Name: high throughput in vitro assessment of latency reversing agents on hiv transcription and splicing
Uploaded: 2019-01-22T14:10:00
Description: Watch this Scientific Journal Video about High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing at JoVE.com

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。

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
<ol><li>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...

<ol>
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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, ?...

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。

<table>
	<tbody>
		<tr>
			<td>Cell culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cells (Human Embryonic Kidney cells)</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td>Replicates vectors carrying the SV40 region of replication.</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM 1x + GlutaMAX-I)</td>
			<td>Gibco</td>
			<td>10569-010</td>
			<td>+ 4.5 g/L D-Glucose + 110 mg/L Sodium Pyruvate</td>
		</tr>
		<tr>
			<td>Fetal Bovine serum</td>
			<td>Gibco</td>
			<td>10099-141</td>
			<td>Origin Australia</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Sigma</td>
			<td>P4458</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (DPBS), no calcium, no magnesium</td>
			<td>Gibco</td>
			<td>14190-136</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue Stain, 0.4%</td>
			<td>Gibco</td>
			<td>15250</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.05%), phenol red</td>
			<td>Gibco</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
			<td>Lipid transfection reagent</td>
		</tr>
		<tr>
			<td>Opti-MEM I (1x) reduced serum medium</td>
			<td>Gibco</td>
			<td>31985-070</td>
			<td>Serum free medium</td>
		</tr>
		<tr>
			<td>NucleoBond Xtra Maxi</td>
			<td>Marcherey-Nagel</td>
			<td>740414.50</td>
			<td></td>
		</tr>
		<tr>
			<td>pEGFP-N1 plasmid</td>
			<td>Clontech (TaKaRa)</td>
			<td>6085-1</td>
			<td>Expression of EGFP in mammalian cells, CMVIE promoter.</td>
		</tr>
		<tr>
			<td>pDsRed-Express-N1</td>
			<td>Clontech (TaKaRa)</td>
			<td>632429</td>
			<td>Expression of DsRed-Express in mammalian cells, CMVIE promoter.</td>
		</tr>
		<tr>
			<td>pLTR.gp140/EGFP.RevD38/DsRed</td>
			<td>Addgene</td>
			<td>115775</td>
			<td></td>
		</tr>
		<tr>
			<td>pCMV-RevNL4.3</td>
			<td>Addgene</td>
			<td>115776</td>
			<td></td>
		</tr>
		<tr>
			<td>pCMV-Tat101AD8-Flag</td>
			<td>Addgene</td>
			<td>115777</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Millipore</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr>
			<td>JQ1(+)</td>
			<td>Cayman Chemical</td>
			<td>11187</td>
			<td>Stock at 10 mM in DMSO; working concentration 1 &#956;M</td>
		</tr>
		<tr>
			<td>JQ1(-)</td>
			<td>Cayman Chemical</td>
			<td>11232</td>
			<td>Stock at 10 mM in DMSO; working concentration 1 &#956;M</td>
		</tr>
		<tr>
			<td>Phorbol Myristate Acetate (PMA)</td>
			<td>Sigma-Aldrich</td>
			<td>16561-29-8</td>
			<td>Stock at 100 &#956;g/mL in DMSO; working concentration 10 nM</td>
		</tr>
		<tr>
			<td>Phytohaemagglutinin (PHA)</td>
			<td>Remel</td>
			<td>HA15/R30852701</td>
			<td>Stock at 1 &#956;g/&#956;L in PBS; working concentration 10 &#956;g/mL</td>
		</tr>
		<tr>
			<td>Vorinostat (VOR)</td>
			<td>Cayman Chemical</td>
			<td>10009929</td>
			<td>Stock at 10 mM in DMSO; working concentration 0.5 &#956;M</td>
		</tr>
		<tr>
			<td>Panobinostat (PAN)</td>
			<td>TRC</td>
			<td>P180500</td>
			<td>Stock at 10 mM in DMSO; working concentration 30 nM</td>
		</tr>
		<tr>
			<td>CellTiter 96 AQueous One Solution Cell Proliferation Assay</td>
			<td>Promega</td>
			<td>63581</td>
			<td></td>
		</tr>
		<tr>
			<td>Venor GeM Classic</td>
			<td>Minerva Biolabs</td>
			<td>11-1100</td>
			<td>Mycoplasma Detection Kit, PCR-based</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Flow cytometry reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LSR Fortessa</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometer (4 lasers-blue, red, violet and yellow)</td>
		</tr>
		<tr>
			<td>LSR II</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometer (3 lasers-blue, red and violet)</td>
		</tr>
		<tr>
			<td>LIVE/DEAD Fixable Near-IR Dead Cell Stain Kit</td>
			<td>Life Technologies</td>
			<td>L34976</td>
			<td>Viability dye: for 633 or 635 nm excitation, 400 assays. Component A and B are both provided in the kit.</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA 0.5M pH8</td>
			<td>Gibco</td>
			<td>15575-038</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde Solution 37/10 (37%)</td>
			<td>Chem-Supply</td>
			<td>FA010</td>
			<td></td>
		</tr>
		<tr>
			<td>BD FACS Diva CS&#38;T Research Beads</td>
			<td>BD Biosciences</td>
			<td>655050</td>
			<td>Calibration beads</td>
		</tr>
		<tr>
			<td>Sphero Rainbow Calibration Particles (8 peaks)</td>
			<td>BD Biosciences</td>
			<td>559123</td>
			<td>3.0 - 3.4 mm</td>
		</tr>
		<tr>
			<td>Sheath solution</td>
			<td>Chem-Supply</td>
			<td>SA046</td>
			<td>90 g NaCl in 10 L water</td>
		</tr>
		<tr>
			<td>HAZ-Tabs</td>
			<td>Guest Medical</td>
			<td>H8801</td>
			<td>Chlorine release tablets for disinfection</td>
		</tr>
		<tr>
			<td>Decon 90</td>
			<td>Decon Laboratories Limited</td>
			<td>N/A</td>
			<td>Concentrated cleaning agents of flow cytometer. Working solution Decon 90 5%.</td>
		</tr>
		<tr>
			<td>Sodium Hypochlorite (12-13% Solution)</td>
			<td>Labco</td>
			<td>SODHYPO-5L</td>
			<td>Concentrated cleaning agents of flow cytometer. Working solution bleach 1%.</td>
		</tr>
		<tr>
			<td>7x</td>
			<td>MPBio</td>
			<td>IM76670</td>
			<td>Concentrated cleaning agents of flow cytometer. Working solution 7x 1%.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Materials</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture flasks (75 cm2, canted neck, cap vented)</td>
			<td>Corning</td>
			<td>430641U</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plates (96 well flat bottom with lid)</td>
			<td>Costar</td>
			<td>3599</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture plates (96 well V-bottom without lid)</td>
			<td>Costar</td>
			<td>3896</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes (10 mL)</td>
			<td>SARSTEDT</td>
			<td>62.9924.284</td>
			<td>100x16 mm</td>
		</tr>
		<tr>
			<td>Centrifuge tubes (50 mL)</td>
			<td>CellStar</td>
			<td>227261</td>
			<td>30x115 mm</td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes (1.5 mL)</td>
			<td>Corning Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipette (25 mL), sterile</td>
			<td>Corning</td>
			<td>CLS4489-200EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipette (10 mL), sterile</td>
			<td>Corning</td>
			<td>CLS4488-200EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological Pipette (5 mL), sterile</td>
			<td>Corning</td>
			<td>CLS4487-200EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent reservoirs (50 mL), sterile</td>
			<td>Corning</td>
			<td>CLS4470-200EA</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Round-Bottom polystyrene test tube, with cell-strainer cap</td>
			<td>Corning</td>
			<td>352235</td>
			<td>12 x 75 mm style, 70 mm</td>
		</tr>
		<tr>
			<td>Nylon Mesh</td>
			<td>SEFAR</td>
			<td>03-100/32</td>
			<td>100 mm</td>
		</tr>
		<tr>
			<td>Titertube Micro test tubes, bulk</td>
			<td>BIO-RAD</td>
			<td>2239391</td>
			<td>microfacs tubes</td>
		</tr>
		<tr>
			<td>5 mL Round-Bottom polystyrene test tube, without cap</td>
			<td>Corning</td>
			<td>352008</td>
			<td>12x75 mm style</td>
		</tr>
		<tr>
			<td>Snap Caps for 12x75 mm Test Tubes</td>
			<td>Corning</td>
			<td>352032</td>
			<td></td>
		</tr>
		<tr>
			<td>Counting chamber, Neubauer improved double net ruling, bright-line (Haemocytometer, LO-Laboroptik)</td>
			<td>ProSciTech</td>
			<td>SVZ4NIOU</td>
			<td>3x3 large squares of 1 mm2; Depth 0.100 mm; volume 0.1 mL; area minimum 0.0025 mm2</td>
		</tr>
		<tr>
			<td>Coverslips (Menzel-Gl&#228;ser)</td>
			<td>Grale Scientific</td>
			<td>HCS2026</td>
			<td>20 x 26 mm</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Nikon TMS</td>
			<td>310528</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge 5810R refrigerated</td>
			<td>Eppendorf</td>
			<td>5811000487</td>
			<td>with rotor A-4-81 including adapters for 15/50 mL conical tubes</td>
		</tr>
		<tr>
			<td>FLUOstar Omega microplate reader</td>
			<td>BMG Labtech</td>
			<td>N/A</td>
			<td>Plate reader for cell proliferation assay. Filter 490 nm.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Softwares</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACS Diva</td>
			<td>BD Biosciences</td>
			<td></td>
			<td>Flow cytometer data acquisition and analysis program, version 8.0.1</td>
		</tr>
		<tr>
			<td>FlowJo</td>
			<td>FlowJo</td>
			<td>FlowJo 10.4.2</td>
			<td>Flow cytometer data analysis program, FlowJo Engine v3.05481</td>
		</tr>
		<tr>
			<td>Omega</td>
			<td>BMG Labtech</td>
			<td></td>
			<td>FLUOstar multi-user reader control, version 5.11</td>
		</tr>
		<tr>
			<td>Omega - Data Analysis</td>
			<td>BMG Labtech</td>
			<td>MARS</td>
			<td>FLUOstar data analysis, version 3.20R2</td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td>Excel:mac 2011</td>
			<td>version 14.0.0</td>
		</tr>
		<tr>
			<td>Prism</td>
			<td>GraphPad</td>
			<td>Prism 7</td>
			<td>version 7.0c</td>
		</tr>
	</tbody>
</table>

high throughput in vitro assessment of latency reversing agents on hiv transcription and splicing

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。

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...

Watch this Scientific Journal Video about High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing at JoVE.com

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

hiv 转录和拼接中延迟逆转剂的高通量体外评价

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。

HIV remains incurable due to the existence of a reservoir of cells that harbors stable and latent form of the virus, which stays invisible to the immune ...

这一程序的总体目标是评估体外，延迟逆转剂对HIV mRNA处理的影响。该协议提供了一种简单、高效和可靠的方法，用于同时评估上帝抵抗军对艾滋病毒转录和拼接的影响。该技术提供了对LRA诱导病毒重新激活和清除潜伏储层的能力的见解。培养细胞后，将2万个细胞放在100微升DMEM培养基中，辅以10%FBS，在96孔平底板的井中放置2万个24小时无抗生素。第二天稀释了双色记者构造。Tat 和 Rev DNA 在 50 微升的血清自由介质中，轻轻混合。轻轻混合脂质试剂，然后在50微升的血清自由介质中每反应稀释0.65微升。轻轻混合，在室温下孵育五分钟。将稀释的脂质试剂与稀释的DNA混合，轻轻混合。在室温下孵育20分钟。然后，在每种井的细胞顶部，将100微升这种脂质试剂DNA复合物滴下。轻轻来回摇动盘子以混合。在加湿的培养箱中孵育板，温度为37摄氏度，二氧化碳含量为5%，为期5小时。在此之后，转染额外的井与100纳米的CMV驱动EGFP和DsRed快速DNA质粒补偿目的。首先，使用生长介质将每个延迟倒转剂稀释到适当的工作状态。使用多通道管头，小心吸气转染介质，代之以含有适当延迟倒转剂的100微升介质。在37%摄氏度的加湿孵化器中孵育，用5%的二氧化碳孵育48小时。要开始染色细胞，请使用多通道管头向每井添加 100 微升 PBS。轻轻管头向上和向下约五次。同时确保避免泡沫，将细胞分离到介质中。接下来，将细胞转移到96孔V型底板的孔中。在500倍重力下离心，在4摄氏度下离心5分钟。在不接触细胞的情况下吸气介质和PBS。用200微升蛋白质和不含血清的PBS至少清洗一次细胞。在500倍重力下离心，在4摄氏度下离心5分钟。倾斜板以丢弃上流液，在不接触细胞的情况下取出洗涤缓冲液。在此稀释蛋白质和血清无色PBS中的固定可行性染料后，以1比1000的比例，准备一个工作的解决方案。在每个井中加入50微升稀释染料，然后管头上下混合。在摄氏四度时孵育。同时使用铝箔防止光线 10 至 15 分钟。然后用150微升的洗涤缓冲液清洗细胞一两次。在此之后，离心机在500倍的重力下，4摄氏度为5分钟，并丢弃上经。要修复细胞，加入100微升新鲜准备的1%甲醛和洗涤缓冲液。并在4摄氏度下孵育，同时保护10至15分钟的光线。在此之后，用100微升的洗涤缓冲液清洗细胞一次或两次。在500倍重力下离心，在4摄氏度下离心5分钟。丢弃上一提液，将细胞颗粒重新在70微升的洗涤缓冲液中。通过运行校准珠检查圆度计设置性能和灵敏度后，使用未污染的样品调整正向和侧散射电压，使主组在屏幕上清晰可辨。使用单个染色样品执行手动或自动补偿，确保 EGFP 阳性总体在 DsRed 探测器中泄漏最少，反之亦然。使用荧光减去一个控件，创建绘图并设置门。接下来，使用未污染的样品调整正向和侧散射电压，使主总体在屏幕上清晰可辨。获取并记录每个样本至少 10，000 个可行的细胞事件。在此之后，使用流式细胞学数据分析软件分析文本协议中概述的数据。HIV-1、未拼接和拼接产品表达的代表性结果表明，JQ1阳性和Tat，显著增加表达EGFP的细胞比例。表示未复制的成绩单。JQ1阳性也被认为显著增加表达DSRed的细胞的百分比。将拼接产品的比例提高至与Tat类似的水平。这证实了JQ1阳性打开HIV转录和拼接的能力。然而，用立体异构体控制JQ1阴性治疗，可消除JQ1对HIV转录和拼接的阳性影响。一旦掌握，这种方法可以单独或组合使用药物进行。测试他们高效协同的能力。这将导致药物和毒性的降低剂量。每次开发后，该技术为在外体患者样本延迟初级模型中测试高效药物组合铺平了道路。

Research

JoVE Journal

Immunology and Infection

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

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

Determination of Molecular Structures of HIV Envelope Glycoproteins using Cryo-Electron Tomography and Automated Sub-tomogram Averaging

使用低温电子断层扫描和自动子断层扫描平均HIV包膜糖蛋白分子结构的测定

Since its discovery nearly 30 years ago, more than 60 million people have been infected with the human immunodeficiency virus (HIV) (www.usaid.gov). The ...

In vitro Uncoating of HIV-1 Cores

In vitro Uncoating of HIV-1 Cores

在体外对HIV - 1核心脱壳

The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

艾滋病毒的成像1信封引起的病毒学突触信号合成脂双层

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

In Vitro Assay to Evaluate the Impact of Immunoregulatory Pathways on HIV-specific CD4 T Cell Effector Function

在体外试验 ，以评估免疫调节途径对HIV特异性CD4 + T细胞效应功能的影响

T cell exhaustion is a major factor in failed pathogen clearance during chronic viral infections. Immunoregulatory pathways, such as PD-1 and IL-10, are ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

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 ...

High Throughput Fluorometric Technique for Assessment of Macrophage Phagocytosis and Actin Polymerization

高通量荧光技术巨噬细胞吞噬功能和肌动蛋白聚合的评估

The goal of fluorometric analysis is to serve as an efficient, cost effective, high throughput method of analyzing phagocytosis and other cellular ...

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

High Throughput Measurement of Extracellular DNA Release and Quantitative NET Formation in Human Neutrophils In Vitro

胞外DNA释放的高通量测量和人中性粒定量NET形成<em&gt;体外</em

Neutrophil granulocytes are the most abundant leukocytes in the human blood. Neutrophils are the first to arrive at the site of infection. Neutrophils ...

High-throughput Gene Tagging in Trypanosoma brucei

High-throughput Gene Tagging in Trypanosoma brucei

高通量基因定位在<em&gt;布氏锥虫</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

测量<em&gt;体外</em&gt; HIV-1 Preintegration配合物的整合活动

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...