这项研究得到了国家癌症研究所的院内研究计划的支持。

1，准备炒股高手的<ol><li>使在DMSO中，待测试的化合物的主股。准备库存为20毫米的标准浓度。 注意：上面的10mM任何浓度都可以使用。可以用来作为阳性对照，以验证该测定化合物包括RAL，EVG和DTG。 </li><li>确保该化合物溶解在DMSO中通过涡旋将溶液多次，持续15秒，并在室温温育1小时。存储的20mM储备溶液在黑暗中在-20℃直至使用。 </li></ol> 2制备的96孔板的化合物筛选<ol><li>选择的细胞系中进行测试（ 例如居屋或TZM-BL），和种子100微升的细胞以4×10 4个细胞/ ml的密度（4000个细胞/孔）在介质（ 例如，Dulbecco改良的Eagle氏或DMEM培养基补充了5％（V / V）胎牛血清，5％新生小牛血清，及青霉素（50单位/毫升）加链霉霉素）。 </li></ol> 3代病毒的股票产生VSV-G假型病毒转染的293细胞（ 如图1的步骤1）32-34。 <ol><li>在当天转染前，板293细胞的直径为100毫米的菜为1.5×10 6个细胞的密度。 </li><li>用磷酸钙法35和4 VSV（pHCMV-G）的微克-对转染当天，转染293细胞16微克野生型或突变的HIV（ΔLUCpNLNgoMIVR）的。 </li><li>磷酸钙沉淀物后约6小时加入，洗两次293细胞用磷酸盐缓冲盐水（PBS）中孵育用新鲜培养基48小时。 [DMEM培养液，用5％（V / V）胎牛血清，5％新生小牛血清，及青霉素（50单位/毫升）加链霉素（50微克/毫升）。 </li><li>从100毫米直径的去除媒体​​收获上清液含有病毒菜，澄清的上清液用低速离心以3000rpm 10分钟后，经过45微米孔径注射器过滤器过滤上清液，治疗与涡轮脱氧核糖核酸酶的上清液30分钟，在RT和在感染测定稀释于介质的上清液用于制备。存储冻结，以等分试样，在-80℃下的病毒上清 注意：p24的上清液中的量是通过使用HIV-1的p24酶联免疫吸附测定试剂盒测定。在p24的浓度是用来控制样品中病毒的量。约500ng的病毒加入到在对前一天感染1.5×10 5个细胞/皿的密度接种在直径60mm的培养皿HOS细胞。经过48小时培养后，收获细胞，通过离心收集，洗涤，再悬浮于100μl的PBS中。添加荧光报告基因检测试剂的量相等，并在部分5.4.1中描述测量荧光素酶活性和5.4.2。由此，经适当稀释的病毒可制成如在步骤4.6中讨论。 </li></ol> 4，化合物筛选的96孔板屏幕的每个化合物一式三份，并平均结果。 注意：对病毒复制的每个化合物的作用是通过归一化到在不存在任何化合物的获得复制的水平校正。 <ol><li>确定经验的浓度范围，以进行筛选。 注意：通常情况下，与11个系列稀释的屏幕是通过将所述化合物通过柱并用7系列稀释屏板式塔由通过排将化合物制成。一式三份集水井必须为无复合控制预留。此外，一列或一行必须保持空白，作为阴性/后台控制。最后，无论是细胞的细胞毒性或感染性测定法将决定如果病毒被添加。 </li><li>准备系列稀释液从​​20mM的储备溶液。取决于浓度的待测试的经验确定的范围内的浓度被选择（如表1所示）。准备在媒体的稀释液在10倍所需的最终浓度， 也就是说 ，如果最终浓度将是100微米，做一个1毫米的工作股票。 注：带IC 50秒以上5-10微米的化合物通常不适合进行药物开发。在最初的实验，我们测试只化合物对野生载体。有前途的化合物，能有效抑制野生型向量，然后对之耐药突变体面板测试。 </li><li>从培养箱中取出96孔板中，并添加要测试的孔中一式三份的化合物的系列稀释液（如示于图1，步骤2）。 注：加入到孔中的体积应为1/10体积的最终浓度。因此，加22微升/孔（终（体积）POST病毒除了是220微升）的感染性测定。对于细胞毒性试验中，添加11微升/孔（终体积为110微升/孔）。 </li><li>返回的96孔板的培养箱中培养。对于细胞毒性屏幕，孵育96孔板48小时在37℃，并在需要的协议4没有进一步的操作：继续进行协议5对于感染性测定，继续按照协议4的说明。 </li><li>感染性测定而已。 至少 3小时温育于37℃下与待分析的化合物后，从孵化器中取出板。 注意：这使得该化合物应采取由前至感染细胞的HIV载体。 </li><li>准备一个股票稀释病毒33，34（通常约1:3），将未经处理的细胞0.2-1.5相对荧光素酶单位（RLUs）之间产生的荧光素酶信号。整个板块将需时约10毫升的稀释的特德病毒。加入100μl病毒到每个孔中，用任何一个8或12的多通道移液器。不添加病毒阴性/背景对照孔。返回板到37℃培养箱中培养48小时。 注：一个1:3股票稀释病毒是典型的稀释会产生基于显示，病毒浓度的上清液约500毫微克1.0毫升p24的测定0.2之间-1.5 RLUs荧光素酶信号。 </li></ol> 5，制备和细胞毒性和感染性的在96孔板测量<ol><li>从孔中吸出培养基（无酚红在媒体可与荧光素酶信号干扰）。使用玻璃移液管与连接到端200μl的移液管尖端。开始在媒体上，慢慢地工作下来朝井底的角落。不花费太多时间的井的底部，或细胞可以从井中移除。 </li><li>加入100μl的PBS辅以0.5米M的MgCl 2至每孔中。这需要立即进行媒体被删除后，使细胞不干燥。 </li><li>仅用于细胞毒性试验，从发光ATP检测法加5毫升底物缓冲提供冻干试剂的每个小瓶。一个小瓶足以用于一个96孔板中。 <ol><li>加入50微升的细胞溶解缓冲液从发光的ATP检测测定每个孔中。摇动的96孔板中以700rpm的转速在室温下用小型恒温5分钟。 </li><li>加入50微升再造ATP荧光检测分析试剂，以除阴性对照/背景井各孔中。摇动以700rpm的转速在室温下用小型恒温5分钟。孵育板在室温下20分钟，以便有时间信号的发展。 </li><li>使用微板光度计读出的96孔板中。 注意：打开的SoftMax Pro的微孔板发光程序。确保光度计设置测量LUMI释光以5个读数/孔的敏感性。 </li></ol></li><li>仅适用于感染性测定，从发光报告基因检测加10毫升底物缓冲提供的冻干试剂的每个小瓶。一个小瓶足以为每个96孔板中。 <ol><li>加入100μl重组荧光报告基因检测试剂的每个孔中。在室温下孵育20分钟，以便有时间信号的发展。 </li><li>采用微孔板发光如第5.3.3进行阅读96孔板。 </li></ol></li></ol> 6，确定CC 50和IC 50值的化合物<ol><li>从转移的微孔板发光的荧光素酶数据到Excel电子表格。 <ol><li>平均都一式三份，并在背景/控制信号的数据的萤光素酶的数据。减去平均一式三份信号在整个浓度范围内的平均背景/控制信号。 </li> &lt;立&gt;规范化背景修正平均信号对活性的浓度范围内，无论是细胞毒性或感染性，在不存在任何化合物，以确定抑制百分数。 注意：抑制百分数被定义为在药物的存在下通过荧光素酶活性在没有药物乘以100除以荧光素酶活性。 </li></ol></li><li>使用Kaleidagraph科学软件程序以获取CC 50和IC 50值<ol><li>传送两个经验确定的浓度范围和抑制百分比荧光素酶活性成Kaleidagraph科学。 </li><li>与x轴的浓度范围内与荧光素酶活性在y轴上的抑制百分比绘制的数据。 </li></ol></li></ol>

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Virol. 83, 11440-11446 (2009).</li><li>Goethals, O., 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=Primary+mutations+selected+in+vitro+with+raltegravir+confer+large+fold+changes+in+susceptibility+to+first-generation+integrase+inhibitors,+but+minor+fold+changes+to+inhibitors+with+second-generation+resistance+profiles.">Primary mutations selected in vitro with raltegravir confer large fold changes in susceptibility to first-generation integrase inhibitors, but minor fold changes to inhibitors with second-generation resistance profiles.</a> Virology. 402, 338-346 (2010).</li><li>Goethals, O., 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=Resistance+mutations+in+human+immunodeficiency+virus+type+1+integrase+selected+with+elvitegravir+confer+reduced+susceptibility+to+a+wide+range+of+integrase+inhibitors.">Resistance mutations in human immunodeficiency virus type 1 integrase selected with elvitegravir confer reduced susceptibility to a wide range of integrase inhibitors.</a> J. Virol. 82, 10366-10374 (2008).</li><li>Canducci, F., 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=Dynamic+patterns+of+human+immunodeficiency+virus+type+1+integrase+gene+evolution+in+patients+failing+raltegravir-based+salvage+therapies.">Dynamic patterns of human immunodeficiency virus type 1 integrase gene evolution in patients failing raltegravir-based salvage therapies.</a> AIDS. 23, 455-460 (2009).</li><li>Ceccherini-Silberstein, F., 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=Characterization+and+structural+analysis+of+HIV-1+integrase+conservation.">Characterization and structural analysis of HIV-1 integrase conservation.</a> AIDS Rev. 11, 17-29 (2009).</li><li>Charpentier, 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=Drug+resistance+profiles+for+the+HIV+integrase+gene+in+patients+failing+raltegravir+salvage+therapy.">Drug resistance profiles for the HIV integrase gene in patients failing raltegravir salvage therapy.</a> HIV Med. 9, 765-770 (2008).</li><li>Julias, J. G., 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=Effects+of+mutations+in+the+G+tract+of+the+human+immunodeficiency+virus+type+1+polypurine+tract+on+virus+replication+and+RNase+H+cleavage.">Effects of mutations in the G tract of the human immunodeficiency virus type 1 polypurine tract on virus replication and RNase H cleavage.</a> J. Virol. 78, 13315-13324 (2004).</li><li>Hare, 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=Structural+and+functional+analyses+of+the+second-generation+integrase+strand+transfer+inhibitor+dolutegravir+(S/GSK1349572).">Structural and functional analyses of the second-generation integrase strand transfer inhibitor dolutegravir (S/GSK1349572).</a> Mol. 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D., 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+novel+polymorphism+at+codon+333+of+human+immunodeficiency+virus+type+1+reverse+transcriptase+can+facilitate+dual+resistance+to+zidovudine+and+L-2',3'-dideoxy-3'-thiacytidine.">A novel polymorphism at codon 333 of human immunodeficiency virus type 1 reverse transcriptase can facilitate dual resistance to zidovudine and L-2',3'-dideoxy-3'-thiacytidine.</a> J. Virol. 72, 5093-5098 (1998).</li><li>Butler, S. L., Hansen, M. S., Bushman, F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+assay+for+HIV+DNA+integration+in+vivo.">A quantitative assay for HIV DNA integration in vivo.</a> Nat. 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作者什么都没有透露。

我们描述了一种快速，有效和可重复的试验，可用于筛选化合物的细胞毒性和它们抑制WT和耐药的HIV-1的复制能力。快速识别化合物，并测试其疗效和毒性的能力是至关重要的新的和改进的药物抗HIV-1的发展。一旦铅化合物被确定，铅化合物的类似物，可以产生并使用相同的测定中测试。该测定是相对简单的。有两个阳性和阴性对照，允许用户来诊断的最常见问题（毒性化合物，存在的问题与载体stock）。使用一式三份组井与没有添加化合物的表示发生病毒感染。细胞毒性的测量是在一个独立的试验中的事实避免误解在荧光素酶的减少所造成的细胞毒性作为病毒复制的特定效果。 的关键步骤在协议中被制备的板，使细胞均匀地分布在孔中，该孔具有适当浓度的待测试的化合物中，加入病毒的相同数量的每个孔中，并测定荧光素酶活性，以确定CC 50和IC 50值。 该法是安全的，定量，重现性好。该测定是安全的，因为该载体是复制缺陷型。该测定是定量的和可重复的，因为它是基于一个单一圆形矢量，表达荧光素酶，它可以准确和方便地进行测定。在多轮病毒复制测定法，测得的IC 50值依赖于病毒生命周期的数目;这是一个特别的问题时，检测涉及WT和药物可能具有显著不同的复制能力，抗病毒。 已经有一些酶检测报告之前，可以可用于筛查中的抑制剂。涉及实时PCR技术来测量整合的DNA检测需要纯化的重组蛋白（S），并且，在一般情况下，两个更多的劳动密集和昂贵36,37。尽管有可能使用酶测定来测量化合物对其它病毒酶（RT和蛋白酶）的影响，每种酶都需要它自己的检测系统。一个圆形矢量测定，如上所述，可以使用，而无需修改，以筛选RT抑制剂。一个类似的试验，可用于筛选蛋白酶抑制剂;然而，在蛋白酶抑制剂的测定法，所述化合物必须被添加到用于制备载体的细胞。一个相关的实验中，使用不同的细胞和载体，也可用于筛选包膜（ENV）和HIV进入融合抑制剂。最后，该法可用于，在更大的规模，利用自动机械手的分配器。因此，该测定可用于筛选化合物的大文库抗WT和米utant艾滋病病毒。然而，事实上该测定可检测的HIV复制抑制剂，作用在病毒生命周期的点，在分析数据的限制不同的阶段。通过自身的测定并没有定义该步骤在生命周期中被阻止的化合物。如果这个问题出现了，这可以通过使用另外测定38的时间来解决，并通过测试对纯化的重组病毒蛋白的化合物。 不幸的是，尽管抗HIV药物的成功，仍然有问题的，这两个电阻和毒性。在没有有效的抗HIV疫苗，有必要不仅要开发新的治疗药物，这将有效地对抗现有药物抗性突变体，还开发预防性药物，可以减少病毒的传播。如果预防性使用抗HIV药物incudes未感染者的治疗，这种方法会放置一个特殊的负担，开发那些有升药ittle或无长期毒性。

的目标在HIV-1病毒的生命周期的几个基本步骤药品的供应，导致药物联合疗法（称为高效抗逆转录病毒疗法，或HAART），极大地提高了治疗的HIV-1感染和长期生存的病人。 HAART治疗，通常使用两种核苷逆转录酶（RT）抑制剂，要么蛋白酶抑制剂或非核苷逆转录酶抑制剂的组合，现在已经转换成一种致命的疾病成为寿命长条件1-5。然而，尽管鸡尾酒疗法在病毒复制的持续抑制了许多成就，但也有局限性。 HAART治疗不根除艾滋病病毒，所以病人没有治愈，治疗是寿命长。有问题的药物的毒性，并与药物抗性菌株的出现。阻力位可以出现于所有的被认可的抗HIV药物，包括新批准的药物，针对HIV整合酶（IN）。耐药性的产生可能贝科利用病毒复制（3×10 -5突变/基地/复制周期误差率）过程中发生的自发突变6。当突变发生在编码基因的反转录病毒药物的目标，这些突变的一小部分会导致在病毒株的敏感性的药物的减少。避免耐药性的发展，药物浓度必须维持在充分地抑制HIV复制的水平。穷坚持治疗方案加剧了这一问题，并可能导致电阻7-9的快速发展。虽然药物浓度可在患者有所不同，因为不同的吸收，代谢，分布和排泄水平，高浓度的药物可导致中毒10。由于继续治疗的病人的生命，大约有抗逆转录酶病毒药物的长期毒性严重的安全问题。抗逆转录病毒药物鸡尾酒疗法中常用的可以有不利影响次出现了发病率，其中的副作用，已危及生命11-15。患者的抵抗力和毒性的发展遇到的问题背后，需要开发新的药物，有效地阻断病毒的很少或根本没有长期毒性的常见耐药菌株的复制。 因此，有必要对一个测定，可以筛选阻断在病毒生命周期的基本步骤很快化合物。在这里，我们描述了可以用来评估化合物和它们的迅速和有效地阻断WT和药物抗性HIV病毒株的复制能力的细胞毒性高效率的检测。我们使用的测定法类似，被开发的病毒从患者16-18分离筛选耐药性的测定法。 虽然可以使用，而无需修改屏幕，可以阻止艾滋病病毒逆转录化合物的分析中，我们将DESCRIBE使用试验，以评估抑制剂。中是一个重要的病毒酶，插入病毒DNA进入细胞基因组19。虽然正在开发一些有前途的抑制剂，其中一些目前正在进行临床试验，只有20的Isentress，21（也称为拉替拉韦或RAL），最近，Elvitegravir（EVG）22和Dolutegravir（DTG）23人被FDA批准。这些化合物具有抗艾滋病病毒B和细胞培养非亚型乙双方及患者24，25。然而，RAL治疗选择为耐药的HIV-1突变体中，包括Y143R，N155H和G140S/Q148H 24，26-31。 N155H和G140S/Q1​​48H也减少了EVG的功效，它强调需要来设计和开发第二代链转移抑制剂（INSTIs）是可以有效对付这些耐药突变。

<table border="0" cellpadding="0" cellspacing="0" width="659">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMSO</td>
			<td style="width:157px;">Sigma</td>
			<td style="width:119px;">D2650</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM</td>
			<td style="width:157px;">Corning Cellgro</td>
			<td style="width:119px;">10-017</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ATPlite Luminescence ATP Detection Assay System</td>
			<td style="width:157px;">Perkin Elmer</td>
			<td style="width:119px;">6016941</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Steady Lite Plus High Sensitivity Luminescence Reporter Gene Assay System</td>
			<td style="width:157px;">Perkin Elmer</td>
			<td style="width:119px;">6016751</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Dulbecco&#39;s PBS</td>
			<td style="width:157px;">Gibco-Life Technologies-Invitrogen</td>
			<td style="width:119px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SpectraMax Gemini EM</td>
			<td style="width:157px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">KaleidaGraph</td>
			<td style="width:157px;">Synergy Software</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Nunc F96 Microwell White Polystyrene Plate</td>
			<td style="width:157px;">Thomas Scientific</td>
			<td style="width:119px;">12-566-26</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Eppendorf Thermomixer Compact</td>
			<td style="width:157px;">Sigma Alrich</td>
			<td style="width:119px;">T1442-1EA</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Turbo Dnase</td>
			<td style="width:157px;">Ambion-Life Technologies-Invitrogen</td>
			<td style="width:119px;">AM2238</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Millex HA Filter Unit, 0.45 &#181;M</td>
			<td style="width:157px;">Millipore</td>
			<td style="width:119px;">SLAHA033SS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Alliance HIV-1 p24 Elisa Kit</td>
			<td style="width:157px;">Perkin Elmer</td>
			<td style="width:119px;">NEK050B001KT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HOS cells</td>
			<td style="width:157px;">ATCC</td>
			<td style="width:119px;">CRL-1543</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TZM-bl cells</td>
			<td style="width:157px;">NIH AIDS Reagent Program</td>
			<td style="width:119px;">8129</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">pNL4.3&#916;Env.LUC</td>
			<td style="width:157px;">NIH-NCI HIV Drug Resistance Program- Hughes Lab</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">VSV-G</td>
			<td style="width:157px;">NIH-NCI HIV Drug Resistance Program- Hughes Lab</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SoftMax Pro</td>
			<td style="width:157px;">Molecular Devices</td>
			<td style="width:119px;">0200-310</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid screening of hiv reverse transcriptase and integrase inhibitors

虽然一些抗HIV药物已被批准，但仍有问题，毒性和耐药性。这表明需要鉴定新的化合物，可以由共同的耐药性的HIV-1毒株具有最小的毒性抑制感染。在这里，我们描述了可以用于快速确定该细胞的细胞毒性和抗WT和突变型病毒株的化合物的功效的高效测定。 所需的目标细胞系接种在96孔板中，并24小时温育后，被测试化合物的连续稀释液加入。没有进一步的操作所必需的细胞的细胞毒性测定法;对于抗HIV测定的任一WT或耐药的HIV-1载体，表达荧光素酶加入到细胞中一个预定量。的细胞毒作用是通过使用一个ATP依赖的发光测定法和感染性化合物的影响测定是通过测定lucife的量测RASE在存在或不存在的推定抑制剂。 此筛选测定需要4天时间来完成和多个化合物可以并行进行筛选。筛选化合物以一式三份，并在数据被归一化到感染性/ ATP水平中不存在目标化合物。这种技术提供了潜在的抗HIV化合物的功效和毒性的快速和准确的测量。

如果该测定（ 图1，步骤1和2）被成功执行，则萤光素酶值应类似于表2中所给出的数据扫描在整个浓度范围内;一种潜在的有效的化合物，就会发现从左至右增加荧光素酶活性，并且控制应该具有最高的荧光素酶的活性。如果荧光素酶活性不超过整个浓度范围0.1相对荧光素酶单位（RLUs），这通常表明，该化合物杀死细胞。如果该荧光素酶的数据是大​​于或等于2.0 RLUs在所有系列稀释，然后在化合物不能够抑制HIV-1感染在测试浓度。 绘制化合物的相对荧光素酶活性的Kaleidagraph科学（ 表3，A部分）的百分比抑制浓度，进行线性回归分析后，会产生类似的结果为t<html>在表3中的B部分所示的软管<img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51400/51400fig1highres.jpg" width="500" /> 图1制备的HIV-1病毒种群和细胞毒性和单轮感染性测定的设置，在步骤1，293T细胞转染与pNL4.3ΔEnv.LUC和VSV-G和培养48小时，以产生病毒34 。病毒收获和储存（冷冻在-80℃下以等分试样），直到它被用在感染性测定。对于步骤2，HOS细胞被接种在96孔板中并孵育24小时。然后将细胞预孵育3小时，待测试化合物的系列稀释液，然后用病毒感染（无论是WT或药物抗性）。 48小时培养后，荧光素酶活性进行测定。 le1.jpg“宽度=”300“/&gt; 表1。药物筛选串行稀释原型。更严格的筛选涉及11个系列稀释它通常开始于10微米，并收于0.0005微米。该系列稀释制备的10倍;有100微升的细胞和100μl的病毒，每孔）。在此体积与以下这些计算，连续稀释将足以为3行的全部96孔板的。细胞毒性实验也同样准备;然而，11系列稀释开始在250微米，并最终在0.05微米。稀释液的只有11微升被加入到孔中含有100微升细胞的板块。 <a href="https://www.jove.com/files/ftp_upload/51400/51400table1.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表2" fo:content-width="6in" src="/files/ftp_upload/51400/51400table2.jpg" width="600" /><br/&gt; 表2。荧光素酶信号数据读出和荧光素酶活性的抑制百分比的测定，数据表显示一组典型的荧光素酶的数据对于一个成功的化合物。该表还显示需要确定荧光素酶活性的抑制百分率和CC 50和IC 50值的额外的计算， <a href="https://www.jove.com/files/ftp_upload/51400/51400table2.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="表3" fo:content-width="6in" src="/files/ftp_upload/51400/51400table3.jpg" width="600" /> 表3。线性回归分析的数据表 。 A部分。作图中使用与在Kaleidagraph科学萤光素酶活性的抑制百分比的浓度范围内会产生相应的抑制曲线。 B部分，该抑制曲线是由3参数定义S形函数拟合线性回归的数据分析18。这个数据表，然后在与Microsoft Excel一起使用来计算了50％， 如 IC 50和CC 50抑制病毒整合和细胞毒性所需的药物浓度。 <a href="https://www.jove.com/files/ftp_upload/51400/51400table3.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a>

Watch this Scientific Journal Video about Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors at JoVE.com

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

这里我们描述了细胞的细胞毒性和单轮感染性测定允许对化合物的快速，准确的筛选以确定它们的细胞毒性（CC 50）和IC 50值对WT和耐药性的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 ...

rapid-screening-of-hiv-reverse-transcriptase-and-integrase-inhibitors

Research

JoVE Journal

Immunology and Infection

17.7K 次观看.  National Cancer Institute. 这里我们描述了细胞的细胞毒性和单轮感染性测定允许对化合物的快速，准确的筛选以确定它们的细胞毒性（CC 50）和IC 50值对WT和耐药性的HIV-1。 

视频: Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

可视化的细胞与细胞转移的艾滋病毒，艾滋病毒和现场共聚焦显微镜的荧光克隆

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

科研

免疫与感染

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

胶粘剂基于磁带的采样和荧光组合原位杂交快速检测沙门氏菌新鲜农产品

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

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 ...

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 ...

Reverse Genetics Mediated Recovery of Infectious Murine Norovirus

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close person-to-person contact cannot be avoided 1, 2. During the last few years an increase in the incidence of outbreaks in hospitals has been reported, causing significant disruptions to their operational capacity as well as large economic losses. The identification of new antiviral approaches has been limited due to the inability of human noroviruses to complete a productive infection in cell culture 3. The recent isolation of a murine norovirus (MNV), closely related to human norovirus 4 but which can be propagated in cells 5 has opened new avenues for the investigation of these pathogens 6, 7. 
MNV replication results in the synthesis of new positive sense genomic and subgenomic RNA molecules, the latter of which corresponds to the last third of the viral genome (Figure 1). MNV contains four different open reading frames (ORFs), of which ORF1 occupies most of the genome and encodes seven non-structural proteins (NS1-7) released from a polyprotein precursor. ORF2 and ORF3 are contained within the subgenomic RNA region and encode the capsid proteins (VP1 and VP2, respectively) (Figure 1). Recently, we have identified that additional ORF4 overlapping ORF2 but in a different reading frame is functional and encodes for a mitochondrial localised virulence factor (VF1) 8.
Replication for positive sense RNA viruses, including noroviruses, takes place in the cytoplasm resulting in the synthesis of new uncapped RNA genomes. To promote viral translation, viruses exploit different strategies aimed at recruiting the cellular protein synthesis machinery 9-11. Interestingly, norovirus translation is driven by the multifunctional viral protein-primer VPg covalently linked to the 5' end of both genomic and subgenomic RNAs 12-14. This sophisticated mechanism of translation is likely to be a major factor in the limited efficiency of viral recovery by conventional reverse genetics approaches. 
Here we report two different strategies based on the generation of murine norovirus-1 (referred to as MNV herewith) transcripts capped at the 5' end. One of the methods involves both in vitro synthesis and capping of viral RNA, whereas the second approach entails the transcription of MNV cDNA in cells expressing T7 RNA polymerase. The availability of these reverse genetics systems for the study of MNV and a small animal model has provided an unprecedented ability to dissect the role of viral sequences in replication and pathogenesis 15-17.

Noroviruses are a major cause of gastroenteritis yet molecular techniques for their characterisation are still relatively new. Here we report two different reverse genetics approaches for the efficient recovery of murine norovirus (MNV), the only member of this genus which can be propagated in cell culture.

反向遗传学感染小鼠诺沃克病毒介导的恢复

Human noroviruses are responsible for most cases of human gastroenteritis (GE) worldwide and are recurrent problem in environments where close ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

高通量筛选RNA病毒的广谱化学抑制剂

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Nucleocapsid Annealing-Mediated Electrophoresis (NAME) Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

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 proteins involved in the regulation of the virus activity recognize several nucleic acids. The nucleocapsid protein NCp7 (NC) is a key protein regulating several processes during virus replication. NC is in fact a chaperone destabilizing the secondary structures of RNA and DNA and facilitating their annealing. The inactivation of NC is a new approach and an interesting target for anti-HIV therapy. The Nucleocapsid Annealing-Mediated Electrophoresis (NAME) assay was developed to identify molecules able to inhibit the melting and annealing of RNA and DNA folded in thermodynamically stable tridimensional conformations, such as hairpin structures of TAR and cTAR elements of HIV, by the nucleocapsid protein of HIV-1. The new assay employs either the recombinant or the synthetic protein, and oligonucleotides without the need of their previous labeling. The analysis of the results is achieved by standard polyacrylamide gel electrophoresis (PAGE) followed by conventional nucleic acid staining. The protocol reported in this work describes how to perform the NAME assay with the full-length protein or its truncated version lacking the basic N-terminal domain, both competent as nucleic acids chaperones, and how to assess the inhibition of NC chaperone activity by a threading intercalator. Moreover, NAME can be performed in two different modes, useful to obtain indications on the putative mechanism of action of the identified NC inhibitors.

Nucleocapsid Annealing-Mediated Electrophoresis NAME Assay Allows the Rapid Identification of HIV-1 Nucleocapsid Inhibitors

This protocol describes NAME, an assay that allows the rapid identification of molecules able to inhibit in vitro the chaperone activities of HIV-1 nucleocapsid protein.

核衣壳退火介导电泳（NAME）分析允许HIV-1核衣壳抑制剂的快速鉴定

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 ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

系统抑制剂靶向细胞内的疗效和毒性筛选<em&gt;结核分枝杆菌</em

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and water-screening methods used currently. While conventional bacterial detection methods, such as polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA) can specifically detect pathogenic contaminants, they require extensive sample preparation and lengthy waiting periods. In addition, these practices demand sophisticated laboratory instruments and settings, and must be executed by trained professionals. Herein, a protocol is proposed for a simpler diagnostic technique that features the unique combination of magnetic and fluorescent parameters in a nanoparticle-based platform. The proposed multiparametric magneto-fluorescent nanosensors (MFnS) can detect E. coli O157:H7 contamination with as little as 1 colony-forming unit present in solution within less than 1 h. Furthermore, the ability of MFnS to remain highly functional in complex media such as milk and lake water has been verified. Additional specificity assays were also used to demonstrate the ability of MFnS to only detect the specific target bacteria, even in the presence of similar bacterial species. The pairing of magnetic and fluorescent modalities allows for the detection and quantification of pathogen contamination in a wide range of concentrations, exhibiting its high performance in both early- and late-stage contamination detection. The effectiveness, affordability, and portability of the MFnS make them an ideal candidate for point-of-care screening for bacterial contaminants in a wide range of settings, from aquatic reservoirs to commercially packaged foods.

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

The overall goal of this protocol is to synthesize functional nanosensors for the portable, cost-effective, and rapid detection of specifically targeted pathogenic bacteria through a combination of magnetic relaxation and fluorescence emission modalities.

利用磁荧光纳米进行食源性致病菌筛选: 快速检测大肠杆菌O157:H7

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared to IN from other retroviruses, PFV IN is more soluble and more amenable to experimental manipulation. Additionally, it is sensitive to clinically relevant human immunodeficiency virus (HIV-1) IN inhibitors, suggesting that the catalytic mechanism of PFV IN is similar to that of HIV-1 IN. IN catalyzes the covalent joining of viral complementary DNA (cDNA) to target DNA in a process called strand transfer. This strand transfer reaction introduces nicks to the target DNA. Analysis of integration reaction products can be confounded by the presence of nucleases that similarly nick DNA. A bacterial nuclease has been shown to co-purify with recombinant PFV IN expressed in Escherichia coli (E. coli). Here we describe a method to isolate PFV IN from the contaminating nuclease by heparin affinity chromatography. Fractions are easily screened for nuclease contamination with a supercoiled plasmid and agarose gel electrophoresis. PFV IN and the contaminating nuclease display alternative affinities for heparin sepharose allowing a nuclease-free preparation of recombinant PFV IN suitable for bulk biochemical or single molecule analysis of integration.

Recombinant prototype foamy virus integrase protein is often contaminated with a bacterial nuclease during purification. This method identifies nuclease contamination and removes it from the final preparation of the enzyme.

重组原型泡沫病毒整合纯化过程中核酸污染的检测与去除

The integrase (IN) protein of the retrovirus prototype foamy virus (PFV) is a model enzyme for studying the mechanism of retroviral integration. Compared ...