Al and Mary Agnes McQuinn, the Richard M. Schulze Foundation, and an NIH Relief Grant from the Mayo Clinic funded representative work described here.

1.克隆的microRNA反应元件到病毒基因组<ol><li> 设计的microRNA反应元件插入。 <ol><li>识别所需的微RNA及其相应的靶序列。几个数据库都可以与成熟的microRNA序列。推荐:http://www.mirbase.org/ 9，27，28，29，30。 </li></ol></li><li> 克隆应答元件到编码的载体基因组或转录物的质粒DNA。 注:对于采用了独特的限制性位点用于插入两个或更多个拷贝的反应的元素是更容易和更灵活。 <ol><li>插入响应元件或使用拼接重叠延伸（SOE）PCR的独特限制性位点。这种方法已经详细31进行了描述。 </li><li>如果使用限制位点用于插入，PUR追商业合成，PAGE纯化的有义和编码由独特的限制性位点（ 图2）的突出端序列侧接插入序列的反义ultramers。 </li><li>结合0.5微克感ultramer，0.5微克的反义ultramer，DNA连接缓冲液为1X的终浓度，并带来至50微升用H 2 O退火ultramers通过孵育在85℃下反应10分钟，然后通过降低温度0.5℃下，每隔30秒，直到反应达到25℃。 </li><li>结合0.5微克载体DNA的编码该新的独特的限制性位点，酶缓冲液，以1倍终浓度，1微升合适的限制性酶，并带来了20微升用H 2 O文摘载体的最终体积在37℃下2小时。净化用琼脂糖凝胶纯化32的线性化的DNA。 </li><li>结扎退火ultramers到消化的载体过夜在16℃温度下用3:1的ultramers:载体的摩尔比和标准连接技术33。 注意:当使用一个单一限制性酶切位点用于插入它可以是更有效的脱磷酸化的消化的载体的端部和磷酸化退火的寡核苷酸的末端。 </li><li>变换连接的DNA到使用热休克方法34大肠杆菌。 注:编码病毒基因组的质粒一般都很大，因此使用优化摄取大量DNA结构可能会增加转化效率的感受态细胞。 </li><li>净化从使用市售的试剂盒纯化35单个菌落的质粒DNA。通过测序插入区域36识别与在正确的方向（ 图2）的微小RNA响应元件的适当克隆。 </li></ol></li></ol> 2.救援微小RNA靶向RNA病毒来回米质粒DNA <ol><li> 在细胞系容许对不表达的同源微RNA（多个）病毒复制救援微小RNA靶向微小核糖核酸病毒。该协议使用H1-HeLa细胞，因为它们不表达的miR-142中，miR-124，或miR-125，但它是不使用H1-HeLa细胞救援所需的。 注意:对传染性病原体的安全处理和处置的所有准则应严格遵守。 <ol><li>板H1-HeLa细胞在6孔板使得它们〜在转染的时间（24小时后播种）80％汇合。在每DMEM中孔2mL板细胞在补充有10％胎牛血清。 </li><li>暖转染试剂至室温。结合250微升无血清培养基，质粒DNA编码微小RNA靶向基因组的2.5微克，和在无菌的离心管转染试剂的7.5微升和吸管轻轻混合。在室温下孵育混合物15-30分钟。 </li><li>吸出从接种的细胞培养基中加入2毫升的新鲜完整的媒体。 </li><li>从步骤2.1.2添加整个转染混合物至6孔板滴的一个孔中。每滴添加到孔的不同区域。 </li><li>孵育所述细胞在37℃，直至细胞病变效应（CPE）或报告蛋白是可检测的（〜24-72小时）。 </li></ol></li><li> 采收和通道获救病毒到新鲜细胞。 <ol><li>在一个6孔板板的细胞，使得它们在感染的时间（24小时后播种）80-90％汇合。 注:扩张或收缩取决于所需的所有后续实验中的病毒量。 </li><li>通过使用橡胶细胞刮棒或橡胶淀刮细胞进入上清液收获各救援良好。轻轻拖曳穿过井底部的刮刀。转移细胞和上清液成低温储存管。 </li><li>冷冻/解冻的样品两次，然后通过沉淀细胞碎片在1200 xg离心离心5分钟，4℃。 </li><li>使用过滤0.2微米的针头式过滤器的两倍澄清的上清液。过滤的救援上清液包含病毒。 注意:滤波器孔径大小可以取决于病毒颗粒的尺寸变化，并且可能不适用于大型病毒。 </li><li>从接种的细胞吸出培养基，用无血清培养基洗一次，并加入1毫升新鲜的无血清培养基的各孔中。 </li><li>加入50微升每过滤救援上清液好，轻轻摇动板均匀地分布病毒。 </li><li>孵育在37℃的板对从各2小时，然后吸出介质以及以除去未掺入的病毒。 </li><li>添加1.5-2毫升每孔新鲜完全培养基中，并在37℃孵育直至CPE或报告蛋白是显而易见的（〜24-48小时）。 </li><li>重复步骤2.2.2至2.2.4。过滤救援上清含有病毒的股票。存储病毒储存在低温储存管一次性使用分装在-80°C。 </li></ol></li></ol> 3.从救援微小RNA靶向病毒体外转录的RNA转录<ol><li>生成从质粒DNA编码使用上游启动子的微小RNA靶向病毒基因组RNA转录物。 注:应用于所有后续步骤，用于产生和保持一个无核酸环境标准的做法。 </li><li>使用转录下游的酶切位点线性化的质粒DNA。结合5微克质粒DNA，1×最终浓度的酶缓冲液，3微升限制酶，并把50微升用H 2 O在37℃下反应3小时。 </li><li>添加的0.5M的EDTA 1/20体积1/10体积的5M NH 4乙酸盐，和2体积的100％乙醇的消化反应并混合。孵育在-20℃1小时至过夜。 </li><li>沉淀10分钟的沉淀的DNA在4℃下17000 xg离心。倒掉supernata核苷酸，重悬在冷的70％乙醇500微升沉淀并通过于4℃在17000 xg离心离心10分钟沉淀DNA。 </li><li> 30秒再次倒掉上清和离心。用吸管除去残留的上清液并空气干燥沉淀。在0.5-1微克/微升的浓度重悬DNA沉淀在无菌无核酸酶的 H 2 O操作。 </li><li>解冻在室温下在体外转录试剂和放置的核糖核苷酸在冰上（在甘油的酶不冻结，并应直接在冰上）。保持反应缓冲液中于室温。 </li><li>通过组合2微升装配在PCR管中的转录反应的每一个的ATP，CTP，GTP，和UTP溶液，2微升10X反应缓冲液，1微克线性化的DNA，2微升酶，并带来对20μL的用核酸酶的终体积-free H 2 O </li><li>孵育在37℃下2小时进行反应。 </li><li> 净化RNA转录。 <b转/&gt;注:在开始，准备用乙醇的指示和预热洗脱溶液至95℃，洗脱步骤洗涤溶液中。 <ol><li>使转录反应以100μL的用洗脱溶液（未预热）。添加粘合溶液浓缩物的350微升和250微升的100％乙醇，以反应和混合。 </li><li>将样品转移到以12,000×g的插入收集管和离心1分钟的过滤器滤芯。丢弃的流量通过。 </li><li>加的洗涤液500μL到滤筒和离心1分钟，在12,000×克弃流过。重复该洗涤步骤一次。弃流过。在12,0000 XG离心1分钟以去除残留的乙醇。 </li><li>滤筒转移到一个干净的收集管，加入预热的洗提溶液的50微升到滤筒。离心12,000×g下1分钟。重复加入100μl洗脱液续的总体积此洗脱步骤癌宁纯化RNA转录。丢弃过滤器滤芯。 </li></ol></li><li>通过测量样品中的260和280nm的吸光度，并使用比尔 - 朗伯定律确定洗脱液中的RNA浓度。 </li><li>评估使用RNA凝胶电泳37 RNA的完整性。参见图3的好和坏RNA的完整性的例子。 RNA应等分成单次使用的管，并储存在-80℃下保持完整性。 </li><li>暖转染试剂至室温。结合250微升无血清培养基，纯化的RNA转录物的2.5微克，升压溶液5μL的，和转染试剂的5微升在无菌的离心管和移液管轻轻混匀。在室温下孵育该混合物2-5分钟。 </li><li>板H1-HeLa细胞在6孔板使得它们〜在转染的时间（24小时后播种）80％汇合。补充有10％的FET中每DMEM中孔2mL板细胞人牛血清。 </li><li>从接种的细胞吸媒体和补充新鲜完整的媒体2毫升。 </li><li>从步骤3.12添加整个转染混合物至6孔板滴的一个孔中。每滴添加到孔的不同区域。 </li><li>孵育所述细胞在37℃，直至细胞病变效应（CPE）或报告蛋白是可检测的（〜24-72小时）。 </li><li>如步骤2.2至2.2.9继续微小RNA靶向病毒抢救。 </li></ol> 4.通过计算滴定病毒储存50％组织培养感染剂量（TCID 50） <ol><li> 板H1-HeLa细胞在96孔板中，每孔10 4个细胞在DMEM补充有10％胎牛血清。孵育细胞在37℃下过夜。 注意:对于许可不表达的microRNA同源病毒复制的细胞滴定病毒。 </li><li> 使病毒的10倍系列稀释液各以1毫升无血清的总体积媒体。 注意:如果需要更精确地使用较低的稀释液。 注意:每次稀释后更改提示，以防止滴度高估病毒能够坚持提示。 </li><li>尖端96孔板的侧面和抽吸媒体从铺板的HeLa细胞。添加每每种病毒稀释度的100微升井到96孔板（每稀释1行）的8孔中。加100μL无血清培养基的无病毒排1和板排12的控制。 注:请始终吸并通过触摸尖端能很好的防止细胞分离介质添加到水井。 </li><li>在37℃下孵育板2小时。前端板的一侧和从孔中吸出介质。 注:每个稀释度排之间变化的提示。 </li><li>添加100微升完全培养基的各孔中。在37℃下孵育板72小时。 </li><li>可视显微镜下孔中，并标记每个井阳性或阴性的CPE。 </li><li>计算用下面的公式每种病毒滴度:螺克10（TCID 50 /毫升）= L + D（S-0.5）+日志10（1 / V）。 L是所测试的最集中的病毒稀释，其中所有孔都是正的负log 10。 D是稀释因子的对数10。 S是个体的比例（PI）的总和。圆周率是一个单独的稀释液（阳性孔/每稀释孔的总金额的金额的计算比例。V是种菌的体积（ml /孔）。 </li></ol><img alt="表格1" src="/files/ftp_upload/55033/55033tbl1.jpg" /> 表1:通过计算TCID 50 量化感染性病毒颗粒 。从细胞中代表性结果感染的一连串的病毒原液的十倍稀释液。 "L"等于4，因为最后的稀释，所有的水井都积极为CPE 10-4。 "D"是10，因为10倍稀释液的对数10被使用，并且因此等于1的"S"是个体的比例的总和。在本实施例的各个比例是1.0，0.875，0.375，0.125，和0的这些的总和（S）为2.375。的"V"是接种在用于最初感染细胞毫升的体积。 5.评估微小RNA靶向功效:单步生长动力学<ol><li>对于该测定控制包括假感染的细胞，未经修饰的病毒和病毒含有非靶向或无功能的响应元素。 </li><li>板H1-HeLa细胞在12孔组织培养板中的时间过程实验，使得它们在感染的时间（24小时后播种）80-90％汇合。在DMEM板细胞在补充有10％胎牛血清以每孔1mL的。 注:推荐到板为每个时间点的单独板。该协议使用7种不同的时间点为病毒与8-12小时的复制周期。不需要H1-HeLa细胞来执行此一SSAY。该测定应在不表达的靶向微小RNA允许细胞来进行。该测定也可以在表达同源微RNA选择性压力下，以评估生长动力学的细胞进行。 </li><li>从水井吸媒体。通过加入0.5毫升无血清培养基中，以每洗井孔，漩涡的板，和从井抽吸介质。加入0.5毫升新鲜无血清培养基的各孔中。 </li><li> 在具有高感染复数感染每个孔（MOI;每个细胞感染性颗粒的数量），以确保所有的细胞被感染。该协议使用3的MOI。 <ol><li>稀释病毒储存在无血清培养基以MOI的浓度= 3每100微升。添加100μL的病毒稀释度的各七个不同的孔中，并在37℃孵育2小时。 </li><li>从所有孔吸媒体，并加入0.5毫升完整的媒体，每孔，轻轻摇摆，然后吸洗两次。 </li></ol></li><li>加入1毫升的complete介质到每个孔中，并孵育在37℃，直到所希望的时间点。 </li><li>在2，4，6，8，10，24和48小时后的感染（每个时间点1孔）收集病毒滴定样本。在阱转移媒体700μL到低温储存管。轻轻移动整个井橡皮刮刀刮去细胞进入剩余的上清液。小区/上清液混合物转移到含有上清液的700微升相应低温储存管。 注意:确保不要从一个刮擦造成污染样品中飞溅期间以及上清液到另一个。转让之前，刮将最大限度地减少飞溅的700微升。 </li><li>在-80℃，将样品直到所有样品的采集。 </li><li>冷冻/解冻样品三次，并通过离心将样品在1200 xg离心在4℃下除去细胞碎片5分钟。 </li><li>滴定病毒作为第4节及以上牛逼比较生长动力学我。 </li></ol> 6.评估微小RNA靶向特异性:病毒传播的测定使用合成的microRNA模仿注:一种合成的microRNA模拟的使用进行显示的microRNA特异性:微RNA为目标的互动。 <ol><li>包括模拟转染，阴性对照microRNA的模拟和实验的microRNA模仿只能控制每一个实验，以评估微小RNA介导的毒性。这也是理想，包括阳性对照（ 即微小RNA靶向基因或基因组）的microRNA模仿的低转染效率可能会导致假阴性。 </li><li>板H1-HeLa细胞在96孔组织培养板上，使得它们在转染的时间（24小时后播种）80-90％汇合。在DMEM板细胞以0.1ml每孔补充有10％胎牛血清。 注意:对于许可不表达同源的microRNA病毒复制的细胞执行此法。 </li><li>暖日Ë转染试剂室温。结合9微升无血清培养基，200nM的每微小RNA模拟库存以及终浓度，0.18微升升压试剂，0.18微升转染试剂混合。在室温下孵育该混合物2-5分钟。 注:所列的卷每口井。建议在一个主混合物被组装为所有孔的转染保持一致性和最小化移液误差。微RNA模拟物的最佳浓度会有所不同。参考随附的微小RNA的制造商的说明模仿一个合理的起始浓度。这将一般为5-200纳米的最终浓度。 </li><li> 从油井吸媒体，并添加92微升的新鲜完全培养基。 注意:如果有样本的显著数，完成组装转染溶液之前这个步骤，该板在37℃下储存直至准备。 </li><li>加入整个转染混合物我n步6.3到细胞逐滴。 </li><li>孵育在37℃下6小时。 </li><li> 感染每口井在低MOI，以确保细胞的比例很低被感染，使病毒传播的分析。该协议使用的0.2的MOI。 <ol><li>稀释病毒储存在无血清培养基以MOI的浓度= 0.2每100微升。从井中取出介质，每孔加入病毒稀释的100微升。 </li><li>孵育在37℃下2小时。 </li></ol></li><li>从每孔吸媒体和补充新鲜完整的媒体的100微升。 </li><li>孵育在37℃进行20-22小时。 </li><li> 确定上清液中的病毒滴定。 <ol><li>收集从每个孔的上清液，用100μL的新鲜完全培养基更换。 注意:如果使用悬浮细胞，在100微升新鲜完全培养基的接下来的步骤重悬细胞沉淀，并返回到用于活力测定样品井。 </li><li>删除cellu通过在4℃以300 xg离心离心5分钟从收集的上清液拉尔碎屑。 </li><li>转移澄清的上清液到新的管中，并在如第4描述不表达同源微RNA允许细胞滴定感染性病毒。 注:保持冰的所有样本，以防止感染的损失。 </li></ol></li><li> 确定细胞的生存能力。 <ol><li>添加MTT10μL的（3-（4,5-二dimethylthiazolyl-2）-2,5-二苯基溴化）每孔试剂。 </li><li>孵育在37℃下2-4小时，直到紫色沉淀是可见的。 </li><li>加100μL洗涤剂试剂。 </li><li>在室温下孵育在黑暗中为2小时。 </li><li>在570 nm处读取所有井的吸收。 注:规范化所有样品模拟转染细胞百分比细胞活力的比较。 </li></ol></li></ol>

The authors have nothing to disclose.

设计，组合物和病毒基因组中的微小RNA应答元件的定位将决定靶向功效和特异性。优化这些将需要试验和错误。然而，设计合理的基础上的RNA结构分析，并在该技术的实施病毒复制和微RNA特征助剂以最少的优化10，11，12，13，38之前的研究。 当发起RE的设...

一个广泛适用，方便，有效的方法，为工程受限取向载体的发展提供了重大机遇，以提高安全性，生物的理解和病毒的治疗效用。一些机制来瞄准病毒嗜包括转导，转录和翻译基础的技术。然而，这些方法都没有普遍适用于所有的载体系统，可能需要在靶细胞缺陷信号传导途径，或需要大的编码序列插入到病毒基因组中。此外，可能会导致该病毒的衰减这些方法中，显著妨碍它们的治疗活性和限制性洞察未修改的系统。 微小RNA是小的（22-25个核苷酸），介导基因沉默在真核细胞中的非编码RNA。通过结合在信使RNA互补的靶序列（反应元件）（mRNA）的微RNA resulti功能<html>纳克在成绩单不稳定，降解或翻译抑制。微RNA通常结合具有部分互补性反应的元件，基因表达的1，2，3，4，5，得到小的修改。在基因表达更显著改变可通过增加反应元件6的互补性来实现。数千成熟微RNA的已在各种细胞和组织类型7，8，9的多种物种和许多表现出差异表达模式的已确定。这些微小RNA签名可通过将完全互补的反应元件到病毒基因组10被利用为病毒扩增的细胞特异性的限制，="外部参照"&gt; 11，12，13。这种微小RNA靶向技术的总体目标是控制一个载体基因组的取向，而不附加衰减。 这种方法用于调节病毒嗜性的实用程序最初被证实在慢病毒载体，以限制在特定的组织14，15，16的转基因表达。这种技术随后被应用于复制型或非复制型病毒载体增强基因治疗以及通过消除在正常组织10，11，12，13不希望的毒性，改善的许多溶瘤病毒的安全性概况，17的繁多。它也被用来生成安全和电子ffective减毒活疫苗，以及改善病毒和疫苗制造工艺18，19，20，21。微RNA靶向载体的可允许在接种疫苗的主机或目标系统的衰减，同时保持生产者系统的野生型的增长水平。微RNA靶向也可以使用通过在一个物种（ 例如人类）限制传输，同时保持其他主机22传输来改善病毒用于研究目的的生物安全。最后，微小RNA-定位可以允许深度通过分离病毒生长23，24，25，26分析病毒的生命周期和发病机理和免疫细胞类型的特定角色。 这种技术提供一个alternative定位方法，很容易实现的，并适用于所有病毒系统。此外，在特定的细胞类型差异表达模式成熟的microRNA的不断扩大，使收集这种技术高度灵活。微RNA为基础的定位已被证明有效的，适用于各种病毒的系统，而不会影响系统的功能。该技术的主要局限性包括试验和错误的优化，为逃逸突变的可能性，并在内源性转录物的潜在的脱靶效应。但是，这些限制一般都可以与优化，合理的反应元件设计克服。正链RNA病毒的趋向是特别响应于微小RNA-靶向由于它们的基因组的正义方向和转录到微小RNA机械的完全细胞质复制周期中的可用性。在这里，我们描述了一个协议，用于产生微小RNA靶向小核糖核酸病毒和experimental方法来验证体外靶向的效率和特异性。

<table><tbody><tr><td>RE encoding Oligonucleotides</td><td>IDT</td><td>PAGE-Purified Ultramer</td><td>Sequence Designed by Investigator</td></tr><tr><td>Oligonucleotides encoding unique restriction site</td><td>IDT</td><td>25nM</td><td>Sequence Designed by Investigator</td></tr><tr><td>Expand High Fidelity PCR Kit</td><td>Sigma Aldrich</td><td>11732641001</td><td>Many other High Fidelity Polymerase PCR kits available</td></tr><tr><td>T4 DNA Ligase System</td><td>NEB</td><td>M0202S</td><td></td></tr><tr><td>MEGAscript Kit</td><td>ThermoFisher Scientific</td><td>AM1333</td><td></td></tr><tr><td>MEGAclear Kit</td><td>ThermoFisher Scientific</td><td>AM1908</td><td></td></tr><tr><td>0.5 M EDTA</td><td>ThermoFisher Scientific</td><td>AM9260G</td><td>RNase-free</td></tr><tr><td>5 M NH&lt;sub&gt;4&lt;sub&gt; Acetate</td><td>ThermoFisher Scientific</td><td>N/A</td><td>Comes in MEGAclear Kit</td></tr><tr><td>Ethanol</td><td>ThermoFisher Scientific</td><td>BP2818100</td><td></td></tr><tr><td>Nuclease-free Water</td><td>Fisher Scientific</td><td>AM9938</td><td></td></tr><tr><td>TransIT-2020 Transfection Reagent</td><td>Mirus</td><td>MIR 5404</td><td></td></tr><tr><td>TransIT-mRNA Transfection Reagent</td><td>Mirus</td><td>MIR 2225</td><td></td></tr><tr><td>0.2 &amp;mu;m syringe filter</td><td>Millipore</td><td>SLGP033RS</td><td></td></tr><tr><td>2 ml Screw-Cap Tubes</td><td>Sarstedt</td><td>72.694.005</td><td></td></tr><tr><td>Cell Scrapers</td><td>Fisher Scientific</td><td>08-100-241</td><td></td></tr><tr><td>MicroRNA Mimics</td><td>Dharmacon</td><td>Varied</td><td></td></tr><tr><td>MTT Cell Proliferation Assay</td><td>ATCC</td><td>30-1010K</td><td></td></tr><tr><td>Subcloning Efficiency DH5&amp;alpha; Competent Cells</td><td>ThermoFisher Scientific</td><td>18265017</td><td></td></tr><tr><td>pBlueScript II Vectors</td><td>Agilent Technologies</td><td>Variable (e.g. 212205)</td><td>There are different plasmids with T7 or T3 promoters and variable cloning sites to enable cloning and RNA transcription.</td></tr></tbody></table>

microrna-based regulation of picornavirus tropism

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and understanding the biological properties of viruses. There are several mechanisms for regulating viral tropism, however they tend to be virus class specific and many result in virus attenuation. Additionally, many viruses, including picornaviruses, exhibit size constraints that do not allow for incorporation of large amounts of foreign genetic material required for some targeting methods. MicroRNAs are short, non-coding RNAs that regulate gene expression in eukaryotic cells by binding complementary target sequences in messenger RNAs, preventing their translation or accelerating their degradation. Different cells exhibit distinct microRNA signatures and many microRNAs serve as biomarkers. These differential expression patterns can be exploited for restricting gene expression in cells that express specific microRNAs while maintaining expression in cells that do not. In regards to regulating viral tropism, sequences complementary to specific microRNAs are incorporated into the viral genome, generally in the 3' non-coding regions, targeting them for destruction in the presence of the cognate microRNAs thus preventing viral gene expression and/or replication. MicroRNA-targeting is a technique that theoretically can be applied to all viral vectors without altering the potency of the virus in the absence of the corresponding microRNAs. Here we describe experimental methods associated with generating a microRNA-targeted picornavirus and evaluating the efficacy and specificity of that targeting in vitro. This protocol is designed for a rapidly replicating virus with a lytic replication cycle, however, modification of the time points analyzed and the specific virus titration readouts used will aid in the adaptation of this protocol to many different viruses.

表1表示对于一个小核糖核酸病毒典型滴定测定的结果，并说明如何计算50％组织培养感染剂量。在这个手稿所述病毒嗜性的基于微RNA调控的整体概念的示意图期间胞内相互作用，用于退火和质粒的插入响应元件的寡核苷酸的适当的设计示于图1的微RNA的取向，以响应元件，和一个编码用于体外转录的微小RNA靶向病毒基因组的质粒DNA的图<strong...

Watch this Scientific Journal Video about MicroRNA-based Regulation of Picornavirus Tropism at JoVE.com

MicroRNA-based Regulation of Picornavirus Tropism

We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle.

小核糖核酸病毒取向的微小RNA基于调控

Cell-specific restriction of viral replication without concomitant attenuation can benefit vaccine development, gene therapy, oncolytic virotherapy, and ...

microrna-based-regulation-of-picornavirus-tropism

Research

JoVE Journal

Immunology and Infection

7.5K 次观看.  Mayo Clinic College of Medicine. We describe here a method for regulating picornavirus tropism by incorporating sequences complementary to specific microRNAs into the viral genome. This protocol can be adapted to all different classes of viruses with modifications based upon the length and nature of their life cycle. 

视频: MicroRNA-based Regulation of Picornavirus Tropism

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6, 7. 
MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8.
Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches. 
Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17.

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

反向遗传学感染小鼠诺沃克病毒介导的恢复

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close ...

科研

免疫与感染

Use of a Recombinant Mosquito Densovirus As a Gene Delivery Vector for the Functional Analysis of Genes in Mosquito Larvae

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method requires a more technically demanding operation and involves complicated procedures, especially when used in larvae due to their small size, relatively thin and fragile cuticle, and high mortality, which limit its application. In contrast, viral vectors for gene delivery have been developed to surmount extracellular and intracellular barriers. These systems have the advantages of easy manipulation, high gene transduction efficiency, long-term maintenance of gene expression, and the ability to produce persistent effects in vivo. Mosquito densoviruses (MDVs) are mosquito-specific, small single-stranded DNA viruses that can effectively deliver foreign nucleic acids into mosquito cells; however, the replacement or insertion of foreign genes to create recombinant viruses typically causes a loss of packaging and/or replication abilities, which is a barrier to the development of these viruses as delivery vectors.
Herein, we report using an artificial intronic small-RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. Detailed procedures for the construction, packaging and quantitative analysis of the rAaeDV vectors, and for larval infection are described.
This study demonstrates, for the first time, the feasibility of developing a non-defective recombinant MDV micro RNA (miRNA) expression system, and thus providing a powerful tool for the functional analysis of genes in mosquito and establishing a basis for the application of viral paratransgenesis for controlling mosquito-borne diseases. We demonstrated that Aedes albopictus 1st instar larvae could be easily and effectively infected by introducing the virus into the water body of the larvae breeding site and that the developed rAaeDVs could be used to overexpress or knock down the expression of a specific target gene in larvae, providing a tool for the functional analysis of mosquito genes.

We report using an artificial intronic small RNA expression strategy to develop a non-defective recombinant Aedes aegypti densovirus (AaeDV) in vivo delivery system. A detailed procedure for the construction, packaging, and quantitative analysis of the rAaeDV vectors as well as for larval infection is described.

应用重组蚊子核作为基因载体, 用于蚊幼虫基因功能分析

In vivo microinjection is the most commonly used gene transfer technique for analyzing the gene functions in individual mosquitoes. However, this method ...

发育生物学

Potato Virus X-Based microRNA Silencing (VbMS) In Potato.

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has been developed and applied for various plant species including Nicotiana benthamiana, tomato, Arabidopsis, cotton, and monocot plants such as wheat and maize. Here, we describe a detailed protocol using PVX-based VbMS vectors to silence endogenous miRNAs in potato. To knock down the expression of a specific miRNA, target mimic (TM) molecules of miRNA of interest are designed, integrated into plant virus vectors, and expressed in potato by Agrobacterium infiltration to bind directly to the endogenous miRNA of interest and block its function.

Potato Virus X-Based microRNA Silencing VbMS In Potato.

We present a detailed protocol for potato virus X (PVX)-based microRNA silencing (VbMS) system to functionally characterize endogenous microRNAs (miRNAs) in potato. Target mimic (TM) molecules of miRNA of interest are integrated into the PVX vector and transiently expressed in potato to silence the target miRNA or miRNA family.

马铃薯病毒基于马铃薯的X型微RNA沉默（VbMS）。

Virus-based microRNA silencing (VbMS) is a rapid and efficient tool for functional characterization of microRNAs (miRNAs) in plants. The VbMS system has ...

环境科学

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

从救援的cDNA重组新城疫病毒

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Monitoring Activation of the Antiviral Pattern Recognition Receptors RIG-I And PKR By Limited Protease Digestion and Native PAGE

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, the PRRs RIG-I and PKR bind to specific viral RNA ligands. This first mediates conformational switching and oligomerization, and then enables activation of an antiviral interferon response. While methods to measure antiviral host gene expression are well established, methods to directly monitor the activation states of RIG-I and PKR are only partially and less well established.
Here, we describe two methods to monitor RIG-I and PKR stimulation upon infection with an established interferon inducer, the Rift Valley fever virus mutant clone 13 (Cl 13). Limited trypsin digestion allows to analyze alterations in protease sensitivity, indicating conformational changes of the PRRs. Trypsin digestion of lysates from mock infected cells results in a rapid degradation of RIG-I and PKR, whereas Cl 13 infection leads to the emergence of a protease-resistant RIG-I fragment. Also PKR shows a virus-induced partial resistance to trypsin digestion, which coincides with its hallmark phosphorylation at Thr 446. The formation of RIG-I and PKR oligomers was validated by native polyacrylamide gel electrophoresis (PAGE). Upon infection, there is a strong accumulation of RIG-I and PKR oligomeric complexes, whereas these proteins remained as monomers in mock infected samples.
Limited protease digestion and native PAGE, both coupled to western blot analysis, allow a sensitive and direct measurement of two diverse steps of RIG-I and PKR activation. These techniques are relatively easy and quick to perform and do not require expensive equipment.

Innate defenses to virus infections are triggered by pattern recognition receptors (PRRs). The two cytoplasmic PRRs RIG-I and PKR bind to viral signature RNAs, change conformation, oligomerize, and activate antiviral signaling. Methods are described which allow to conveniently monitor the conformational switching and the oligomerization of these cytoplasmic PRRs.

在抗病毒模式识别受体RIG-I和PKR通过有限蛋白酶消化和Native PAGE监测激活

Host defenses to virus infection are dependent on a rapid detection by pattern recognition receptors (PRRs) of the innate immune system. In the cytoplasm, ...

A Reporter Assay to Analyze Intronic microRNA Maturation in Mammalian Cells

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves different RNA-binding proteins in the nucleus. Mature miRNAs frequently mediate gene silencing, and this has become an important tool for comprehending post-transcriptional events. Besides that, it can be explored as a promising methodology for gene therapies. However, there is currently a lack of direct methods for assessing miRNA expression in mammalian cell cultures. Here, we describe an efficient and simple method that aids in determining miRNA biogenesis and maturation through confirmation of its interaction with target sequences. Also, this system allows the separation of exogenous miRNA maturation from its endogenous activity using a doxycycline-inducible promoter capable of controlling primary miRNA (pri-miRNA) transcription with high efficiency and low cost. This tool also allows modulation with RNA-binding proteins in a separate plasmid. In addition to its use with a variety of different miRNAs and their respective targets, it can be adapted to different cell lines, provided these are amenable to transfection.

We developed an intronic microRNA biogenesis reporter assay to be used in cells in vitro with four plasmids: one with intronic miRNA, one with the target, one to overexpress a regulatory protein, and one for Renilla luciferase. The miRNA was processed and could control luciferase expression by binding to the target sequence.

一种报告基因检测法，用于分析哺乳动物细胞中内旋微RNA成熟

MicroRNAs (miRNAs) are short RNA molecules that are widespread in eukaryotes. Most miRNAs are transcribed from introns, and their maturation involves ...

癌症研究

Genome-wide Screen for miRNA Targets Using the MISSION Target ID Library

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, genome-wide cDNA library cloned into the 3'UTR downstream from the dual-selection fusion protein, thymidine kinase-zeocin (TKzeo). The first round of selection is for stable transformants, followed with introduction of a miRNA of interest, and finally, selecting for cDNAs containing the miRNA's target. Selected cDNAs are identified by sequencing (see Figure 1-3 for Target ID Library Workflow and details).
To ensure broad coverage of the human transcriptome, Target ID Library cDNAs were generated via oligo-dT priming using a pool of total RNA prepared from multiple human tissues and cell lines. Resulting cDNA range from 0.5 to 4 kb, with an average size of 1.2 kb, and were cloned into the p3&#900;TKzeo dual-selection plasmid (see Figure 4 for plasmid map). The gene targets represented in the library can be found on the Sigma-Aldrich webpage. Results from Illumina sequencing (Table 3), show that the library includes 16,922 of the 21,518 unique genes in UCSC RefGene (79%), or 14,000 genes with 10 or more reads (66%).

The Target ID Library is a plasmid-based, genome-wide collection of cloned cDNA used to identify miRNA targets. Here we demonstrate its use and application.

miRNA的目标使用的任务目标ID库的全基因组屏幕

The Target ID Library is designed to assist in discovery and identification of microRNA (miRNA) targets. The Target ID Library is a plasmid-based, ...

生物学

Identifying Targets of Human microRNAs with the LightSwitch Luciferase Assay System using 3'UTR-reporter Constructs and a microRNA Mimic in Adherent Cells

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes. More than 700 human miRNAs have been identified so far with each having up to hundreds of unique target mRNAs. Computational tools, expression and proteomics assays, and chromatin-immunoprecipitation-based techniques provide important clues for identifying mRNAs that are direct targets of a particular miRNA. In addition, 3'UTR-reporter assays have become an important component of thorough miRNA target studies because they provide functional evidence for and quantitate the effects of specific miRNA-3'UTR interactions in a cell-based system. To enable more researchers to leverage 3'UTR-reporter assays and to support the scale-up of such assays to high-throughput levels, we have created a genome-wide collection of human 3'UTR luciferase reporters in the highly-optimized LightSwitch Luciferase Assay System. The system also includes synthetic miRNA target reporter constructs for use as positive controls, various endogenous 3'UTR reporter constructs, and a series of standardized experimental protocols.
Here we describe a method for co-transfection of individual 3'UTR-reporter constructs along with a miRNA mimic that is efficient, reproducible, and amenable to high-throughput analysis.

MicroRNAs (miRNAs) are important regulators of gene expression and have been shown to play a role in numerous biological processes. To better understand miRNA-UTR interactions, we have created a genome-wide collection of 3 UTR luciferase reporters paired with a novel luciferase gene and assay reagent, the LightSwitch system.

模仿人类小分子RNA的LightSwitch萤光素酶检测系统使用3&#39;UTR记者构造的microRNA识别目标在贴壁细胞

MicroRNAs (miRNAs) are important regulators of gene expression and play a role in many biological processes.  More than 700 human miRNAs have been ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

虫媒病毒感染作为鉴定病毒免疫调节剂和宿主抗病毒因子的筛选工具

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Confocal Imaging of Double-Stranded RNA and Pattern Recognition Receptors in Negative-Sense RNA Virus Infection

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors (PRRs) such as the retinoic acid (RIG-I) like receptors (RLRs) RIG-I and melanoma differentiation-associated protein 5 (MDA-5) leads to the induction of the innate immune response. The formation and intracellular distribution of dsRNA in positive-sense RNA virus infection has been well characterized by microscopy. Many negative-sense RNA viruses, including some arenaviruses, trigger the innate immune response during infection. However, negative-sense RNA viruses were thought to produce low levels of dsRNA, which hinders the imaging study of PRR recognition of viral dsRNA. Additionally, infection experiments with highly pathogenic arenaviruses must be performed in high containment biosafety level facilities (BSL-4). The interaction between viral RNA and PRRs for highly pathogenic RNA virus is largely unknown due to the additional technical challenges that researchers need to face in the BSL-4 facilities. Recently, a monoclonal antibody (Mab) (clone 9D5) originally used for pan-enterovirus detection has been found to specifically detect dsRNA with a higher sensitivity than the traditional J2 or K1 anti-dsRNA antibodies. Herein, by utilizing the 9D5 antibody, we describe a confocal microscopy protocol that has been used successfully to visualize dsRNA, viral protein and PRR simultaneously in individual cells infected by arenavirus. The protocol is also suitable for imaging studies of dsRNA and PRR distribution in pathogenic arenavirus infected cells in BSL4 facilities.

Double-stranded RNA produced during RNA virus replication can be recognized by pattern recognition receptors to induce an innate immune response. For negative-sense RNA viruses, the interaction between the low-level dsRNA and PRRs remains unclear. We have developed a confocal microscopy method to visualize arenavirus dsRNA and PRR in individual cells.

微链 rna 的共聚焦成像和模式识别受体在负感 rna 病毒感染中的应用

Double-stranded (ds) RNA is produced as a replicative intermediate during RNA virus infection. Recognition of dsRNA by host pattern recognition receptors ...