这里介绍的工作是由卫生研究院（CIHR）加拿大研究院的支持（授予DCB-120266，PPP-133377和HBF-348967到AG）。

1.细胞转染与<ol><li>培养的HEK 293T细胞在Dulbecco改进的Eagle培养基（DMEM）中补充有10％胎牛血清（FBS）和1％青霉素/链霉素。制备在细胞培养基中的2×10 5个细胞/ ml悬浮液。加500，100和1000微升的细胞悬液以每孔24孔，96孔和12孔板，用于病毒生产，细胞生存力和免疫激活测定法，分别为（ 图2A）。 </li><li>轻轻摇动平板，用5％的CO 2孵育他们O / N在37℃。生长细胞50-70％汇合。 </li><li>根据计划，转染，在1.5ml微管（例如， 图2B）准备测试的RNA及其控制的稀释液。 <ol><li>对于病毒生成测定，准备HIV-1表达质粒的10纳克/微升稀释并加入10微升至每管。下一个准备测试RNA的5微米稀释和阴性对照RNA.添加2.5或10微升各试验RNA和阴性对照稀释到相应管25或100nM的最终浓度。 </li><li>对于细胞存活力测定法，制备试验RNA的5微米稀释和10毫克/毫升稀释的阳性对照RNA，低分子量聚I的:C。添加2微升试验RNA或阳性对照RNA稀释至用于分别100nM或200微克/毫升的最终浓度，相应的管中。 </li><li>对于免疫激活法，加入20微升试验RNA或步骤1.3.2准备阳性对照RNA稀释。为分别100nM或200微克/毫升的最终浓度，相应的管中。 注意:对于测试RNA表达质粒，在地方试验RNA的5μM稀释准备10毫微克/微升稀释液，给25和100最终金额纳克步1.3.1。和100纳克的步骤1.3.2。 1.3.3和。 </li></ol></li><li>添加50，25或75微升的DMEM为病毒督促各转管，减税，细胞活力和免疫激活测定，分别。用于病毒生产测定，带来细胞和制备转染管到生物安全水平3（BSL3）实验室下一步骤之前。 </li><li>添加2微升转染试剂依次向转染管和孵育15至20分钟以使配合物形成。见材料的表的具体的转染试剂中使用。 </li><li>从每个微管逐滴向在细胞培养板中的相应位置添加整个转染混合物。轻轻摇动并在37℃用5％CO 2孵育48小时将板。 </li><li>测量在细胞培养板（ 图2C）的HIV-1的生产（第2部分），细胞存活率（第3节）和免疫激活（第4节）。 </li></ol> 2.病毒生成测定<ol><li>除去从培养箱24孔细胞培养板，并轻轻摇动BSL3细胞培养内板引擎盖。转移150微升上清液从每孔在96孔平底板，这将被用于量化的HIV-1的生产相应的井。 注意:普通的病毒定量测定包括测定HIV-1的衣壳蛋白（p24的）通过酶联免疫吸附测定（ELISA）20，量化通过逆转录聚合酶链反应（RT-PCR）21病毒RNA的表达，并测量活性的HIV-1逆转录酶。步骤2.2至2.5解释的方法来量化HIV-1 RT活性13,15,16。 </li><li>转印5微升上清液在一个96孔板的相应孔，含有25微升的病毒破坏鸡尾酒15（ 表1）。在室温下孵育5分钟该混合物，将板转移到放射性工作站。 注:步骤2.2。只有必要的，如果一个放射性工作站不在BSL3实验室提供。如果板不需要从除去BSL3实验室，继续执行步骤2.3。和到位的25微升放射性鸡尾酒的加入50微升放射性/病毒破坏的鸡尾酒（ 表1）。 </li><li>制备放射性鸡尾酒15（ 表1），并添加25微升病毒上清和破坏鸡尾酒的每个孔中。孵育所述板在37℃下2小时。 </li><li>点5微升中的玻璃纤维二乙基氨基乙基（DEAE）滤纸纸，将反应混合物到相应的平方和允许斑点干燥10分钟。现货每隔广场反应混合物中，以便没有两个样品直接房客彼此。这有助于在步骤2.6确定每分钟计数（CPM），以避免样品之间交叉。 </li><li>洗纸5倍5分钟用2×盐水柠檬酸钠（SSC）缓冲液（ 表1），随后是两个1分钟洗涤用95％的乙醇。允许论文干，封住他们的样品袋。 </li><li>剪辑取样袋合作ntaining的纸滤纸成磁带，将磁带插入微板闪烁计数器。设置计数器读取的cpm为32磷与设置在实验中使用的批次[32 P] dTTP各基准日期。选择使用板地图读取和启动计数器进行采样。 </li><li>为任何病毒定量方法中，通过相邻的阴性对照划分为每个测试的RNA得到的值再乘以100这个值来得到的HIV-1生产的抑制百分率的测试RNA的每个重复。比较不同浓度不同的测试的RNA的结果的一个例子在图3中提供。 </li></ol> 3.细胞活力检测<ol><li>从培养箱中取出96孔板，并添加20微升5mg / ml的MTT（3- [4.5二甲基-2-噻唑基] -2,5-二苯基-2H-四唑鎓溴化物）的Dulbecco氏磷酸盐缓冲盐水稀释（ DPBS）到每个孔中。孵育板˚F或在37℃下3小时。 </li><li>添加150微升酸化异丙醇与去污剂（1％NP-40，4毫HCl的异丙醇）至各孔，孵育所述板在室温2小时。 </li><li>确定在微孔板分光光度计570nm处的吸光度。 </li><li>除以由相邻的转染控制每个采样得到的值计算每个阳性对照，检测RNA相对MTT代谢。比较不同测试RNA和阳性对照结果的一个例子在图4中提供。 </li></ol> 4.免疫活化分析<ol><li>从培养箱中取出12孔板并吸出培养基。轻轻用DPBS洗涤细胞两次，并添加70微升的冷裂解缓冲液22（包括蛋白酶和磷酸酶抑制剂， 表1）向每个孔中。孵育在冰上10分钟的板。 </li><li>转移细胞裂解液微管浸入快速冻结他们管在液氮中。允许样品解冻，并重复2更冻融循环，共3。 </li><li>离心15分钟的裂解物在4℃，15700 XG，以沉淀细胞碎片。将上清转移到新的微管中，并确定使用考马斯蓝（布拉德福德）方法23,24蛋白质浓度。 </li><li>解决75微克蛋白的从每个样品在10％变性聚丙烯酰胺凝胶并转移到硝酸纤维素膜上如前所述25,26。 </li><li>以下电泳并转移到膜上，通过丽春红S（ 表1）为1分钟，随后在双蒸馏水洗涤孵育膜揭示蛋白质带。使用带和蛋白质梯作为指导，在80和55 kDa的切割膜。 注意:在步骤4.4的样品可以在16×18 CM短运行2凝胶至34 kDa的，所以，它很容易在规定的位置上切割膜，并通过不切感兴趣的乐队。可替代地，多个凝胶可以用相同的样品来运行，以避免切割膜。 </li><li>洗出用含有0.05％吐温20（TBST， 表1）的Tris缓冲盐水的Ponceau S染色。添加TBST用5％无脂牛奶以完全覆盖膜。孵育在RT膜搅拌1小时。 </li><li>孵育膜O / N在TBST用3％牛血清白蛋白（BSA）和抗体在1000稀释到1对ADAR1（110和150 kDa的），磷酸PKR（62 kDa的），和磷酸化IRF3（47 kDa的），用于分别的顶部，中部和底部片膜。 </li><li>洗涤该膜5倍5分钟用TBST和在TBST用5％无脂奶和过氧化物酶标记的山羊抗兔次级抗体（在5000稀释到1）1小时孵育。 </li><li>洗涤该膜5×5分钟用TBST和应用电化学发光（ECL）溶液可视化上，根据制造商的说明电影的频带。 </li><li>可视化上薄膜的蛋白质条带后，洗膜的中部和底部件10分钟与抗体反萃液。孵育针对总PKR膜O / N在TBST用3％BSA和抗体（在500 1）和IRF3（在1000 1），用于分别的中部和底部件。重复步骤4.8。和4.9。使用过氧化物酶标记的山羊抗小鼠代替用于PKR膜（中）的抗 - 兔二级抗体。 </li><li>洗底部片膜5倍的5分钟用TBST和用抗肌动蛋白的抗体（在5000稀释到1）孵育，用3％BSA的TBST将膜1小时。重复步骤4.8。和4.9。使用过氧化物酶标记的山羊抗小鼠代替抗兔二级抗体。比较不同测试RNA和阳性对照结果的一个例子在图5中提供。参见材料的表中使用的特异性抗体。 </li></ol>

<ol>
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The authors have nothing to disclose.

用HEK293T细胞（ 图2）进行描述的HIV-1的生产法和类似用于筛选HIV-1RNA有效核酶13，shRNA的10,29，siRNA的30，和U1i的RNA 11,31靶位点测定。使用不同的方法来量化的HIV-1的生产，大多数研究已经测量的病毒生产48小时与候选的RNA的HIV-1的表达质粒共转染之后。继生产HIV-1的，不成熟的病毒粒子经历的多聚蛋白的蛋白裂解由HIV-1蛋白酶变得成熟的病毒粒子，能够感染新的细胞。在步骤2.2中所述的HIV-1逆转录酶活性测定。至2.5。，无论是生产和成熟步骤进行计算，因为逆转录酶只在成熟病毒粒子的活性。与此相反，衣壳蛋白和病毒RNA可以存在于成熟和未成熟的病毒粒子。因此，量化的HIV-1的生产通过的p24 ELISA或RT-PCR米唉错过了本地化HIV-1病毒颗粒32和抑制加格处理除HIV-1蛋白的表达13基于反义分子对HIV-1复制周期的成熟步骤行事的治疗RNA的作用，如核酶， 31。的病毒的生产检测的限制是所用的细胞不表达HIV-1的条目的相应受体，并不能用于评估治疗性RNA的对HIV-1进入或整合的影响。 为了评估抗HIV-1在整个复制周期的RNA的影响，各种HIV-1感染模型已经在不同的T淋巴细胞的细胞系或原血细胞5,33使用。因为这些细胞系都难以转染，劳动密集型的方法，例如慢病毒载体基因插入或适体/肽偶联是必要的，以提供足够量的抗HIV-1的RNA，观察对HIV-1复制的效果。这种限制使得迪fficult迅速比较特定抗HIV-1 RNA的变体之间的构效关系或执行大规模筛选，以确定该最佳目标的网站的新类抗HIV-1 RNA的。而病毒产生检测是有益的，用于识别在支持HIV-1复制容易转染细胞系，如TZM-BL细胞新的抗HIV-1的RNA分子，可替代的细胞模型，将用于筛选有用的抗HIV-1的RNA靶向在复制周期中的其他步骤，例如进入和融合。 取决于类抗HIV-1 RNA的，毒性的几个潜在的机制已经描述。例如，基于反义RNA可以对包含相同或相似的序列，以它们的预期目标部位中的HIV-1 RNA的细胞的RNA的脱靶效应。类似地，RNA适体，反式激活响应元件（TAR）或冯响应元件（RRE）仿照，可以影响细胞PR的功能oteins诸如TAR RNA结合蛋白（TRBP）34。的shRNA和siRNA的有增加的潜力通过隔离的RNAi蛋白35和一些的U1i分子影响RNA干扰途径已经显示通过隔离U1小核RNA蛋白复合36的蛋白来影响细胞的RNA的剪接和加工。虽然其中一些效果可以通过精心的设计被最小化，在用于新的抗HIV-1的RNA分子的屏幕的细胞毒性的测量是在从进一步的发展潜力的脱靶效应不含分子是有用的。此外，脱靶效应可能间接地抑制HIV-1的产生，使细胞毒性在验证新的抗HIV-1 HIV-1的生产屏幕画面识别分子的疗效的重要测量。 细胞活力检测步骤3.1描述。 3.4。是已经被用于筛选多样的抗病毒分子的标准测定法的变化<html>第37条 。该测定测量的NAD的活性（P）H-依赖的细胞酶的MTT试剂减少至其不溶性紫色形式，甲。该协议从先前公布的38方法改编和几 ​​个套件采用MTT或其他密切相关的试剂可用。虽然以测定在用于筛选抗HIV-1的RNA的细胞系的细胞活力是重要的，但应注意的是，不同的细胞系在其朝向RNA诱导的毒性的敏感性而变化。例如，Reynolds 等人证明了Dicer酶底物siRNA的有在HEK293T细胞在MCF7，DU145和HeLa S3细胞39对细胞活力没有影响，但显著降低细胞生存力。对于本文所述的测定法中，HEK293T细胞被用作一个例子（ 图4）;然而，同样的测定也已在MCF7细胞进行，以评估在细胞系即小RNA诱导的毒性16更敏感的潜在毒性。 e_content"&gt;由于抗HIV-1的RNA可以不被处理并呈现给适应性免疫系统，它们被认为是免疫原性更低相比抗HIV-1的蛋白质或肽，然而，这取决于它们的序列或结构，它们能够引起先天的免疫应答，和几个免疫传感器和信号通路已经确定，可以对小RNA（在40中综述）反应。在此描述的免疫激活测定法中，磷酸化的PKR和IRF3的水平与小RNA作为转染的细胞进行了比较PKR或TLR3活化的指示，分别，两者的RNA传感器存在于广泛的细胞系和其活化可导致产生1型干扰素，并在PKR，在翻译一个关闭的情况下，为了评价对于抗HIV-1 RNA的潜在激活生产型的1干扰素由替代途径，干扰素刺激的基因ADAR1（P150）的水平在与转染的细胞进行了比较抗HIV-1 RNA的。这表现在长的dsRNA正控制的效果，聚I:C，所有这些反应都在HEK293T细胞活性（ 图5）和在MCF7细胞16中观察到类似的效果。由于这些反应可能抑制HIV-1的产生在没有对细胞活力的影响，免疫激活检测提供了新的抗HIV-1 RNA的功效额外的验证。可以添加到该评价进一步测量包括测量产生炎性细胞因子和类型在细胞培养物上清液1干扰素，和测量的更干扰素刺激基因的表达。由于艾滋病毒的靶细胞，如CD4 + T细胞和巨噬细胞可能表达不同水平的先天免疫传感器，从在此协议中描述的测定法鉴定的候选也应该在这些细胞类型的潜在免疫刺激进行评价。 对于所有的试验都描述D，用于获得可再现和精确的结果的临界步骤是DNA或RNA转染管的制备（步骤1.3）。质粒DNA或RNA应是高纯度的与浓度精确地确定。这也很关键，包括适当的控制。对于病毒生成测定评估新的测试RNA表达质粒，适当的阴性对照是空表达质粒。基于反义的RNA，表达非靶向的RNA的附加阴性对照质粒也应包含在内。在13提供了用于非靶向核酶序列和shRNA不影响HIV的产生。对于测试的siRNAs，可以使用的唯一的阴性对照是一个非靶向RNA和用于病毒生产测定中16，提供了一种适当的非靶向siRNA序列。对细胞活力和免疫激活测定中，与阴性对照应细胞单独用转染试剂处理，它为critical该阳性对照如聚I:C被包括，以确认该细胞是响应于RNA诱导的毒性或免疫激活。用于RNA表达质粒，也应包括在空质粒，以确保载体本身并不具有对细胞的毒性作用。 总体而言，本文所描述的测定法代表朝向识别为有效和安全使用的RNA疗法中的HIV-1基因或药物疗法的第一步。为分子靶向HIV-1RNA，同样重要的是要考虑其目标站点中的HIV-1流行株，以及详细的方法保护，以计算序列保守先前已发表​​15。向前移动的分子进入临床试验，长期毒性和功效研究应在人原代细胞和动物模型中进行，以确认所识别的候选将是安全的，并在临床上是有效的。

的当前的HIV-1治疗的局限性在于，它们必须长期施用，以防止疾病的进展。 HIV-1的抗T淋巴细胞，或造血干细胞的移植，有可能提供HIV-1复制的长期控制在没有药物治疗1,2的电位，也可以是实现一种HIV-1治疗的有效方法3。渲染到HIV-1复制抗性细胞的一种方法是自体移植4中插入一个或多个基因编码抗-HIV-1的RNA或肽进入受感染个体的细胞。几个候选抗HIV-1基因已被设计有一些进入临床试验在两个5或3个6的组合，以防止HIV-1的抗性的发展的任何单一基因。 抗HIV-1 RNA的顶端候选组合的基因治疗中，由于它们的低电势以引起免疫反应和，因为它们从很短的基因序列转录。一些抗HIV-1的RNA已被设计为靶向病毒进入和整合。然而，大多数的抗HIV-1的RNA靶向病毒生命周期积分后步骤（ 图1）。后的整合酶抑制剂包括诱饵的RNA，针对HIV-1调控蛋白达或REV 1，和基于反义RNA的，在HIV-1 RNA针对不同的网站，如核酶7，8的shRNA的U1i和RNA的9。已用于比较的抗HIV-1 RNA的功效的方法包括监测病毒复制与编码基因候选RNA和测定用表达候选RNA的质粒瞬时转染细胞的病毒的生产和HIV-1的表达质粒转染细胞10 -13。我们先前使用过的HIV-1的生产试验，筛选新的核酶靶位点13-15 HIV-1 RNA。这些方法，至今已细化到优化的RNA的格式干扰分子从质粒DNA表达为一个shRNA的或作为合成的siRNA 16传递。该测定测量生产的人胚胎肾（HEK）293T细胞成熟的病毒，并且可以用于比较针对在HIV-1复制周期积分后步骤（ 图1）抑制剂的影响。对于面向预集成的步骤抑制剂，需要替代的试验，如TZM-BL细胞感染性测定17来评价抗病毒疗效。 在临床的抗HIV-1 RNA的递送主要的安全问题包括对人类RNA或蛋白质，以及天然免疫传感器的激活潜在的脱靶效应。为了评估抗HIV-1 siRNA的毒性，我们使用在不同的细胞系16的细胞活力测定。我们还测量了双链RNA的免疫传感器的激活，RNA活化蛋白激酶R（PKR）和Toll样受体3（TLR3），以及移干扰素的PRESSION刺激基因，ADAR1 P150。这些测定可用于证实抗HIV-1 RNA的效力不因对细胞活力或免疫传感器活化间接影响。他们也是不包括候选人的RNA与进一步发展的潜在的毒性非常有用。 在下面的协议，过程，以确定新的治疗RNA和优化现有的格式被描述。该方法可用于HIV-1复制的筛选基于RNA后整合抑制剂和可适于筛选其他集成后抑制剂如小分子靶向启介导的病毒RNA 18或CRISPR的出口/设计CAS大全MSDS系统到目标集成HIV-1 DNA 19。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>DMEM HyClone</td>
			<td>GE Healthcare</td>
			<td>SH30243.01</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS HyClone</td>
			<td>GE Healthcare</td>
			<td>SH30396.03</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin Gibco</td>
			<td>Thermo Fisher</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture plates, 96 well, 24 well, 6 well.</td>
			<td>Corning</td>
			<td>353075, 353047, 353043</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tubes Axygen</td>
			<td>Corning</td>
			<td>311-08-051</td>
			<td></td>
		</tr>
		<tr>
			<td>Low molecular weight Poly I:C</td>
			<td>InvivoGen</td>
			<td>3182-29-6</td>
			<td></td>
		</tr>
		<tr>
			<td>DharmaFECT-1</td>
			<td>Dharmacon</td>
			<td>T-2001-01</td>
			<td>transfection reagent for synthetic RNAs</td>
		</tr>
		<tr>
			<td>TransIT-LT1</td>
			<td>Mirus</td>
			<td>MIR 2300</td>
			<td>transfection reagent for RNA expression plasmids</td>
		</tr>
		<tr>
			<td>Nonidet P40 (NP-40)</td>
			<td>USB</td>
			<td>19628</td>
			<td></td>
		</tr>
		<tr>
			<td>[32P]dTTP</td>
			<td>Perkin Elmer</td>
			<td>BLU505H</td>
			<td></td>
		</tr>
		<tr>
			<td>poly(A) RNA template&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>10108626001</td>
			<td></td>
		</tr>
		<tr>
			<td>oligo(dT)12-18 DNA primer</td>
			<td>Thermo Fisher</td>
			<td>18418-012</td>
			<td></td>
		</tr>
		<tr>
			<td>DEAE filtermat paper&#160;</td>
			<td>Perkin Elmer</td>
			<td>1450-522</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate scintillation counter</td>
			<td>Perkin Elmer</td>
			<td>1450-024</td>
			<td></td>
		</tr>
		<tr>
			<td>MTT</td>
			<td>Sigma-Aldrich</td>
			<td>M-2128</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS HyClone</td>
			<td>GE Healthcare</td>
			<td>SH30028.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Microplate spectrophotometer</td>
			<td>Bio-rad</td>
			<td>1706930</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysis buffer tablets</td>
			<td>Roche</td>
			<td>4693159001, 4906837001</td>
			<td>protease and phosphatase inhibitors</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td>Eppendorf</td>
			<td>5415R</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford reagent</td>
			<td>Bio-rad</td>
			<td>500-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel running chamber</td>
			<td>Hoefer</td>
			<td>SE600</td>
			<td></td>
		</tr>
		<tr>
			<td>Semi-dry transfer cell</td>
			<td>Bio-rad</td>
			<td>1703940</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein ladder EZ-Run</td>
			<td>Thermo Fisher</td>
			<td>BP3603-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane</td>
			<td>Bio-rad</td>
			<td>162-0094</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A9647-1006</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody stripping solution</td>
			<td>Millipore</td>
			<td>2504</td>
			<td></td>
		</tr>
		<tr>
			<td>ECL - Pierce</td>
			<td>Thermo Fisher&#160;</td>
			<td>PI32106</td>
			<td></td>
		</tr>
		<tr>
			<td>ADAR1 antibody</td>
			<td></td>
			<td></td>
			<td>from Dr. B.L. Bass</td>
		</tr>
		<tr>
			<td>phospho-T446-PKR antibody</td>
			<td>Abcam</td>
			<td>ab32036</td>
			<td></td>
		</tr>
		<tr>
			<td>phospho-S396-IRF3 antibody</td>
			<td>Cell Signaling</td>
			<td>4947</td>
			<td></td>
		</tr>
		<tr>
			<td>PKR antibody</td>
			<td></td>
			<td></td>
			<td>from Dr. A. Hovanessian</td>
		</tr>
		<tr>
			<td>IRF3 antibody</td>
			<td>Cell Signaling</td>
			<td>11904</td>
			<td></td>
		</tr>
		<tr>
			<td>Actin antibody</td>
			<td>Millipore</td>
			<td>MAB1501</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase-labeled goat anti-rabbit</td>
			<td>KPL</td>
			<td>474-1506</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxidase-labeled goat anti-mouse</td>
			<td>KPL</td>
			<td>474-1806</td>
			<td></td>
		</tr>
		<tr>
			<td>Ponceau S&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>6226-79-5</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

的程序的一般示意性示出在图2中为三个测试RNA和在图2B中提供了一个对照RNA的一例的转染方案。用于病毒生产和细胞生存力测定法，所读出的每个测试构建体是标准化为阴性对照。重复转染在集，使得每个测试RNA被归一化到其相邻的阴性对照。这样做是为了避免与配位和转染之间的时间，这可能会导致如果，例如，所有的阴性对照的是第一转染不准确的数据。因为HIV-1的可影响免疫应答27,28，细胞存活率和免疫激活测定法不加入一种HIV-1表达质粒的完成。 要识别RNA干扰分子的最佳长度在针对HIV-1 RNA保守位点（1498至1415年在HIV-1株NL4-3）13 19，一组的siRNA的设计（ 图3A）。使用HIV-1的生产试验中，相比于用一个长的Dicer底物无义的siRNA转染的细胞的RT活性的百分比（SINS）计算用于与在不同量的共转染用100ng的质粒中的每个测试的siRNA表达转染的细胞HIV-1毒株NL4-3（ 图3B）。 使用细胞生存力测定法，MTT法的百分比代谢物用于与在图3B中确定的最有效的siRNA转染的细胞来确定。数据是在单独用转染试剂（ 图4）处理的细胞中标准化为MTT代谢。长双链RNA（聚I:C）降低细胞活力;然而，其效果是只在评估的最高剂量显著。观察到的siRNA靶向1498 SI细胞活力无显著减少<html> TE在HIV-1 RNA，不论其长度。使用免疫激活测定法中，相同的一组RNA的评价其潜在引发免疫应答（ 图5）。在聚我条件:C活化ADAR1 P150的表达和PKR和IRF3的诱导的磷酸化，可以为任何测试RNA的被观察到的免疫活化标记没有显著效果。 <img alt="图1" src="/files/ftp_upload/54486/54486fig1.jpg" /> 在HIV-1复制周期之前和之后的集成步骤 如图1 示意图。预集成（1-3）和集成后（4-7）中的HIV-1复制周期的步骤示于概述框。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54486/54486fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 2"SRC ="/文件/ ftp_upload / 54486 / 54486fig2.jpg"/&gt; 图2 示意图病毒产生，细胞生存力和免疫激活测定的:（A）板贴壁细胞和培养24小时。细胞接种在24-，96-和分别在500，100 12-孔板和1000微升的细胞培养基的病毒生产，细胞生存力和免疫激活测定法。 （ 二 ）准备转管，转染细胞和文化48小时。为一组三个测试的RNA（RNA1-3）的示例的转染方案被示为具有适当的控制。转染过程也示出。 （C）读出。读出的病毒生产，细胞活力和免疫激活检测都写了详细的第2，第3和第4解释，分别为。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54486/54486fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> &lt;/ P&gt; <img alt="图3" src="/files/ftp_upload/54486/54486fig3.jpg" /> 图3. 测试的siRNAs对HIV-1的产生。（A）具有不同的长度和对称性的siRNA设计。在HIV-1 RNA（1498目标站点）的保守序列用于设计对称和非对称的siRNA（si1498-）不同长度（17至29个核苷酸义链）。预期的意义上（顶部，黑）和反义（下部，红色）链表示。 （B）由si1498长度变种HIV-1产生抑制。在与对称或不对称si1498长度变体转染的HEK293T细胞的上清液的HIV-1 RT活性的百分比（％）抑制示出相对于一个长的Dicer底物无义的siRNA（SINS）。各试验的RNA相比以不同剂量在两到三个重复的至少两个独立转染的罪。数据是位于expressed为平均值±标准误差手段（中小型企业）。这个数字已经从16修改，版权美国微生物学会。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54486/54486fig3large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54486/54486fig4.jpg" /> 图4. si1498长度对细胞生存力的变体的HEK293T细胞用聚I转染:C，在50和200微克/毫升在100nM si1498的（pIC 50和PIC200）和不同的长度和格式。 MTT的％代谢相对于用单独的转染试剂（模拟，M）在三个独立转染两到三次重复处理的细胞进行了测定。数据表示为平均值±中小企业。未配对的学生t检验来确定是否不同转染significantlY（*，P &lt;0.05）降低细胞活力相对于模拟转染的细胞。这个数字已经从16修改，版权美国微生物学会。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54486/54486fig4large.jpg" target="_blank">请点击此处查看本图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54486/54486fig5.jpg" /> 变体在 图5 si1498长度的影响先天免疫反应。HEK293T细胞用pIC 50，PIC200和不同的长度和在100nM si1498格式转染。 PKR和TLR3的激活分别用测量磷酸化PKR和IRF3，评估。通过测量相对组成型表达的变异，ADAR1 P110的ISG ADAR1 P150水平评估干扰素刺激基因（ISG）的表达。肌动蛋白的表达被用作上样对照。这FIGURË已经从16修改，版权美国微生物学会。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54486/54486fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 缓冲器/试剂名称 </td><td> 组成 </td></tr><tr><td> 病毒破坏鸡尾酒 </td><td> 60mM的Tris-盐酸（距离的1M的Tris-HCl，pH值7.8），75mM的氯化钾，5mM的MgCl 2的，1.04毫摩尔EDTA，1％NP-40。 </td></tr><tr><td> 放射性鸡尾酒 </td><td> 60mM的Tris-盐酸（距离的1M的Tris-HCl，pH值7.8），75mM的氯化钾，5mM的MgCl 2的，1.04毫摩尔EDTA，10微克/毫升多聚（A），0.33微克/毫升寡聚dT。在使用前立即补充:8二硫苏糖醇（DTT，C 4 H 10 O 2 S 2）和5微升[32 P] dTTP的（3000居里/毫摩尔）每次500微升鸡尾酒。 </td></tr><tr><td> 放射性/病毒破坏鸡尾酒 </td><td> 60mM的Tris-盐酸（距离的1M的Tris-HCl，pH值7.8），75mM的氯化钾，5mM的MgCl 2的，1.04毫摩尔EDTA，0.1％NP-40，5微克/毫升多聚（A），0.16微克/毫升寡胸苷。在使用前立即加入:8二硫苏糖醇（DTT，C 4 H 10 O 2 S 2）和5μl[32 P] dTTP的（3000居里/毫摩尔），用于将每1ml鸡尾酒。 </td></tr><tr><td> 2X SSC </td><td> 17.53克NaCl和8.82克柠檬酸钠- 2H 1 LH 2 O 2 O </td></tr><tr><td> 裂解缓冲液 </td><td>的50mM的Tris-HCl（从1M的Tris-盐酸，pH为7.4），150 mM氯化钠，5mM的EDTA（来自0.5M的，pH值8.0），10％体积/体积的甘油，1％体积/体积的NP-40。 </td></tr><tr><td> 丽春红 </td><td> 2.5克丽春红S和5ml乙酸在500毫升H 2 O </td></tr><tr><td> TBST </td&gt; <td> 6.05克的Tris，8.76克氯化钠和1ml Tween 20的1 LH 2 O </td></tr></tbody></table>提供非商业缓冲液和试剂的成分配方的所有非商业缓冲液和试剂:表1。

Watch this Scientific Journal Video about Evaluation of the Efficacy And Toxicity of RNAs Targeting HIV-1 Production for Use in Gene or Drug Therapy at JoVE.com

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

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 ...

evaluation-efficacy-toxicity-rnas-targeting-hiv-1-production-for-use

Nanyang Technological University

Research

JoVE Journal

Immunology and Infection

7.8K 次观看.  Lady Davis Institute for Medical Research. 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. 

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

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

Generation of Multivirus-specific T Cells to Prevent/treat Viral Infections after Allogeneic Hematopoietic Stem Cell Transplant

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully generated and infused T-cells specific for Epstein Barr virus (EBV), cytomegalovirus (CMV) and Adenovirus (Adv) using monocytes and EBV-transformed lymphoblastoid cell (EBV-LCL) gene-modified with an adenovirus vector as antigen presenting cells (APCs). As few as 2x105/kg trivirus-specific cytotoxic T lymphocytes (CTL) proliferated by several logs after infusion and appeared to prevent and treat even severe viral disease resistant to other available therapies. The broader implementation of this encouraging approach is limited by high production costs, complexity of manufacture and the prolonged time (4-6 weeks for EBV-LCL generation, and 4-8 weeks for CTL manufacture &#x2013; total 10-14 weeks) for preparation. To overcome these limitations we have developed a new, GMP-compliant CTL production protocol. First, in place of adenovectors to stimulate T-cells we use dendritic cells (DCs) nucleofected with DNA plasmids encoding LMP2, EBNA1 and BZLF1 (EBV), Hexon and Penton (Adv), and pp65 and IE1 (CMV) as antigen-presenting cells. These APCs reactivate T cells specific for all the stimulating antigens. Second, culture of activated T-cells in the presence of IL-4 (1,000U/ml) and IL-7 (10ng/ml) increases and sustains the repertoire and frequency of specific T cells in our lines. Third, we have used a new, gas permeable culture device (G-Rex) that promotes the expansion and survival of large cell numbers after a single stimulation, thus removing the requirement for EBV-LCLs and reducing technician intervention. By implementing these changes we can now produce multispecific CTL targeting EBV, CMV, and Adv at a cost per 106 cells that is reduced by &gt;90%, and in just 10 days rather than 10 weeks using an approach that may be extended to additional protective viral antigens. Our FDA-approved approach should be of value for prophylactic and treatment applications for high risk allogeneic HSCT recipients.

A rapid, simple and cost-effective protocol for the generation of donor-derived multivirus-specific CTLs (rCTL) for infusion to allogeneic hematopoietic stem cell transplant (HSCT) recipients at risk of developing CMV, Adv or EBV infections. This manufacturing process is GMP-compliant and should ensure the broader implementation of T-cell immunotherapy beyond specialized centers.

Multivirus特异性T细胞的生成，预防/治疗异基因造血干细胞移植后的病毒感染

Viral infections cause morbidity and mortality in allogeneic hematopoietic stem cell transplant (HSCT) recipients. We and others have successfully ...

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 ...

In vitro Uncoating of HIV-1 Cores

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 composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core.
Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection.
HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37&deg;C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating.

In vitro Uncoating of HIV-1 Cores

Uncoating is an essential step in the early phase of the HIV-1 life cycle and is defined as the disassembly of the capsid shell and the release of the viral ribonucleoprotein complex (vRNP). Here, we demonstrate techniques for isolating intact cores from HIV-1 virions and for quantifying their uncoating in vitro.

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

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

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 ...

Conformational Evaluation of HIV-1 Trimeric Envelope Glycoproteins Using a Cell-based ELISA Assay

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins are able to fuel the fusion process through their conformational changes could lead to the design of better, more effective immunogens for vaccine strategies. Here we describe a cell-based ELISA assay that allows studying the recognition of trimeric HIV-1 Env by monoclonal antibodies. Following expression of HIV-1 trimeric Env at the surface of transfected cells, conformation specific anti-Env antibodies are incubated with the cells. A horseradish peroxidase-conjugated secondary antibody and a simple chemiluminescence reaction are then used to detect bound antibodies. This system is highly flexible and can detect Env conformational changes induced by soluble CD4 or cellular proteins. It requires minimal amount of material and no highly-specialized equipment or know-how. Thus, this technique can be established for medium to high throughput screening of antigens and antibodies, such as newly-isolated antibodies.

Understanding viral surface antigens conformations is required to evaluate antibody neutralization and guide the design of effective vaccine immunogens. Here we describe a cell-based ELISA assay that allows the study of the recognition of trimeric HIV-1 Env expressed at the surface of transfected cells by specific anti-Env antibodies.

HIV-1的三聚体包膜糖蛋白的构象评估使用基于细胞的ELISA检测

HIV-1 envelope glycoproteins (Env) mediate viral entry into target cells and are essential to the infectious cycle. Understanding how those glycoproteins ...

High-throughput Quantitative Real-time RT-PCR Assay for Determining Expression Profiles of Types I and III Interferon Subtypes

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific locked nucleic acid (LNA) and molecular beacon (MB) probes, transcript standards, automated multichannel pipetting, and plate drying. Determining expression among the type I interferons (IFN), especially the twelve IFN-&#945; subtypes, is limited by their shared sequence identity; likewise, the sequences of the type III IFN, especially IFN-&#955;2 and -&#955;3, are highly similar. This assay provides a reliable, reproducible, and relatively inexpensive means to analyze the expression of the seventeen interferon subtype transcripts.

This protocol describes a high-throughput qRT-PCR assay for the analysis of type I and III IFN expression signatures. The assay discriminates single base pair differences between the highly similar transcripts of these genes. Through batch assembly and robotic pipetting, the assays are consistent and reproducible.

高通量实时定量RT-PCR检测方法的确定I型和III型干扰素亚型的表达谱

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

从HIV-1阳性者的血浆分离外来体的

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

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

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 viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

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

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

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 ...

Evaluation of Host-Pathogen Responses and Vaccine Efficacy in Mice

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a striking increase in life expectancy around the globe. Nonetheless, determining vaccine efficacy remains a challenge. Emerging evidence suggests that the current acellular vaccine (aPV) for Bordetella pertussis (B. pertussis) induces suboptimal immunity. Therefore, a major challenge is designing a next-generation vaccine that induces protective immunity without the adverse side effects of a whole-cell vaccine (wPV). Here we describe a protocol that we used to test the efficacy of a promising, novel adjuvant that skews immune responses to a protective Th1/Th17 phenotype and promotes a better clearance of a B. pertussis challenge from the murine respiratory tract. This article describes the protocol for mouse immunization, bacterial inoculation, tissue harvesting, and analysis of immune responses. Using this method, within our model, we have successfully elucidated crucial mechanisms elicited by a promising, next-generation acellular pertussis vaccine. This method can be applied to any infectious disease model in order to determine vaccine efficacy.

Here we present an elegant protocol for in vivo evaluation of vaccine effectiveness and host immune responses. This protocol can be adapted for vaccine models that study viral, bacterial, or parasitic pathogens.

宿主病原学反应及疫苗效果评价

Vaccines are a 20th century medical marvel. They have dramatically reduced the morbidity and mortality caused by infectious diseases and contributed to a ...