我们要感谢穆罕默德·萨利姆，精武和克里希南Raghunathan有益的讨论。在拍摄中我们也感谢普里亚Begani寻求帮助。这项工作是由美国国立卫生研究院授予R01 AI071727（以AO）和R01 GM110052（SLV到）的支持。

第1天:使用基于裂解物，小麦胚芽体外转录翻译系统的Gag蛋白表达 1，HIV-1加格的制备<ol><li>从冰柜中取出商业麦胚CECF试剂盒所有试剂（小麦胚芽裂解物储存在-80ºC;在-20ºC其他试剂）。解冻他们在冰上并如表1所示混合组分。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 混合-1（进料混合） </td><td></td></tr><tr><td>进料液</td><td> 900微升</td></tr><tr><td>氨基酸</td><td> 80微升</td></tr><tr><td>蛋氨酸</td><td> 20微升</td></tr><tr><td> 总 </td><td> 1000微升 </td></tr><tr><td></td><td></td></tr><tr><td> 混合-2（裂解液混合） </str翁&gt; </td><td></td></tr><tr><td>小麦胚芽裂解液</td><td> 15微升</td></tr><tr><td>核糖核酸酶自由水</td><td> 7微升</td></tr><tr><td>氨基酸</td><td> 4微升</td></tr><tr><td>蛋氨酸</td><td> 1微升</td></tr><tr><td>质粒DNA的TE缓冲液（1微克/微升）** </td><td> 4微升</td></tr><tr><td>反应缓冲液</td><td> 15微升</td></tr><tr><td> RNA酶抑制剂* </td><td> 4微升</td></tr><tr><td> 总 </td><td> 50微升 </td></tr></tbody></table> 表1:小麦胚芽反应所需要的混合物组成。 注意:如果该实验的目的是检验的RNA去除被RNA酶上的Gag膜结合的效果，替换无RNase水核糖核酸酶抑制剂。质粒DNA应根据制造商的指令免于杂质。然而，对lasmids使用常规制备，但不无内毒素，质粒分离试剂盒已被成功使用。制造商的指令还建议使用溶解于无核酸酶的水的DNA。如果使用悬浮于TE [10mM的Tris-盐酸含有1mM EDTA（pH7.4）中的DNA，应避免使用低浓度的DNA，因为EDTA于TE可能影响产量。溶于TE缓冲液以0.8-1微克/微升的浓度范围的质粒已经成功地被使用。 <ol start="2"><li>首先添加料混合物（混合-1）插入反应盒（透明室）。然后将溶胞产物混合物（混合-2）添加到另一个室（红色）。不引入任何气泡，同时加入混合物到各自室中，因为这可能会导致溶液组分低效交换，从而降低反应效率。 </li><li>覆盖随试剂盒提供的粘合膜的腔室。 </li><li>插入反应盒插入盒座和incubaTE的Eppendorf恒温R.装配24小时24ºC以900rpm的恒定速度晃动</li></ol>第2天:准备GUVs由Electroformation和收获小麦胚芽裂解液 2. GUVs的制备<ol><li>等分试样的脂质溶解于无机溶剂，例如在与有聚四氟乙烯衬里衬和用Parafilm包裹螺旋盖玻璃小瓶氯仿或氯仿的混合物和甲醇。处理脂类汉密尔顿式玻璃注射器，如果可用，耐氯仿的塑料提示。取出含有脂质用于从-20 C冷柜GUV制备玻璃小瓶中。 <ol><li>一旦小瓶平衡至室温，确认通过视觉检查该脂质悬浮液是清楚的。血脂，特别是酸性脂类，如棕榈酰油酰磷脂酰丝氨酸（POPS）和PI（4,5）P2的质量，是成功的重要实验。不要使用油脂是在等分试样的时间超过2个月。 </li></ol></li><li>预暖的热块到所需的温度。该温度必须高于脂质（多个）具有最高的Tm的熔化温度（T M）高至少5ºC。 </li><li>计算根据所需摩尔脂质比所需要的脂质的卷。用于研究的HIV-1的Gag这里结合，使用以下摩尔比示于表2。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 脂质组合 </td><td> 摩尔比 </td><td> 体积（微升） </td><td> 股权集中度 </td></tr><tr><td> POPC + POPS +澈</td><td> 46.​​6 + 23.3 + 30 </td><td> 19.74 + 10.18 + 6.57 </td><td> POPC，POPS，澈:10毫克/毫升</td></tr><tr><td rowspan="2"> POPC + POPS +澈+脑-PI（4,5）P2 </td><td行跨度="2"&gt; 40 + 20 + 30 + 10 </td><td rowspan="2"> 16.11 + 8.3 + 6.25 + 58.19 </td><td> POPC，POPS，澈:10毫克/毫升</td></tr><tr><td>脑-PI（4,5）P 2:1毫克/毫升</td></tr></tbody></table> 表2:不同血脂卷用来获取GUVs。 <ol start="4"><li>添加脂质的所需卷到一个干净的螺旋盖玻璃小瓶。 </li><li>使用ITO涂布的尺寸为25×50×1.1 立方毫米和电阻70-100Ω（如在产品目录中所述）的幻灯片。将两个ITO镀膜载玻片干净的长椅上。每个electroformation室需要两个铟锡氧化物（ITO）涂覆的玻璃载片。验证该玻璃载片的导电面朝上，通过使用万用表检查的电阻。它应该显示的约200Ω的值。确保幻灯片的表面是干净的通过使用不起毛的抹布70％酒精轻轻擦拭。 </li><li>清洗由里注射器的针的外表面用氯仿nsing它和将散热块上的注射器。 </li><li>将带有导电朝上加热块上的新的ITO膜的玻璃载玻片，并允许它平衡至所需的温度通常为约2-3分钟。不重复使用在ITO涂覆的玻璃载片的潜在风险，即在载玻片在ITO涂层可能清洁程序期间被损坏。 </li><li>使用40-50微升脂质混合物的体积为均匀，但快于载玻片上的脂质的扩频每个幻灯片。调整用合适的有机溶剂的总体积，例如氯仿。 <ol><li>放置在载玻片脂质混合物的总体积的一半，并立即使用清洁注射器（步骤2.6），留下一个均匀的脂质膜（整个滑动2-3次移动所述针）传播脂质。轻轻（但很快）传播所述脂质膜以避免对薄涂敷ITO任何损坏。非均匀扩散或蔓延粗糙可能导致次优产量或GUVs的计算的异质性。 </li><li>因此，通过检查在继续下一步骤之前传播之后脂质膜确保均匀扩散。如果薄膜的不透明度出现过度不均匀，采用了新的幻灯片重做的过程。 </li></ol></li><li>放置一个培养皿与涂布面朝上内侧被覆滑动。 </li><li>重复步骤2.6-2.9使用另一ITO涂布滑动的混合物的其余部分。 </li><li>放置在常规的真空干燥室含有幻灯片60-90分钟以除去有机溶剂任何微量培养皿。 </li><li>在此过程中，培养箱设定为相同的温度，该加热块（65ºC在大多数情况下），并预温300毫在孵化蔗糖溶液。 </li><li>干燥后，将培养皿在板凳上，并放置在载玻片上清洁的表面。 </li><li>轻轻擦拭用70％的乙醇和干燥它完井将PDMS垫片（ 图1）tely。 </li><li>将其放置在脂质包被的载玻片， 如图1和轻轻用力按下，使该垫圈形成水密封。确认不存在与PDMS垫片和滑动之间的间隙。这导致了浅腔用于保存蔗糖溶液的形成。 </li></ol><img alt="图1" src="/files/ftp_upload/54293/54293fig1.jpg" /> 图1:用于electroformation在ITO镀膜玻璃幻灯片的版式 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54293/54293fig1large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <ol start="16"><li>填充预热蔗糖溶液的腔室，没有任何气泡。 </li><li>在上面放置另一涂覆滑板，使一个紧密的密封。确保有没有气泡。 如图2的最终组装应出现。紧固室全光照摹长尾夹。 </li></ol><img alt="图2" src="/files/ftp_upload/54293/54293fig2.jpg" /> 图2:在electroformation室（侧视）的示意图 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54293/54293fig2large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <ol start="18"><li>吸去除多余的蔗糖和清洁使用不起毛的抹布的幻灯片。紧滑动到铜条使得导电两侧都与酒吧均匀接触。确保载玻片只有一个导电侧是与一个铜条接触。 </li><li>连接铜排使用鳄鱼夹的函数发生器的输出。放置组件培养箱内，以允许组件平衡至所需温度。 </li><li>应用10Hz的正弦波频率和1伏90分钟的电位差。 EnsurË，电压为1V通过万用表， 如图3，继续进行90分钟的处理。 </li></ol><img alt="图3" src="/files/ftp_upload/54293/54293fig3.jpg" /> 图3:在从万用表的电极放置用于测量频率和电流的位置 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54293/54293fig3large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <ol start="21"><li> 90分钟后，将频率缓慢降低至2赫兹，并继续electroformation另外10分钟。在这段时间，收获的体外翻译反应（步骤3.1-3.2）。 </li><li>关闭功能发生器并允许组件（通过关闭培养箱和离开培养箱门稍微打开）冷却到室温缓慢。 </li><li>冷却后，断开电缆连接到发电机从滑动组件。 GUVs可能是非常脆弱的，因此，需要进行很温和处理。仔细把装配了替补席上。 </li><li>轻轻取出使用镊子顶部滑动拆卸室。收获用裁员1000微升尖，并转移到一个干净的试管蔗糖含停牌GUVs。轻拿轻放管以防止GUVs中断。 </li><li>立即使用GUVs。当在室温下保存GUVs是稳定的收获后至少90分钟。 </li></ol> 3.收获体外翻译反应<ol><li>收获使用微量成管含有从红色室所需的蛋白质， 在体外翻译反应。 </li><li>要删除聚集的蛋白质，在16,200 XG自旋裂解液15分钟，在台式离心机，并小心地将上清转移到另一管。 </li></ol> 4. GUV结合分析<ol><li>使用切尖，混合51;含有翻译的蛋白质和5μlGUVs的在管体外翻译反应的升和在室温下孵育2-3分钟。 </li><li>用一个干净的盖玻片一个小孔（3-4毫米直径）附加PDMS表，并确保密封轻轻并牢牢压在盖玻片。 </li><li>从步骤4.1 10微升的混合物放入小孔。 </li><li>图像在室温下倒置外延或共聚焦荧光显微镜下的混合物。 注意:为了防止蒸发，在成像腔室可以由盖玻片覆盖。 </li></ol>

<ol>
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C., 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=Unilamellar+vesicle+formation+and+encapsulation+by+microfluidic+jetting.">Unilamellar vesicle formation and encapsulation by microfluidic jetting.</a> Proc Natl Acad Sci U S A. 105 (12), 4697-4702 (2008).</li><li>Yamauchi, 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=The+consensus+motif+for+N-myristoylation+of+plant+proteins+in+a+wheat+germ+cell-free+translation+system.">The consensus motif for N-myristoylation of plant proteins in a wheat germ cell-free translation system.</a> FEBS J. 277 (17), 3596-3607 (2010).</li><li>Olety, B., Veatch, S. L., Ono, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol-(4,5)-Bisphosphate+Acyl+Chains+Differentiate+Membrane+Binding+of+HIV-1+Gag+from+That+of+the+Phospholipase+Cdelta1+Pleckstrin+Homology+Domain.">Phosphatidylinositol-(4,5)-Bisphosphate Acyl Chains Differentiate Membrane Binding of HIV-1 Gag from That of the Phospholipase Cdelta1 Pleckstrin Homology Domain.</a> J Virol. 89 (15), 7861-7873 (2015).</li><li>Morales-Penningston, N. F., 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=GUV+preparation+and+imaging:+minimizing+artifacts.">GUV preparation and imaging: minimizing artifacts.</a> Biochim Biophys Acta. 1798 (7), 1324-1332 (2010).</li><li>Veatch, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electro-formation+and+fluorescence+microscopy+of+giant+vesicles+with+coexisting+liquid+phases.">Electro-formation and fluorescence microscopy of giant vesicles with coexisting liquid phases.</a> Methods Mol Biol. 398, 59-72 (2007).</li><li>Ayuyan, A. G., Cohen, F. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+peroxides+promote+large+rafts:+effects+of+excitation+of+probes+in+fluorescence+microscopy+and+electrochemical+reactions+during+vesicle+formation.">Lipid peroxides promote large rafts: effects of excitation of probes in fluorescence microscopy and electrochemical reactions during vesicle formation.</a> Biophys J. 91 (6), 2172-2183 (2006).</li><li>Montes, L. R., 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=Electroformation+of+giant+unilamellar+vesicles+from+native+membranes+and+organic+lipid+mixtures+for+the+study+of+lipid+domains+under+physiological+ionic-strength+conditions.">Electroformation of giant unilamellar vesicles from native membranes and organic lipid mixtures for the study of lipid domains under physiological ionic-strength conditions.</a> Methods Mol Biol. 606, 105-114 (2010).</li><li>Olety, B., Ono, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+played+by+acidic+lipids+in+HIV-1+Gag+membrane+binding.">Roles played by acidic lipids in HIV-1 Gag membrane binding.</a> Virus Res. 193, 108-115 (2014).</li><li>Keller, H., Krausslich, H. G., Schwille, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multimerizable+HIV+Gag+derivative+binds+to+the+liquid-disordered+phase+in+model+membranes.">Multimerizable HIV Gag derivative binds to the liquid-disordered phase in model membranes.</a> Cell Microbiol. 15 (2), 237-247 (2013).</li><li>Carlson, L. A., Hurley, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+reconstitution+of+the+ordered+assembly+of+the+endosomal+sorting+complex+required+for+transport+at+membrane-bound+HIV-1+Gag+clusters.">In vitro reconstitution of the ordered assembly of the endosomal sorting complex required for transport at membrane-bound HIV-1 Gag clusters.</a> Proc Natl Acad Sci U S A. 109 (42), 16928-16933 (2012).</li><li>Gui, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+minimal+in+vitro+system+for+analyzing+HIV-1+Gag-mediated+budding.">A novel minimal in vitro system for analyzing HIV-1 Gag-mediated budding.</a> J Biol Phys. 41 (2), 135-149 (2015).</li><li>Prevost, C., 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=IRSp53+senses+negative+membrane+curvature+and+phase+separates+along+membrane+tubules.">IRSp53 senses negative membrane curvature and phase separates along membrane tubules.</a> Nat Commun. 6, 8529 (2015).</li></ol>

We declare that we have no conflicting interests.

如上所述GUV结合测定法提供了在场景中其他蛋白质 - 类脂相互作用测定法有其局限性一个很好的选择。该测定允许我们检查与脂质双层上下文天然长酰基链肉豆蔻酰化全长Gag和酸性脂质之间的相互作用，并通过加格脂质的高密度蔗糖梯度或其它结合后处理没有冗长浮选离心这样做复合物。另一个优点是，加格的行为可以在复杂的混合物中进行检验（ 例如 ，在细胞溶质组分的存在），这是?...

人类免疫缺陷病毒1型（HIV-1）是一种有包膜的病毒，在大多数细胞类型组装在和从质膜（PM）的芽。 HIV-1病毒粒子的装配是由称为PR55 的Gag（GAG）的55 kDa的病毒核心蛋白驱动。加格被合成为四个主要的结构域，即，矩阵，衣壳，核衣壳和P6，以及两个间隔肽SP1和SP2组成的前体多蛋白。在装配过程中，矩阵（MA）域负责加格靶向组装现场，衣壳（CA）域介导的Gag Gag的相互作用，核衣壳（NC）域募集病毒基因组RNA和P6新兵宿主因素，援助从质膜病毒颗粒断裂。加格也通过加入在其N-末端14个碳的脂肪酸或肉豆蔻部分的经历共翻译修饰。 膜的Gag的结合是用于病毒装配的一项基本要求，因为mutants是在膜结合缺陷不能产生病毒颗粒。我们和其他人已经表明，膜是由马域内偶信号介导的Gag的结合:一种介导与脂质双层和称为高碱性区域MA域内的碱性残基簇（疏水相互作用的N-末端肉豆蔻基HBR）与上下午1-4酸性脂相互作用。使用polyphosphoinositide 5-磷酸酶IV（5ptaseIV），其催化PM-特定酸性磷脂phosphatidylinositol-的水解酶的异位表达Gag的膜相互作用的研究（4,5）-bisphosphate [PI（4,5）P 2]到磷脂酰肌醇-4-磷酸，在细胞中建议的Gag-PM定位由PI（4,5）P2 3,5-介导的。但是， 在体外研究旨在理解的Gag-PI（4,5）P2的更多的机械细节2相互作用已被证明是具有挑战性的原因很多。对于例如，全长的纯化肉豆蔻酰化的HIV-1 Gag的生化实验已经至少部分技术上难以由于加格的倾向纯化期间聚集。因此，HIV-1 Gag的截短形式，如秘耳-MA或秘耳-MA-CA，或者非肉豆蔻酰化形式已被频繁地研究，必要的Gag纯化而使用（ 如 ，核磁共振，蛋白足迹，和表面等离子体共振6-9）。备选地，耦合体外转录-翻译反应已被用于产生其它生化研究1,2-全长肉豆蔻酰化的HIV-1 Gag的。典型地，在这个系统中，一个编码的Gag质粒被转录并用真核细胞裂解物（ 例如 ，兔网织红细胞裂解物），缺少任何细胞膜和信使RNA的但含有用于转录和翻译机器翻译。反应后，将含有Gag的细胞裂解物与我混合mbranes与脂类加格相互作用的分析。除了 ​​易于制备全长肉豆蔻酰化的Gag的，使用体外转录翻译系统的方法具有的Gag合成和随后的膜结合反应发生在一个"真核细胞溶胶状"环境，可能更好地代表生理条件的优点。此属性促成了研究，结果表明绑定MA域RNA分子调节加格在竞争方式1,2,10-12结合酸性血脂。然而，由于在这些细胞裂解物获得的Gag蛋白质的总量不高，与放射性标记的氨基酸的蛋白质的代谢标记是必要的它们的检测。 根据不同的方法来测量的Gag - 脂质相互作用，已经使用了各种膜制剂。每一种方法都有其优势和局限性。大多数基于核磁共振的检验都要求短酰基利用脂质链是水溶性（ 例如 ，C 4和C8-PI（4,5）P 2）6,8。而核磁共振方法来测试的Gag的结合具有在细胞中发现的长酰基链正在开发中的脂质，它们迄今已8,13只有在用肉豆蔻酰化或nonmyristoylated MA。可替代地，从脂质制备具有天然长度的酰基链的脂质体已在生化方法如脂质体浮选或荧光脂质体珠粒结合测定2,3,10,14-16使用。然而，在这些测定中使用脂质体具有的直径小，因而它们的膜有陡峭的和积极的曲率。与此相反，在颗粒组装的在HIV-1感染的细胞中的早期阶段，加格结合到PM，这是上的Gag的规模几乎平坦，并随后出芽期间诱导负曲率。因此，陡峭，正曲率脂质体膜可能不是理想的脂质双层研究加格脂相互作用。至于脂质体海军报通货膨胀测定，另一个潜在需要注意的是加格脂质复合高渗性蔗糖梯度离心过程中暴露可能影响实验结果。为缓解这些限制，并提供一个互补的实验系统中，为GAG结合到巨单层囊泡（GUVs）测定法已被开发，近年来。 GUVs是单脂双层囊泡，其直径延伸至几十微米。因此，这些膜的曲率类似于上的Gag的规模的PM。此外，由于它的大尺寸，这使得在光学显微镜目视检查，膜在混合时可以在不加格 - 脂质复合物的后续处理可以很容易地确定的荧光标记或标记的Gag蛋白结合这些囊泡。 在这里，我们描述了一个协议，以研究HIV-1 Gag的膜使用从electroformation方法获得GUVs约束力。各种方法，如温和水合，凝胶辅助水合，麦克风rofluidic喷射和electroformation 17-22已被用来获取GUVs。对于这里所描述的协议中，electroformation方法在形成具有酸性脂质和其相对的易用性，而不需要昂贵的设置的GUVs使用主要是因为它的效率。自的Gag的可视化就必须荧光报道，黄色荧光蛋白（YFP）的基因加入到的Gag（加格-YFP）的C末端。的gag-YFP蛋白通过在小麦胚芽裂解物的体外转录和翻译反应的基础上连续交换连续流（CECF）技术获得的。在该技术中，既除去反应和反应底物和能量分量的供给抑制副产物的在基于透析的机制得以实现。这些反应中，使用T7启动子的控制下编码的Gag-YFP的质粒。值得注意的是，如上文所示，小麦胚芽裂解支持豆蔻无需额外的元件23,24。使用这种方法，就已经可以得到足够数量的全长上GUV膜24肉豆蔻酰化的Gag-YFP为加格的可视化。在这里，我们描述了与HIV-1 GAG结合到PI协议（4,5）P2含GUV膜可以无需冗长后续处理以下结合反应，并提出，这种方法已经存在补充加格膜结合试验，并可以进行检查扩展进一步了解HIV-1的Gag膜结合。

<table><tbody><tr><td>Digital Multimeter</td><td>Meterman</td><td>30XR</td><td>A generic multimeter that can measure resistance and volts will serve the purpose.</td></tr><tr><td>Function Generator</td><td>Instek</td><td>GFG-8216A</td><td>A generic function generator that is capable of generating a sine wave at 10 Hz and 1 V is sufficient.</td></tr><tr><td>ITO coated glass slides</td><td>Delta Technologies, Loveland, CO</td><td>CG-90IN</td><td></td></tr><tr><td>Incubator</td><td>Hoefer</td><td></td><td>Any incubator that can accurately maintain temperature will be sufficient</td></tr><tr><td>Vacuum chamber</td><td>Nalgene</td><td></td><td></td></tr><tr><td>Thermomixer R</td><td>Eppendorf</td><td>21516-166</td><td></td></tr><tr><td>Syringe</td><td>Hamilton</td><td>80400</td><td>Gauge 22S, Syringe number 702</td></tr><tr><td>PDMS</td><td>Sylgard elastomer base kit, Dow-Corning</td><td>Sylgard, 184</td><td></td></tr><tr><td>RTS 100 Wheat Germ CECF Kit</td><td>BiotechRabbit,&lt;br/&gt; Berlin, Germany</td><td>BR1401001</td><td></td></tr><tr><td>DiD</td><td>Life Technologies, Carlsbad, CA&amp;nbsp;</td><td>D7757</td><td></td></tr><tr><td>POPC</td><td>Avanti Polar Lipids, Alabaster, AL&amp;nbsp;</td><td>850457C</td><td></td></tr><tr><td>POPS</td><td>Avanti Polar Lipids</td><td>840034C</td><td></td></tr><tr><td>Cholesterol</td><td>Avanti Polar Lipids</td><td>700041P</td><td></td></tr><tr><td>Brain-PI(4,5)P&lt;sub&gt;2&lt;/sub&gt;&amp;nbsp;</td><td>Avanti Polar Lipids</td><td>840046X</td><td></td></tr></tbody></table>

visualization of hiv-1 gag binding to giant unilamellar vesicle (guv) membranes

HIV-1的结构蛋白，PR55 的Gag（或加格），在病毒组装过程中结合到在细胞质膜。加格膜结合是病毒颗粒形成的一个重要步骤，因为在加格膜结合导致病毒颗粒生产严重受损的缺陷。获得的Gag-脂质膜相互作用的机理的细节，基于核磁共振，蛋白足迹，表面等离子体共振，脂质体浮选离心或荧光脂质珠体外方法结合迄今已开发的。然而，每一个的这些体外方法有其局限性。为了克服这些局限性，并提供一个互补的方式来先前建立的方法，我们开发了一种体外测定，其中HIV-1 Gag和脂质膜之间的相互作用发生在一个"细胞样"的环境。在该测定中，GAG结合到脂质膜在视觉上分析使用YFP - taggeð加格在小麦胚芽合成基于体外翻译系统，并通过electroformation技术制备GUVs。在这里，我们描述了背景和协议，以获得必要的测定肉豆蔻酰化全长的Gag蛋白和GUV膜和检测的Gag-GUV通过显微镜约束力。

<html>使用上述协议中，我们制备POPC + POPS +澈组成GUVs（摩尔比:4.66:2.33:3）。该组合物被选为大约反映PM的PS和胆固醇的浓度。稳健高效加格结合仅观察到时，大脑PI（4,5）P 2被纳入POPC + POPS +澈混合物（POPC + POPS +澈+脑-PI（4,5）P2 [摩尔比:4:2:3:1]在本实施例中）（ 图4，比较面板B和D与A和C）。的Gag-YFP结合POPC +组成GUVs POPS +澈+脑-PI（4,5）P 2由?...

Watch this Scientific Journal Video about Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle GUV Membranes at JoVE.com

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle GUV Membranes

We illustrate here an in vitro membrane binding assay in which interactions between HIV-1 Gag and lipid membranes are visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique.

HIV-1 Gag的可视化绑定到巨人的单片囊泡（GUV）膜

The structural protein of HIV-1, Pr55Gag (or Gag), binds to the plasma membrane in cells during the virus assembly process. Membrane binding of Gag is an ...

visualization-hiv-1-gag-binding-to-giant-unilamellar-vesicle-guv

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle (GUV) Membranes

8.8K 次观看.  University of Michigan Medical School. We illustrate here an in vitro membrane binding assay in which interactions between HIV-1 Gag and lipid membranes are visually analyzed using YFP-tagged Gag synthesized in a wheat germ-based in vitro translation system and GUVs prepared by an electroformation technique. 

视频: Visualization of HIV-1 Gag Binding to Giant Unilamellar Vesicle GUV Membranes

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

可视化的细胞与细胞转移的艾滋病毒，艾滋病毒和现场共聚焦显微镜的荧光克隆

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

科研

免疫与感染

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) <a href="http://www.usaid.gov">(www.usaid.gov)</a>. The virus infects and destroys CD4+ T-cells thereby crippling the immune system, and causing an acquired immunodeficiency syndrome (AIDS) 2. Infection begins when the HIV Envelope glycoprotein "spike" makes contact with the CD4 receptor on the surface of the CD4+ T-cell. This interaction induces a conformational change in the spike, which promotes interaction with a second cell surface co-receptor 5,9. The significance of these protein interactions in the HIV infection pathway makes them of profound importance in fundamental HIV research, and in the pursuit of an HIV vaccine.
The need to better understand the molecular-scale interactions of HIV cell contact and neutralization motivated the development of a technique to determine the structures of the HIV spike interacting with cell surface receptor proteins and molecules that block infection. Using cryo-electron tomography and 3D image processing, we recently demonstrated the ability to determine such structures on the surface of native virus, at &tilde;20 &Aring; resolution 9,14. This approach is not limited to resolving HIV Envelope structures, and can be extended to other viral membrane proteins and proteins reconstituted on a liposome. In this protocol, we describe how to obtain structures of HIV envelope glycoproteins starting from purified HIV virions and proceeding stepwise through preparing vitrified samples, collecting, cryo-electron microscopy data, reconstituting and processing 3D data volumes, averaging and classifying 3D protein subvolumes, and interpreting results to produce a protein model. The computational aspects of our approach were adapted into modules that can be accessed and executed remotely using the Biowulf GNU/Linux parallel processing cluster at the NIH <a href="http://biowulf.nih.gov">(http://biowulf.nih.gov)</a>. This remote access, combined with low-cost computer hardware and high-speed network access, has made possible the involvement of researchers and students working from school or home.

The protocol describes a high-throughput approach to determining structures of membrane proteins using cryo-electron tomography and 3D image processing. It covers the details of specimen preparation, data collection, data processing and interpretation, and concludes with the production of a representative target for the approach, the HIV-1 Envelope glycoprotein. These computational procedures are designed in a way that enables researchers and students to work remotely and contribute to data processing and structural analysis.

使用低温电子断层扫描和自动子断层扫描平均HIV包膜糖蛋白分子结构的测定

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

艾滋病毒的成像1信封引起的病毒学突触信号合成脂双层

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

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 ...

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

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

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

吞噬体偏移和速度与HIV-1感染的活小学的人巨噬细胞测量

Macrophages are phagocytic cells that play a major role at the crossroads between innate and specific immunity. They can be infected by the human ...

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 ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

荧光检测病毒RNA<em&gt;原位</em&gt;杂交（FISH）

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

生物学

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs)

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma membrane and the numerous factors that influence protein behavior in live cells. Giant unilamellar vesicles (GUVs) are a biomimetic and highly tunable in vitro model system for investigating protein-membrane interactions and probing protein behavior in a precise, stimulus-dependent manner. In this protocol, we present an inexpensive and effective method for fabricating GUVs with the human serotonin 1A receptor (5-HT1AR) stably integrated in the membrane. We fabricate GUVs using a modified hydrogel swelling method; by depositing a lipid film on top of a mixture of agarose and 5-HT1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT1AR incorporated into the membrane. These GUVs can then be used to examine protein-membrane interactions and localization behavior via microscopy. Ultimately, this protocol can advance our understanding of the functionality of integral membrane proteins, providing profound physiological insight.

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors GPCRs

This protocol utilizes agarose swelling as a powerful and generalizable technique for incorporating integral membrane proteins (IMPs) into giant unilamellar lipid vesicles (GUVs), as described here for the reconstitution of the human 1A serotonin receptor protein (5-HT1AR), one of the classes of pharmacologically important G protein-coupled receptors.

构建含有G蛋白偶联受体（GPCR）的脂质膜模型

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma ...

生物工程

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

从巨 Unilamellar 泡中提取膜纳米管

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

Single-Cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that enable sensitive and simultaneous labeling of viral nucleic acid and protein. Conventional DNA fluorescence in situ hybridization (FISH) methods are often not compatible with immunostaining. We have therefore developed an imaging approach, MICDDRP (multiplex immunofluorescent cell-based detection of DNA, RNA and protein), which enables simultaneous single-cell visualization of DNA, RNA, and protein. Compared to conventional DNA FISH, MICDDRP utilizes branched DNA (bDNA) ISH technology, which dramatically improves oligonucleotide probe sensitivity and detection. Small modifications of MICDDRP enable imaging of viral proteins concomitantly with nucleic acids (RNA or DNA) of different strandedness. We have applied these protocols to study the life cycles of multiple viral pathogens, including human immunodeficiency virus (HIV)-1, human T-lymphotropic virus (HTLV)-1, hepatitis B virus (HBV), hepatitis C virus (HCV), Zika virus (ZKV), and influenza A virus (IAV). We demonstrated that we can efficiently label viral nucleic acids and proteins across a diverse range of viruses. These studies can provide us with improved mechanistic understanding of multiple viral systems, and in addition, serve as a template for application of multiplexed fluorescence imaging of DNA, RNA, and protein across a broad spectrum of cellular systems.

Presented here is a protocol for a fluorescence imaging approach, multiplex immunofluorescent cell-based detection of DNA, RNA, and protein (MICDDRP), a method capable of simultaneous fluorescence single-cell visualization of viral protein and nucleic acids of different type and strandedness. This approach can be applied to a diverse range of systems.

单细胞多重荧光成像，可可视化病毒核酸和蛋白质，并监测 HIV、HTLV、HBV、HCV、寨卡病毒和流感感染

Capturing the dynamic replication and assembly processes of viruses has been hindered by the lack of robust in situ hybridization (ISH) technologies that ...