作者非常感谢鲍勃·菲舍尔（LV，DIR，NIAID，NIH），谁担任解说员，以及奥斯汀Athman（RTB，DIR，NIAID，NIH）和梅根·摩根（DOHS，口服补液盐，外径，​​美国国立卫生研究院）的他们的帮助，使得电影伴随这个手稿。此外，我们还要感谢佳佳Groseth（LV，DIR，NIAID，NIH）的手稿批判性阅读。这项研究是由美国国立卫生研究院，NIAID的院内研究计划的支持。

1，拆分生产细胞的trVLPs的初始生产<ol><li>从80-90％汇合的293细胞培养在75cm移除介质2烧瓶中的高葡萄糖Dulbecco改良的Eagle培养基（DMEM）中，用10％胎牛血清（FBS），2mM的L-谷氨酰胺和1x青霉素/链霉素（DMEM10 ）。用10毫升磷酸盐缓冲盐水（PBS）洗涤细胞两次，注意不要移去细胞，并加入2 ml胰蛋白酶-EDTA细胞中。 </li><li>孵育的细胞在室温下直到细胞显示显著舍时，在显微镜（约30秒）下观察。打跑细胞通过点击烧瓶中，加入8毫升DMEM10。彻底轻轻吹打向上悬浮细胞和向下直到在显微镜下观察时，单细胞悬浮液中观察到。 </li><li>算使用自动细胞计数器中的细胞。稀释细胞至每毫升2×10 5个细胞于DMEM10。加入2 ml的细胞悬浮液，每孔为6孔板中（4×10每孔5个细胞）。 </li><li>培养在湿润的组织培养箱中的板在37℃下用5％的CO 2。 </li></ol> 2，转生产细胞的trVLPs的初始生产<ol><li>分裂的细胞（参见图5为实验定时的概述）24小时后，吸移管的质粒DNA 15（用于量见表1）进入无菌2毫升离心管使用过滤提示。添加每孔100μlOptiMEM中的DNA。旋涡混合短暂，轻轻用微量降速管。如果多口井是具有相同质粒转染中，mastermix几个井可。 </li><li>使用前短暂涡旋过境LT1小瓶。每孔加入7.5微升过境LT1到稀释的DNA。然后振荡混合物中，小心不要在离心管的盖，收集的液体，并孵育15分钟，在室温下进行。 </li><li>轻轻混匀转染通过上下吹打复杂。添加100μl的转染复合物滴加到每孔中。岩盘向前/向后，并从一侧到另一侧分发染物。不摇动板，因为这将导致不均匀的转染复合物的扩散。 </li><li>返回的细胞培养箱。 </li><li> 24小时后，取出，从细胞上清液。加入4毫升的DMEM，5％FBS，2mM的L-谷氨酰胺，1×青霉素/链霉素（DMEM5）至细胞中。这个步骤可以在没有孔的干燥时间进行长达3个孔，假设孔含有相同的样品（否则，由于在本系统中trVLPs的自放大性质，交叉污染会成为一个问题，并这一步应该在同一时间内做了一个很好）。 </li><li>返回的细胞培养箱。 </li></ol> 3，制备的靶细胞<ol><li>如在2毫升DMEM10第1节，播种4×10 5细胞每孔的分裂293细胞6孔板中。 </li><li>靶细胞的裂解后24小时，并在感染前24小时，如用DNA在第2条所述的转染靶细胞的量在表1中 ，每孔4.5微升全顺LT1。 </li></ol> 4，感染生产细胞的靶细胞与收获<ol><li>传送来自生产细胞的上清液至15ml管中。取出并使用真空软管丢弃任何剩余的上清液。加入250μl格洛裂解缓冲液的细胞。 </li><li>离心上清为5分钟，在800 XG和室温下以清除细胞碎片的样品。 </li><li>从目标小区中的一个以及除去上清液。小心加3毫升清生产者细胞上清液针对使用最慢的速度吸管细胞。避免直接以避免破坏细胞单层吸取到细胞上。这个步骤有一次做一个孔 。 </li><li>当所有的样品已经反橱，返回靶细胞培养箱允许trVLPs的沉淀靶细胞和感染。 </li><li>孵化后，在室温下在格洛裂解缓冲液15分钟，重新悬浮在使用微量设定为150微升裂解缓冲液的生产者细胞中，并将样品转移到2ml离心管。在这一点上，裂解物可在-80℃下作为在第6部分所述冻结，或者直接测定。 </li><li>感染后24小时，从靶细胞除去上清液。添加4毫升DMEM5每孔的细胞。这个步骤可以在短时间内完成最多为三个井，如果将孔含有相同的样品（否则，由于在本系统中trVLPs的自放大性质，交叉污染会成为一个问题，并且该步骤应在同一时间做了一个很好）。 </li></ol> 5，收获靶细胞的单循环感染<ol><li>如果只有一个感染周期应评估，在72小时后感染除去叔他上清液用真空软管的靶细胞。 </li><li>在第4节中描述的生产者细胞收获细胞在250微升格洛裂解液。 </li></ol>嘉实目标6细胞与trVLPs连续传代<ol><li>如果trVLPs要连续传代，在部分3中记载，使它们准备用于第一组靶细胞（参见图5）的感染在感染后72小时，准备一组新的靶细胞。 </li><li>感染的新的一组的靶细胞如（代替生产细胞的使用第一组靶细胞）在第4说明。 </li><li>重复这些步骤，每72小时。 </li></ol>记者活动7分析<ol><li>如果细胞裂解液进行冷冻，解冻它们在室温下进行。保证样品已经达到了之前的测量室温。 </li><li>解冻所需的海肾格洛分析缓冲液（最好是冷冻ì量（每个样品40微升）Ñ​​等分）在室温下进行。确保缓冲区已到达之前测量室温。 </li><li>添加海肾格洛衬底1/100卷到海肾格洛测定缓冲液中，得到的Renilla Glo试剂，并通过涡旋混合。移液管将40μl的海肾Glo试剂的成为白色的96孔板。 </li><li>加入40μl样品的海肾-Glo试剂。等待10分钟，然后用1秒的积分时间测量的光度计的样品。 </li></ol>

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作者宣称，他们没有竞争的经济利益。

在这个手稿中描述的tetracistronic trVLP法允许埃博拉病毒的生命周期模型在几个传染周期。重要的是，在该系统中所产生的trVLPs不包含的遗传信息的核衣壳蛋白NP，VP35，VP30，和L，它们一起构成了埃博拉病毒基因组的大约60％，并且为病毒复制所必需的。相反，这些蛋白质具有要在靶细胞中从表达载体中提供反式 ，和不表达所有4这些蛋白质的任何感染是流产。重要的是，没有任何证据基因重组的丝状病毒，并有在tetracistronic微型基因组中的表达质粒的核衣壳蛋白之间共享没有同源区。因此，既没有任何实际的证据，也没有能够提供用于产生全长埃博拉病毒基因组中，然后可能潜在导致的可能的任何理论基础生产感染埃博拉病毒，使该系统安全BSL2条件下使用。 有在tetracistronic trVLP测定的两个关键步骤是通过实验条件的影响， 即生产trVLPs的，并且靶细胞与这些trVLPs的感染。生产trVLPs的是依赖于高水平的小基因组的复制和转录，这反过来又依赖于具有高转染效率的。转染效率可以通过包括-L控制，其中L的表达式质粒被交换，用于编码绿色荧光蛋白的表达质粒容易评估。在这些条件下的转染率应通过用荧光显微镜观察后24小时转染检查超过50％。此外，细胞必须是自由的支原体，因为在我们的经验支原体污染显着地削弱了小基因组的复制和转录（未发表数据）。由于它们的高transfectability 293 CELLS是trVLP测定所选择的细胞系;然而，这些细胞是相对较差易感的（虽然不完全折射）感染埃博拉病毒16。的附着因子，如添-1的表达增强了293细胞trVLPs约100倍的病毒感染，而且是对本系统的在几个传代的成功是至关重要的。 而tetracistronic trVLPs不是对自己的自我复制，它们是自我复制的细胞中表达的核衣壳蛋白。因此，预防措施，必须进行，以避免含有不同trVLPs（ 例如 ，用在小基因组不同的突变）的孔之间的交叉污染。从更技术的角度来看，由于在该测定中（约4原木）正信号和负信号之间的较大差异，样品之间的串扰测量萤光素酶活性时，可以是一个问题;然而，这可以容易地通过在留下一空的以及各样品之间避免用于测定萤光素酶活性的96孔板。 有众多的tetracistronic trVLPs可能的应用。显然，它们非常适合于学习纤维病毒颗粒的进入，因为感染性trVLPs有传染性丝状病毒14的典型螺纹状结构，并含有相同的病毒成分为纤维病毒颗粒。重要的是，它们不采用假型病毒颗粒或病毒样颗粒，如逆转录病毒颗粒轴承GP 1,2，或重组病毒，例如表达GP 1,2重组水泡性口炎病毒时包含的其他病毒元件，就是这种情况。用293细胞作为靶细胞在此系统中，当用于附连的因素的要求，可被利用来筛选并调查这些附着因子的作用，而其它细胞因子，如那些参与基因组复制和转录中的作用以及形态发生和芽丁，可以利用RNAi技术进行研究。在对病毒生命周期中的病毒蛋白的突变的影响也被研究，但人们必须记住，而VP40，GP 1,2，和VP24病毒转录后表达的其它病毒蛋白的表达，从表达式实现质粒，使得由于这些蛋白质的表达的影响必须考虑。此外，应注意，在小基因组突变不显著改变小基因组的长度，因为报告基因活性是直接受微型基因组的长度15。最后，由于tetracistronic微型基因组的病毒基因组的类似物携带病毒基因，以及由病毒聚合酶复合物将被复制，它也应该可以研究在BSL2条件下反应的突变，这些基因的进化。因此，当进一步研究，但仍然需要在这个方向上，它应该是可能的，例如，以引入不理想在微型基因组，然后通过trVLPs内的基因突变，直到免费突变出现。 具有要牢记的tetracistronic trVLP测定的一个限制是，当它的模型大部分病毒生命周期中的，在靶细胞中初级转录不受该系统模型中，由于靶细胞具有表达反式的核衣壳蛋白为了使trVLPs复制。如果初级转录必须被评估，因此能够使用幼稚靶细胞10;然而，在这种情况下，没有基因组复制发生在靶细胞中，并没有进一步感染trVLPs产生，中止感染。这就是不能在没有完全呈现trVLPs自我复制，通过包括基因的核衣壳蛋白进入微型基因组，这将实际上把它们变成感染性重组埃博拉病毒克服的主要问题。事实上，EbolA型病毒表达荧光素酶或其他的记者已经生成，并且可以用来评估和研究基因组的复制和转录17,18;然而，它们的使用被限制为BSL4实验室。此外，它必须牢记，而VP40，GP 1,2，和VP24是从病毒基因组中的模拟表示，其在微型基因组的位置（第2 次 ，第3 次和第 4 次转录单元）是不相同的它们在病毒基因组中的位置（第3 次 ， 第 4 次和第 6 次转录单位），这可以相对于影响他们的绝对表达水平，以及它们的相对表达水平到另一个。 总体而言，tetracistronic trVLP测定表示为可用日期埃博拉病毒的最全面的生命周期模型系统，并允许基因组的复制和转录，颗粒形态和出芽的造型，以及附接和连接尝试进入靶细胞在多个传染性周期。因此，它具有巨大的潜力，在调查的埃博拉病毒BSL2条件下的生物学用途。

埃博拉病毒是一种严重出血热在人类和非人类灵长类动物具有高达90％的致死率在人类爆发1的病原体。虽然已显著进步，近年来在开发疫苗以及具体的治疗方法（2,3审查），这些都没有批准供人类使用。埃博拉病毒颗粒的特性螺纹状外观具有约1μm的长度和直径为96至98纳米4。甲核壳形式的病毒颗粒的核心，包含:1）将非分段单链负链RNA基因组，其编码的7埃博拉病毒的基因（ 图1），2）的核蛋白NP，其encapsidates的基因组中，3个）病毒聚合酶复合物组成的聚合酶L和其辅因子VP35的，和4）的转录激活VP30。此外，最近已表明，该蛋白VP24也与核衣壳相关第4。核衣壳由矩阵空间，其中的基质蛋白VP40，它负责的形态发生和病毒颗粒的出芽，位于包围。病毒颗粒进一步包封，并嵌入在该包络线是唯一的表面蛋白GP 1,2，其负责病毒附着和进入。 与感染埃博拉病毒的工作，必须在以下级生物安全（BSL）4个条件，制约着这项工作在世界范围内几设施的最高封闭性实验室进行。为了研究这些病毒的生物学或BSL2条件下，开发新的治疗中，研究人员依赖或者在埃博拉病毒蛋白的重组表达，或对生命周期建模系统，这两者都可以用在不存在感染埃博拉病毒的被加工。埃博拉病毒蛋白的重组表达是无论是从表达质粒或病毒载体来实现的。该战略的一个特殊情况是基于病毒以外埃博拉病毒产生的病毒颗粒或病毒样颗粒（最常用的逆转录病毒或疱疹性口炎病毒）中的重组的存在表示GP 1,2，导致产生假型颗粒，它可以用来研究丝状病毒和屏幕的输入过程进入抑制剂5。备选地，重组病毒（ 例如 ，水泡性口炎病毒），其编码的自身糖蛋白，埃博拉病毒GP 1,2代替可以生成并用来研究下的生物安全级别2的条件6病毒条目。 生命周期建模的系统的反向遗传学系统具有使用截断埃博拉病毒基因组的类似物（小基因组），其最初是从cDNA制备，然后复制，并通过在反式提供埃博拉病毒蛋白转录的形式。埃博拉病毒的第一个微型基因组系统的开发超过15年的前7，并已被用于研究埃博拉病毒基因组的复制和转录（在8,9审查）。在这个系统中的单顺反子微型基因组由单报告基因由埃博拉病毒基因组的末端非编码区侧翼的（所谓的片头和片尾）（ 图1）（使用T7 RNA聚合酶，通常由转录）的表达在哺乳动物细胞中连同病毒蛋白L，VP35，VP30和NP。小基因组是由NP包被，然后复制，并且通过使用定位在片头和片尾的顺式作用信号的其他核衣壳蛋白的转录，导致报告基因活性，反映了病毒的生命周期（ 图2）这两个方面。 为了建模的附加步骤的病毒的生命周期，转录和复制能力的病毒样颗粒（trVLP）系统已被开发，这是基于经典微型基因组的系统的，但配备了A其他病毒蛋白VP24，VP40和GP 1,2的表达质粒10,11 dditional表达。 VP40的存在导致形成trVLPs的，其承受的GP 1,2在其表面上，并进行内部含有小基因组，核衣壳。这些trVLPs可用于感染靶细胞，或已pretransfected与表达质粒为L，VP35，VP30和NP，以促进内trVLPs 11复制进入靶细胞的小基因组和转录，或者是幼稚的靶细胞（ 即没有埃博拉病毒蛋白的质粒驱动的表达）10。这导致了报告基因活性的靶细胞，这反映了在生产细胞，形态发生和trVLPs的出芽，其进入靶细胞的小基因组的复制，和1）中pretransfected靶细胞的情况下也基因组复制和次要转录（ 即转录病毒蛋白PRODuced在靶细胞）中的靶细胞，或在幼稚的靶细胞也初级转录（ 即转录由内trVLPs进入靶细胞的病毒蛋白）（ 图3）的情况下，2）。重要的是，这些系统只被用来模拟一个感染周期，并依靠所有的病毒蛋白，其在VP24和VP40的情况下是特别成问题的过表达，因为这些蛋白质已被证明是基因组复制的强负调节和转录的质粒12,13过度表达时。另外，在这些系统中产生trVLP制剂含有非感染性颗粒的比例高，有引发传染病trVLPs 14的生化分析的挑战。 为了克服这些问题，我们最近开发了一种tetracistronic微型基因组系统中，除了报告基因，还包含VP40，GP 1,2和编码该基因VP24（ 图1）。类似于传统的单顺反子微型基因组的系统，该系统导致产生trVLPs能够感染靶细胞（ 图4）15。然而，与此相反的古典微型基因组系统，VP40，GP 1,2，和VP24是病毒基因组的转录后产生的，而不是从质粒被过表达。其结果是，这些蛋白质的动力学和表达水平的更紧密地模仿那些在病毒生命周期的过程中发现的，因此感染性非传染性trVLPs的比率增加约500倍，在本系统15。另外，在使用本系统，有可能连续通道tetracistronic含有小基因组-trVLPs，造型多种传染性周期。因此，tetracistronic trVLPs是目前最全面的生命周期建模系统可用来研究BSL2条件下，埃博拉病毒生物学。在这里，我们提供了利用电脑的详细信息上本系统中，以及在预期的结果。

<table><tbody><tr><td>75 cm&lt;sup&gt;2&lt;/sup&gt; Cell culture flask</td><td>Corning&amp;nbsp;</td><td>430641</td><td></td></tr><tr><td>DMEM</td><td>Sigma</td><td>D6546</td><td>preheat to 37 &amp;deg;C prior to use</td></tr><tr><td>FBS</td><td>Life Technologies</td><td>26140-079</td><td>heat inactivate 30 min @ 56 &amp;deg;C</td></tr><tr><td>L-Glutamine</td><td>Life Technologies</td><td>25030-081</td><td>100x</td></tr><tr><td>Pen/strep</td><td>Life Technologies</td><td>15070-063</td><td>100x</td></tr><tr><td>0.25% Trypsin-EDTA</td><td>Life Technologies</td><td>25200-056</td><td></td></tr><tr><td>PBS</td><td></td><td></td><td>8 g NaCl, 0.2 g KCl, 1.44 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 0.24 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, H&lt;sub&gt;2&lt;/sub&gt;O add 1 L; autoclave and store at room temperature&amp;nbsp;</td></tr><tr><td>6-well Plates</td><td>Costar</td><td>3516</td><td></td></tr><tr><td>Opti-MEM I</td><td>Life Technologies</td><td>31985-070</td><td></td></tr><tr><td>Transit LT1</td><td>Mirus</td><td>MIR 2300</td><td></td></tr><tr><td>Glo lysis buffer</td><td>Promega</td><td>E2661</td><td></td></tr><tr><td>Renilla Glo luciferase assay system</td><td>Promega</td><td>E2720</td><td></td></tr><tr><td>96-well Assay plate (white)</td><td>Costar</td><td>3912</td><td></td></tr><tr><td>Modulus microplate luminometer</td><td>Turner Microsystems</td><td>998-9300</td><td></td></tr></tbody></table>

modeling the lifecycle of ebola virus under biosafety level 2 conditions with virus-like particles containing tetracistronic minigenomes

埃博拉病毒导致人类和非人类灵长类动物严重的出血热，与病死率高达90％。没有批准的疫苗或特定治疗这些病毒引起的疾病，并与感染性埃博拉病毒的工作被限制为4级生物安全实验室，显著限制于这些病毒的研究。生命周期建模系统模式下的生物安全二级条件，病毒的生命周期;然而，直到最近这种系统已被限制在病毒生命周期中的任一单独的方面，或一个单一的感染周期。 Tetracistronic小基因组，其由埃博拉病毒的非编码区，报告基因，和三个埃博拉病毒基因参与形态，出芽和条目（VP40，GP 1,2，和VP24），可用于产生复制和转录-competent病毒样颗粒（trVLPs）含有这些小基因组。这些trVLPs可连续感染细胞表达的埃博拉病毒病毒蛋白负责基因组复制和转录，使我们能够安全模式下的生物安全2级条件的多个感染周期。重要的是，此系统的病毒组分仅仅是来自于埃博拉病毒，而不是来自其他病毒（如，例如，在采用假型病毒的系统的情况下），VP40，GP 1,2和VP24，并 ​​且未在该系统中过表达，使得它非常适合于学习形态，萌芽和进入，但其他方面的病毒生命周期的，如基因组复制和转录，也与该系统进行建模。因此，tetracistronic trVLP法代表了最全面的生命周期建模系统埃博拉病毒，并在调查埃博拉病毒在未来的生物巨大的潜力以供使用。这里，我们提供了对使用本系统的详细信息，以及关于预期的结果。

293生产细胞表达质粒编码的埃博拉病毒核衣壳蛋白的NP，VP35，VP30和L，A tetracistronic微型基因组和配件T7聚合酶导致微型基因组复制和转录和记者的活动，很容易检测到72小时（ 图6的转）。重要的是，观察到的报告基因活性（10 5.6相对发光单位（RLU））超过阴性对照中的表达质粒编码L的从转染（10 3 RLU）由2个以上的日志中省略的这一点。活动即使在没有升观察到的水平低很可能是由于在小基因组质粒一个神秘的推手。感染了从生产者细胞的上清液trVLPs靶细胞相比，生产细胞实际显示有所增加报告基因活性的水平，和值在10 6和10 7 RLUs达到，这取决于通道。与此相反，瓦特母鸡-L控制生产细胞的上清液，传代到靶细胞（表达所有核衣壳蛋白，包括L）时，报告基因活性不超过光度计（约10 2 RLU）的背景噪声。 该tetracistronic trVLP法可用于研究埃博拉病毒的生命周期。例如，293细胞，trVLPs的感染依赖于附着因子的存在，如添-1 16，它必须在反式在这些细胞中提供。因此，在一个tetracistronic trVLP试验中报告基因活性的下降在大约100倍的靶细胞是在没有添-1（ 图7A）的观察。特定（病毒或细胞）的蛋白质在病毒生命周期中的作用也可以使用RNAi技术一起使用这种方法进行评估。作为一个例子，在以约100倍的报告基因活性的下降RNAi介导的下调Ļ结果，反映的L中的核心作用复制和转录（ 图7B）7。另外，也可以直接操纵微型基因组，以评估突变对病毒生命周期的影响。作为一个例子，当VP24表达来自小基因组，通过引入3个终止密码子紧接在起始密码子下游的取消，在生产者细胞中报告基因活性没有显着的变化;然而，在靶细胞中报告基因活性是由约20倍的降低一个通路，以及两个通道后400倍后，表明VP24在生产感染性trVLPs（ 图7C）15的作用。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/52381/52381fig1highres.jpg" width="500" /> 图1的结构埃博拉病毒的基因组，以及单顺反子和tetracistronic小基因组中，编码区为埃博拉病毒蛋白都显示为红色（NP，VP35，VP30，和L），黄色（VP40和VP24）或蓝色（GP 1,2）拖曳。非编码区（NCR的）示于绿色的，具有所指示的片头和片尾的区域。下标表示该病毒无碳复写纸被用于连接的编码区。对于记者（REP）的编码区中示出紫色。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/52381/52381fig2highres.jpg" width="500" /> 图2:单顺反子微型基因组检测。细胞转染表达质粒的埃博拉病毒核衣壳蛋白（NP，VP35，VP30，L），A单顺反子微型基因组（MG）和T7聚合酶。小基因组是由T7聚合酶（一）首先转录为在相同的方向在病毒基因组（病毒RNA），然后由NP（B）包被Âunencapsidated小基因组RNA。这个包被病毒RNA是通过互补的微型基因组RNA的复制（的cRNA）中间体ediate（c）中，然后转录成mRNA的记者（d）该被翻译成报告蛋白（e）所示。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/52381/52381fig3highres.jpg" width="500" /> 用单顺反子微型基因组如图3 trVLP测定。将细胞转染表达质粒的小基因组测定组分（埃博拉病毒核衣壳蛋白NP，VP35，VP30，L，A单顺反子微型基因组和T7聚合酶）以及VP40，GP 1 1,2和 VP24。这导致了包括含有小基因组，核衣壳（六）trVLPs的形成。这些trVLPs可以再感染的靶细胞（克），它要么pretransfected与表达质粒NP，VP35，VP30，和L（顶部），因此，复制和次要转录（四）导致报告基因的表达（e）中，或幼稚靶细胞（底部），从而导致最小的初级转录igenomes（H），也导致了报告基因的表达（E）。 <img alt="图4" fo:content-width="5in" src="/files/ftp_upload/52381/52381fig4highres.jpg" width="500" /> 图与tetracistronic微型基因组4 trVLP检测。细胞转染表达质粒的埃博拉病毒核衣壳蛋白（NP，VP35，VP30，L），A tetracistronic微型基因组（MG）和T7聚合酶。初始转录（一），包壳（B），基因组复制（C）和转录（D）以及翻译（五）如发生在单顺反子小基因组分析。然而，除了报告mRNA，mRNA表达的VP40，GP 1,2和VP24也从tetracistronic微型基因组转录，导致trVLPs的形成（f）所示。这些trVLPs感染已pretransfected与表达质粒的核衣壳蛋白NP，VP35，VP30和L，以及蜂窝式埃博拉病毒的靶细胞附着因子添-1，从而导致基因组复制trVLPs可用于感染新鲜的靶细胞和转录和生产。 <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/52381/52381fig5highres.jpg" width="500" /> 图5，定时，连续3通路A tetracistronic trVLP测定。为种子细胞（S），转染细胞（T），感染细胞（I），中变化（C）和收获的细胞和trVLPs（H）的日子对于三个连续的通道指示（用箭头表示）。 <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/52381/52381fig6highres.jpg" width="500" /> 在tetracistronic trVLP试验观察报告基因活性的图6典型的水平。继protoc进行了5通路A tetracistronic trVLP分析在这个手稿OL概述。作为阴性对照的表达质粒编码L的距离P0生产细胞的转染中省略（ - L）。靶细胞在通道P1至P5被转染的表达质粒对所有核衣壳蛋白，包括L.光度计的背景噪声被表示为虚线。装置和从3个独立实验4次生物学重复的标准偏差显示。 <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/52381/52381fig7highres.jpg" width="500" /> 图7评估各种病毒和细胞因子的使用tetracistronic trVLPs病毒生命周期的影响。 （一）添-1作为附件的因素。这是pretransfected与核衣壳蛋白和带或不带蒂姆- 1（15描述）的质粒编码的编码表达质粒感染TET靶细胞racistronic trVLPs。在p1的靶细胞72小时后的感染报告基因活性进行测定。装置和从4个独立实验4次生物学重复的标准偏差是针对靶细胞被pretransfected与表达质粒的核衣壳成分，添-1和miRNA的图示L于基因组的复制和转录的RNAi敲低的（B）的影响。 L（反-L）或不相关的蛋白（抗GFP）（在15中所述）感染了tetracistronic trVLPs。在p1的靶细胞72小时后的感染报告基因活性进行测定。手段和2个独立实验5生物学重复的标准偏差表示。（三）影响的突变在trVLP感染的微型基因组，使用一个野生型微型基因组（4cis-WT），或在一个小基因组执行了tetracistronic trVLP分析其中3个终止密码子已立即推出了VP24的起始密码子后，废除表达O上本蛋白但引入在P0，P1和P2的问候长度，核酸组合物，二级结构等记者活性只有很小的变化以小基因组，测定转染/感染后72小时。装置和从3个独立实验3次生物学重复的标准偏差显示。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr height="19"><td height="19" style="height:19px;width:115px;"></td><td style="width:127px;"> 生产者细胞（P0） </td><td style="width:180px;"> 靶细胞（P1及更高版本） </td></tr><tr height="19"><td height="19" style="height:19px;">将pCAGGS-NP </td><td> 125 </td><td> 125 </td></tr><tr height="19"><td height="19" style="height:19px;">将pCAGGS-VP35 </td><td> 125 </td><td> 125 </td></tr><tr height="19"><td height="19" style="height:19px;">将pCAGGS-VP30 </td><td> 75 </td><td> 75 </td></tr><tr height="19"><td heiGHT ="19"的风格="高度:19px;"&gt;将pCAGGS-L </td><td>千</td><td>千</td></tr><tr height="19"><td height="19" style="height:19px;"> p4cis，病毒RNA，它们间</td><td> 250 </td><td> - </td></tr><tr height="19"><td height="19" style="height:19px;">将pCAGGS-T7 </td><td> 250 </td><td> - </td></tr><tr height="19"><td height="19" style="height:19px;">将pCAGGS-TIM1 </td><td> - </td><td> 250 </td></tr></tbody></table> 表1中的DNA量进行转染。需要生产者和靶细胞的转染每种质粒的量示于每一个6孔板的纳克。所有质粒在瓦等[15]描述。

Watch this Scientific Journal Video about Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes at JoVE.com

Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

与感染埃博拉病毒的工作仅限于生物安全4级实验室。 Tetracistronic复制和转录能力的病毒样颗粒含有微型基因组 - （trVLPs）代表一个生命周期建模系统，使我们能够安全地模拟多种传染病周期下的生物安全级别2的条件下，对​​埃博拉病毒组件完全依赖。

造型埃博拉病毒的生命周期在生物安全二级条件下的病毒样颗粒包含Tetracistronic小基因组

Ebola viruses cause severe hemorrhagic fevers in humans and non-human primates, with case fatality rates as high as 90%. There are no approved vaccines or ...

modeling-lifecycle-ebola-virus-under-biosafety-level-2-conditions

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

36.1K 次观看.  National Institute of Allergy and Infectious Diseases, National Institutes of Health. 与感染埃博拉病毒的工作仅限于生物安全4级实验室。 Tetracistronic复制和转录能力的病毒样颗粒含有微型基因组 - （trVLPs）代表一个生命周期建模系统，使我们能够安全地模拟多种传染病周期下的生物安全级别2的条件下，对​​埃博拉病毒组件完全依赖。 

视频: Modeling The Lifecycle Of Ebola Virus Under Biosafety Level 2 Conditions With Virus-like Particles Containing Tetracistronic Minigenomes

Packaging HIV- or FIV-based Lentivector Expression Constructs &amp; Transduction of VSV-G Pseudotyped Viral Particles

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient expression of effectors. Packaging lentiviral expression constructs into pseudoviral particles, however, enables up to 100% transduction, even with difficult-to-transfect cells, such as primary, stem, and differentiated cells. Moreover, the lentiviral delivery does not produce the specific cellular responses typically associated with chemical transfections, such as cell death resulting from toxicity of the transfection reagent 1, 2. When transduced into target cells, the lentiviral construct integrates into genomic DNA and provides stable expression of the small hairpin RNA (shRNA), cDNA, microRNA or reporter gene 3, 4. Target cells stably expressing the effector molecule can be isolated using a selectable marker contained in the expression vector construct such as puromycin or GFP. After pseudoviral particles infect target cells, they cannot replicate within target cells because the viral structural genes are absent and the long terminal repeats (LTRs) are designed to be self-inactivating upon transduction 5, 6.
There are three main components necessary for efficient lentiviral packaging 1, 5, 6, 7. 
1. The lentiviral expression vector that contains some of the genetic elements required for packaging, stable integration of the viral expression construct into genomic DNA, and expression of the effector or reporter. 
2. The lentiviral packaging plasmids that provide the proteins essential for transcription and packaging of an RNA copy of the expression construct into recombinant pseudoviral particles. This protocol uses the pPACK plasmids (SBI) that encode for gag, pol, and rev from the HIV or FIV genome and Vesicular Stomatitis Virus g protein (VSV-G) for the viral coat protein. 
3. 293TN producer cells (derived from HEK293 cells) that express the SV40 large T antigen, which is required for high-titer lentiviral production and a neomycin resistance gene, useful for reselecting the cells for maintenance.
An overview of the viral production protocol can be seen in Figure 1. Viral production starts by co-transfecting 293TN producer cells with the lentiviral expression vector and the packaging plasmids. Viral particles are secreted into the media. After 48-72 hours the cell culture media is harvested. Cellular debris is removed from the cell culture media, and the viral particles are precipitated by centrifugation with PEG-it for concentration. Produced lentiviral particles are then titered and can be used to transduce target cells. Details of viral titering are not included in this protocol, but can be found at:
<a href="http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf" target="_blank">http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf</a> 8.
This protocol has been optimized using the specific products indicated. Other reagents may be substituted, but the same results cannot be guaranteed.

Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles

Lentiviral expression vectors are the most effective vehicles for stably expressing different effector molecules or reporter constructs in dividing and non-dividing mammalian cells and whole organisms. Here we provide a protocol on how to package lentivector expression constructs in pseudoviral particles and to transduce target cells using the pseudoviral particles.

包装艾滋病毒或FIV的基于Lentivector表达结构与VSV-G假型病毒颗粒转导

As with standard plasmid vectors, it is possible to transfect lentivectors in plasmid form into cells with low-to-medium efficiency to obtain transient ...

科研

免疫与感染

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for safe and effective therapies tailored to individual tumor immune profiles. Oncolytic viruses enable the induction of anti-tumor immune responses as well as tumor-restricted gene expression. This protocol describes the generation and ex vivo analysis of immunomodulatory oncolytic vectors. Focusing on measles vaccine viruses encoding bispecific T cell engagers as an example, the general methodology can be adapted to other virus species and transgenes. The presented workflow includes the design, cloning, rescue, and propagation of recombinant viruses. Assays to analyze replication kinetics and lytic activity of the vector as well as functionality of the isolated immunomodulator ex vivo are included, thus facilitating the generation of novel agents for further development in preclinical models and ultimately clinical translation.

This protocol describes a detailed workflow for the generation and ex vivo characterization of oncolytic viruses for expression of immunomodulators, using measles viruses encoding bispecific T cell engagers as an example. Application and adaptation to other vector platforms and transgenes will accelerate the development of novel immunovirotherapeutics for clinical translation.

肿瘤靶向免疫调节副病毒的设计与评价

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

癌症研究

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

病毒遗传背景明确的神经回路跟踪

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

神经科学

Safety Precautions and Operating Procedures in an (A)BSL-4 Laboratory: 2. General Practices

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory with non-high-consequence pathogens will take significantly longer in a BSL-4 setting. This increased time requirement is due to a multitude of factors that are aimed at protecting the researcher from laboratory-acquired infections, the work environment from potential contamination and the local community from possible release of high-consequence pathogens. Inside the laboratory, movement is restricted due to air hoses attached to the mandatory full-body safety suits. In addition, disinfection of every item that is removed from Class II biosafety cabinets (BSCs) is required. Laboratory specialists must be trained in the practices of the BSL-4 laboratory and must show high proficiency in the skills they are performing. The focus of this article is to outline proper procedures and techniques to ensure laboratory biosafety and experimental accuracy using a standard viral plaque assay as an example procedure. In particular, proper techniques to work safely in a BSL-4 environment when performing an experiment will be visually emphasized. These techniques include: setting up a Class II BSC for experiments, proper cleaning of the Class II BSC when finished working, waste management and safe disposal of waste generated inside a BSL-4 laboratory, and the removal of inactivated samples from inside a BSL-4 laboratory to the BSL-2 laboratory.

Safety Precautions and Operating Procedures in an ABSL-4 Laboratory: 2. General Practices

Performing viral assays in a BSL-4 laboratory is more involved compared to work in a BSL-2 laboratory due to required additional safety precautions. Here, we present an overview of practices and procedures used inside a BSL-4 laboratory illustrating proper Class II biosafety cabinet usage, waste management/disposal, and sample removal.

安全注意事项和操作步骤在（A）BSL-4实验室:2.常规做法

Work in a biosafety level 4 (BSL-4) containment laboratory requires time and great attention to detail. The same work that is done in a BSL-2 laboratory ...

Surface Functionalization of Hepatitis E Virus Nanoparticles Using Chemical Conjugation Methods

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of various diseases. This application relies on genetic modification, self-assembly, and cysteine conjugation to fulfill the tumor-targeting application of recombinant VLPs. Compared with genetic modification alone, chemical conjugation of foreign peptides to VLPs offers a significant advantage because it allows a variety of entities, such as synthetic peptides or oligosaccharides, to be conjugated to the surface of VLPs in a modulated and flexible manner without alteration of the VLP assembly.
Here, we demonstrate how to use the hepatitis E virus nanoparticle (HEVNP), a modularized theranostic capsule, as a multifunctional delivery carrier. Functions of HEVNPs include tissue-targeting, imaging, and therapeutic delivery. Based on the well-established structural research of HEVNP, the structurally independent and surface-exposed residues were selected for cysteine replacement as conjugation sites for maleimide-linked chemical groups via thiol-selective linkages. One particular cysteine-modified HEVNP (a Cys replacement of the asparagine at 573 aa (HEVNP-573C)) was conjugated to a breast cancer cell-specific ligand, LXY30 and labeled with near-infrared (NIR) fluorescence dye (Cy5.5), rendering the tumor-targeted HEVNPs as effective diagnostic capsules (LXY30-HEVNP-Cy5.5). Similar engineering strategies can be employed with other macromolecular complexes with well-known atomic structures to explore potential applications in theranostic delivery.

We have engineered the capsid protein of hepatitis E virus as a theranostic nanoparticle (HEVNP). HEVNP self-assembles into a stable icosahedral cage in mucosal delivery. Here, we describe the modification of HEVNPs for tumor targeting by mutating surface-exposed residues to cysteines, which conjugate synthetic ligands that specifically bind tumor cells.

化学共轭法在戊型肝炎病毒纳米粒子表面功能化中的应用

Virus-like particles (VLPs) have been used as nanocarriers to display foreign epitopes and/or deliver small molecules in the detection and treatment of ...

生物工程

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

增强生物安全与聚合物复合物Ecotropic慢病毒的人体细胞的转导

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

生物学

Production of Pseudotyped Particles to Study Highly Pathogenic Coronaviruses in a Biosafety Level 2 Setting

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase reporter, using a simple transfection procedure of the widely available HEK-293T cell line. Once formed and released from producer cells, these pseudovirions incorporate a luciferase reporter gene. Since they only contain the heterologous coronavirus spike protein on their surface, the particles behave like their native coronavirus counterparts for entry steps. As such, they are the excellent surrogates of native virions for studying viral entry into host cells. Upon successful entry and infection into target cells, the luciferase reporter gets integrated into the host cell genome and is expressed. Using a simple luciferase assay, transduced cells can be easily quantified. An important advantage of the procedure is that it can be performed in biosafety level 2 (BSL-2) facilities instead of BSL-3 facilities required for work with highly pathogenic coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV) and severe acute respiratory syndrome coronavirus (SARS-CoV). Another benefit comes from its versatility as it can be applied to envelope proteins belonging to all three classes of viral fusion proteins, such as the class I influenza hemagglutinin (HA) and Ebola virus glycoprotein (GP), the class II Semliki forest virus E1 protein, or the class III vesicular stomatitis virus G glycoprotein. A limitation of the methodology is that it can only recapitulate virus entry steps mediated by the envelope protein being investigated. For studying other viral life cycle steps, other methods are required. Examples of the many applications these pseudotype particles can be used in include investigation of host cell susceptibility and tropism and testing the effects of virus entry inhibitors to dissect viral entry pathways used.

Here, we present a protocol to generate pseudotyped particles in a BSL-2 setting incorporating the spike protein of the highly pathogenic viruses Middle East respiratory syndrome and severe acute respiratory syndrome coronaviruses. These pseudotyped particles contain a luciferase reporter gene allowing quantification of virus entry into target host cells.

在生物安全2级环境中制备假型颗粒研究高致病性冠状病毒

The protocol aims to generate coronavirus (CoV) spike (S) fusion protein pseudotyped particles with a murine leukemia virus (MLV) core and luciferase ...

Generation, Amplification, and Titration of Recombinant Respiratory Syncytial Viruses

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, both to decipher viral replication mechanisms and to study antivirals or provide development platform for vaccines. The construction and manipulation of a reverse genetic system for a negative-strand RNA virus such as a respiratory syncytial virus (RSV), however, remains delicate and requires special know-how. The RSV genome is a single-strand, negative-sense RNA of about 15 kb that serves as a template for both viral RNA replication and transcription. Our reverse genetics system uses a cDNA copy of the human RSV long strain genome (HRSV). This cDNA, as well as cDNAs encoding viral proteins of the polymerase complex (L, P, N, and M2-1), are placed in individual expression vectors under T7 polymerase control sequences. The transfection of these elements in BSR-T7/5 cells, which stably express T7 polymerase, allows the cytoplasmic replication and transcription of the recombinant RSV, giving rise to genetically modified virions. A new RSV, which is present at the cell surface and in the culture supernatant of BSRT7/5, is gathered to infect human HEp-2 cells for viral amplification. Two or three rounds of amplification are needed to obtain viral stocks containing 1 x 106 to 1 x 107 plaque-forming units (PFU)/mL. Methods for the optimal harvesting, freezing, and titration of viral stocks are described here in detail. We illustrate the protocol presented here by creating two recombinant viruses respectively expressing free green fluorescent protein (GFP) (RSV-GFP) or viral M2-1 fused to GFP (RSV-M2-1-GFP). We show how to use RSV-GFP to quantify RSV replication and the RSV-M2-1-GFP to visualize viral structures, as well as viral protein dynamics in live cells, by using video microscopy techniques.

We describe a method for generating and amplifying genetically modified respiratory syncytial viruses (RSVs) and an optimized plaque assay for RSVs. We illustrate this protocol by creating two recombinant viruses that respectively allow quantification of RSV replication and live analysis of RSV inclusion bodies and inclusion bodies-associated granules dynamics.

重组呼吸道合胞病毒的生成、扩增和滴定

The use of recombinant viruses has become crucial in basic or applied virology. Reverse genetics has been proven to be an extremely powerful technology, ...

Production of High-Titer Infectious Influenza Pseudotyped Particles with Envelope Glycoproteins from Highly Pathogenic H5N1 and Avian H7N9 Viruses

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious public health issues and suggest the possibility of an epidemic. However, our molecular understanding of the virus is rudimentary, and it is necessary to study the biological properties of its envelope proteins as therapeutic targets and to develop strategies to control infection. We developed a solid viral pseudotyped particle (pp) platform to study avian influenza virus, including the functional analysis of its hemagglutinin (HA) and neuraminidase (NA) envelope glycoproteins, the reassortment characteristics of the HAs and NAs, receptors, tropisms, neutralizing antibodies, diagnosis, infectivity, for the purposes of drug development and vaccine design. Here, we describe an experimental procedure to establish pps with the envelope glycoproteins (HA, NA) from two influenza A strains (HAPI H5N1 and 2013 avian H7N9). Their generation is based on the capacity of some viruses, such as murine leukemia virus (MLV), to incorporate envelope glycoproteins into a pp. In addition, we also detail how these pps are quantified with RT-qPCR, and the infectivity detection of native and mismatched virus pps depending on the origin of the HAs and NAs. This system is highly flexible and adaptable and can be used to establish viral pps with envelope glycoproteins that can be incorporated in any other type of enveloped virus. Thus, this viral particle platform can be used to study wild viruses in many research investigations.

This protocol describes an experimental process to produce high-titer infectious viral pseudotyped particles (pp) with envelope glycoproteins from two influenza A strains and how to determine their infectivity. This protocol is highly adaptable to develop pps of any other type of enveloped viruses with different envelope glycoproteins.

从高致病性H5N1病毒和禽流感病毒中生产含有包络糖蛋白的高蒂特感染性流感伪型颗粒

The occasional direct transmission of the highly pathogenic avian influenza A virus H5N1 (HPAI H5N1) and H7N9 to humans and their lethality are serious ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

在EMBL的MultiBac蛋白质复杂的生产平台

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...