我们要承认在履行机构的工作人员和科学家，他们在开发和测试这些协议的支持。

1。 293TN细胞与包装质粒转染和表达建设<ol><li>种子7.0 - 8.0×10 6 293TN细胞每15厘米2 20毫升含DMEM培养基辅以4毫米L-谷氨酰胺，4.5 g / L葡萄糖，不含抗生素的胎牛血清（10％）的文化媒介文化板。成长为18-24小时，在37°C 5％CO 2，使转染时细胞密度达到60-80％汇合。在某些情况下，它可能是必要的种子细胞的几个板块，以获得足够高的靶细胞传导滴度。 </li><li>对于每一个细胞板，添加1.6毫升的无血清DMEM蒸压2的毫升Eppendorff管。加入45μL的lentivector pPACKH1或pPACKF1和4.5微克建设吸取了DMEM培养液和混合。 </li><li>添加到同一个管55μLPureFection。涡为10秒。孵育的DMEM-质粒，PureFection混合物在室温温度f或15分钟。 </li><li>添加下拉明智和漩涡，轻轻均匀地分散在整个板块的组织培养板的DMEM培养液，质粒PureFection混合物。返回菜的孵化器和5％的CO 2增长在37°C。 </li><li>有没有必要改变转染后的媒体。如果您lentivector结构表示如GFP荧光蛋白基因编码，检查你的细胞，显微镜下转染后12-24小时。你应该能够看到90％的GFP阳性细胞，表明转染成功。 </li><li>收集到50毫升的无菌离心管细胞培养上清后48和72小时。现在这种细胞培养上清含有传染性pseudoviral颗粒。 （BSL-2类安全工作的建议指引。）离心15分钟，在室温下沉淀细胞碎片1500 XG。病毒上清转移到一个新的管，确保不打扰颗粒。 </li><li> 293TN细胞病毒包装已使用应弃置在生物危害容器，是不被用于进一步几轮病毒包装。 </li></ol> 2。病毒上清的浓度<ol><li>新增1卷，冷（4℃）聚乙二醇病毒降水解慢病毒颗粒含上清，每4卷。大量的慢病毒颗粒的沉淀，可以实现与康宁250毫升聚丙烯离心管。反相几次管混合解决方案，但不涡混合物。 </li><li>冷藏至少12小时的解决方案（长达4天是可以接受的），在4°C。不要摇晃或旋转管在制冷步骤。 </li><li>离心30分钟，1500 XG上清/聚乙二醇混合物在4°C。慢病毒颗粒离心后，会出现如米色或白色颗粒在试管底部。倒出来的supernataNT。 </li><li>取下吸液的所有痕迹，注意不要打扰在颗粒沉淀的慢病毒颗粒。残留的PEG的痕迹，它会稀释病毒（从而降低效价），但不会影响靶细胞的完整性，因为聚乙二醇是惰性的，而不是有毒。 </li><li>用冷（4℃）重悬，并结合原体积的1/100的慢病毒颗粒，无菌磷酸盐缓冲生理盐水或培养液含有25毫米的HEPES缓冲。 </li><li>分装到无菌低温瓶，直到准备使用储存于-70℃。 </li></ol> 3。靶细胞病毒滴定和传导<ol><li>我们建议滴定前转导细胞的利益目标和适当的感染复数（MOI）计算实验之间的一致性的靶细胞的慢病毒颗粒。病毒滴定，我们建议使用HT1080细胞容易感染积极控制线。 PL缓解注意到，滴定协议，涉及与你已经打包的慢病毒颗粒的小等分转导HT1080细胞。滴度是已知的，一旦靶细胞的利益也可以转产生的慢病毒颗粒。一个建议滴定协议，可以发现在： <a href="http://www.systembio.com/downloads/manual_titer_kit_web.pdf" target="_blank">http://www.systembio.com/downloads/manual_titer_kit_web.pdf</a> 7。 </li><li>板50000每口井的目标细胞的利息在24孔板中的细胞培养基。使用这些细胞通常培养英寸在其指定的培养条件下生长细胞过夜的媒体。细胞应该是转天之间的50至70％汇合。 </li><li>转天，吸从细胞中的媒体。培养基与TransDux结合终浓度为1×（例如，2.5μL培养基TransDux到500μL）。然后转移的TransDux-CELL培养基混合，以每口井。 </li><li>吸取了适当的病毒量，对应到每口井的靶细胞的最佳教学语言。如果最佳的教学语言是未知的，我们建议尝试了各种不同的水分（如莫伊容易感染的组织培养细胞，1，2和5和1，10和20为更难以内政部感染原代细胞）。如果慢病毒颗粒的浓度不知道（通过病毒滴定协议确定），不同的病毒量，可以尝试，如1，2和5μL的病毒。病毒与靶细胞可以保持长达72小时的接触。 </li><li>进一步的实验与转靶细胞的利益可能会开始后，转72个小时，一旦病毒结构已整合到宿主细胞基因组。更改的介质，并根据他们的具体需要，通过靶细胞的利益。 </li></ol> 4。代表结果<p类=“jove_step”的293TN细胞转染 293TN细胞密度开始是成功的病毒包装的关键。 293TN细胞必须融合在60 -80％之间的转天（ 图2）。 293TN细胞小于60％汇合，使他们成长为几个小时，然后再尝试转染。如果293TN细胞&gt; 80％汇合，他们应该被转染前replated。 293TN细胞至少有90％如果使用lentivector表达GFP，应该荧光转染后24小时绿色，表明成功转染（ 图3）。如果低于90％的细胞GFP阳性，不进行下一个步骤，因为这表明，没有成功，转染和病毒滴度低。相反，板新293TN细胞并重新启动，使确定，293TN细胞在适当的细胞密度是前染。 293TN细胞可能会拉断的组织培养皿转染后48-72小时。这不会造成负面影响的慢病毒颗粒的生产。 滴定与HT1080细胞履行机构的全球超速滴定试剂盒，根据制造商的指示，可以计算出病毒滴度。该系统采用定量PCR WPRE序列的病毒整合到HT1080细胞的慢病毒载体来衡量和标准曲线进行比较，根据基因组参考序列，精确地测量每毫升传染性单位（图4）。 转导的靶细胞如果使用慢病毒颗粒表达组成性激活的GFP标记，GFP阳性细胞转导12-24小时内开始出现，如果传导反应工作。 GFP表达后应继续稳定的慢病毒结构集成到T他宿主细胞基因组。如果用不同的水分传导，GFP荧光应约匹配的教学语言，在低水分表明GFP荧光和较高的水分表明增加了GFP荧光（ 图5）。 <img src="/files/ftp_upload/3171/3171fig1.jpg" alt="图1" /> 图1。概述病毒的生产协议。混合的lentivector和pPACK包装质粒，并共同为293TN细胞转染。 48 -72小时后，收集病毒上清，用PEG，它沉淀病毒颗粒。滴定慢病毒颗粒后，他们可以使用转导靶细胞的利益。 <a href="http://www.jove.com/files/ftp_upload/3171/3171fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img src="/files/ftp_upload/3171/3171fig2.jpg" alt="图2" /> 图2。期293TN细胞图像的对比度。细胞density应该是60-80％之间融合转一天。 <img src="/files/ftp_upload/3171/3171fig3.jpg" alt="图3" /> 图3。293TN细胞GFP阳性的荧光图像与Purefection，pPACKH1和结构lentivector编码绿色荧光蛋白转染后24小时。 <img src="/files/ftp_upload/3171/3171fig4.jpg" alt="图4" /> 图4。WPRE qPCR的结果和标准曲线，利用SBI的全球超速滴定套件来计算MOI和相应的滴度包装病毒颗粒。 <img src="/files/ftp_upload/3171/3171fig5.jpg" alt="图5" /> 图5。HT1080细胞荧光图像转慢病毒的数量不断增加。图片是从72小时后转。

<ol>
	<li>Cann, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> RNA Viruses. A Practical Approach. , (2000).</li><li>Gould, D. J., Favorov, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vectors+for+the+treatment+of+autoimmune+diseases.">Vectors for the treatment of autoimmune diseases.</a> Gene Therapy. 10, 912-927 (2003).</li><li>Naldini, L., Blomer, U., Gage, F. H., Trono, D., Verma, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transfer,+integration,+and+sustained+long-term+expression+of+the+transgene+in+adult+rat+brains+injected+with+a+lentiviral+vector.">Efficient transfer, integration, and sustained long-term expression of the transgene in adult rat brains injected with a lentiviral vector.</a> Proc. Natl. Acad. Sci. U.S.A. 93, 11382-11388 (1996).</li><li>Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F. 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+vivo+gene+delivery+and+stable+transduction+of+nondividing+cells+by+a+lentiviral+vector.">In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.</a> Science. 272, 263-267 (1996).</li><li>Dull, T., Zufferey, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+third-generation+lentivirus+vector+with+a+conditional+packaging+system.">A third-generation lentivirus vector with a conditional packaging system.</a> J. Virol. 72, 8463-8471 (1998).</li><li>Federico, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Lentivirus Gene Engineering Protocols. , (2003).</li><li>Curran, M. A., Kaiser, S. M., Achacoso, P. L., Nolan, G. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+transduction+of+nondividing+cells+by+optimized+feline+immunodeficiency+virus+vectors.">Efficient transduction of nondividing cells by optimized feline immunodeficiency virus vectors.</a> Mol. Ther. 1, 31-38 (2000).</li><li>Kahlig, K. M., Saridey, S. K., Kaja, A., Daniels, M. A., George, A. L., Wilson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+transposon-mediated+stable+gene+transfer+in+human+cells.">Multiplexed transposon-mediated stable gene transfer in human cells.</a> Proc. Natl. Acad. Sci. U.S.A. 107 (4), 1343-1348 (2010).</li><li>Kim, A., Pyykko, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size+matters:+versatile+use+of+PiggyBac+transposons+as+a+genetic+manipulation+tool.">Size matters: versatile use of PiggyBac transposons as a genetic manipulation tool.</a> Mol. Cell Biochem. , (2011).</li></ol>

生产和自由地进入这个视频文章是由系统Biosciences公司的赞助。

此病毒的包装和对靶细胞的转​​导协议进行了优化与SBI的病毒包装试剂的使用。其他试剂替代，可能会影响协议的结果。该协议只进行了优化慢病毒生产，未必适合生产病毒或pseudoviruses的其他类型。 病毒包装的成功取决于几个关键步骤。 293TN细胞表达SV40大T抗原，分泌到细胞培养上清的慢病毒颗粒，这是绝对必要的。 293TN细胞在转染时的密度，是为了为PureFection试剂，特别是重要的，充分的lentivector和包装质粒进入细胞转移。添加PureFection混合物彻底旋流板下拉明智地组织培养板，是重要的，分散的包装质粒和lentivector构建整个细胞。未能适当分发的载体，可能会导致生产不足的慢病毒颗粒。病毒的包装反应，可以扩大生产高滴度的病毒，组织培养皿的表面面积的基础上。一个可以选择每293TN细胞结构，需要包装的几个组织培养皿板。病毒上清的浓度，尤其是有助于建立高滴度的病毒。可以结合多个板细胞病毒上清，并集中在体内实验中使用的，或难以转导的细胞。 重要的是计算准确的教学语言使用的靶细胞转导滴定产生的慢病毒颗粒。了解教学语言，用于在一个给定的传导，使解决事件，是不会成功的转。例如，使用的教学语言是过低可能会导致在极少数的目标细胞表达的利益结构。使用1教学语言，是太高了，但是，可能会导致靶细胞，表明压力的迹象。我们的目标是使用1内政部最大化的利益结构的表达，同时保持健康的细胞。虽然我们建议qPCR可以基于的滴定协议措施，在HT1080细胞的慢病毒结构的综合副本，也可以用于其他滴定方法，如P24 ELISA或GFP阳性细胞的百分比估计。 成功转导的靶细胞也取决于几个关键因素。 TransDux是一个独特的感染试剂，使转导效率高，但就是没有细胞毒性。可以用来代替TransDux凝聚胺，这往往是有毒的靶细胞。在靶细胞的转​​列入TransDux帮助，以消除病毒颗粒的指控，并允许进入细胞的病毒第由于TransDux是没有毒性的细胞，可以让慢病毒颗粒与细胞接触中保持长达72小时（已整合到宿主细胞基因组病毒结构的时间），从而增加了概率病毒粒子进入细胞。对于一些难以转导的细胞，转导，可重复连续几天，以提高转染效率。例如，转导，第1天的细胞，使细胞得到休息，第2天，然后再次转导第3天的细胞。重要的是等待72小时后，最后传递开始之前选择，进一步的实验或靶细胞的特性。 高效的病毒包装也依赖于慢病毒结构的大小，5&#39;和3&#39;LTR地区之间。慢病毒构造的这一部分应该是大约900 KB或更少，这相当于本地的艾滋病毒基因组的大小。超过5月的9 KB增加生产pseudoviral颗粒滴度。 piggyBac转座子载体，如新技术，可靠，稳定的外源基因整合到100 KB通过一个单一的转让。这种方法可用于超过大小限制的情况下，它是可能的转染靶细胞，慢病毒载体的结构，并提供病毒包装步骤9,10不需要额外的好处。 粒子产生的pseudoviral使用此协议是传染病，因为它们可以感染大多数哺乳动物细胞类型。然而在本协议所使用的产品，是第三代，他们生产的非复制和自我失活pseudoviruses BIOSAFE。因此，一旦转成靶细胞或动物，靶细胞或动物是没有传染性或传染性。其他不属于第三代BIOSAFE lentivector质粒换人是可能的，虽然不是从生物安全的建议的观点。应进行病毒生产协议，并随后转导的靶细胞在生物安全等级2，根据NIH的准则11，和研究人员应该处理的病毒粒子时，总是穿白大褂和手套。使用293TN生产者细胞，病毒颗粒，并一次性移液器管应弃置在适当的生物危害容器。可重复使用的移液器和玻璃器皿应该用漂白粉进行消毒和灭菌，以减少人员暴露。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM高糖培养</td><td> Gibco公司</td><td> 11995-073 </td><td></td></tr><tr><td> 293TN细胞</td><td>履行机构</td><td> LV900A-1 </td><td></td></tr><tr><td> pPACKH1 </td><td>履行机构</td><td> LV500A-1 </td><td>为艾滋病毒为主lentivectors </td></tr><tr><td> pPACKF1 </td><td>履行机构</td><td> LV100A-1 </td><td>为FIV的基于lentivectors </td></tr><tr><td>任何慢病毒载体</td><td>履行机构</td><td>各个</td><td>基因的shRNA，小分子RNA，转录记者</td></tr><tr><td> PureFection </td><td>履行机构</td><td> LV750A-1 LV750A-5 </td><td></td></tr><tr><td>聚乙二醇</td><td>履行机构</td><td> LV810A-1 LV825A-1 </td><td></td></tr><tr><td>全球超速滴定试剂盒</td><td>履行机构</td><td> LV961A-1 </td><td>在这个协议没有说明，但需要滴度病毒颗粒</td></tr><tr><td> TransDux </td><td>履行机构</td><td> LV850A-1 </td><td></td></tr></tbody></table>

packaging hiv- or fiv-based lentivector expression constructs &amp; transduction of vsv-g pseudotyped viral particles

作为标准的载体，它是可能的转染效率低到中等的细胞载体形式的lentivectors获得瞬时表达效应。然而，包装成慢病毒表达pseudoviral颗粒结构，可实现高达100％的转导，甚至小学，干，分化的细胞，如难以转染细胞。此外，慢病毒传递不会产生通常与化学转染，如从转染试剂1，2的毒性导致细胞死亡相关的特定细胞的反应。当进入靶细胞转，慢病毒结构整合到基因组DNA，并提供小发夹RNA（shRNA的），3基因的cDNA，小分子RNA或记者，4稳定表达。可以使用一个可选的标记，如嘌呤或GFP的表达载体的构建中分离靶细胞稳定表达的效应分子。经过PSeudoviral颗粒感染靶细胞，靶细胞内不能复制，因为病毒的结构基因是没有长末端重复序列（公升）被设计成自我失活后转5，6。 有高效的慢病毒包装1，5，6，7所必需的三个主要组成部分。 <ol><li>慢病毒表达载体，其中包含一些包装所需的遗传因素，病毒表达的稳定整合到基因组DNA构建，表达的效应或记者。 </li><li>提供蛋白质必不可少的转录和表达的RNA副本包装的慢病毒包装质粒构建成重组pseudoviral颗粒。此协议使用从艾滋病毒或FIV的基因组和水泡性口炎病毒G蛋白（VSV-G的）pPACK质粒（SBI）的编码为插科打诨，POL，转病毒外壳蛋白。 </li><li> 293TN产生<html> R细胞（来自HEK293细胞）表达SV40大T抗原，这是需要高滴度的慢病毒的生产和新霉素抗性基因，有助于维护细胞重新选择。 </li></ol>病毒生产协议概述图1中可以看出。病毒性生产开始由慢病毒表达载体和包装质粒共转染的的293TN生产者细胞。病毒颗粒分泌到媒体。 48-72小时后收获细胞培养基。从细胞培养基，细胞碎片和病毒颗粒浓度的聚乙二醇，离心沉淀。产生慢病毒颗粒滴度，可用于转导靶细胞。病毒滴定的细节是不包括在此协议，但可以发现： ANK“&gt; http://www.systembio.com/downloads/global_titer_kit_web_090710.pdf 8。 该协议已被优化使用所表示的特定产品。其他试剂可代替，但不能保证相同的结果。

Watch this Scientific Journal Video about Packaging HIV- or FIV-based Lentivector Expression Constructs & Transduction of VSV-G Pseudotyped Viral Particles at JoVE.com

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

慢病毒表达载体的稳定表达不同的效应分子的分裂和非分裂哺乳动物细胞和整个生物体或记者构造最有效的车辆。在这里，我们提供了关于如何打包pseudoviral颗粒lentivector表达结构和转导靶细胞使用pseudoviral颗粒协议。

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 ...

packaging-hiv-or-fiv-based-lentivector-expression-constructs

Research

JoVE Journal

Immunology and Infection

35.0K 次观看.  System Biosciences. 慢病毒表达载体的稳定表达不同的效应分子的分裂和非分裂哺乳动物细胞和整个生物体或记者构造最有效的车辆。在这里，我们提供了关于如何打包pseudoviral颗粒lentivector表达结构和转导靶细胞使用pseudoviral颗粒协议。

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

Genotypic Inference of HIV-1 Tropism Using Population-based Sequencing of V3

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her viral population uses the CCR5 coreceptor for cellular entry, and not an alternative coreceptor. One approach to tropism testing is to examine the sequence of the V3 region of the HIV envelope, which interacts with the coreceptor.
Methods: Viral RNA is extracted from blood plasma. The V3 region is amplified in triplicate with nested reverse transcriptase-PCR. The amplifications are then sequenced and analyzed using the software, RE_Call. Sequences are then submitted to a bioinformatic algorithm such as geno2pheno to infer viral tropism from the V3 region. Sequences are inferred to be non-R5 if their geno2pheno false positive rate falls below 5.75%. If any one of the three sequences from a sample is inferred to be non-R5, the patient is unlikely to respond to a CCR5-antagonist.

HIV tropism can be inferred from the V3 region of the viral envelope. V3 is PCR amplified in triplicate using nested RT-PCR, sequenced, and interpreted using bioinformatic software. Samples with with 1 or more sequence(s) with low g2P scores are classified as non-R5 virus.

V3的HIV - 1取向的基因型推理使用人口为基础的测序

Background: Prior to receiving a drug from CCR5-antagonist class in HIV therapy, a patient must undergo an HIV tropism test to confirm that his or her ...

科研

免疫与感染

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

在艾滋病毒感染者病毒载量低于检测限制标准的临床实验放大和量化HIV - 1 RNA

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

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

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

HIV逆转录酶和整合酶抑制剂的快速筛选

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

Vaccinia Reporter Viruses for Quantifying Viral Function at All Stages of Gene Expression

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. The family also includes the smallpox virus, Variola. Due to the complexity of poxvirus replication, many questions still remain regarding their gene expression strategy. In this article we describe the conceptualization and usage of recombinant vaccinia viruses that enable real-time measurement of single and multiple stages of viral gene expression in a high-throughput format. This is enabled through the use of spectrally distinct fluorescent proteins as reporters for each of three stages of viral replication. These viruses provide a high signal-to-noise ratio while retaining stage specific expression patterns, enabling plate-based assays and microscopic observations of virus propagation and replication. These tools have uses for antiviral discovery, studies of the virus-host interaction, and evolutionary biology.

We describe the usage of a fluorescent reporter vaccinia virus that enables real-time measurement of viral infectivity and gene expression through the stage-specific expression of spectrally distinct reporter fluorophores. We detail a plate-based method for accurately identifying the stage at which virus replication is affected in response to small molecule inhibition.

牛痘病毒记者量化病毒功能在基因表达的各个阶段

Poxviruses are a family of double stranded DNA viruses that include active human pathogens such as monkeypox, molluscum contagiousum, and Contagalo virus. ...

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

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

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

RNA的疗效和毒性的评价基因或药物治疗靶向HIV-1的生产使用

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

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be difficult. To overcome this challenge, a methodology combining indexed flow cytometry and high-throughput multiplexed quantitative polymerase chain reaction (qPCR) was developed. The objective was to identify and characterize simian immunodeficiency virus (SIV)-infected cells present within rhesus macaques. Through quantitation of surface protein by fluorescence-activated cell sorting (FACS) and mRNA by qPCR, virus-infected cells are identified by viral gene expression, which is combined with host gene and protein measurements to create a multidimensional profile. We term the approach, targeted Single-Cell Proteo-transcriptional Evaluation, or tSCEPTRE. To perform the method, viable cells are stained with fluorescent antibodies specific for surface markers used for FACS isolation of a cell subset and/or downstream phenotypic analysis. Single cells are sorted followed by immediate lysis, multiplex reverse transcription (RT), PCR pre-amplification, and high throughput qPCR of up to 96 transcripts. FACS measurements are recorded at the time of sorting and subsequently linked to the gene expression data by well position to create a combined protein and transcriptional profile. To study SIV-infected cells directly ex vivo, cells were identified by qPCR detection of multiple viral RNA species. The combination of viral transcripts and the quantity of each provide a framework for classifying cells into distinct stages of the viral life cycle (e.g., productive versus non-productive). Moreover, tSCEPTRE of SIV+ cells were compared to uninfected cells isolated from the same specimen to assess differentially expressed host genes and proteins. The analysis revealed previously unappreciated viral RNA expression heterogeneity among infected cells as well as in vivo SIV-mediated post-transcriptional gene regulation with single-cell resolution. The tSCEPTRE method is relevant for the analysis of any cell population amenable to identification by expression of surface protein marker(s), host or pathogen gene(s), or combinations thereof.

Single-cell Quantitation of mRNA and Surface Protein Expression in Simian Immunodeficiency Virus-infected CD4+ T Cells Isolated from Rhesus macaques

Described is a methodology to quantitate the expression of 96 genes and 18 surface proteins by single cells ex vivo, allowing for the identification of differentially expressed genes and proteins in virus-infected cells relative to uninfected cells. We apply the approach to study SIV-infected CD4+ T cells isolated from rhesus macaques.

猕猴免疫缺陷病毒感染 CD4+ T 细胞单细胞定量的 mRNA 和表面蛋白的表达

Single-cell analysis is an important tool for dissecting heterogeneous populations of cells. The identification and isolation of rare cells can be ...

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 ...

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 ...

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 ...

评估假型病毒对 SARS-CoV-2 的血清中和

Pseudotyped Viruses As a Molecular Tool to Monitor Humoral Immune Responses Against SARS-CoV-2 Via Neutralization Assay

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in addition to their better-known use in gene therapy for the delivery of a gene of interest. PVs are replication defective because the viral genome is divided into different plasmids that are not incorporated into the PVs. This safe and versatile system allows the use of PVs in biosafety level 2 laboratories. Here, we present a general methodology to produce lentiviral PVs based on three plasmids as mentioned here: (1) the backbone plasmid carrying the reporter gene needed to monitor the infection; (2) the packaging plasmid carrying the genes for all the structural proteins needed to generate the PVs; (3) the envelope surface glycoprotein expression plasmid that determines virus tropism and mediates viral entry into the host cell. In this work, SARS-CoV-2 Spike is the envelope glycoprotein used for the production of non-replicative SARS-CoV-2 pseudotyped lentiviruses.
Briefly, packaging cells (HEK293T) were co-transfected with the three different plasmids using standard methods. After 48 h, the supernatant containing the PVs was harvested, filtered, and stored at -80 &#176;C. The infectivity of SARS-CoV-2 PVs was tested by studying the expression of the reporter gene (luciferase) in a target cell line 48 h after infection. The higher the value for relative luminescence units (RLUs), the higher the infection/transduction rate. Furthermore, the infectious PVs were added to the serially diluted serum samples to study the neutralization process of pseudoviruses' entry into target cells, measured as the reduction in RLU intensity: lower values corresponding to high neutralizing activity.

作者聚焦：研究宿主-病毒与假型病毒的相互作用 

Pseudotyped viruses (PVs) are replication-defective virions that are used to study host-virus interactions under safer conditions than handling authentic viruses. Presented here is a detailed protocol that shows how SARS-CoV-2 PVs can be used to test the neutralizing ability of patients' serum after COVID-19 vaccination.

假型病毒作为通过中和测定监测针对 SARS-CoV-2 的体液免疫反应的分子工具

Pseudotyped viruses (PVs) are molecular tools that can be used to study host-virus interactions and to test the neutralizing ability of serum samples, in ...