hiv 转录和拼接中延迟逆转剂的高通量体外评价
Summary
通过测试干预措施对艾滋病毒转录和剪接的影响, 描述并应用了一种用于艾滋病毒高效再激活和清除的高通量协议。给出了延迟反转剂对 ltr 驱动的转录和剪接的影响的代表性结果。
Abstract
由于存在着大量的细胞, 这些细胞储存着病毒的稳定和潜在形式, 免疫系统对此仍然是看不见的, 而且目前的抗逆转录病毒疗法没有针对艾滋病毒。转录和剪接已被证明可以加强 hiv-1 延迟静止 cd4 + t 细胞。在 “冲击和杀死” 方法中使用潜伏期反转剂 (lra) 引起的潜伏期逆转已被广泛研究, 试图清除这个水库, 但由于缺乏足够的小分子, 可以有效地干扰这个水库。这里介绍的协议提供了一种可靠、高效地评估 hiv 转录和剪接延迟逆转剂 (lra) 的方法。这种方法是基于使用 cytometry 驱动的双色记者, 可以同时测量上帝军的影响转录和剪接流细胞学。这里描述的协议对粘附细胞以及悬浮细胞来说都是足够的。它对于在高吞吐量系统中测试大量药物非常有用。该方法在技术上易于实现, 成本效益高。此外, 流式细胞仪的使用可以同时评估细胞的活力, 从而评估药物毒性。
Introduction
尽管有效的长期抗逆转录病毒疗法, 艾滋病病毒仍然在潜在的状态作为一个综合提供在记忆 cd4+ t 细胞1。hiv-1 5 长端重复 (ltr) 启动子的染色质结构和表观遗传修饰, 如基因组化和 dna 甲基转移酶 (dnmt) 和组蛋白去乙酰化酶 (hdac) 等修饰, 是导致导致转录抑制, 从而整合后潜伏期2,3。对多种潜伏期逆转剂 (lra) 进行了研究, 以研究其诱导病毒体外和体内产生病毒的有效性, 这些药物可诱导病毒在体外和体内产生, 这些药物的作用是诱导潜在感染的静息 cd4 + t 细胞4、5、6、7 ,8。在测试的 lra 中, hdaci (hdac 抑制剂) 和 bet 溴瘤抑制剂 (betis) 分别诱导染色质去密度和释放阳性转录伸长率因子 b (p-tefb), 从而导致随后缓解转录在 5 ‘ ltr 的镇压和激活艾滋病毒表达9,10,11, 12,13.然而, 这些 lra 所实现的再激活幅度有限, 因为在体内14, 15 观察到的细胞相关的未切片 hiv mrna (美国 rna) 仅略有增加, 表明病毒转录。重要的是, 这些 lra 也未能导致潜在感染细胞频率的降低。
hiv 的表达可能会受到效率低下的拼接16以及多重拼接 hiv rna (ms rna)17的核出口缺陷的进一步限制。因此, 需要确定更有效且可能影响病毒生产后集成的不同方面的新类型 lra。此外, 还需要开发新的检测方法, 帮助定义最佳化合物, 以有效地反向延迟。
在这里, 提出了一个协议, 其中采用高通量的方法, 功能评估干预措施对艾滋病毒 ltr 驱动转录和拼接的影响。总之, 一种新的 ltr 驱动的双色记者系统rev 38/dsred (图 1) 用于通过流式细胞术评估 hiv 的再激活。在本荧光记者中, 无拼接 hiv mrna (4kb) 的表达导致绿色荧光蛋白 (egfp) 表达增强, 而拼接 mrna (2kb) 的表达会导致争议病毒 sp.红色 (dsred) 荧光蛋白的表达。简单地说, 我们使用了荧光 env-egfp 融合蛋白, gp140unc。egfp, 其中 egfp 的编码序列被放置在框架与一个未切割和截断的形式的信封 (环境)。引入了一些改变, 以消融可防止 env 分离成 gp120 和 gp41-egfp, 并在跨膜域之前截断 gp120 蛋白, 从而形成可溶性 env 类似物, 从而促进正确折叠和egfp 的表达。在细胞内的表达时, rev 定位到细胞核, 通过与 rev 反应元件 (rre) 的相互作用, 介导 4 kb env mrna 的核细胞质出口。env 的截断不会损害位于 gp120 和 gp120 之间的 rre 和 a7 3 ‘ 拼接站点。在该系统中, 在 hiv-1 拼接供体 4 (sd4) 和拼接受体 7 (sa7) 处进行拼接, 导致产生一种 2kb mrna 编码的非功能性 rev 蛋白, 该蛋白编码在氨基酸38融合到 dsred 荧光蛋白时被截断,。简单地说 , dsred 入到在氨基酸 38 的 rev 的第 2外显子重叠引伸 18 。为了促进无拼接 mrna 的核出口, 将哺乳动物表达载体编码 rev (pcmv-revnl4.3) 与荧光记者结构 (图 2) 共同转染。这里描述的这个独特的记者结构是有用的高通量评估的 hiv 转录和拼接, 而无需使用病毒载体。
Protocol
请注意:克隆、转化和测序程序在其他地方讨论 18、19。本文的协议从哺乳动物表达载体的转染开始 (图 3)。 1. 采用双色记者构建 hek293t 细胞转染 在 dulbecco 的改良鹰培养基 (dmem) 中培养 hek293t 细胞, 并在37°c 的 5% co 2 孵化器中补充 10% (v/v) 胎儿牛血清 (fbs)、青霉素 (100 v/v) 和链霉素 (100μgml).<su…
Representative Results
图 5显示了用溴蒙丹抑制剂 jq1 治疗后 hiv-1 无拼接 (egfp) 和拼接 (dsred) 产品的表达的代表性结果。JQ1(+) 和 tat 都显著增加了表达 egfp 的细胞百分比 (分别为2.18 和 4.13 fc, 高于 dmso; n = 3) 表明没有拼接的转录。此外, JQ1(+) 显著提高了表达 dsred (46.6 fc 高于 dmso) 的细胞比例, 以及拼接产品 (7.37 fc 高于 dmso) 的比例, 达到与 tat (分别为29.6 和 5.83 fc 高于 dmso) 确认…
Discussion
鉴于在体内测量病毒再激活的困难, 为了研究 hiv 潜伏期, 包括潜伏期感染的 t 细胞系 (j-lats、ach2、u1)、主要的静息感染模型 (o ‘ deherty, lewin, greene 和脊柱模型) 或预激活的 cd4 + t 细胞 (sahu, marini, planelles, siliciano, karn 模型) 与单轮或复制主管记者病毒22。为了模拟静止 cd4+ t 细胞中 hiv 潜伏期的生理条件, 随后出现了双荧光记者系统, 如 ivan sadowski 小组开发的 rgh (红绿艾滋病毒) 记者, ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了澳大利亚国家住房改革委员会项目赠款 app1129320 和 app1052979 方案赠款的支持。我们感谢亚当·惠特利博士、玛丽娜·亚历山大博士、珍妮·安德森博士和米歇尔·y·李先生为圆满完成这项工作提供了必要的结构和建议。我们还感谢 dmi 流动设施工作人员在维持本研究中使用的流式细胞仪方面提供的建议和慷慨的协助。
Materials
| Cell culture | |||
| HEK293T cells (Human Embryonic Kidney cells) | ATCC | CRL-3216 | Replicates vectors carrying the SV40 region of replication. |
| Dulbecco's Modified Eagle's Medium (DMEM 1x + GlutaMAX-I) | Gibco | 10569-010 | + 4.5 g/L D-Glucose + 110 mg/L Sodium Pyruvate |
| Fetal Bovine serum | Gibco | 10099-141 | Origin Australia |
| Penicillin-Streptomycin | Sigma | P4458 | |
| Dulbecco's phosphate buffered saline (DPBS), no calcium, no magnesium | Gibco | 14190-136 | |
| Trypan blue Stain, 0.4% | Gibco | 15250 | |
| Trypsin-EDTA (0.05%), phenol red | Gibco | 25300054 | |
| Lipofectamine 2000 | Invitrogen | 11668-019 | Lipid transfection reagent |
| Opti-MEM I (1x) reduced serum medium | Gibco | 31985-070 | Serum free medium |
| NucleoBond Xtra Maxi | Marcherey-Nagel | 740414.50 | |
| pEGFP-N1 plasmid | Clontech (TaKaRa) | 6085-1 | Expression of EGFP in mammalian cells, CMVIE promoter. |
| pDsRed-Express-N1 | Clontech (TaKaRa) | 632429 | Expression of DsRed-Express in mammalian cells, CMVIE promoter. |
| pLTR.gp140/EGFP.RevD38/DsRed | Addgene | 115775 | |
| pCMV-RevNL4.3 | Addgene | 115776 | |
| pCMV-Tat101AD8-Flag | Addgene | 115777 | |
| Dimethyl sulfoxide (DMSO) | Millipore | 67-68-5 | |
| JQ1(+) | Cayman Chemical | 11187 | Stock at 10 mM in DMSO; working concentration 1 μM |
| JQ1(-) | Cayman Chemical | 11232 | Stock at 10 mM in DMSO; working concentration 1 μM |
| Phorbol Myristate Acetate (PMA) | Sigma-Aldrich | 16561-29-8 | Stock at 100 μg/mL in DMSO; working concentration 10 nM |
| Phytohaemagglutinin (PHA) | Remel | HA15/R30852701 | Stock at 1 μg/μL in PBS; working concentration 10 μg/mL |
| Vorinostat (VOR) | Cayman Chemical | 10009929 | Stock at 10 mM in DMSO; working concentration 0.5 μM |
| Panobinostat (PAN) | TRC | P180500 | Stock at 10 mM in DMSO; working concentration 30 nM |
| CellTiter 96 AQueous One Solution Cell Proliferation Assay | Promega | 63581 | |
| Venor GeM Classic | Minerva Biolabs | 11-1100 | Mycoplasma Detection Kit, PCR-based |
| Name | Company | Catalog Number | Comments |
| Flow cytometry reagents | |||
| LSR Fortessa | BD Biosciences | Flow cytometer (4 lasers-blue, red, violet and yellow) | |
| LSR II | BD Biosciences | Flow cytometer (3 lasers-blue, red and violet) | |
| LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit | Life Technologies | L34976 | Viability dye: for 633 or 635 nm excitation, 400 assays. Component A and B are both provided in the kit. |
| Bovine Serum Albumin | Sigma | A2153 | |
| EDTA 0.5M pH8 | Gibco | 15575-038 | |
| Formaldehyde Solution 37/10 (37%) | Chem-Supply | FA010 | |
| BD FACS Diva CS&T Research Beads | BD Biosciences | 655050 | Calibration beads |
| Sphero Rainbow Calibration Particles (8 peaks) | BD Biosciences | 559123 | 3.0 – 3.4 mm |
| Sheath solution | Chem-Supply | SA046 | 90 g NaCl in 10 L water |
| HAZ-Tabs | Guest Medical | H8801 | Chlorine release tablets for disinfection |
| Decon 90 | Decon Laboratories Limited | N/A | Concentrated cleaning agents of flow cytometer. Working solution Decon 90 5%. |
| Sodium Hypochlorite (12-13% Solution) | Labco | SODHYPO-5L | Concentrated cleaning agents of flow cytometer. Working solution bleach 1%. |
| 7x | MPBio | IM76670 | Concentrated cleaning agents of flow cytometer. Working solution 7x 1%. |
| Name | Company | Catalog Number | Comments |
| Materials | |||
| Tissue culture flasks (75 cm2, canted neck, cap vented) | Corning | 430641U | |
| Tissue culture plates (96 well flat bottom with lid) | Costar | 3599 | |
| Tissue culture plates (96 well V-bottom without lid) | Costar | 3896 | |
| Centrifuge tubes (10 mL) | SARSTEDT | 62.9924.284 | 100×16 mm |
| Centrifuge tubes (50 mL) | CellStar | 227261 | 30×115 mm |
| Microcentrifuge tubes (1.5 mL) | Corning Axygen | MCT-150-C | |
| Serological Pipette (25 mL), sterile | Corning | CLS4489-200EA | |
| Serological Pipette (10 mL), sterile | Corning | CLS4488-200EA | |
| Serological Pipette (5 mL), sterile | Corning | CLS4487-200EA | |
| Reagent reservoirs (50 mL), sterile | Corning | CLS4470-200EA | |
| 5 mL Round-Bottom polystyrene test tube, with cell-strainer cap | Corning | 352235 | 12 x 75 mm style, 70 mm |
| Nylon Mesh | SEFAR | 03-100/32 | 100 mm |
| Titertube Micro test tubes, bulk | BIO-RAD | 2239391 | microfacs tubes |
| 5 mL Round-Bottom polystyrene test tube, without cap | Corning | 352008 | 12×75 mm style |
| Snap Caps for 12×75 mm Test Tubes | Corning | 352032 | |
| Counting chamber, Neubauer improved double net ruling, bright-line (Haemocytometer, LO-Laboroptik) | ProSciTech | SVZ4NIOU | 3×3 large squares of 1 mm2; Depth 0.100 mm; volume 0.1 mL; area minimum 0.0025 mm2 |
| Coverslips (Menzel-Gläser) | Grale Scientific | HCS2026 | 20 x 26 mm |
| Microscope | Nikon TMS | 310528 | |
| Centrifuge 5810R refrigerated | Eppendorf | 5811000487 | with rotor A-4-81 including adapters for 15/50 mL conical tubes |
| FLUOstar Omega microplate reader | BMG Labtech | N/A | Plate reader for cell proliferation assay. Filter 490 nm. |
| Name | Company | Catalog Number | Comments |
| Softwares | |||
| FACS Diva | BD Biosciences | Flow cytometer data acquisition and analysis program, version 8.0.1 | |
| FlowJo | FlowJo | FlowJo 10.4.2 | Flow cytometer data analysis program, FlowJo Engine v3.05481 |
| Omega | BMG Labtech | FLUOstar multi-user reader control, version 5.11 | |
| Omega – Data Analysis | BMG Labtech | MARS | FLUOstar data analysis, version 3.20R2 |
| Microsoft Excel | Microsoft | Excel:mac 2011 | version 14.0.0 |
| Prism | GraphPad | Prism 7 | version 7.0c |
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Cite This Article
Khoury, G., Purcell, D. F. High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing. J. Vis. Exp. (143), e58753, doi:10.3791/58753 (2019).