行使用侧翼病毒LTR（LTR-R和LTR-U的引物）的一个短区域的引物，以选择性地扩增从第一ロ集成VDNA序列第二轮的qPCRUND PCR产物。以确定集成VDNA拷贝数，生成通过使用已知拷贝数的10倍系列稀释的标准曲线（ 例如，10 0 - 10 8）的HIV-1的分子克隆。 <ol><li>组装，如表5中描述的第二轮的qPCR混合物。组装不含模板DNA的所有部件的主混合物，小心地分配25μL的等分试样到96孔PCR反应板孔的适当数量，然后添加未纯化的第一轮的5微升PCR产物或无核酸酶水（控制）。一式三份执行所有的qPCR反应。 </li><li>加载PCR板放入实时PCR仪，并使用表6中所建议的循环条件进行定量PCR。 </li><li>使用合适软件进行数据分析。 </li></ol></li></ol>

<ol>
	<li>Sattentau, Q. J., Weiss, 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=The+CD4+antigen:+physiological+ligand+and+HIV+receptor.">The CD4 antigen: physiological ligand and HIV receptor.</a> Cell. 52 (5), 631-633 (1988).</li><li>Wilen, C. B., Tilton, J. C., Doms, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+HIV+entry.">Molecular mechanisms of HIV entry.</a> Adv Exp Med Biol. 726, 223-242 (2012).</li><li>Bowerman, B., Brown, P. O., Bishop, J. M., Varmus, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+nucleoprotein+complex+mediates+the+integration+of+retroviral+DNA.">A nucleoprotein complex mediates the integration of retroviral DNA.</a> Genes Dev. 3 (4), 469-478 (1989).</li><li>Bukrinsky, M. I., Sharova, N., Dempsey, M. P., 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=Active+nuclear+import+of+human+immunodeficiency+virus+type+1+preintegration+complexes.">Active nuclear import of human immunodeficiency virus type 1 preintegration complexes.</a> Proc Natl Acad Sci. 9 (14), 6580-6584 (1992).</li><li>Hansen, M. S., Smith, G. J., Kafri, T., Molteni, V., Siegel, J. S., Bushman, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+complexes+derived+from+HIV+vectors+for+rapid+assays+in+vitro.">Integration complexes derived from HIV vectors for rapid assays in vitro.</a> Nature biotechnol. 17 (6), 578-582 (1999).</li><li>Farnet, C. M., Haseltine, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+human+immunodeficiency+virus+type+1+DNA+in+vitro.">Integration of human immunodeficiency virus type 1 DNA in vitro.</a> Proc Natl Acad Sci. 87 (11), 4164-4168 (1990).</li><li>Fujiwara, T., Mizuuchi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+DNA+integration:+structure+of+an+integration+intermediate.">Retroviral DNA integration: structure of an integration intermediate.</a> Cell. 54 (4), 497-504 (1988).</li><li>Brown, P. O., Bowerman, B., Varmus, H. E., Bishop, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Correct+integration+of+retroviral+DNA+in+vitro.">Correct integration of retroviral DNA in vitro.</a> Cell. 49 (3), 347-356 (1987).</li><li>Lewinski, M. K., Bushman, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retroviral+DNA+integration--mechanism+and+consequences.">Retroviral DNA integration--mechanism and consequences.</a> Adv Genet. 55, 147-181 (2005).</li><li>Engelman, A., Mizuuchi, K., Craigie, R. <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+DNA+integration:+mechanism+of+viral+DNA+cleavage+and+DNA+strand+transfer.">HIV-1 DNA integration: mechanism of viral DNA cleavage and DNA strand transfer.</a> Cell. 67 (6), 1211-1221 (1991).</li><li>Goff, S. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetics+of+retroviral+integration.">Genetics of retroviral integration.</a> Ann rev gen. 26, 527-544 (1992).</li><li>Panganiban, A. T., Temin, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+terminal+nucleotides+of+retrovirus+DNA+are+required+for+integration+but+not+virus+production.">The terminal nucleotides of retrovirus DNA are required for integration but not virus production.</a> Nature. 306 (5939), 155-160 (1983).</li><li>Bieniasz, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+HIV-1+virion+genesis.">The cell biology of HIV-1 virion genesis.</a> Cell host microbe. 5 (6), 550-558 (2009).</li><li>Sherman, P. A., Fyfe, 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=Human+immunodeficiency+virus+integration+protein+expressed+in+Escherichia+coli+possesses+selective+DNA+cleaving+activity.">Human immunodeficiency virus integration protein expressed in Escherichia coli possesses selective DNA cleaving activity.</a> Proc Natl Acad Sci. 87 (13), 5119-5123 (1990).</li><li>Katzman, M., Katz, R. A., Skalka, A. M., Leis, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+avian+retroviral+integration+protein+cleaves+the+terminal+sequences+of+linear+viral+DNA+at+the+in+vivo+sites+of+integration.">The avian retroviral integration protein cleaves the terminal sequences of linear viral DNA at the in vivo sites of integration.</a> J Virol. 63 (12), 5319-5327 (1989).</li><li>Craigie, R., Mizuuchi, K., Bushman, F. D., Engelman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+in+vitro+assay+for+HIV+DNA+integration.">A rapid in vitro assay for HIV DNA integration.</a> Nucl acid res. 19 (10), 2729-2734 (1991).</li><li>Bushman, F. D., Craigie, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activities+of+human+immunodeficiency+virus+(HIV)+integration+protein+in+vitro:+specific+cleavage+and+integration+of+HIV+DNA.">Activities of human immunodeficiency virus (HIV) integration protein in vitro: specific cleavage and integration of HIV DNA.</a> Proc Natl Acad Sci. 88 (4), 1339-1343 (1991).</li><li>Pauza, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+bases+are+deleted+from+the+termini+of+HIV-1+linear+DNA+during+integrative+recombination.">Two bases are deleted from the termini of HIV-1 linear DNA during integrative recombination.</a> Virology. 179 (2), 886-889 (1990).</li><li>Engelman, 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+analysis+of+HIV-1+preintegration+complexes.">Isolation and analysis of HIV-1 preintegration complexes.</a> Meth Mol Biol. 485, 135-149 (2009).</li><li>Engelman, A., Oztop, I., Vandegraaff, N., Raghavendra, N. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+analysis+of+HIV-1+preintegration+complexes.">Quantitative analysis of HIV-1 preintegration complexes.</a> Methods. 47 (4), 283-290 (2009).</li><li>Addai, A. B., Pandhare, J., Paromov, V., Mantri, C. K., Pratap, S., Dash, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cocaine+modulates+HIV-1+integration+in+primary+CD4++T+cells:+implications+in+HIV-1+pathogenesis+in+drug-abusing+patients.">Cocaine modulates HIV-1 integration in primary CD4+ T cells: implications in HIV-1 pathogenesis in drug-abusing patients.</a> J Leuk Biol. 97 (4), 779-790 (2015).</li><li>Wehrly, K., Chesebro, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p24+antigen+capture+assay+for+quantification+of+human+immunodeficiency+virus+using+readily+available+inexpensive+reagents.">p24 antigen capture assay for quantification of human immunodeficiency virus using readily available inexpensive reagents.</a> Methods. 12 (4), 288-293 (1997).</li></ol>

作者宣称没有竞争的金融和非金融利益。

逆转录病毒的PIC的生化分析提供了重要的见解逆转录病毒DNA整合的机制。逆转录病毒的PIC的融合活性的测定可通过空斑形成试验，Southern印迹分析，和嵌套的qPCR来实现。空斑试验的实验策略是费力并且利用测量积分6，7，18的间接方法。 Southern印迹的检测方法衡量直接集成，但需要较大量的PIC 6，7，18。 该报告详细介绍了体外测定HIV-1 PIC-相关整合活动嵌套的qPCR的策略。主要的优势，这种方法的意义在于每反应4相对少量的PIC准备的要求，5，19。这种方法的最重要的步骤是一种高滴度的病毒感染的产生和PIC制备的处理。这里应注意的是，在第一轮PCR涉及一体化结的指数扩增。因此，在第二轮中的qPCR确定综合VDNA的拷贝数是半定量，因此，具有不给的PIC-相关VDNA拷贝措施的局限性。然而，在PIC相关VDNA可以通过qPCR的DNA的特定LTR引物的PIC纯化来确定。集成VDNA的比率号码复制到相应的PIC相关VDNA可提供有关在体外结合反应的整合效率的信息。这种方法可以通过将选择和其他底漆等异源靶DNA在两个方向来检测的整合进行修改。总之，该方法这里描述的定量测定HIV-1图片的融合活性，可用于研究开发的逆转录病毒整合的生化细节的理解通过。

在靶细胞中HIV-1复制涉及多个步骤，大致分为两个阶段:早期和晚期的事件。当HIV-1顺序地结合靶细胞表面受体CD4和两个共同受体（CCR5或CXCR4）1的一个早期的事件开始。因此，该病毒细胞膜熔丝，病毒衣壳（CA）的芯被释放到细胞质2。在CA芯含有病毒基因组的单链RNA（ssRNA）和几种蛋白质，包括病毒和起源细胞。在CA相关病毒蛋白包括逆转录酶（RT）和整合酶（IN），在病毒复制的早期事件介导的关键步骤的两种酶。 RT和在核蛋白复合体，即反转录复合物（RTC）和一体化的预复合物（PIC），分别为3，4，5的上下文功能&lt;SUP&gt;，6。所述病毒ssRNA基因组海港末端重复（R）相邻的元件以特有的序列U5（在5&#39;端）和U3（在3&#39;端）的端部。 RTC的转换病毒ssRNA基因组中双链（DS）的DNA拷贝（VDNA）。此逆转录过程也导致U5和U3序列的重复，从而产生在VDNA 7,8的两端长末端重复序列（LTR）。接着，在PIC相关中催化两个顺序的生化反应，使所述VDNA的整合到宿主染色体9，10，11。首先，在细胞质中，在多聚体接合两者的LTR 12和从每个3&#39;-末端的裂解二核苷酸。此3&#39;末端加工在每个3&#39;端的VDNA的（CA OH）产生羟基。接着，PIC投放到细胞核，其中IN使用VDNA CA OH作为亲核切断染色体DNA的两条链以交错的方式。同时，在协调拼接的VDNA两端在染色体DNA上的相反链所得到的磷酸二酯键。以下这个链转移步骤，所述宿主细胞机器在整合位点的连接点除去在VDNA的5&#39;端和修理随后的单链缺口两个未配对核苷酸。因此，早期事件达到高潮与建立的HIV-1基因组的集成DNA拷贝（前病毒）。原病毒提供了高效的病毒基因表达，从而启动后的事件，包括病毒编码的蛋白质的表达的合适的环境;未成熟病毒的组装;而崭露头角，释放和成熟为感染性病毒体13。 纯化的重组逆转录病毒在有资格进行， 在体外 ，两个3&#39;-末端加工和链转移活动外源提供的LTR状衬底的DNA。使用这样的纯化的重组中的生化研究已经揭示VDNA积分14，15，16，17的关键方面。然而，不同于在天然感染，这在体外生化反应不支持基板DNA导入靶DNA的两端的协调一致的集成。与此相反，从急性感染的细胞中分离逆转录病毒的PIC协调一致内源性VDNA两端整合到异源靶DNA。这导致了广泛的采用和I N体外集成（IVI）测定后续改进使用分离的天然照片。 在首次报道逆转录病毒的IVI测定法，从感染了重组小鼠细胞胞质提取物白血病病毒（MLV）窝藏在其LTR 大肠杆菌supF基因担任活动的源，和一个突变λ噬菌体缺陷在裂解生长作为目标的DNA外源提供的DNA中。重组MLV的DNA成功整合复制到噬菌体DNA和随后supF表达导致的重组噬菌体的噬斑形成能力的恢复。然而，该实验策略是一种间接的方式费力和措施集成。为了解决这样的警告，一个Southern印迹间接末端标记测定法，其中量化的PIC-相关联的IVI活性内源性VDNA纳入外源线性噬菌体phiX174的DNA的量度，被开发6，7，18。虽然这种方法提供了积分事件的直接量度，需要较高量的PIC的准备，这仍然是一个技术挑战性努力。为了规避这一点，只需要适量的PIC的巢式PCR为基础的测定法开发的4，5，19。 在这份报告中，我们描述的更新版本基于PCR技术在设计来概括自然感染过程中出现的HIV-1 PIC介导的整合活动体外方法嵌套。这种方法使用的HIV-1感染靶细胞的胞质提取物作为内源性的PIC活性的源，其与实时定量聚合酶链式反应（qPCR的）测量。隔离PIC和测量它们的整合活动的程序改编，经过修改，从由恩格尔曼实验室20发表协议。在该方法中，在PIC相关积分活性是通过提供外源的线性靶DNA 21，并从得到的DNA产物发起这种融合反应中纯化，并用作用于基于PCR的随后的量化的起始原料。在第一轮常规PCR，病毒靶DNA结是通过使用合适的引物扩增。在第二轮的qPCR，特定LTR-引物用于特异性富集来自第一轮PCR产物的VDNA人口。请参见该方法的详细描述的协议部分。 在用反转录病毒的PIC 体外研究已经导致显著进展中的逆转录病毒DNA整合机制的理解以及在抑制剂的开发。根据最近的更加注重映射HIV-1的整合位点，确定的病毒和宿主因子的HIV-1整合的作用，并朝向打击抗药性发展新的抗病毒药物，我们设想以增加的兴趣，并广泛使用的IVI测定的一样，本报告中所述的。这反过来会导致在该方法将来精炼，从而多样化其应用的范围。

<table><tbody><tr><td>&lt;p&gt;Fetal Bovine Serum (FBS)&lt;/p&gt;</td><td> &lt;p&gt;GIBCO/Invitrogen&lt;/p&gt; </td><td> &lt;p&gt;10437-028&lt;/p&gt; </td><td></td></tr><tr><td>&lt;p&gt;Heat-inactivated Fetal Bovine Serum (Hi-FBS)&lt;/p&gt;</td><td> &lt;p&gt;GIBCO/Invitrogen&lt;/p&gt; </td><td> &lt;p&gt;10438-026&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Dulbecco&amp;rsquo;s modified Eagle&amp;rsquo;s medium (DMEM)&lt;/p&gt; &lt;p&gt;&lt;/p&gt; </td><td> &lt;p&gt;GIBCO/Invitrogen&lt;/p&gt; </td><td> &lt;p&gt;11995-065&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Roswell Park Memorial Institute (RPMI) 1640 medium&lt;/p&gt; </td><td> &lt;p&gt;GIBCO/Invitrogen&lt;/p&gt; </td><td> &lt;p&gt;11875-093&lt;/p&gt; </td><td></td></tr><tr><td>&lt;p&gt;Phosphate-buffered Saline (PBS) (1x)&lt;/p&gt;</td><td> &lt;p&gt;GIBCO/Invitrogen&lt;/p&gt; </td><td> &lt;p&gt;20012-027&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Trypsin-EDTA (0.25%)&lt;/p&gt; &lt;p&gt;&lt;/p&gt; </td><td> &lt;p&gt;GIBCO/Invitrogen&lt;/p&gt; </td><td> &lt;p&gt;25200-056&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Penicillin-Streptomycin solution (100x)&lt;/p&gt; </td><td> &lt;p&gt;Cellgro/Mediatech&lt;/p&gt; </td><td> &lt;p&gt;30-002-CI&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;HEK293T cells (293T)&lt;/p&gt; </td><td> &lt;p&gt;American Type Culture Collection (ATCC)&lt;/p&gt; </td><td> &lt;p&gt;CRL-3216&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;HIV-1 proviral molecular clone pNL4-3&lt;/p&gt; </td><td> &lt;p&gt;NIH AIDS Research and Reference Reagent Program&lt;/p&gt; &lt;p&gt;&lt;/p&gt; </td><td> &lt;p&gt;114&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;PolyFect transfection reagent&lt;/p&gt; </td><td> &lt;p&gt;Qiagen&lt;/p&gt; </td><td> &lt;p&gt;301107&lt;/p&gt; </td><td></td></tr><tr><td>&lt;p&gt;DNase 1&lt;/p&gt; &lt;p&gt;&lt;/p&gt;</td><td> &lt;p&gt;Calbiochem/Merckmillipore&lt;/p&gt; </td><td> &lt;p&gt;260913&lt;/p&gt; </td><td></td></tr><tr><td>&lt;p&gt;Sup-T1 cells&lt;/p&gt;</td><td> &lt;p&gt;American Type Culture Collection (ATCC)&lt;/p&gt; </td><td> &lt;p&gt;CRL-1942&lt;/p&gt; &lt;p&gt;&lt;/p&gt; </td><td></td></tr><tr><td>&lt;p&gt;HEPES pH 7.2 - 7.5&lt;/p&gt; &lt;p&gt;(ready-to-use solution)&lt;/p&gt;</td><td> &lt;p&gt;GIBCO/Invitrogen&lt;/p&gt; </td><td> &lt;p&gt;15630-080&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Potassium chloride (KCl) solution&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;60142&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Magnesium chloride (MgCl&lt;sub&gt;2&lt;/sub&gt;) solution&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;M1028&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Dithiothreitol (DTT)&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;43815&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Aprotinin&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;A6106&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Digitonin&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;D141&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;RNase A&lt;/p&gt; </td><td> &lt;p&gt;Invitrogen&lt;/p&gt; </td><td> &lt;p&gt;12091-021&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Sucrose&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;S0389&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;phiX174 Replicative Form 1 DNA&lt;/p&gt; </td><td> &lt;p&gt;Promega&lt;/p&gt; </td><td> &lt;p&gt;D1531&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;&lt;u&gt;PhiX174-F primer:&lt;/u&gt;&lt;/p&gt; &lt;p&gt;5&amp;rsquo;-CGCTTCCATGACGCAGAAGTT-3&amp;rsquo;&lt;/p&gt; &lt;p&gt;&lt;/p&gt; &lt;p&gt;&lt;u&gt;PhiX174-R primer:&lt;/u&gt;&lt;/p&gt; &lt;p&gt;5&amp;rsquo;-CACTGACCCTCAGCAATCTTA-3&amp;rsquo;&lt;/p&gt; &lt;p&gt;&lt;/p&gt; &lt;p&gt;&lt;/p&gt; </td><td> &lt;p&gt;Invitrogen and/or IDT-DNA&lt;/p&gt; </td><td> &lt;p&gt;&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;dNTP mix&lt;/p&gt; </td><td> &lt;p&gt;Promega&lt;/p&gt; </td><td> &lt;p&gt;U1515&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Go Taq DNA Polymerase&lt;/p&gt; </td><td> &lt;p&gt;Promega&lt;/p&gt; </td><td> &lt;p&gt;M3005&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Qiaquick gel extraction kit&lt;/p&gt; </td><td> &lt;p&gt;Qiagen&lt;/p&gt; </td><td> &lt;p&gt;28706&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Ultrapure distilled water&lt;/p&gt; </td><td> &lt;p&gt;Invitrogen&lt;/p&gt; </td><td> &lt;p&gt;10977-015&lt;/p&gt; </td><td></td></tr><tr><td>&lt;p&gt;Tris-EDTA (TE) buffer (100x)&lt;/p&gt;</td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;T9285&lt;/p&gt; </td><td></td></tr><tr><td>&lt;p&gt;Sodium Dodecyl Sulphate (SDS)&lt;/p&gt;</td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;L3771&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Ethylenediamine tetraacetic acid, disodium salt (EDTA) solution, 0.5 M, pH 8.0&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;E7889&lt;/p&gt; </td><td></td></tr><tr><td>&lt;p&gt;Proteinase K (20 mg/mL ready-to-use solution)&lt;/p&gt;</td><td> &lt;p&gt;Qiagen&lt;/p&gt; </td><td> &lt;p&gt;19131&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Phenol (equilibrated with 10&amp;nbsp;mM Tris HCl, pH&amp;nbsp;8.0 and 1&amp;nbsp;mM EDTA)&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;P4557&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;Chloroform&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;288306&lt;/p&gt; </td><td></td></tr><tr><td>&lt;p&gt;Glycogen (5 mg/mL solution)&lt;/p&gt;</td><td> &lt;p&gt;Ambion/Invitrogen&lt;/p&gt; </td><td> &lt;p&gt;AM9510&lt;/p&gt; </td><td></td></tr><tr><td>&lt;p&gt;Sodium acetate (3 M, pH 5.2)&lt;/p&gt;</td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td>&lt;p&gt;S7899&lt;/p&gt;</td><td></td></tr><tr><td> &lt;p&gt;Ethanol&lt;/p&gt; </td><td> &lt;p&gt;Sigma-Aldrich&lt;/p&gt; </td><td> &lt;p&gt;E7023&lt;/p&gt; </td><td></td></tr><tr><td>&lt;p&gt;&lt;u&gt;First round LTR primer&lt;/u&gt;:&lt;/p&gt; &lt;p&gt;5&amp;rsquo;-GTGCGCGCTTCAGCAAG-3&amp;rsquo;&lt;/p&gt; &lt;p&gt;&lt;u&gt;First round phiX174 primer&lt;/u&gt;:&lt;/p&gt; &lt;p&gt;5&amp;rsquo;-CACTGACCCTCAGCAATCTTA-3&amp;rsquo;&lt;/p&gt;</td><td> &lt;p&gt;Invitrogen and/or IDT-DNA&lt;/p&gt; </td><td> &lt;p&gt;---&lt;/p&gt; </td><td></td></tr><tr><td>&lt;p&gt;&lt;u&gt;Second round LTR-R primer&lt;/u&gt;:&lt;/p&gt; &lt;p&gt;5&amp;rsquo;-TCTGGCTAACTAGGGAACCCA-3&amp;rsquo;&lt;/p&gt; &lt;p&gt;&lt;u&gt;Second round LTR-U primer&lt;/u&gt;:&lt;/p&gt; &lt;p&gt;5&amp;rsquo;-CTGACTAAAAGGGTCTGAGG-3&amp;rsquo;&lt;/p&gt; &lt;p&gt;&lt;/p&gt; &lt;p&gt;&lt;u&gt;Second round Taqman probe&lt;/u&gt;:&lt;/p&gt; &lt;p&gt;&amp;nbsp;5&amp;rsquo;-6- FAM/TTAAGCCTCAATAAAGCTTGC&lt;/p&gt; &lt;p&gt;CTTGAGTGC/6-TAMRA/-3&amp;rsquo;&lt;/p&gt;</td><td> &lt;p&gt;Invitrogen and/or IDT-DNA&lt;/p&gt; </td><td> &lt;p&gt;---&lt;/p&gt; </td><td></td></tr><tr><td> &lt;p&gt;iQ Supermix&lt;/p&gt; </td><td> &lt;p&gt;Bio-Rad&lt;/p&gt; </td><td> &lt;p&gt;170-8862&lt;/p&gt; </td><td></td></tr></tbody></table>

measurement of in vitro integration activity of hiv-1 preintegration complexes

HIV-1包膜蛋白的接合靶细胞表面，这导致病毒细胞膜融合，随后通过释放病毒衣壳（CA）的芯的进入细胞质上的同源受体。接着，病毒逆转录酶（RT），如一个同名核蛋白复合物的一部分被称为反转录复合物（RTC），转换病毒单链RNA基因组中的双链DNA拷贝（VDNA）。这导致了另一个核蛋白复合物中的生物合成，称为集成预复合物（PIC），所述VDNA和相关病毒蛋白和宿主因素组成。在PIC相关病毒整合酶（IN）的编排一体化VDNA成在时间上和空间上调节两步骤过程宿主染色体DNA。首先，在IN处理VDNA的3&#39;端在细胞质中，第二，在PIC投放到细胞核后，它介导的加工VDNA整合到染色体DNA中。太平洋岛国隔离从急性感染HIV-1的靶细胞在体外功能，因为它们有能力相关VDNA集成到一个外源添加的异源靶DNA。这种PIC-基于体外整合实验已显著贡献描绘逆转录病毒整合的机械细节和发现IN抑制剂。在这份报告中，我们在阐述一个更新的HIV-1检测PIC，它采用嵌套实时定量聚合酶链反应（定量PCR）为基础的策略，用于测量分离的天然太平洋岛国的体外整合活动。

HIV-1图片的分离 用于HIV-1图片由急性感染SUP-T1细胞分离协议的原理图如图1所示。这个协议是从由恩格尔曼等人描述的方法得到。 19，20。的HIV-1 PIC可被组装在感染的细胞和被包括在逆转录VDNA的核蛋白复合物，病毒蛋白，和细胞因子19,20。本报告中所描述的方法使用的HIV-1感染的细胞的细胞质提取物作为PIC相关整合活性的来源。 在 HIV-1图片的 体外 活性融合 如图2所示。此方法利用测量的PIC相关IVI活性的嵌套基于定量PCR策略，和程序适于与修改，从由恩格尔曼实验室19，20，21公布的协议。在该方法中，在PIC相关积分活性是通过提供异源线性靶DNA 21启动，并从该积分反应得到的DNA产物被纯化并用作用于基于PCR的随后的量化的起始原料。在第一轮常规PCR，病毒靶DNA结使用合适的引物扩增。在第二轮的qPCR，特定LTR-引物用于特异性富集来自第一轮PCR产物的VDNA人口。请参阅协议小号挠度这个方法的详细描述。 采用的HIV-1的PIC在图3中所示执行从IVI测定代表性结果。 SUP-T1细胞（80×10 6个细胞/感染）用30mL分别spinoculated DNA酶1处理的HIV-1（〜50微克的p24当量）在不存在或为1μM的HIV-1的存在下，在抑制剂雷特格韦在480 XG和25℃2小时。细胞随后在37℃，5小时和5％的CO 2中培养。在协议部分所述的PIC是孤立的。的IVI反应45分钟进行的，每个都使用200微升的PIC的和靶DNA的600纳克（通过PCR从phiX174 DNA中扩增2 kb的DNA片段），在37℃下。该反应通过蛋白酶K处理，在SDS和EDTA的存在下，在56°CO / N脱蛋白质。用苯酚两次反应产物提取，一旦与酚:氯仿（1:1）的混合物，并且一旦用氯仿。接着，将DNA用乙醇沉淀在盐和共沉淀剂糖原的存在，并且该DNA沉淀用80％乙醇洗涤一次。再悬浮于无核酸酶的水加入50μL纯化的DNA。嵌套的qPCR策略被用来确定整合到phiX174的DNA VDNA的数目。在第一轮常规PCR，以对phiX174靶DNA和病毒LTR区序列互补的引物用于从积分反应DNA产物的5微升扩增集成病毒靶DNA结。在第二轮的qPCR，LTR-跨越引物用于只放大来自第一轮扩增产物的5微升和从HIV特异性DNA序列10倍已知拷贝数的连续稀释液（10 8 -10 0） HIV-1的分子克隆的（标准）。 所有的qPCR反应一式三份进行，并且WER数据e。使用商业软件进行分析。从标准得出的值进行作图，以产生标准曲线，从该集成VDNA拷贝数进行内插。误差棒代表标准偏差。如在图3中所示，所述的HIV-1的PIC鲁棒整合相关VDNA入靶DNA（泳道5）。重要的是，太平洋岛国的IVI效率在补充用IN抑制剂雷特格韦（泳道6）的反应被大大降低。这强烈属性在该试验中对HIV-1测得的整合活动。此外，含有未感染的细胞的细胞质溶解产物的对照反应（泳道1），单独的PIC（泳道2），目标单独的DNA（泳道3），和单独的缓冲液（泳道4）没有表现出任何整合活性。此外，排除了在第一轮PCR Taq聚合酶（泳道7）的控制并没有扩增一体化产品。值得注意的是，从太平洋岛国单独控制（泳道2）中的数据表明，该嵌套的qPCR策略specifi凯莉测量集成VDNA而不是PIC-相关VDNA。总的来说，这些数据表明PIC相关整合活性的测量。 <img alt="图1" src="/files/ftp_upload/54581/54581fig1.jpg" /> 图1:协议HIV-1图片的制备方法的示意图。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54581/54581fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 含太平洋岛国细胞质提取物是由HIV-1感染的SUP-T1细胞中分离。这些PIC被用作融合活性的体外整合测定的来源。 <img alt="图2" src="/files/ftp_upload/54581/54581fig2.jpg" /> 图2:在体外示意图 </e<html> M&gt; HIV-1图片的整合分析。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54581/54581fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 积分测定法采用的HIV-1 PIC和异源靶phiX174的DNA 在体外进行。集成的HIV-1 DNA用巢式PCR的策略，其中，所述第一轮常规PCR放大来自哪些特定VDNA-序列在第二轮的qPCR优先扩增整合结测量。 <img alt="图3" src="/files/ftp_upload/54581/54581fig3.jpg" /> 图3:HIV-1 PIC-相关VDNA整合活动。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54581/54581fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> "FO:保持-together.within页="1"&gt; HIV-1的PIC-相关整合酶活性测定的集成VDNA拷贝数通过使用的10倍连续稀释绘制从产生的标准曲线得到的值来确定的代表性结果。一种HIV-1分子克隆的公知拷贝数。所有反应一式三份进行，并使用商业软件进行分析的数据。误差棒代表标准偏差。 <table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 零件 </td><td> 股票 </td><td> 最终体积为50微升反应 </td></tr><tr><td> phiX174质粒DNA </td><td> 10毫微克/μL </td><td> 5.0μL </td></tr><tr><td> GoTaq反应缓冲液</td><td> 5X </td><td> 10.0微升</td></tr><tr><td>的dNTP核苷酸混合物</td><td>每10毫</td&gt; <td> 1.0μL </td></tr><tr><td> phiX174-F引物</td><td> 10μM </td><td> 2.5微升</td></tr><tr><td> phiX174-R引物</td><td> 10μM </td><td> 2.5微升</td></tr><tr><td> GoTaq DNA聚合酶</td><td> （5U /μL） </td><td> 0.25微升</td></tr><tr><td>不含核酸酶的蒸馏水</td><td> - </td><td> 28.75微升</td></tr></tbody></table> 表格1。 <table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 95℃= 2分钟</td></tr><tr><td>周期34重复: 95℃= 30秒 53℃= 30秒 72°C = 2分钟</td></tr><tr><td> 72℃= 10分钟</td></tr><tr><td> 4°C =保留</td></tr></tbody></table> 表2中。 <tab乐FO:保together.within页="1"FO:保持与 - next.within页="总是"&gt; <tbody><tr><td> 零件 </td><td> 股票 </td><td> 最终体积为50微升反应 </td></tr><tr><td> IVI反应DNA产物</td><td> - </td><td> 5.0μL </td></tr><tr><td> GoTaq反应缓冲液</td><td> 5X </td><td> 10.0微升</td></tr><tr><td>的dNTP核苷酸混合物</td><td>每10毫</td><td> 1.0μL </td></tr><tr><td> LTR底漆</td><td> 10μM </td><td> 2.5微升</td></tr><tr><td> phiX174底漆</td><td> 10μM </td><td> 2.5微升</td></tr><tr><td> GoTaq DNA聚合酶</td><td> （5U /μL） </td><td> 0.25微升</td></tr><tr><td>不含核酸酶的蒸馏水</td><td> - </td><td> 28.75微升</td></tr></tbody></table> 表3。 <table fo:kEEP-together.within页="1"FO:保持与 - next.within页="总是"&gt; <tbody><tr><td> 95℃= 5分钟</td></tr><tr><td> 23周重复: 94℃= 30秒 55°C = 30秒 72°C = 4分钟</td></tr><tr><td> 72℃= 10分钟</td></tr><tr><td> 4°C =保留</td></tr></tbody></table> 表4。 <table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 零件 </td><td> 股票 </td><td> 最终体积为30微升反应 </td></tr><tr><td> 第一轮PCR产物</td><td> - </td><td> 5.0μL </td></tr><tr><td> LTR-R引物</td><td> 10μM </td><td> 0.9μL </td></tr><tr><td> LTR-U底漆</td><td> 10μM </td><td> 0.9μL </td></tr><tr> &lt;TD&gt; TaqMan探针</td><td> 10μM </td><td> 0.3μL </td></tr><tr><td>智商超混合液</td><td> 2倍</td><td> 15.0微升</td></tr><tr><td>不含核酸酶的蒸馏水</td><td> - </td><td> 7.9μL </td></tr></tbody></table> 表5中。 <table fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 95℃= 3分钟</td></tr><tr><td>周期39重复: 94℃= 15秒 58℃= 30秒 72℃= 30秒 读板</td></tr></tbody></table> 表6。

Watch this Scientific Journal Video about Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes at JoVE.com

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 ...

measurement-vitro-integration-activity-hiv-1-preintegration

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

7.5K 次观看.  Meharry Medical College. 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. 

视频: Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

Bidirectional Retroviral Integration Site PCR Methodology and Quantitative Data Analysis Workflow

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral gene therapy studies, IS assays, in combination with next-generation sequencing, have been used as a cell-tracking tool to characterize clonal stem cell populations sharing the same IS. For the accurate comparison of repopulating stem cell clones within and across different samples, the detection sensitivity, data reproducibility, and high-throughput capacity of the assay are among the most important assay qualities. This work provides a detailed protocol and data analysis workflow for bidirectional IS analysis. The bidirectional assay can simultaneously sequence both upstream and downstream vector-host junctions. Compared to conventional unidirectional IS sequencing approaches, the bidirectional approach significantly improves IS detection rates and the characterization of integration events at both ends of the target DNA. The data analysis pipeline described here accurately identifies and enumerates identical IS sequences through multiple steps of comparison that map IS sequences onto the reference genome and determine sequencing errors. Using an optimized assay procedure, we have recently published the detailed repopulation patterns of thousands of Hematopoietic Stem Cell (HSC) clones following transplant in rhesus macaques, demonstrating for the first time the precise time point of HSC repopulation and the functional heterogeneity of HSCs in the primate system. The following protocol describes the step-by-step experimental procedure and data analysis workflow that accurately identifies and quantifies identical IS sequences.

This manuscript describes the experimental procedure and software analysis for a bidirectional integration site assay that can simultaneously analyze upstream and downstream vector-host junction DNA. Bidirectional PCR products can be used for any downstream sequencing platform. The resulting data are useful for a high-throughput, quantitative comparison of integrated DNA targets.

双向逆转录病毒整合位点PCR方法和定量数据分析工作流程

Integration Site (IS) assays are a critical component of the study of retroviral integration sites and their biological significance. In recent retroviral ...

科研

遗传学

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 ...

免疫与感染

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are upregulated upon this ongoing antigen exposure and contribute to loss of proliferation, reduced cytolytic function, and impaired cytokine production by CD4 and CD8 T cells. In the murine model of LCMV infection, administration of blocking antibodies against these two pathways augmented T cell responses. However, there is currently no in vitro assay to measure the impact of such blockade on cytokine secretion in cells from human samples. Our protocol and experimental approach enable us to accurately and efficiently quantify the restoration of cytokine production by HIV-specific CD4 T cells from HIV infected subjects.
Here, we depict an in vitro experimental design that enables measurements of cytokine secretion by HIV-specific CD4 T cells and their impact on other cell subsets. CD8 T cells were depleted from whole blood and remaining PBMCs were isolated via Ficoll separation method. CD8-depleted PBMCs were then incubated with blocking antibodies against PD-L1 and/or IL-10R&#945; and, after stimulation with an HIV-1 Gag peptide pool, cells were incubated at 37 &#176;C, 5% CO2. After 48 hr, supernatant was collected for cytokine analysis by beads arrays and cell pellets were collected for either phenotypic analysis using flow cytometry or transcriptional analysis using qRT-PCR. For more detailed analysis, different cell populations were obtained by selective subset depletion from PBMCs or by sorting using flow cytometry before being assessed in the same assays. These methods provide a highly sensitive and specific approach to determine the modulation of cytokine production by antigen-specific T-helper cells and to determine functional interactions between different populations of immune cells.

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

We developed an in vitro assay to investigate the role of immunoregulatory pathways in the regulation of cytokine secretion by HIV-specific CD4 T cells.

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

HIV逆转录酶和整合酶抑制剂的快速筛选

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

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

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

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

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

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

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

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

成对增长竞争测定法测定人类免疫缺陷病毒的复制健身

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

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

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

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

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

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

Assembly and Purification of Prototype Foamy Virus Intasomes

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an integrase (IN) enzyme that catalyzes the covalent joining of viral to host DNA, which is known as strand transfer. Integration may be modeled in vitro with recombinant retroviral IN and DNA oligomers mimicking the ends of the viral genome. In order to more closely recapitulate the integration reaction that occurs in vivo, integration complexes are assembled from recombinant IN and synthetic oligomers by dialysis in a reduced salt concentration buffer. The integration complex, called an intasome, may be purified by size exclusion chromatography. In the case of prototype foamy virus (PFV), the intasome is a tetramer of IN and two DNA oligomers and is readily separated from monomeric IN and free oligomer DNA. The integration efficiency of PFV intasomes may be assayed under a variety of experimental conditions to better understand the dynamics and mechanics of retroviral integration.

Recombinant retroviral integrase and DNA oligomers mimicking viral DNA ends can form an enzymatically active complex known as an intasome. Intasomes may be used for biochemical, structural, and kinetic studies. This protocol details how to assemble and purify prototype foamy virus intasomes.

泡沫病毒 Intasomes 原型的组装与纯化

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

高通量的高效液氧活化病毒膜融合检测 HIV-1 病毒膜融合抑制剂的研究

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

<meta charset="utf8"></head><body>研究 T 细胞 HIV-1 感染期间的 ER 应激和 UPR 激活

Measuring Endoplasmic Reticulum Stress and Unfolded Protein Response in HIV-1 Infected T-Cells and Analyzing its Role in HIV-1 Replication

Viral infections can cause Endoplasmic Reticulum (ER) stress due to abnormal protein accumulation, leading to Unfolded Protein Response (UPR). Viruses have developed strategies to manipulate the host UPR, but there is a lack of detailed understanding of UPR modulation and its functional significance during HIV-1 infection in the literature. In this context, the current article describes the protocols used in our laboratory to measure ER stress levels and UPR during HIV-1 infection in T-cells and the effect of UPR on viral replication and infectivity. 
Thioflavin T (ThT) staining is a relatively new method used to detect ER stress in the cells by detecting protein aggregates. Here, we have illustrated the protocol for ThT staining in HIV-1 infected cells to detect and quantify ER stress. Moreover, ER stress was also detected indirectly by measuring the levels of UPR markers such as BiP, phosphorylated IRE1, PERK, and eIF2&#945;, splicing of XBP1, cleavage of ATF6, ATF4, CHOP, and GADD34 in HIV-1 infected cells, using conventional immunoblotting and quantitative reverse transcription polymerase chain reaction (RT-PCR). We have found that the ThT-fluorescence correlates with the indicators of UPR activation. This article also demonstrates the protocols to analyze the impact of ER stress and UPR modulation on HIV-1 replication by knockdown experiments as well as the use of pharmacological molecules. The effect of UPR on HIV-1 gene expression/replication and virus production was analyzed by Luciferase reporter assays and p24 antigen capture ELISA, respectively, whereas the effect on virion infectivity was analyzed by staining of infected reporter cells. Collectively, this set of methods provides a comprehensive understanding of the Unfolded Protein Response pathways during HIV-1 infection, revealing its intricate dynamics.

<meta charset="utf8"></head><body>作者聚焦:探索未折叠蛋白反应在 HIV-1 复制和感染中的作用

Here, we describe some established methods to determine endoplasmic reticulum (ER) stress and unfolded protein response (UPR) activation, with particular emphasis on HIV-1 infection. This article also describes a set of protocols to investigate the effect of ER stress/UPR on HIV-1 replication and virion infectivity.

测量 HIV-1 感染的 T 细胞中的内质网应激和未折叠蛋白反应并分析其在 HIV-1 复制中的作用

Viral infections can cause Endoplasmic Reticulum (ER) stress due to abnormal protein accumulation, leading to Unfolded Protein Response (UPR). Viruses ...

测量

摘要

探索更多视频

双向逆转录病毒整合位点PCR方法和定量数据分析工作流程

在体外对HIV - 1核心脱壳

在体外试验，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

HIV逆转录酶和整合酶抑制剂的快速筛选

限制性内切酶克隆方法，以评估

成对增长竞争测定法测定人类免疫缺陷病毒的复制健身

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

泡沫病毒 Intasomes 原型的组装与纯化

高通量的高效液氧活化病毒膜融合检测 HIV-1 病毒膜融合抑制剂的研究

测量 HIV-1 感染的 T 细胞中的内质网应激和未折叠蛋白反应并分析其在 HIV-1 复制中的作用

测量

摘要

探索更多视频

双向逆转录病毒整合位点PCR方法和定量数据分析工作流程

在体外对HIV - 1核心脱壳

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

HIV逆转录酶和整合酶抑制剂的快速筛选

限制性内切酶克隆方法，以评估

成对增长竞争测定法测定人类免疫缺陷病毒的复制健身

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

泡沫病毒 Intasomes 原型的组装与纯化

高通量的高效液氧活化病毒膜融合检测 HIV-1 病毒膜融合抑制剂的研究

测量 HIV-1 感染的 T 细胞中的内质网应激和未折叠蛋白反应并分析其在 HIV-1 复制中的作用

在体外试验，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响