Name: a high-throughput cre-lox activated viral membrane fusion assay to identify inhibitors of hiv-1 viral membrane fusion
Uploaded: 2018-08-14T14:00:00
Description: Watch this Scientific Journal Video about A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion at JoVE.com

这项研究得到了 nih/NIAID AI112423 和 nih/研究院 GM113885 给本杰明. 陈和 nih/NIAID K08-AI120806 的资助。我们想感谢在西奈山院长的流式细胞术核心的伊坎医学院。

1. 目标细胞线的生成
注意: 此步骤是可选的;如果使用现有的 RG 目标单元格行, 请从步骤2开始。
<ol><li>Cotransfect 70% 汇合 10 cm 板293T 细胞11 [在10毫升 Dulbecco 的被改进的老鹰的媒介 (DMEM) 与10% 胎牛血清 (血清)] 与 pMSCV-loxp-dsRed-loxp-eGFP-普洛 WPRE10和 pCL-10A112 (包装质粒) 在一个1:1 比例 (20 µg 总) 使用钙基转染13。...

<ol>
	<li>Esposito, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high+throughput+Cre-lox+activated+viral+membrane+fusion+assay+identifies+pharmacological+inhibitors+of+HIV+entry.">A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry.</a> Virology. 490, 6-16 (2016).</li><li>Cavrois, M., De Noronha, C., Greene, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+sensitive+and+specific+enzyme-based+assay+detecting+HIV-1+virion+fusion+in+primary+T+lymphocytes.">A sensitive and specific enzyme-based assay detecting HIV-1 virion fusion in primary T lymphocytes.</a> Nature Biotechnology. 20, 1151-1154 (2002).</li><li>Huerta, L., Lamoyi, E., Baez-Saldana, A., Larralde, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+immunodeficiency+virus+envelope-dependent+cell-cell+fusion:+a+quantitative+fluorescence+cytometric+assay.">Human immunodeficiency virus envelope-dependent cell-cell fusion: a quantitative fluorescence cytometric assay.</a> Cytometry. 47, 100-106 (2002).</li><li>Sakamoto, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+HIV+cell-cell+fusion+assay+by+using+two+genetically+modified+HeLa+cell+lines+and+reporter+gene.">Establishment of an HIV cell-cell fusion assay by using two genetically modified HeLa cell lines and reporter gene.</a> Journal of Virological Methods. 114, 159-166 (2003).</li><li>Herschhorn, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+inducible+cell-cell+fusion+system+with+integrated+ability+to+measure+the+efficiency+and+specificity+of+HIV-1+entry+inhibitors.">An inducible cell-cell fusion system with integrated ability to measure the efficiency and specificity of HIV-1 entry inhibitors.</a> PLoS One. 6, e26731 (2011).</li><li>Swartz, T. H., Esposito, A. M., Durham, N. D., Hartmann, B. M., Chen, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=P2X-selective+purinergic+antagonists+are+strong+inhibitors+of+HIV-1+fusion+during+both+cell-to-cell+and+cell-free+infection.">P2X-selective purinergic antagonists are strong inhibitors of HIV-1 fusion during both cell-to-cell and cell-free infection.</a> Journal of Virology. 88, 11504-11515 (2014).</li><li>Marin, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+HIV-Cell+Fusion+Assay+for+Discovery+of+Virus+Entry+Inhibitors.">High-Throughput HIV-Cell Fusion Assay for Discovery of Virus Entry Inhibitors.</a> Assay and Drug Development Technologies. 13, 155-166 (2015).</li><li>Giroud, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+and+Functional+Profiling+of+Small-Molecule+HIV-1+Entry+and+Fusion+Inhibitors.">Screening and Functional Profiling of Small-Molecule HIV-1 Entry and Fusion Inhibitors.</a> Assay and Drug Development Technologies. 15, 53-63 (2017).</li><li>Hubner, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequence+of+human+immunodeficiency+virus+type+1+(HIV-1)+Gag+localization+and+oligomerization+monitored+with+live+confocal+imaging+of+a+replication-competent,+fluorescently+tagged+HIV-1.">Sequence of human immunodeficiency virus type 1 (HIV-1) Gag localization and oligomerization monitored with live confocal imaging of a replication-competent, fluorescently tagged HIV-1.</a> Journal of Virology. 81, 12596-12607 (2007).</li><li>Koo, B. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Controlled+gene+expression+in+primary+Lgr5+organoid+cultures.">Controlled gene expression in primary Lgr5 organoid cultures.</a> Nature Methods. 9, 81-83 (2011).</li><li>Pear, W. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+high-titer+helper-free+retroviruses+by+transient+transfection.">Production of high-titer helper-free retroviruses by transient transfection.</a> Proceedings of the National Academy of Sciences. 90, 8392-8396 (1993).</li><li>Naviaux, R. K., Costanzi, E., Haas, M., Verma, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pCL+vector+system:+Rapid+production+of+helper-free,+high+titer,+recombinant+retroviruses.">The pCL vector system: Rapid production of helper-free, high titer, recombinant retroviruses.</a> Journal of Virology. 70, 5701-5705 (1996).</li><li>Kingston, R. E., Chen, C. A., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+Phosphate+Transfection.">Calcium Phosphate Transfection.</a> Current Protocols in Immunology. 10, 10-13 (2001).</li></ol>

地球复兴社区的实验证明对筛选可能抑制病毒复制融合步骤的药物候选者非常有用。在执行此检测时, 获得良好信号的最重要步骤类似于大多数病毒感染化验。第一个关键步骤是产生高质量的病毒的高滴度。这一步要求293T 细胞频繁传代 (至少每48小时 1x), 这样它们就不会变成 overconfluent 并聚集在一起。此外, 它可能值得尝试不同的转染方法, 因为一些研究人员发现, 磷酸钙转染提供了更好的结果, 而...

新的抗逆转录病毒疗法的需要, 促使开发高通量的屏幕抑制剂的 HIV-1 进入。地球复兴社区报告的研究是为了识别细胞-细胞感染系统融合步骤中病毒进入的抑制剂, 具体测量与宿主细胞膜1的病毒膜融合。研究了一种新的抑制剂的筛选, 具体表现在 HIV-1 感染的早期阶段, 直至病毒膜融合点。测量细胞感染的一个挑战是最初的接种含有受感染的供体细胞和 ininfected 靶细胞, 因此测量新的感染很难从输入细胞中辨别出来。理想的系统将涉及一个外源基因标记, 可诱导的目标细胞感染的启动, 并没有出现在捐献细胞。一种流行的病毒含量混合检测基于病毒蛋白-酶融合, 砰-Vpr, 可以有效, 但有限制高通量, 细胞筛选2。这种检测包括一个β-酶 (砰) 报告基因, 融合到 HIV-1 Vpr, 包装成病毒, 并交付到靶细胞后, 病毒膜融合。基质 CCF2-AM 被装入靶细胞的细胞质中, 在卵裂时进行荧光转移。CCF2-AM 基板成本高昂, 可用于高吞吐量屏幕。为了区分供体和靶细胞, 有必要对目标种群进行染色标记。最后, 该协议需要几个洗涤和孵化步骤, 这可能是繁重和昂贵的测试大量的化合物。
以地球复兴社区为解决这些问题的方法, 开发了高通量的细胞间融合抑制剂筛选。此系统不需要将基板加载到单元格中。该检测也可用于无细胞研究, 并适用于其他病毒融合研究使用伪类型 HIV-1 地球复兴社区病毒粒子。艾滋病病毒的细胞融合检测可以测量病毒的 HIV-1 蛋白介导的细胞融合, 然而这些不使用实际的病毒粒子作为地球复兴社区的实验3,4,5。这种检测可以用流式细胞术或荧光显微镜阅读。它已成功地用于筛选 FDA 图书馆以及一个小图书馆的嘌呤抑制剂1,6。其他调查人员还通过一种经过改进和优化的高通量融合检测发现了 HIV-1 融合中的嘌呤抑制剂, 报告将病毒封装的β-酶转移到细胞质7、8.
地球复兴社区病毒的设计与 iGFP 病毒类似的方法, 插入 recombinase 之间的基质和衣壳, 两侧由 HIV-1 蛋白酶网站9。当 HIV-1 蛋白酶在新生的病毒微粒中激活时, 该酶是作为插入在 polyprotein 中的前驱体而制成的。因此,通过病毒膜融合过程将蛋白酶活化的 HIV-1 粒子的含量传递到目标细胞中, 该方法的交付是依赖的。这里提供了两个版本的协议。首先使用 HIV-1 感染的人群感染靶细胞, 研究 HIV 的直接细胞间传播。第二个版本使用无细胞病毒来研究无细胞病毒感染。细胞对细胞的透射检测需要7天的时间完成从细胞解冻的当天, 或5天, 如果细胞已经解冻和传代。如果细胞需要解冻, 如果细胞解冻和传代, 可以在5天内进行无细胞感染检测。还可以使用 Clevers 实验室10创建的质粒, 在所需的单元格类型中生成一个 rg (红到绿) 目标单元格线 (如果未使用现有的 rg 目标单元格线)。建议在本试验中采用病毒和病毒表达细胞进行适当的生物安全防护。我们在 BSL2+ 组织培养设施中进行这种检测的传染性部分。细胞固定后, 可在标准流式细胞仪和显微术中进行分析。
在这里, 我们描述了这种检测的应用, 以筛选新的化合物, 抑制 HIV-1 病毒膜融合 (图 1)。嘌呤受体是亲炎症介质。我们的实验室已经证明, 非选择性的嘌呤受体抑制剂作为 HIV-1 病毒膜融合的抑制剂6。我们报告说, 这种检测在高通量的利用可以识别新的 HIV-1 病毒膜融合抑制剂。我们证明, 嘌呤类受体抑制剂是一种新型的 HIV-1 病毒膜融合抑制剂。

<table>
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)</td>
			<td>Sigma Aldrich</td>
			<td>D5546</td>
			<td>Media for 293 Cells</td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>Sigma Aldrich</td>
			<td>R0883</td>
			<td>Media for Jurkats</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum Albumin</td>
			<td>Gibco</td>
			<td>16140-071</td>
			<td>Serum for 293 Cells</td>
		</tr>
		<tr>
			<td>Cosmic Calf Serum</td>
			<td>Hyclone</td>
			<td>SH30087.03</td>
			<td>Serum for Jurkat Cells</td>
		</tr>
		<tr>
			<td>Hyclone Pennecillin Streptomycin solution</td>
			<td>GE Healthcare Life sciences</td>
			<td>SV30010</td>
			<td>Penn/Strep used in both media</td>
		</tr>
		<tr>
			<td>T75 Flasks</td>
			<td>Corning</td>
			<td>3073</td>
			<td>Used for Culture of Jurkat Cells</td>
		</tr>
		<tr>
			<td>10cm Tissue Culture Plates</td>
			<td>Corning</td>
			<td>430167</td>
			<td>Used for Culture of 293 Cells</td>
		</tr>
		<tr>
			<td>96 Well Plates (tissue culture Treated)</td>
			<td>Corning</td>
			<td>3595</td>
			<td>Used for fusion assay</td>
		</tr>
		<tr>
			<td>Polyjet Transfection Reagent</td>
			<td>Signagen</td>
			<td>SL100688</td>
			<td>Used to transfect 293 Cells</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Sigma Aldrich</td>
			<td>D8537-100ML</td>
			<td>Used in wash steps</td>
		</tr>
		<tr>
			<td>Hyclone Trypsin Protease</td>
			<td>GE Healthcare Life sciences</td>
			<td>SH30042.01</td>
			<td>For Trypsinization of 293 cells</td>
		</tr>
		<tr>
			<td>Amaxa Cell Line Nucleofector Kit V</td>
			<td>Lonza</td>
			<td>VACA-1003</td>
			<td>For Nucleofection of Jurkats</td>
		</tr>
		<tr>
			<td>Ficoll-Paque plus</td>
			<td>GE Healthcare Life sciences</td>
			<td>17144002</td>
			<td>For Nucleofection of Jurkats</td>
		</tr>
		<tr>
			<td>Serological Pipettes</td>
			<td>Fisher Brand</td>
			<td>13-678-11E</td>
			<td>For all tissue culture</td>
		</tr>
		<tr>
			<td>Pipettor Tips</td>
			<td>Denville Scientific</td>
			<td>P3020-CPS</td>
			<td>For all tissue culture and liquid handling steps</td>
		</tr>
		<tr>
			<td>Millex Syringe Filter (0.45 micron)</td>
			<td>Millipore</td>
			<td>SLHA033</td>
			<td>For filtration of virus</td>
		</tr>
		<tr>
			<td>BD Slip Tip Sterile syringes</td>
			<td>BD Diagnostics</td>
			<td>309656</td>
			<td>For filtration of virus</td>
		</tr>
		<tr>
			<td>Amaxa Nucleofector</td>
			<td>Lonza</td>
			<td>2b</td>
			<td>for Nucleofection of Jurkats (various models available)</td>
		</tr>
		<tr>
			<td>BD Fortessa Flow Cytometer</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>for flow cytometry analyss of samples</td>
		</tr>
		<tr>
			<td>Tissue Culture Hood</td>
			<td>Various models</td>
			<td>Fortessa 2</td>
			<td></td>
		</tr>
		<tr>
			<td>pMSCV-loxp-dsRed-loxp-eGFP-Puro-W PRE</td>
			<td>Addgene</td>
			<td>32702</td>
			<td>Koo BK et al. Controlled gene expression in primary Lgr5 organoid cultures. Nature Methods 9:81-83 (2011).</td>
		</tr>
		<tr>
			<td>pCL10a1</td>
			<td>Novus Bio</td>
			<td>NBP2-29542</td>
			<td>Naviaux, RK, Costanzi, E, Haas, M and Verma, I. The pCL vector system: Rapid production of helper-free, high titer, recombinant retroviruses. Journal of Virology 70: 5701-5705 (1996)</td>
		</tr>
		<tr>
			<td>Gag-iCre</td>
			<td>Benjamin Chen Lab</td>
			<td></td>
			<td>Esposito AM et al. A high throughput Cre-lox activated viral membrane fusion assay identifies pharmacological inhibitors of HIV entry. Virology 490:6-16 (2016).</td>
		</tr>
	</tbody>
</table>

a high-throughput cre-lox activated viral membrane fusion assay to identify inhibitors of hiv-1 viral membrane fusion

本试验旨在通过流式细胞术或荧光显微镜检测绿色荧光蛋白 (GFP) 的表达, 具体报告 HIV-1 融合。一个 HIV-1 报告病毒 (HIV-1 地球复兴社区) 是通过插入 recombinase 到 HIV-1 基因组之间的母体和外壳蛋白的 polyprotein。这导致了 recombinase 的包装成病毒微粒, 然后被释放入目标细胞线稳定地表达 recombinase 激活的红色荧光蛋白 (RFP) 到 GFP 交换盒。在基本状态下, 此卡带只表示 RFP。在 recombinase 进入目标细胞后, RFP 由 loxP 站点两侧切除, 导致 GFP 表达。该方法可用于检测无细胞和细胞对细胞感染系统中的任何病毒进入抑制剂 (特别是融合步骤), 并被用来鉴定一类嘌呤受体拮抗剂作为新的 HIV-1 病毒膜融合抑制剂。

未感染的 RG Jurkat 细胞展示了一个低级别的背景 GFP 信号 (0.3%) 与一个非常强的 RFP 信号 (图 2A, 未感染的专栏)。地球复兴社区的感染导致 GFP 信号 (24.9%) 的增加, 而 HIV-1 融合抑制剂 AMD3100 (20 µM) 的存在抑制了这一信号的发展, 使其下降到未感染的背景水平 (0.34%)。当使用反向转录抑制剂 AZT (2 µM) 的后融合事件抑制剂时, 信号不受显著影响 (23.5%), 表明该?...

Watch this Scientific Journal Video about A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion at JoVE.com

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

通过流式细胞术或荧光显微镜检测绿色荧光蛋白的表达, 我们描述了以细胞为基础的 HIV-1 融合报告。它可以用来测试病毒进入的抑制剂 (特别是在融合步骤) 在无细胞和细胞对细胞感染系统。

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

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Current investigations into phage-host interactions are dependent on extrapolating knowledge from (meta)genomes. Interestingly, 60 - 95% of all phage ...

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The Development of Lyophilized Loop-mediated Isothermal Amplification Reagents for the Detection of Coxiella burnetii

冻干环介导等温扩增试剂的检测中的发展<em&gt;贝氏柯克斯体</em

Coxiella burnetii, the agent causing Q fever, is an obligate intracellular bacterium. PCR based diagnostic assays have been developed for detecting C. ...

Measuring Influenza Neuraminidase Inhibition Antibody Titers by Enzyme-linked Lectin Assay

通过酶联凝集素测定来测量神经氨酸酶流感抑制抗体滴度

Antibodies to neuraminidase (NA), the second most abundant surface protein on influenza virus, contribute toward protection against influenza. Traditional ...

Swab Sampling Method for the Detection of Human Norovirus on Surfaces

人类诺沃克病毒的表面上检测拭子采样方法

Human noroviruses are a leading cause of epidemic and sporadic gastroenteritis worldwide. Because most infections are either spread directly via the ...