Name: nucleocapsid annealing-mediated electrophoresis (name) assay allows the rapid identification of hiv-1 nucleocapsid inhibitors
Uploaded: 2015-01-19T15:00:00.000+00:00
Duration: 8 min 33 s
Description: Watch this Scientific Journal Video about Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors at JoVE.com

This work was supported by Ministero degli Affari Esteri (MAE, DRPG, Unit&#224; per la cooperazione scientifica e tecnologica, Grant: PGR Italy-USA) and by MIUR, Progetti di Ricerca di Interesse Nazionale (Grant: 2010W2KM5L_006).

1.准备材料，核酸和蛋白质<ol><li>核酸<ol><li>高压灭菌1.5 ml微量离心管，并将它们存储在无菌容器中。执行使用无菌过滤嘴，以消除污染物质，即核酸从实验室耗材每一步。 </li><li>制备的Tris-HCl的10mM缓冲液，pH 7.5在DEPC-处理的水和过滤用0.22μm孔径过滤器的溶液。 </li><li>注:所谓的"焦油"的寡核苷酸对应于短（29聚体）的RNA序列5&#39;-GGCAGAUCUGAGCCUGGGAGCUCUCUGCC-3&#39;15而CTAR是它的DNA的互补序列5&#39;-GGCAGAGAGCTCCCAGGCTCAGATCTGCC-3&#39;。 <ol><li>在Tris缓冲上述溶解tar和CTAR（1.1.2），使100μM储备液。商店CTAR原液在-20℃（等分可保存几个月在这些条件下）。对于长期储存的RNA，使西藏自治区的20微升等分原液，使用真空浓缩离心机干燥每个等分试样并将其储存在-80℃。新鲜的使用之前，重新悬浮于20μlDEPC处理的水每TAR等分。 注:工作TAR等分可以储存在-20℃下两周。 </li></ol></li></ol></li><li> NC蛋白和（12-55）NC肽<ol><li>制备全长重组的NC蛋白作为报道16储存于-20℃下储备溶液以等分试样。确定使用的消光系数的确切蛋白质浓度使用UV-Vis分光光度计在6410 M -1 -1 280纳米。 </li><li>重悬合成的（12-55）的NC肽的Tris-HCl 10mM的pH为7.5和原液中的等分试样储存在-20℃。确定使用的消光系数上的紫外可见分光光度计的正确的肽浓度的5700 M 280纳米-1 -1。 注:（12-55）NC肽，获得HPLC纯化和lyophilized出含有两当量的氯化锌的溶液。 </li></ol></li><li>化合物1 <ol><li>称重约1mg冻干化合物1的使用分析天平和溶解于100微升的100％DMSO中，巧称重，以得到高浓度（≈10毫摩尔）储液。利用其消光系数确定其每紫外可见分光光度计的确切化合物浓度（在354纳米:11387 M -1厘米-1）。储存在-20℃，使用前将储备溶液在黑暗中。 </li></ol></li></ol> 2.设置凝胶的凝胶设备和铸造了<ol><li>要设置的凝胶，冲洗两个板（一长一短的），用70％的乙醇，让它们干燥，然后放置两个1毫米垫片沿长板的长边;与短板覆盖它，并确保对准两个板块在底部。 </li><li>投凝胶，遵循人联党提供的说明利尔（不同的供应商使用略有不同的设备;三明治夹和堆栈各铸造装置提供）。在所有情况下，确保夹具，堆栈和垫圈是干净，并去除丙烯酰胺的痕迹由以前的用户留下。 </li><li>将在铸造支架组装凝胶三明治并按照供应商的具体说明。 注意:一般清洁硅胶垫片在浇铸槽的底部，确保良好的密封，并有助于避免泄漏浇注的凝胶时。 </li><li>要检查是否有泄漏，使用玻璃板之间的移液管倒入蒸馏水。加水填满三明治，等待几分钟，以确保没有泄漏发生。如果夹心是正确地组装时，除去水，并插入一个滤纸两个玻璃之间以干燥的眼镜。取出纸张，现在夹心准备凝胶浇注。 </li><li>倾凝胶在室温（RT，25℃）。由于聚丙烯酰胺是高度神经，戴手套。我们在非变性（天然）条件E连拍12％聚丙烯酰胺凝胶进行NAME检测。 <ol><li>准备12％凝胶溶液:混合12毫升40％丙烯酰胺/双丙烯酰胺（19/1）解决方案，4毫升TBE 10x缓冲液（10X:的Tris-HCl 890毫米，890硼酸毫米，EDTA 20毫米）和24毫升超纯水。加入400微升APS和50微升立即使用前TEMED。混合溶液中，并迅速地用一个吸管倒出溶液倒在玻璃板之间。引入梳玻璃板之间，避免气泡。添加凝胶溶液以完全填充夹心。 </li></ol></li><li>让凝胶聚合​​45分钟到1小时。马上装样前，慢慢地取出梳子和水井冲洗用蒸馏水彻底。 </li><li>经过聚合和水井冲洗步骤，请按照供应商的具体说明，将凝胶三明治垂直电泳设备。如果冷却系统存在，请务必将凝胶体系的心病冷却芯的矩形的位置。 </li><li>如果系统被设计为处理一个以上的凝胶，但只有一个凝胶具有要运行，附加一个凝胶夹层作为缓冲坝至冷却芯的另一侧，以形成完整的上缓冲液槽。 注意:如果大坝放置不正确，上缓冲区将泄漏可能停止运行凝胶。 </li><li>制备2.5升的TBE的运行缓冲液（1×:Tris-盐酸89毫，硼酸89毫米，EDTA 2毫摩尔）的去离子水。置相距约500毫升的TBE缓冲液用于上缓冲室和倾其余进入下部缓冲室。倒入剩余500毫升TBE缓冲液到上部缓冲室。 </li><li>放置在下部缓冲室的顶部的盖子，以使阳极和阴极是在适当的位置。 </li><li>连接冷却芯，如果存在的话，以一个水龙头用软管。填充芯用自来水，将作为电泳期间散热器，并允许通过选在芯自来水循环rophoresis。 </li></ol> 3.核衣壳退火介电泳（NAME）分析<ol><li>缓冲区的制备<ol><li>制备TNMG缓冲液（1×:的Tris-HCl的10mM，NaCl的20mM，镁（CLO 4）2 1毫米，pH值7.5）在DEPC-处理的水和过滤用0.22μm孔径过滤器的溶液。 注:TNMG缓冲器可以存储在4℃至7天。为了获得最佳的折叠和退火效果，建议使用新鲜的缓冲区。 </li><li>制备的凝胶上样缓冲液含有SDS（GLB SDS:Tris-盐酸100mM的，EDTA 4mM的，50％w / v的甘油，2％w / v的SDS，0.05％w / v的溴酚蓝）于DEPC处理过的水。 注:GLB有助于跟踪多远寡核苷酸的样本正在运行到凝胶系统，并允许下沉样品进入凝胶。除SDS至GLB的是NAME的测定法的最佳成功的一个关键步骤。如果没有遵循此步骤中，它不可能区分核酸频带中戈升系统（ 图2）。 </li></ol></li><li>控制准备<ol><li>连续稀释的寡核苷酸股票方案，并使用新鲜的10微米的解决方案，以准备10微升的TAR的1微米的解决方案。准备以相同的方式，单独地，将10μl一个1μM的CTAR在TNMG 1X缓冲液中。加热每个管至95℃进行5分钟，然后离开冷却至室温，以便TAR和CTAR承担其茎 - 膨出环结构。 </li><li>准备1微米的解决方案混合TAR / CTAR作为控制。拌1微升10μM的TAR用1微升10μM的CTAR在TNMG 1X，在10微升的总体积。由管加热至95℃5分钟变性寡核苷酸和离开它缓慢冷却至室温，以便TAR退火至其互补序列CTAR并形成双链杂交TAR / CTAR。 </li><li>当溶液冷却至室温，进行短旋转离心。加入3微升GLB SDS的，吸管EAC内管3倍H液，并把该管在冰上。请对照样品稳定在冰上，直到加载一步。 </li></ol></li><li>样品制备，分析NC蛋白和（12-55）NC肽活性<ol><li>用2.5微升各样品新鲜制备4μM的寡核苷酸溶液中。总是准备每个解决方案有点过量，防止移液错误。在TNMG 1X缓冲折32.5微升的TAR的4微米的解决方案，并单独CTAR作为报告的编制控制上面提到的（步骤3.2.1）。 </li><li>稀两者的NC蛋白的原液和（12-55）肽的NC至80微米的溶液用milliQ水中。 </li><li>当TAR和CTAR溶液冷却至RT，执行这两个管的短旋转离心，并开始将样品管的制备。 </li><li>准备12个空0.5毫升蒸压管。放置3排4管在机架的实验室样品。每行表示，分别来分析个样品ê全长的NC蛋白，没有蛋白质样品和样品分析（12-55）的NC肽活性。 </li><li>总是随时更换不同的试剂使用技巧。 </li><li>加入每管2.5微升折叠TAR和2.5微升折叠CTAR的。加入4微升DEPC处理过的水的样本（12-55）NC数控和分析。添加5微升DEPC处理水的其他样品。 </li><li>加入1μl的80μMNC或1微升的80微米（12-55）的NC的样品，分别为NC或（12-55）的NC分析。 </li><li>加立即3微升GLB的SDS于时间0分钟的样品（ 图1中报告为0&#39;），移液内每个管3倍溶液中，并且最后把该管在冰上。重复所有样品和对照这一步。保持稳定的样品在冰上，直到加载一步。 </li><li>后15分钟，30分钟和1小时温育在室温下，加入3微升GLB的SDS，吸管内EAC溶液ħ管3倍，并把该管在冰上。重复所有样品和对照，分别此步骤中，并保持在冰上，直到装载步骤。 </li><li>取下凝胶装置的盖。负载13微升的每个样品放入孔中形成的通过铸造凝胶具有良好的成形梳如前所述。加载以下报道于图1的顺序中的样品。 </li><li>取代凝胶装置的盖。附加凝胶装置的引线到电源。检查电极插入到电源中正确的插槽。 </li><li>打开电源并运行凝胶2小时和30分钟，在200伏的恒定电压，直至染料已迁移至1.5厘米凝胶的底部的上方。 </li></ol></li><li>样品制备，分析蒽醌活动的NC和（12-55）NC <ol><li>稀释的化合物1母液。制备10微升的500微米，250微米，50微米，5微米稀释化合物的&lt;STRONG&gt; 1的超纯水。 </li><li>准备对照样品上述报告（第3.2段）。 </li><li>用2.5微升各样品新鲜制备4μM的寡核苷酸溶液中。总是准备每个解决方案有点过量，防止移液错误。在TNMG 1X缓冲倍CTAR 27.5微升的TAR的4微米的解决方案，并分别，如上面提到的（步骤3.2.1）。 </li><li>稀两者的NC蛋白的原液和（12-55）肽的NC至80微米的解决方案中的水。 </li><li>当TAR和CTAR溶液冷却至RT，执行这两个管的短旋转离心，并开始样品的制备。 </li><li>准备20空0.5毫升蒸压管。将2行，每行10管机架的实验室样品。 </li><li>总是随时更换不同的试剂使用技巧。 </li><li>加入第一排的每个管2.5微升折叠TAR的。加在第二行的每一个管，用2.5微升折叠CTAR的。 </li><li&gt;添加2微升合适的化合物 1稀释TAR和CTAR（从5微米到500微米的稀释浓度增加）。样品在更换水化合物的数控的在不存在化合物的分析和（12-55）的NC。孵育样品15分钟，在室温。 </li><li>孵育15分钟后，混合CTAR样品（第二排的管）与相应的TAR样品（第一行的管）。 </li><li>加入1μl的80μMNC或1微升80微米（12-55）NC的样品NC或（12-55）NC分析，分别。 </li><li>孵育15分钟后，加入3微升GLB SDS，和吸取各管3倍，并把该管在冰上。重复这个步骤，分别使用样品为NC和（12-55）的NC分析。保持稳定的样品在冰上，直到加载一步。 </li><li>取下凝胶装置的盖。装载13微升各样品放入孔中。加载样品下列报道中所示的顺序URE 3B。 </li><li>取代凝胶装置的盖。附加凝胶装置的引线到电源。检查电极插入到电源中正确的插槽。 </li><li>打开电源并运行凝胶2小时和30分钟，在200伏的恒定电压，直至染料已迁移至1.5厘米凝胶的底部的上方。 </li></ol></li><li>样品制备:低聚预培养模式VS NC-预培养模式<ol><li>淡化复合 1母液。制备10微升的500微米，250微米，50微米，5微米稀释化合物1的milliQ水中。 </li><li>准备对照样品上述报告（第3.2段）。 </li><li>用2.5微升4μM的寡核苷酸溶液对每个样品。总是准备每个解决方案有点过量，防止移液错误。在TNMG 1X缓冲折27.5微升的TAR的4微米的解决方案，并单独CTAR作为上述（步骤3.20.1。）。 </li><li>稀两者的NC蛋白的原液和（12-55）肽的NC至80微米的解决方案中的水。 </li><li>当TAR和CTAR溶液冷却至RT，执行这两个管的短旋转离心，并开始样品的制备。 </li><li>总是更换每次试剂被改变的提示，或者如果包含在反应管中的任何其他液体或溶液被触摸。 </li><li>从这一步骤确保清楚标示并分离样品的两个不同的预孵育步骤。 <ol><li>低聚预培养模式<ol><li>准备10个空0.5毫升蒸压管。将2排5管在机架的实验室样品。 </li><li>加在第一行2.5微升折叠TAR中的每个管中。加在第二行2.5微升折叠CTAR中的每个管中。 </li><li>加入2微升合适的化合物 1稀释TAR和CTAR（从5微米到500微米的稀释浓度增加）。样本中替换水复合数控在没有化合物的分析。孵育样品15分钟，在室温。 </li><li>孵育15分钟后，取CTAR样品（第二行），并把它们与相应的TAR样品（第一行）。 </li><li>加入1μl的80μM的NC向每个样品孵育样品15分钟，在室温。 </li><li>孵育15分钟后，加入3微升GLB SDS，和吸取各管3倍，并把该管在冰上。保持稳定的样品在冰上，直到加载一步。 </li></ol></li><li> NC-预​​培养模式<ol><li>准备5空0.5毫升高压管，并将其放置在机架的实验室样品。 </li><li>加在各管4微升的适当化合物1的稀释（增加浓度从5微米到500微米的稀释度）。样本中替换水复合数控在没有化合物的分析。 </li><li>加在每个管1微升80μ，M的NC孵育样品15分钟，在室温。 </li><li>混合15微升折叠的TAR用15微升折叠CTAR中的管，并使用在下一步此TAR + CTAR溶液。 </li><li>孵育15分钟后，添加到每个样品取5μlTAR + CTAR混合溶液的孵育样品15分钟，在室温。 </li><li>孵育15分钟后，加入3微升GLB SDS，和吸取各管3倍，并把该管在冰上。保持稳定的样品在冰上，直到加载一步。 注意:从步骤3.5.8上分析可以与所有样本（来自3.5.7恢复）进行。 </li></ol></li></ol></li><li>取下凝胶装置的盖。装载13微升各样品放入孔中。加载以下报告于图4的顺序采样。 </li><li>取代凝胶装置的盖。附加凝胶装置的引线到电源。检查电极插入到电源增刊正确的插槽年。 </li><li>打开电源并运行凝胶2小时和30分钟，在200伏的恒定电压，直至染料已迁移至1.5厘米凝胶的底部的上方。 </li></ol></li></ol> 4.取出凝胶和凝胶染色<ol><li>电泳后，关闭电源，并拔下电器导线。关闭冷却系统的水龙头，并从核心拔下软管。 </li><li>去掉盖子，并且，如果将系统冷却，取出冷却芯和倒出从两个腔室的缓冲液中。 </li><li>轻轻拆开从装置凝胶夹层和从玻璃板上除去所有夹子。以从板除去凝胶，推衬垫出到所述板的一侧的一个和扭转它轻轻拉起和离开上部玻璃板。抓住凝胶的两个角，然后将其关闭轻轻地从玻璃中删除。 </li><li>将凝胶在一个合适的容器中与所需的核酸染色溶液30-45分钟，同时搅拌。 </li><li>染色后，把一个透射成象系统上的凝胶，以检测核酸的荧光信号中的凝胶体系。 </li></ol>

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	<li>Druillennec, 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=A+mimic+of+HIV-1+nucleocapsid+protein+impairs+reverse+transcription+and+displays+antiviral+activity.">A mimic of HIV-1 nucleocapsid protein impairs reverse transcription and displays antiviral activity.</a> Proc Natl Acad Sci USA. 96, 4886-4891 (1999).</li><li>Darlix, J. L., Garrido, J. L., Morellet, N., Mely, Y., de Rocquigny, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties,+functions,+and+drug+targeting+of+the+multifunctional+nucleocapsid+protein+of+the+human+immunodeficiency+virus.">Properties, functions, and drug targeting of the multifunctional nucleocapsid protein of the human immunodeficiency virus.</a> Adv Pharmacol. 55, 299-346 (2007).</li><li>Thomas, J. A., Gorelick, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nucleocapsid+protein+function+in+early+infection+processes.">Nucleocapsid protein function in early infection processes.</a> Virus Res. 134, 39-63 (2008).</li><li>Mirambeau, G., Lyonnais, S., Gorelick, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Features,+processing+states,+and+heterologous+protein+interactions+in+the+modulation+of+the+retroviral+nucleocapsid+protein+function.">Features, processing states, and heterologous protein interactions in the modulation of the retroviral nucleocapsid protein function.</a> RNA Biol. 7, 724-734 (2010).</li><li>Zeng, Y., 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=Probing+nucleation,+reverse+annealing,+and+chaperone+function+along+the+reaction+path+of+HIV-1+single-strand+transfer.">Probing nucleation, reverse annealing, and chaperone function along the reaction path of HIV-1 single-strand transfer.</a> Proc Natl Acad Sci USA. 104, 12651-12656 (2007).</li><li>Basu, V. 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=Strand+transfer+events+during+HIV-1+reverse+transcription.">Strand transfer events during HIV-1 reverse transcription.</a> Virus Res. 134, 19-38 (2008).</li><li>Guo, J., Henderson, L. E., Bess, J., Kane, B., Levin, J. G. <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+nucleocapsid+protein+promotes+efficient+strand+transfer+and+specific+viral+DNA+synthesis+by+inhibiting+TAR-dependent+self-priming+from+minus-strand+strong-stop+DNA.">Human immunodeficiency virus type 1 nucleocapsid protein promotes efficient strand transfer and specific viral DNA synthesis by inhibiting TAR-dependent self-priming from minus-strand strong-stop DNA.</a> J Virol. 71, 5178-5188 (1997).</li><li>Godet, J., Mely, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biophysical+studies+of+the+nucleic+acid+chaperone+properties+of+the+HIV-1+nucleocapsid+protein.">Biophysical studies of the nucleic acid chaperone properties of the HIV-1 nucleocapsid protein.</a> RNA Biol. 7, 687-699 (2010).</li><li>Darlix, J. 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=Flexible+nature+and+specific+functions+of+the+HIV-1+nucleocapsid+protein.">Flexible nature and specific functions of the HIV-1 nucleocapsid protein.</a> J Mol Biol. 410, 565-581 (2011).</li><li>Lapadat-Tapolsky, M., Pernelle, C., Borie, C., Darlix, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+nucleic+acid+annealing+activities+of+nucleocapsid+protein+from+HIV-1.">Analysis of the nucleic acid annealing activities of nucleocapsid protein from HIV-1.</a> Nucleic Acids Res. 23, 2434-2441 (1995).</li><li>Vo, M. -. N., Barany, G., Rouzina, I., Musier-Forsyth, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanistic+studies+of+mini-TAR+RNA/DNA+annealing+in+the+absence+and+presence+of+HIV-1+nucleocapsid+protein.">Mechanistic studies of mini-TAR RNA/DNA annealing in the absence and presence of HIV-1 nucleocapsid protein.</a> J Mol Biol. 363, 244-261 (2006).</li><li>Beltz, H., 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=Structural+determinants+of+HIV-1+nucleocapsid+protein+for+cTAR+DNA+binding+and+destabilization,+and+correlation+with+inhibition+of+self-primed+DNA+synthesis.">Structural determinants of HIV-1 nucleocapsid protein for cTAR DNA binding and destabilization, and correlation with inhibition of self-primed DNA synthesis.</a> J Mol Biol. 348, 1113-1126 (2005).</li><li>Mori, 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=Nucleocapsid+Protein:+a+Desirable+Target+for+Future+Therapies+against+HIV-1.">Nucleocapsid Protein: a Desirable Target for Future Therapies against HIV-1.</a> Curr Top Microbiol Immunol. , .</li><li>Sosic, 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=Design,+synthesis+and+biological+evaluation+of+TAR+and+cTAR+binders+as+HIV-1+nucleocapsid+inhibitors.">Design, synthesis and biological evaluation of TAR and cTAR binders as HIV-1 nucleocapsid inhibitors.</a> MedChemComm. 4, 1388-1393 (2013).</li><li>Tabarrini, O., 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=Studies+of+Anti-HIV+Transcription+Inhibitor+Quinolones:+Identification+of+Potent+N1-Vinyl+Derivatives.">Studies of Anti-HIV Transcription Inhibitor Quinolones: Identification of Potent N1-Vinyl Derivatives.</a> Chemmedchem. 5, 1880-1892 (2010).</li><li>Hagan, N., Fabris, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+mass+spectrometric+determination+of+the+stoichiometry+and+binding+affinity+of+the+complexes+between+nucleocapsid+protein+and+RNA+stem-loop+hairpins+of+the+HIV-1+Psi-recognition+element.">Direct mass spectrometric determination of the stoichiometry and binding affinity of the complexes between nucleocapsid protein and RNA stem-loop hairpins of the HIV-1 Psi-recognition element.</a> Biochemistry. 42, 10736-10745 (2003).</li><li>Ivanyi-Nagy, R., Davidovic, L., Khandjian, E. W., Darlix, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disordered+RNA+chaperone+proteins:+from+functions+to+disease.">Disordered RNA chaperone proteins: from functions to disease.</a> Cell Mol Life Sciences: CMLS. 62, 1409-1417 (2005).</li><li>Zagotto, G., 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=Tuning+G-quadruplex+vs+double-stranded+DNA+recognition+in+regioisomeric+lysyl-peptidyl-anthraquinone+conjugates.">Tuning G-quadruplex vs double-stranded DNA recognition in regioisomeric lysyl-peptidyl-anthraquinone conjugates.</a> Bioconjug Chem. 22, 2126-2135 (2011).</li><li>Carlson, C. B., Vuyisich, M., Gooch, B. D., Beal, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferred+RNA+binding+sites+for+a+threading+intercalator+revealed+by+in+vitro+evolution.">Preferred RNA binding sites for a threading intercalator revealed by in vitro evolution.</a> Chem Biol. 10, 663-672 (2003).</li><li>Gooch, B. D., Beal, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recognition+of+duplex+RNA+by+helix-threading+peptides.">Recognition of duplex RNA by helix-threading peptides.</a> J Am Chem Soc. 126, 10603-10610 (2004).</li><li>Kanevsky, I., 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=Structural+determinants+of+TAR+RNA-DNA+annealing+in+the+absence+and+presence+of+HIV-1+nucleocapsid+protein.">Structural determinants of TAR RNA-DNA annealing in the absence and presence of HIV-1 nucleocapsid protein.</a> Nucleic Acids Res. 39, 8148-8162 (2011).</li></ol>

The authors declare that they have no competing financial interests.

NAME是一个测定，它允许快速评估的HIV-1，在搜索的新的抗HIV药物的有趣靶的核衣壳蛋白的分子伴侣活性的抑制。数控退火快，并在室温的核酸，其稳定的折叠，否则将需要的热变性，在高温度，随后小心退火。所述测定法可以与任一全长NC或与截断（12-55）的NC来执行:在两种情况下，两个核酸（RNA和其互补DNA复制）正在迅速退火。这与文献观察与截短的NC肽一致:退火由CTAR的杆，随后通过其互补心尖循环，这是一个薄弱的NC结合位点相互作用的有效熔化开始，随着TAR心尖环的互补序列，通过几个不稳定的中间体，以扩展混合退火结束了。21 NC是一个非常稳定的PROTEIN和一些问题，而可能发生在执行NAME。这是非常重要的但添加新鲜制备的SDS凝胶加样缓冲液用于使反应停止:如果这是不实现的，该凝胶不会显示该核酸频带在正确的位置上。事实上，NC是核酸结合蛋白，从而正确地可视化的TAR / CTAR异源通过凝胶电泳SDS必须存在于凝胶上样缓冲液使蛋白质变性并从它的紧配合物与核酸释放出来。这也是该实验的可能的故障， 即转移带的凝胶运行期间的形成。这种效果是特别明显，当全长的NC被采用，如在图2中 ，并且被链接到的高碱性的N-末端尾部的存在下，具有上的核酸的强聚集效应。 的终端基本尾部的存在使得在退火反应更难以被抑制，因为显示中的n 图3使用化合物1，一个代表性的螺纹嵌入， 即嵌入剂，是稳定的CTAR TAR和茎-环结构特别有效。事实上，这种类型的相互作用与核酸时凸起和循环中断的双螺旋的连续性，从而允许更简单的螺纹的大体积取代基成碱基对是有利的。 TAR和CTAR由这些核酸结合剂稳定化之前由所增加的两个寡核苷酸的熔融温度的测定分析。14此外，增加的熔化温度与由HIV-1的NC催化退火的抑制相关，导致异源TAR / CTAR的减小形成和指示线程嵌入剂是一类有趣的粘合剂用于RNA的动态结构，如TAR元件14 作用的机制调用这些COMPOunds可以通过执行在不同替代格式的名称测定法来进一步证实， 如图4:在嵌入剂的情况下，退火异源的抑制增强当药物孵育与两折叠的寡核苷酸。具有不同的作用机制，如在NC蛋白的直接结合剂作用的化合物，就不太受不同预培养模式。在这两种方法，因此可用于筛选化合物文库数控抑制剂的识别名试验进行的，并且还同时搜索针对NC抗HIV药物评估阳性命中的动作的推定机理。显然，声称对确定NC抑制剂的作用的具体机制的时候使用单独这些技术是不够的，需要有其他合适的生化检测，以维持工作的前提。14 最后，它必须在名称使用高度强调纯化的酶，无论是重组的或合成的，得到由所述测试化合物的" 体外 "抑制数控宝贵信息。这是"力量和弱点"的测定:NAME能很好地配合虚拟筛选和分子模拟方法在药物研究计划的预备步骤，从而能够快速构建和分析构效关系，并最终重定向的合成和药物设计。然而，如用在HIV药物开发项目其它生化测定法，名称将不给于推定的抑制剂在病毒感染的细胞的影响的信息。

人类免疫缺陷病毒1型（HIV-1）的核衣壳蛋白NCp7（NC）]是紧密基因组RNA的成熟的感染性病毒相关联的小，碱性蛋白，演奏作为反转录过程中的辅因子在病毒复制中的关键作用，基因组的识别，和包装。1-3数控充当核酸分子伴侣催化稳定的核酸结构的不稳定和互补序列的退火。其核酸聚集活性主要存在于该11个氨基酸的N-末端结构域，而双工不稳定活动已经映射到其锌指结构（12-55肽）4的带正电荷的蛋白降低退火反应的静电屏障和增加的这两个独立的互补序列走到一起的速度。 折叠稳定的构象核酸要求NC的伴侣活动facilitatË其退火5，这是在逆转录，其中NC是在链转移事件是至关重要的特别重要的:在所述负链转移， 负链 DNA的停止 ，只是retrotranscribed，必须转 ​​移并退火到互补序列中的R区在RNA基因组的3&#39;末端的模板。6虽然热力学上是有利地，该反应并不广泛在没有数控的发生是由于反式激活响应区 （TAR）RNA的稳定结构在R区域的存在，必须被关联到它的DNA复制（CTAR DNA）。7，带有特征茎环构象CTAR和TAR是，事实上，高度结构化的区域。数控蛋白质变性这些发夹，并促进负链通过增加分子间退火的互补的核酸链之间的速率传输。 TAR和CTAR数控退火机制已经彻底调查，并去刻划在几个研究论文TAR退火法和所提出的方案中描述了极高的评价。8-11汇总，数控动摇的稳定的RNA如TAR-RNA的二级结构，动摇其互补序列，CTAR-DNA的二级结构和促进的RNA / DNA的通向TAR / CTAR异源双链体的形成10,11的退火反应。其结果是，HIV复制过程中的链转移步骤是有利的。12 数控是为新的抗病毒剂，因为诱导朝数控小分子的潜在的干扰将导致减少的病毒基因组的逆转录作为妥协的NC活动的结果的发展的一个有吸引力的目标。2,13这种方法能最终导致成功的抗HIV药物的开发。在筛选数控的过程抑制剂14，我们开发依靠公知的特性的测定10,11核衣壳有效地破坏和退火互补的寡核苷酸。我们把它叫做ñucleocapsid一个 nnealing- M ediatedËlectrophoresis（NAME）检测。 NAME测定使用数控介导的互补核酸稳定地结构化的副本之间的退火，在我们的情况下，对应于与它的互补DNA序列（CTAR），得到混合的TAR / CTAR异源一TAR-RNA序列的顶端部分的寡核苷酸的。在数控存在焦油/ CTAR退火反应的分析可以通过天然聚丙烯酰胺凝胶电泳（PAGE）进行调查。 此协议说明如何执行名称测定快速退火在室温下寡核苷酸折叠在稳定的发夹结构，如何分析实验的反应和可能的故障诊断的结果。核酸的放射性标记不请求，并且检测系统，效对寡核苷酸的频带可以通过常规染色的方法进行。的测定法中，其采用任一重组全长蛋白或NC的截短的合成版本，允许螺纹嵌入抑制NC，证实与强结合到RNA和DNA的活性的鉴定。14

<table><tbody><tr><td>cTAR, 29-mer DNA oligo. Desalted. HPLC purified.</td><td>Metabion International AG</td><td></td><td>Store the stock&amp;nbsp; solution at -20 &amp;deg;C&amp;nbsp;</td></tr><tr><td>TAR, 29-mer RNA oligo. Desalted. HPLC purified.&amp;nbsp;&amp;nbsp;</td><td>Metabion International AG</td><td></td><td>Store the stock aliquot at -80 &amp;deg;C&amp;nbsp;</td></tr><tr><td>(12-55)NC peptide</td><td>EspiKem srl</td><td></td><td>Store the stock solution at -20 &amp;deg;C</td></tr><tr><td>1.5 ml tubes</td><td>Eppendorf</td><td>0030 120 086</td><td>Autoclave before use</td></tr><tr><td>0.5 ml tubes</td><td>Eppendorf</td><td>0030 121 023</td><td>Autoclave before use</td></tr><tr><td>Biosphere Filter Tips 0.1-10 &amp;micro;l</td><td>Sarstedt</td><td>701,130,210</td><td></td></tr><tr><td>Biosphere Filter Tips 2-20 &amp;micro;l</td><td>Sarstedt</td><td>70,760,213</td><td></td></tr><tr><td>Biosphere Filter Tips 200&amp;nbsp;&amp;micro;l</td><td>Sarstedt</td><td>70,760,213</td><td></td></tr><tr><td>Syringe Filter 0.22&amp;nbsp;&amp;micro;m</td><td>Biosigma srl</td><td>51,798</td><td></td></tr><tr><td>Ethanol</td><td>Carlo Erba</td><td>414631</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate</td><td>Sigma-Aldrich</td><td>71725-50G</td><td></td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma-Aldrich</td><td>D8418-1L</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma-Aldrich</td><td>71376-1KG</td><td></td></tr><tr><td>Boric Acid</td><td>Sigma-Aldrich</td><td>B0252-2.5KG</td><td></td></tr><tr><td>Ethylenediaminetetraacetic Acid</td><td>Sigma-Aldrich</td><td>E5134-500G</td><td></td></tr><tr><td>Magnesium Perchlorate</td><td>Sigma-Aldrich</td><td>222283-100G</td><td>Store the 1&amp;nbsp;M solution at -20&amp;nbsp;&amp;deg;C</td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>49770-1L</td><td></td></tr><tr><td>Bromophenol Blue</td><td>GE Healthcare Life Sciences</td><td>17-1329-01</td><td></td></tr><tr><td>&lt;em&gt;N,N,N&amp;prime;,N&amp;prime;&lt;/em&gt;-Tetramethylethylenediamine</td><td>Sigma-Aldrich</td><td>T9281-100ML</td><td></td></tr><tr><td>Ammonium Persulfate</td><td>Sigma-Aldrich</td><td>A7460-500G</td><td>Prepare the 10% solution freshly before use</td></tr><tr><td>Acrylamide-bis ready-to-use solution 40% (19:1)</td><td>Merck Millipore</td><td>1006401000</td><td>Polyacrylamide is highly neurotoxic: wear gloves during the gel casting</td></tr><tr><td>Tris ultrapure</td><td>Applichem</td><td>A1086,1000</td><td></td></tr><tr><td>DEPC-treated water</td><td>Ambion</td><td>AM9922</td><td></td></tr><tr><td>Centrifuge 5415R</td><td>Eppendorf</td><td>22,621,408</td><td></td></tr><tr><td>NanoDrop 1000 spectrometer</td><td>Thermo Scientific</td><td></td><td></td></tr><tr><td>UV-VIS spectrometer Lambda 20</td><td>Perkin Elmer</td><td></td><td></td></tr><tr><td>Protean II xi Cell</td><td>Bio-Rad</td><td>165-1803</td><td></td></tr><tr><td>PowerPac Basic Power Supply</td><td>Bio-Rad</td><td>164-5050</td><td></td></tr><tr><td>SybrGreen II RNA Gel Staining</td><td>Lonza</td><td>50523</td><td>Make the 1/10,000 dilution in TBE 1x. Store it in the dark at 4&amp;nbsp;&amp;deg;C for two weeks.</td></tr><tr><td>Geliance 600 Imaging System</td><td>Perkin Elmer</td><td></td><td></td></tr></tbody></table>

nucleocapsid annealing-mediated electrophoresis (name) assay allows the rapid identification of hiv-1 nucleocapsid inhibitors

RNA或DNA折叠稳定立体折叠是在抗肿瘤或抗病毒的药物的发展感兴趣的目标。在HIV-1的情况下，所涉及的病毒活性的调节病毒蛋白识别几种核酸。核衣壳蛋白NCp7（NC）是一种关键蛋白调节病毒复制过程中的几个过程。数控实际上是分子伴侣的不稳定的RNA和DNA及促进其退火的二级结构。 NC的失活是一种新的方法和抗HIV治疗的一个有趣的目标。 N 个 ucleocapsid 一个 nnealing- M ediatedËlectrophoresis（NAME）法的开发是为了找出分子能抑制RNA和DNA折叠热力学稳定的三维构象，如TAR的发夹结构和艾滋病CTAR元素的熔化和退火，由HIV-1的核衣壳蛋白。新的检测采用要么重新combinant或合成的蛋白质，以及寡核苷酸没有他们以前的标签的需要。结果的分析是通过标准的聚丙烯酰胺凝胶电泳（PAGE），随后通过常规的核酸染色来实现。报道在这项工作中的协议描述了如何执行与全长蛋白质或其截短形式缺少基本的N-末端结构域的名称测定中，两个主管核酸分子伴侣，以及如何通过评估的NC伴侣活性的抑制线程嵌入。此外，名称可以在两种不同的模式来执行的，有用的，以获得指示所识别的NC抑制剂的作用的推定机理。

图1显示了核蛋白介导退火电泳（NAME）测定的代表性的结果，与全长重组蛋白进行ⅰ），ⅱ）无蛋白质， 即混合CTAR + TAR和孵育达1小时，在室温， 以及 iii ）与（12-55）的NC，合成肽缺乏N-末端结构域的11个氨基酸进行同样的试验。所述预折叠TAR和CTAR混合并退火异源TAR的形成/ CTAR在（ 图1的左车道）的全长重组数控存在下监测，在不存在的蛋白质（ 图1中的中央车道），并且在截断（12-55）的NC肽（右车道在图1中存在）。控件:折叠CTAR，折叠TAR和混合退火（TAR / CTAR），通过TAR的稳定折叠结构CTAR的热变性之后的缓慢annealin获得克14 数控变性两个稳定的核酸序列插入延长异源双链螺旋的能力是显而易见的:全长的NC蛋白立即导致焦油/ CTAR杂交体的形成:0&#39;表示的最小时间除数控之间传递样品和另外的凝胶加样缓冲液，其中停止所述反应;完全形成15&#39;孵育时间后已达到。在退火异源的强度的增加平行于所述折叠CTAR和TAR寡核苷酸的强度的降低。的异源双链体的形成也实现了截短形式的存在， 即在（12-55）的NC，证实了合成肽的生物活性。在不存在的蛋白质或异源双链体的形成没有观察到肽的，和TAR和CTAR寡核苷酸不会退火在室温下进异源（ 图1）。在所有的实验中，快速形成不稳定的中间体（在图1"的中间复合物"），该随时间降低的观察，始终与提出通过CTAR的发夹和TAR直到正确的核酸折叠链交换的机制。17 实验的可能排除在凝胶缓冲液缺乏SDS的。在没有SDS在GLB的反应的结果示于图2和比较用GLB + SDS的结果。所述预折叠TAR和CTAR混合并温育3小时，在室温的3小时，在37℃下用重组全长的NC蛋白。温育后，将样品分成两半:二分之一的凝胶加样缓冲液不含SDS（GLB）加入，同时于另一半GLB是用SDS（GLB SDS）。样品装载在凝胶上，通过运行该凝胶后的核酸带的位置是可视化染料染色。当数控是存在的，但将样品装入GLB，所有核酸条带向上移动:这是预期并指示蛋白质和核酸之间的强的结合。与GLB SDS的结果示于凝胶的最右边:加入的SDS变性的蛋白质和释放从稳定络合物与蛋白质的核酸，从而允许与对照的比较。 该名称测定法用于评估线程嵌入稳定动态核酸的结构和抑制数控伴侣活性的能力。14线程嵌入是由位于环系统的相对位点笨重侧链，如蒽醌取代的平面芳烃分子在图3A中所示。18，这些嵌入剂能够通过的核酸双螺旋螺纹，最后定位其侧链在双重的每个槽螺旋。19,20 图3B示出了在化合物1中，公知的螺纹嵌入，18的在截短的肽的全长的NC的存在或存在下NAME测定的结果。在这两种情况下，焦油/ CTAR混合由数控的形成是通过增加嵌入的浓度，同时，在同一时间，不含焦油和CTAR增加的量减少。缺乏N末端尾部（12-55NC）该蛋白质的截短形式是其活性的抑制更敏感:该差异与所有迄今测试的化合物进行了验证。 该名称测定可以执行两种方式，或者通过预孵育试验化合物用NC，随后加入折叠寡核苷酸（NC-预培养模式），或通过与折叠核酸底物预先培养15分钟，试验化合物中，随后加入的NC和进一步温育15分钟（低聚PReincubation模式）。虽然整体的温育时间是在两种不同的模式是相同的，并加入NC的前折叠寡核苷酸的预培养可以有更多的时间用于嵌入到折叠核酸的穿线。这导致它们的动态结构的更强的稳定和成更容易抑制数控伴侣的活动。因此NAME测定在两种模式的分析增强了数控抑制剂的作用机制的差异， 如图4:当化合物1是由NAME在寡聚预培养方式分析（ 图4，泳道左侧）它显示了数控介导退火的有效抑制，而低得多的抑制是在NC-预培养模式观察（ 图4，泳道在右侧）。请注意，为抑制浓度而筛选组相关的化合物与该测定并在这些条件下，每个experi的确定精神疾病，必须至少使用一式三份紧密间隔的浓度（特别是围绕所述IC 50值）在14来执行。 <img alt="图1" src="/files/ftp_upload/52474/52474fig1highres.jpg" /> 图1:用全长NC和与截断（12-55）的NC折叠TAR和CTAR，各1μM的名称测定中，孵育用重组NC或与（12-55）NC（寡聚/ NC = 1 / 8）为0分钟，15分钟，30分钟和1小时。 TAR和CTAR孵育在不存在蛋白（0分钟，15分钟，30分钟和1个小时）被用作阴性对照。折叠TAR，折叠CTAR和混合TAR / CTAR热变性，随后通过体外退火用作对照后获得的。该反应然后用GLB SDS停止。样品解决上在TBE 1×12％聚丙烯酰胺凝胶;凝胶上的电泳核酸后进行染色，并在检测到的透射。 <html><img alt="图2" src="/files/ftp_upload/52474/52474fig2highres.jpg" /> 图2:SDS的影响凝胶缓冲液（GLB）在分析TAR / CTAR退火由NC TAR和CTAR，预先折叠的TNMG 1倍，分别加入全长重组NC（寡/ NC = 1/8）3小时，在室温下或在37℃。 GLB或GLB SDS然后加入到培养与NC的样品。控制:TAR，CTAR和退火混合TAR / CTAR（各1μM）。电泳是使用在TBE 1×12％聚丙烯酰胺凝胶进行的;凝胶上的电泳核酸后进行染色，并在检测到的透射。这个数字已被修改，从图S4的:蓝宝石上硅集成电路，A. 等人的设计，合成和TAR和CTAR粘合剂为HIV-1核衣壳抑制剂的生物学评价MedChemComm 4，1388年至1393年，DOI:10.1039 / c3md00212h（2013年） 。 "&gt; <img alt="图3" src="/files/ftp_upload/52474/52474fig3highres.jpg" /> 图3:螺纹嵌入1（B）的（A）的化学结构，作用线程嵌入1由NAME检测评估。折叠TAR和CTAR，每1微米，孵育在化合物1的指定浓度与全长NC或与（12-55）的NC的存在下，每8微米。折叠TAR，折叠CTAR和扩展异源TAR / CTAR用作对照。该反应然后用GLB SDS停止。样品解决上在TBE 1×12％聚丙烯酰胺凝胶;凝胶上的电泳核酸后进行染色，并在检测到的透射。 <img alt="图4" src="/files/ftp_upload/52474/52474fig4highres.jpg" /> 图4:低聚预培养与NC-预培养模式:在NAME实验效果 低聚预孵育国防部 。E:折叠TAR和折叠CTAR，每1微米，是在全长的NC（8微米） 的NC-预孵育的存在下预温育与螺纹嵌入1所示浓度的15分钟，然后附加15分钟模式 :在指定浓度的螺纹嵌入1是在折叠TAR的存在下预温育与全长的NC（8微米）15分钟，然后额外15分钟，并折叠CTAR，每1微米。折叠TAR，折叠CTAR和扩展异源TAR / CTAR用作对照。所有的反应使用GLB SDS终于停了下来。样品解决上在TBE 1×12％聚丙烯酰胺凝胶;凝胶上的电泳核酸后进行染色，并在检测到的透射。

Watch this Scientific Journal Video about Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors at JoVE.com

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

核衣壳退火介导电泳（NAME）分析允许HIV-1核衣壳抑制剂的快速鉴定

RNA or DNA folded in stable tridimensional folding are interesting targets in the development of antitumor or antiviral drugs. In the case of HIV-1, viral ...

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Research

JoVE Journal

Immunology and Infection

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

8.9K 次观看.  University of Padova. This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

视频: Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

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 ...

科研

免疫与感染

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

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

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

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

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

遗传学

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 ...

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 ...

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 ...

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

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

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

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

测量<em&gt;体外</em&gt; HIV-1 Preintegration配合物的整合活动

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine detection of the virus. The current detection and quantification techniques, which rely primarily on nucleic acid amplification, do not discriminate infectious from non-infectious viral particles. The purpose of this article is to present specific details on recent advances in techniques used together in order to acquire further information on the infectivity status of viral particles. One technique involves assessing binding of a norovirus ssDNA aptamer to capsids. Aptamers have the advantage of being easily synthesized and modified, and are inexpensive and stable. Another technique, dynamic light scattering (DLS), has the advantage of observing capsid behavior in solution. Electron microscopy allows for visualization of the structural integrity of the viral capsids. Although promising, there are some drawbacks to each technique, such as non-specific aptamer binding to positively-charged molecules from sample matrices, requirement of purified capsid for DLS, and poor sensitivity for electron microscopy. Nonetheless, when these techniques are used in combination, the body of data produced provides more comprehensive information on norovirus capsid integrity that can be used to infer infectivity, information which is essential for accurate evaluation of inactivation methods or interpretation of virus detection. This article provides protocols for using these methods to discriminate infectious human norovirus particles.

Alternative In Vitro Methods for the Determination of Viral Capsid Structural Integrity

Routine detection methods utilizing viral genome amplification are limited by their inability to discriminate infectious from non-infectious particles. The purpose of this article is to provide detailed protocols for alternative methods to aid in discrimination of infectious norovirus particles using aptamer binding, dynamic light scattering, and transmission electron microscopy.

替代体外测定病毒衣壳结构完整性的方法

Human norovirus exacts considerable public health and economic losses worldwide. Emerging in vitro cultivation advances are not yet applicable for routine ...

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 ...

Identification of Nucleolar Factors During HIV-1 Replication Through Rev Immunoprecipitation and Mass Spectrometry

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious cycle further requires the formation of viral/host protein complexes with HIV-1 RNA to regulate the splicing and enable nucleocytoplasmic transport. The HIV-1 Rev protein accomplishes the nuclear export of HIV-1 mRNAs through multimerization with intronic cis-acting targets - the Rev response element (RRE). A nucleolar localization signal (NoLS) exists within the COOH-terminus of the Rev arginine-rich motif (ARM), allowing the accumulation of Rev/RRE complexes in the nucleolus. Nucleolar factors are speculated to support the HIV-1 infectious cycle through various other functions in addition to mediating mRNA-independent nuclear export and splicing. We describe an immunoprecipitation method of wild-type (WT) Rev in comparison to Rev nucleolar mutations (deletion and single-point Rev-NoLS mutations) in the presence of HIV-1 replication for mass spectrometry. Nucleolar factors implicated in the nucleocytoplasmic transport (nucleophosmin B23 and nucleolin C23), as well as cellular splicing factors, lose interaction with Rev in the presence of Rev-NoLS mutations. Various other nucleolar factors, such as snoRNA C/D box 58, are identified to lose interaction with Rev mutations, yet their function in the HIV-1 replication cycle remain unknown. The results presented here demonstrate the use of this approach for the identification of viral/host nucleolar factors that maintain the HIV-1 infectious cycle. The concepts used in this approach are applicable to other viral and disease models requiring the characterization of understudied pathways.

Here we describe Rev immunoprecipitation in the presence of HIV-1 replication for mass spectrometry. The methods described can be used for the identification of nucleolar factors involved in the HIV-1 infectious cycle and are applicable to other disease models for the characterization of understudied pathways.

通过转版免疫沉淀和质谱测定,在HIV-1复制过程中核子因子的识别

The HIV-1 infectious cycle requires viral protein interactions with host factors to facilitate viral replication, packaging, and release. The infectious ...

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle (VLP)-Based Vaccines Using a Capture Assay

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate antigen-displaying VLPs. Surface antigen-specific antibodies are coupled to, and thus immobilized on a solid and insoluble matrix such as beads. Due to their high affinity to the target antigen, these antibodies can capture VLPs decorated with the cognate antigen anchored in the membrane envelope of the VLPs. This protocol describes the binding of antigen-specific antibodies to protein A- or G-conjugated magnetic beads. In our study, human immunodeficiency virus (HIV)-derived VLPs formed by the group-specific antigen (Gag) viral core precursor protein p55 Gag and displaying the envelope glycoproteins (Env) of HIV are examined. The VLPs are captured utilizing broadly neutralizing antibodies (bNAbs) directed against neutralization-sensitive epitopes in Env. The VLP capture assay outlined here represents a sensitive and easy-to-perform method to demonstrate that (i) the VLPs are decorated with the respective target antigen, (ii) the surface antigen retained its structural integrity as demonstrated by the epitope-specific binding of bNAbs used in the assay and (iii) the structural integrity of the VLPs revealed by the detection of Gag proteins in a subsequent Western blot-analysis. Consequently, the utilization of bNAbs for immunoprecipitation facilitates a prediction of whether VLP vaccines will be able to elicit a neutralizing B cell response in vaccinated humans. We anticipate that this protocol will furnish other researchers with a valuable and straightforward experimental approach to examine potential VLP-based vaccines.

Detection of Neutralization-sensitive Epitopes in Antigens Displayed on Virus-Like Particle VLP-Based Vaccines Using a Capture Assay

Here, we present a protocol to detect neutralization epitopes on antigen-displaying virus-like particles (VLPs). Immunoprecipitation of the human immunodeficiency virus (HIV)-derived VLPs is performed using envelope glycoproteins-specific monoclonal antibodies coupled to protein G-conjugated magnetic beads. Captured VLPs are subsequently subjected to SDS-PAGE and Western blot-analysis employing viral core protein Gag-specific antibodies.

使用捕获测定法检测基于病毒样颗粒（VLP）的疫苗上显示的抗原中和敏感表位

The virus-like particle (VLP) capture assay is an immunoprecipitation method, commonly known as a &#39;pull-down assay&#39; used to purify and isolate ...