These studies were funded by US Public Health Services grants P01AI057005, R01AI047734, R01AI111806 and the Functional Profiling and Computational Biology Core of the University of Washington&#8217;s Center for AIDS Research (P30 AI027757).

<ol>
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作者宣称，他们有没有竞争的财务权益。这里表达的观点是作者的，不应被解释为官员或代表美国国防部部或陆军部的意见。

提出的方案包括两个主要部分:施工重组HIV-1分子克隆和增长竞争分析。为了区分这两种病毒在一个双重感染的细胞培养，但重要的是相互竞争的分子克隆含有序列标签，这可以通过一个RT-qPCR的引物 - 探针测定法或通过Sanger测序来检测。这个协议利用了VIFA和VIFB标签，它们占据的HIV-1的NL4-3 的vif同一区域和编码相同氨基酸序列，但相差6同义突变。这些突变显示不影响 病毒复制健身20。在这个协议中使用的基于PCR的克隆方法在选择的克隆位点提供了更多的灵活性，相对于限制性位点克隆。然而，PCR克隆的效率降低为插入尺寸增大。插入大小的电流限制为5〜34 KB。用于基于PCR的克隆和位点定向mutagenesis时，使用高保真DNA聚合酶的关键是减少的额外的突变的可能性。使用得到的产品足够量的所需要的PCR循环的最小数目的还建议。根据我们的经验，我们没有发现这里所描述的基于PCR的克隆和位点定向诱变步骤后的任何额外突变。尽管如此，PCR产物应测序，以检查是否有任何不希望的突变。理想的情况下，整个的HIV编码区应当重新测序。 至少三个采样时间点应该在指数生长期9内进行检查。该病毒生长动力学必须首先使用每日取样，以确定适当的培养周期和采样时间点的成长竞争建立。影响病毒生长动力学的一个因素是感染复数（MOI）的多重性。该协议使用的0.005共MOI为monoinfection和增长的竞争，因为它被证明产生更多的RO胸围结果低于9的MOI。尽管如此，低的MOI可用于在必要时，得到较长的指数增长阶段，但在结果的一致性为代价的。这个协议建议的50:50的初始感染比率，假定的病毒适应度差异一般未知预先。然而，使用不相等的输入比率是合适的时，有初步数据表明在病毒复制动力学显著差异。在这些情况下，以70:30的感染比率，推荐以允许大型健身差的检测，其中所述不太适合病毒被放置在过量9。 该协议确定TCID 50，病毒生长动力学，以及用于执行使用HIV-1的B亚型的生长竞争试验进行了优化，NL4-3 COTB-p24和来自单一供体的外周血单个核细胞的PBMC已经证明一致易感性艾滋毒1感染在体外 。培养期并在这个协议中提出的采样时间点都可能是适合研究HIV-1的M组病毒在人PBMC。使用的PBMC来自单一来源，强烈建议，以获得一致的结果作为病毒复制可以在不同的供体细胞35而变化。尽管不太希望，PBMC中从多个供体汇集的可以用作一个替代条件是相同的池用于所有的实验。另一种替代方法是使用细胞系。这里介绍的协议被成功用于与T细胞系CEMx174 23,36。但是，重要的是，细胞接种的号码重新优化以实现一致的细胞生长。病毒生长动力学，还必须重新建立的，因为它很可能在不同的细胞系而异，在生长竞争步骤，以确定适当的采样时间点。 两种不同的方法来确定病毒比率用于计算健身都包含在protoc醇。第一种方法使用RT-qPCR的测量病毒cDNA拷贝数在每个采样时间点。病毒复制健身然后从病毒比计算，是根据cDNA的拷贝数。可替代地，病毒比率可以基于色谱峰高在VifAB标签位点的比值来确定。这两种方法产生了类似的结果（ 图4）。色谱峰高度的方法可以适用于其它HIV-1病毒株未经改造的序列标签。用于RT-qPCR的，使用引物或探针来区分病毒变异体必须首先仔细评估（见20）。然而，RT-qPCR的提供用少量的病毒RNA，如那些从指数生长期中的第一个时间点样品的灵敏度。病毒cDNA的直接测量还可以检测可能产生的RNA提取和cDNA合成技术问题。利用分子克隆与序列标签提供一个具有成本效益的解决方案牛逼澳RT-qPCR的方法，因为只需要两对引物和探针来研究多种病毒。这一策略也避免了高峰高度变化的顺序问题色谱图，由于邻近的基地37，测序完成在该研究中所有的病毒在同一网站。制备用于RT-qPCR的和Sanger测序PCR产物也可以是与其他方法使用，以确定病毒的比例如本体测序单个克隆12,13，HTA 4,7,17,19，或寡核苷酸连接测定法（OLA）38 。

病毒复制健身被定义为病毒，以产生感染性子代在给定的环境1的能力，并在确定病毒的变体在群体水平的患病率随着时间的推移2的一个重要因素。作为体内健身研究是不致病的人类病毒，如HIV-1，各种体外和可行体外复制健身测定已开发研究从耐药性和免疫逃避突变，上位和进化所产生的健身效果病毒的人群3-6。在不同的健身测定法，生长竞争分析，确认得到的健身差别更敏感和有效的措施，为两个或更多的病毒变体竞争恰恰相同的环境条件下相同的细胞群，如发生在体内 1,7,8。开始增长竞争实验，几个variab前莱斯需要确定，包括使用不同感染多重性（MOI），病毒输入比，以及分析用取样定时。我们已经研究了这些参数对病毒生长动力学和上的竞争实验的结果的影响，并在细胞培养9确定所需的HIV-1的健身鲁棒测量的关键因素。 除了测定变量，有各种各样的用于定量在生长竞争实验的病毒变体的方法。散装10,11或克隆测序12,13已被用于确定基于在感兴趣的位点（多个）核苷酸频率竞争病毒的比例。相对适合从改变这个比例随着时间的推移得到。此方法是方便的DNA测序服务广泛提供。并行等位基因特异性测序（PASS）方法能够测序在多个位点和重组体14的检测</sup&gt;，但它也需要专门开发的试剂和检测系统。本质上，开发了这些方法，研究病毒株具有少量的在感兴趣的区域的核苷酸差异。其他方法使用小报道基因15或同义突变4,16-18作为标记来区分竞争病毒通过测序，异源跟踪测定（HTA）4,7,17,19或逆转录定量实时聚合酶链反应（RT-qPCR的）15,16,18,20，所有这些可以由适用于研究竞争菌株无关的序列相似性。一个附加的步骤是必需的，以引入标记插入病毒基因组和RT-qPCR分析也需要特定的试剂和仪器。我们已发现，散装Sanger测序产量比较的结果9。 继增长的竞争，病毒复制健身是作为相对健身，或T之间的比值健身WO病毒变种。病毒的相对适合度可以被定义为一种病毒变体通过在接种最初的比例或以两种相互竞争的病毒之间的净生长速率差标准化的最终比例。我们发现，后一方法中，使用仅在指数生长期纵向的数据点，产生了最健壮的结果9,20。 在体外实验健身主要用于研究生物克隆6-8和HIV-1感染性克隆的分子。后者，是适合于遗传操作，通常用于研究对从特定的突变或感兴趣3-5,21,22特定序列的健身效果。以下协议描述的工作流程从构建新的全长感染HIV-1的分子克隆用HIV-1载体含有序列标签，引入利益的突变，使得病毒的股票，建立的病毒生长动力学的角度，在执行的生长竞争测定和计算相对适合度（ 图1）。 使用我们的程序优化，我们创建了三个重组HIV-1突变体和确定其复制健身。首先构建通过更换pNL4-3，含有HIV-1毒株实验室的NL4-3全长基因组感染性的质粒的HIV-1 的gag-P24基因区域，用合成COTB（中心最先进重组分子克隆树，B亚型）GAG-P24序列23创建原型株。单个氨基酸变化（T186M，T242N和I256V），然后介绍了创建三个突变克隆。每个突变体竞争对原型病毒，观察在给定的遗传背景各突变的适合度的影响。这三种突变体展示不同的复制fsitness水平从轻微到比原型病毒显著降低。的T242N突变先前报道具有适度fitneSS花费24-26，类似于在这项研究中所示的结果。其他两个突变的适合度以前没有报道。

<table><tbody><tr><td>Construction of recombinant clones</td><td></td><td></td><td></td></tr><tr><td>Chimeric primer/Mutagenic/Sequencing primers</td><td>IDT</td><td></td><td>Custom DNA oligos</td></tr><tr><td>pNL4-3VifA and pNL4-3VifB plasmid</td><td></td><td></td><td>Please contact Dr. James I Mullins (jmullins@uw.edu)</td></tr><tr><td>High-Fidelity DNA polymerase</td><td>Thermo Scientific</td><td>F-549S</td><td></td></tr><tr><td>High-fidelity buffer</td><td>Thermo Scientific</td><td>F-549S</td><td></td></tr><tr><td>dNTP</td><td>Bioline</td><td>Bio-39026</td><td></td></tr><tr><td>Thermal cycler</td><td>Life Technologies</td><td></td><td>Applied Biosystems&amp;reg; GeneAmp&amp;reg; PCR System 9700</td></tr><tr><td>DNA loading dye</td><td>Thermo Scientific</td><td>R0631</td><td></td></tr><tr><td>1 kb Plus DNA ladder</td><td>Invitrogen</td><td>10787-018</td><td></td></tr><tr><td>QIAquick PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>QIAquick gel extraction kit</td><td>Qiagen</td><td>28704</td><td></td></tr><tr><td>DpnI enzyme</td><td>New England Biolabs</td><td>R0176S</td><td></td></tr><tr><td>TOP10 chemically competent &lt;em&gt;Escherichia coli&lt;/em&gt;</td><td>Invitrogen</td><td>C4040-10</td><td></td></tr><tr><td>Luria Broth base powder</td><td>Invitrogen</td><td>12795-084</td><td></td></tr><tr><td>Carbenicillin</td><td>Research Products International</td><td>C46000-25.0</td><td></td></tr><tr><td>QIAprep Spin Miniprep kit&amp;nbsp;</td><td>Qiagen</td><td>27104</td><td></td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>Qiagen</td><td>12362</td><td></td></tr><tr><td>Transfection</td><td></td><td></td><td></td></tr><tr><td>X-tremeGENE 9 DNA transfection reagent</td><td>Roche</td><td>6365787001</td><td></td></tr><tr><td>HEK 293T-17</td><td>ATCC</td><td>CRL-11268</td><td>http://www.atcc.org/</td></tr><tr><td>0.22 &amp;#956;m filter top tube</td><td>VWR International</td><td>89220-716&amp;nbsp;</td><td></td></tr><tr><td>Cell culture</td><td></td><td></td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Life Technologies</td><td>10566016</td><td></td></tr><tr><td>Iscove&amp;prime;s Modified Dulbecco&amp;prime;s Medium (IMDM)</td><td>Life Technologies</td><td>31980-030</td><td></td></tr><tr><td>RPMI-1640 media</td><td>Life Technologies</td><td>61870-036</td><td></td></tr><tr><td>Phytohemagglutinin (PHA)</td><td>Thermo Scientific</td><td>R30852801</td><td></td></tr><tr><td>Human Interleukin-2</td><td>Roche</td><td>11147528001</td><td></td></tr><tr><td>Fetal bovine serum</td><td>JR Scientific</td><td>43640</td><td></td></tr><tr><td>Penicillin and Streptomycin</td><td>Corning Cellgro</td><td>30-001-CI</td><td></td></tr><tr><td>6 well plate</td><td>VWR Scientific</td><td>73520-906</td><td></td></tr><tr><td>48 well plate</td><td>VWR Scientific</td><td>62407-338</td><td></td></tr><tr><td>96 well flat-bottomed plate</td><td>ISC Bioexpress</td><td>T-3015-4</td><td></td></tr><tr><td>96 well round-bottomed plate</td><td>BD Falcon</td><td>353077</td><td></td></tr><tr><td>1.5 ml Microcentrifuge Tube</td><td>Mt. Baker Bio</td><td>MBD-1500</td><td></td></tr><tr><td>1.5 ml tubes w/ O-ring</td><td>VWR Scientific</td><td>89004-290</td><td></td></tr><tr><td>50 ml conical tube</td><td>ISC Bioexpress</td><td>C-3317-6</td><td></td></tr><tr><td>ELISA</td><td></td><td></td><td></td></tr><tr><td>Triton-X 100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Phosphate buffer saline (PBS)</td><td>Invitrogen</td><td>14190-250</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Sigma-Aldrich</td><td>F0392</td><td></td></tr><tr><td>glycerol</td><td>Sigma-Aldrich</td><td>G5516</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P1379</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>Sigma-Aldrich</td><td>A2153</td><td></td></tr><tr><td>Rabbit anti-HIV-1 SF2 p24 antiserum</td><td>NIH AIDS Reagent Program</td><td>4250</td><td></td></tr><tr><td>Goat anti-rabbit HRP</td><td>KPL</td><td>474-1516</td><td></td></tr><tr><td>TMB Microwell Peroxidase Substrate</td><td>KPL</td><td>52-00-01</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Fisher Scientific</td><td>A300-500</td><td>Causes severe burns by all exposure routes. Use personal protective equipment. Use only under a chemical fume hood. Wash off immediately with plenty of water for at least 15 min.</td></tr><tr><td>HIV p24 standard</td><td>AIDS and Cancer Virus program (NCI-Frederick)</td><td></td><td>For order details please contact Julian Bess, Jr. (bessjw@mail.nih.gov); http://ncifrederick.cancer.gov/Programs/Science/Acvp/bio/Bess.aspx</td></tr><tr><td>Microplate reader</td><td>Molecular Devices</td><td></td><td>VMax Kinetic ELISA Microplate Reader</td></tr><tr><td>RNA isolation cDNA synthesis</td><td></td><td></td><td></td></tr><tr><td>QIAxtractor</td><td>Qiagen</td><td></td><td>http://www.qiagen.com/products/catalog/automated-solutions/sample-prep/qiaxtractor</td></tr><tr><td>QIAamp Viral RNA Mini Kit</td><td>Qiagen</td><td>52906</td><td></td></tr><tr><td>dNTP</td><td>Bioline</td><td>Bio-39026</td><td></td></tr><tr><td>SuperscriptIII</td><td>Invitrogen</td><td>18080-085</td><td></td></tr><tr><td>First-strand buffer&amp;nbsp;</td><td>Invitrogen</td><td>18080-085</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Invitrogen</td><td>18080-085</td><td></td></tr><tr><td>Rnase inhibitor</td><td>Roche</td><td>3335402001</td><td></td></tr><tr><td>RnaseH</td><td>Invitrogen</td><td>18021-071</td><td></td></tr><tr><td>qPCR</td><td></td><td></td><td></td></tr><tr><td>Real-time qPCR machine</td><td>Life Technologies</td><td></td><td>Applied Biosystems 7300 Real-Time PCR&amp;nbsp;</td></tr><tr><td>TaqMan&amp;reg; Gene Expression Master Mix</td><td>Life Technologies</td><td>4369016</td><td></td></tr><tr><td>Forward, reverse primer</td><td>IDT</td><td></td><td>Custom order</td></tr><tr><td>Probe</td><td>Life Technologies</td><td></td><td>Custom order</td></tr><tr><td>optical 96 well reaction plate</td><td>Life Technologies</td><td>I19N3Q216</td><td></td></tr><tr><td>PCR+sequencing</td><td></td><td></td><td></td></tr><tr><td>Taq DNA polymerase (Biolase)</td><td>Bioline</td><td>Bio-21043</td><td></td></tr><tr><td>NH4 buffer&amp;nbsp;</td><td>Bioline</td><td>Bio-21043</td><td></td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Bioline</td><td>Bio-21043</td><td></td></tr><tr><td>Sequencing primer</td><td>IDT</td><td></td><td>Custom order</td></tr><tr><td>QIAxcel</td><td>Qiagen</td><td></td><td>http://www.qiagen.com/products/catalog/automated-solutions/detection-and-analysis/qiaxcel-advanced-system</td></tr><tr><td>DNA sequencing service provider</td><td>http://www.htseq.org/</td><td></td><td></td></tr><tr><td>GRC web tool</td><td>http://indra.mullins.microbiol.washington.edu/grc/</td><td></td><td></td></tr><tr><td>ChromatQuan web tool</td><td>http://indra.mullins.microbiol.washington.edu/cgi-bin/chromatquant.cgi</td><td></td><td></td></tr></tbody></table>

pairwise growth competition assay for determining the replication fitness of human immunodeficiency viruses

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to extrapolate viral replication fitness, ranging from the number of viral particles per infectious unit, growth rate in cell culture, and relative fitness derived from multiple-cycle growth competition assays. Growth competition assays provide a particularly sensitive measurement of fitness since the viruses are competing for cellular targets under identical growth conditions. There are several experimental factors to consider when conducting growth competition assays, including the multiplicity of infection (MOI), sampling times, and viral detection and fitness calculation methods. Each factor can affect the end result and hence must be considered carefully during the experimental design. The protocol presented here includes steps from constructing a new recombinant HIV-1 clone to performing growth competition assays and analyzing the experimental results. This protocol utilizes experimental parameter values previously shown to yield consistent and robust results. Alternatives are discussed, as some parameters need to be adjusted according to the cell type and viruses being studied. The protocol contains two alternative viral detection methods to provide flexibility as the availability of instruments, reagents and expertise varies between laboratories.

研究的单氨基酸改变的适应度的影响在HIV-1的Gag-p24的，我们使用寡核苷酸定向诱变，以引入突变到含有HIV-1的COTB-p24的基因23,32,33一个pNL4-3质粒。通过293T细胞的转染产生和48小时后收获病毒母液。我们估计每个病毒的股票的50％组织培养感染剂量（TCID 50）的里德- Muench的方法31。原型的TCID 50和突变病毒介于10月4日至10月五日国际单位/毫升（ 图3A）。 的重组病毒的生长动力学建立在PBMC中从以0.005的MOI单一供体。 RT-qPCR方法每天测量病毒cDNA拷贝数6天。所有突变体和病毒的原型成倍增长2日和4日，随后病毒增长放缓，由病毒RNA拷贝数的增加降低斜率（指示与&lt;STRONG&gt;图3B）。在cDNA中的减少复制0天（相应于接种物）和第2天之间数是由于病毒的吸收，细胞和由第1天洗涤（步骤5.1.6）去除未结合的病毒粒子。在指数生长期中，所有三个突变体具有更慢的生长速率（ 克 ）比原型病毒（ 图3C）。 所有这三个突变体对竞争的原型病毒生长竞争测定为0.005，总MOI。在双重感染病毒培养生长动力学是类似于monoinfection的;病毒指数生长期为2和4天之间，和病毒生长达到约5天（ 图4A）平台期。 病毒生长速率的差异来源于在病毒比率随时间的变化。基于参考的cDNA拷贝数和利用RT-qPCR的，突变的病毒和通过比较峰高的病毒比计算在序列色谱图在核苷酸位点区分这两种病毒（ 图4B）。使用峰高和病毒RNA的拷贝数的方法来确定的生长速率的差异，得到了类似的结果（ 图4C）。所有三种突变体具有比原型病毒与具有最低健身（ 图4C）的突变I256V下复制健身。 <img alt="图1" src="/files/ftp_upload/52610/52610fig1.jpg" /> 图1:在这个文件中提出的协议流程图的HIV-1重组克隆病毒制剂协议建设的关注个股病毒和生成。健身化验协议是建立病毒的生长动力学，并确定相对病毒健身。虚线表示的协议备选流。例如，一个突变可直接引入HIV-1的NL4-3分子克隆，但不产生重组​​克隆。 <img alt="图2" src="/files/ftp_upload/52610/52610fig2.jpg" /> 图2:采用重叠延伸PCR（A）的HIV-1 NL4-3 COTB的Gag-P24重组分子克隆施工图设计嵌合引物。 5&#39;引物半部包含NL4-3矢量序列和3&#39;半部分包含插入序列的两端。 （B）的嵌合引物用于扩增插入片段，COTB的Gag的p24。 （C）的插入片段被用作引物用于第二次PCR以产生新的重组质粒。 <img alt="图3" src="/files/ftp_upload/52610/52610fig3.jpg" /> 图3:外周血单个核细胞中的病毒生长特点 （ 一 ）登录10 TCID 50 </sUB&gt;病毒股票。 （ 二）monoinfections病毒生长动力学开始以MOI = 0.005。 （C）的病毒生长率超过六天，包括从面板（B）中得到的指数生长期（2-4天）。显示的值代表从一个实验的三次重复的平均值。误差线代表95％置信区间。 <img alt="图4" src="/files/ftp_upload/52610/52610fig4.jpg" /> 图4:病毒生长和相对适合测定的 （A）双重感染PBMC培养物的病毒生长的影响。的T242N和原型病毒（B）比率使用RT-qPCR的或测序峰高度的方法测定。 （C）的突变和双重感染的文化原型病毒之间的病毒净增长速度的差异（D），如图（A）。分别计算F D值ROM中的（B）示出的病毒比数据。显示的值代表从一个实验的三次重复的平均值。误差线代表95％置信区间。

Watch this Scientific Journal Video about Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses at JoVE.com

Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

成对增长竞争测定法测定人类免疫缺陷病毒的复制健身

In vitro fitness assays are essential tools for determining viral replication fitness for viruses such as HIV-1. Various measurements have been used to ...

pairwise-growth-competition-assay-for-determining-replication-fitness

Research

JoVE Journal

Immunology and Infection

11.1K 次观看.  University of Washington. Growth competition between nearly isogenic viruses provides a sensitive measurement for determining relative replication fitness. The protocols described here include the construction of recombinant HIV-1 clones, virus propagation and growth competition and analysis methods optimized to yield sensitive and consistent results.

视频: Pairwise Growth Competition Assay for Determining the Replication Fitness of Human Immunodeficiency Viruses

Amplification of Near Full-length HIV-1 Proviruses for Next-Generation Sequencing

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near full-length (intact and defective), HIV-1 proviruses. FLIPS allows determination of the genetic composition of integrated HIV-1 within a cell population. Through identifying defects within HIV-1 proviral sequences that arise during reverse transcription, such as large internal deletions, deleterious stop codons/hypermutation, frameshift mutations, and mutations/deletions in cis acting elements required for virion maturation, FLIPS can identify integrated proviruses incapable of replication. The FLIPS assay can be utilized to identify HIV-1 proviruses that lack these defects and are&#160;therefore potentially replication-competent. The FLIPS protocol involves: lysis of HIV-1 infected cells, nested PCR of near full-length HIV-1 proviruses (using primers targeted to the HIV-1 5&#39; and 3&#39; LTR), DNA purification and quantification, library preparation for Next-generation Sequencing (NGS), NGS, de novo assembly of proviral contigs, and a simple process of elimination for identifying replication-competent proviruses. FLIPS provides advantages over traditional methods designed to sequence integrated HIV-1 proviruses, such as single-proviral sequencing. FLIPS amplifies and sequences near full-length proviruses enabling replication competency to be determined, and also uses fewer amplification primers, preventing the consequences of primer mismatches. FLIPS is a useful tool for understanding the genetic landscape of integrated HIV-1 proviruses, especially within the latent reservoir, however, its utilization can extend to any application in which the genetic composition of integrated HIV-1 is required.

Full-length individual proviral sequencing (FLIPS) provides an efficient and high-throughput method for the amplification and sequencing of single, near full-length (intact and defective) HIV-1 proviruses and allows for determination of their potential replication-competency. FLIPS overcomes limitations of previous assays designed to sequence the latent HIV-1 reservoir.

用于下一代测序的近全长 HIV-1 Proviruses 的扩增

The Full-Length Individual Proviral Sequencing (FLIPS) assay is an efficient and high-throughput method designed to amplify and sequence single, near ...

科研

遗传学

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

基于 crispr-cas9 的竞争检测对遗传依赖性的调查

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

癌症研究

Next-generation Viral RNA/DNA in situ Hybridization Applications in Human Immunodeficiency Virus/Simian Immunodeficiency Virus Research

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important insights into physiological processes and disease pathogenesis. In situ hybridization (ISH) has been used for many years to assess the location of cells infected by viruses, but recently a next-generation ISH approach was developed with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. This next-generation ISH is based on an approach like branched PCR, but performed in situ and is more facile, sensitive, and reproducible than classical ISH methods or in situ PCR approaches in routinely detecting RNA or DNA in formalin-fixed paraffin embedded (FFPE) tissues. For the last several years our laboratory has been applying this ISH platform for the detection of human immunodeficiency (HIV) and simian immunodeficiency (SIV) viral RNA (vRNA) and/or viral DNA (vDNA) positive cells within a multitude of FFPE tissues. With this detailed technical manuscript, we would like to share our knowledge and advice with all individuals interested in using next-generation ISH in their research.

Here we present a next-generation in situ hybridization assay to identify specific viral RNA or DNA sequences in formalin-fixed paraffin embedded (FFPE) tissues. This approach allows visualization of low copies of RNA and DNA in less than 24 h with very high sensitivity and specificity.

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important ...

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

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 new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant 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 ...

免疫与感染

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target conserved portions of the HIV-1 genome that escape with difficulty. Sequence changes attributed to cellular immune pressure arise across the genome during infection, and if found within conserved regions of the genome such as Gag, can affect the ability of the virus to replicate in vitro. Transmission of HLA-linked polymorphisms in Gag to HLA-mismatched recipients has been associated with reduced set point viral loads. We hypothesized this may be due to a reduced replication capacity of the virus. Here we present a novel method for assessing the in vitro replication of HIV-1 as influenced by the gag gene isolated from acute time points from subtype C infected Zambians. This method uses restriction enzyme based cloning to insert the gag gene into a common subtype C HIV-1 proviral backbone, MJ4. This makes it more appropriate to the study of subtype C sequences than previous recombination based methods that have assessed the in vitro replication of chronically derived gag-pro sequences. Nevertheless, the protocol could be readily modified for studies of viruses from other subtypes. Moreover, this protocol details a robust and reproducible method for assessing the replication capacity of the Gag-MJ4 chimeric viruses on a CEM-based T cell line. This method was utilized for the study of Gag-MJ4 chimeric viruses derived from 149 subtype C acutely infected Zambians, and has allowed for the identification of residues in Gag that affect replication. More importantly, the implementation of this technique has facilitated a deeper understanding of how viral replication defines parameters of early HIV-1 pathogenesis such as set point viral load and longitudinal CD4+ T cell decline.

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

HIV-1 pathogenesis is defined by both viral characteristics and host genetic factors. Here we describe a robust method that allows for reproducible measurements to assess the impact of the gag gene sequence variation on the in vitro replication capacity of the virus.

限制性内切酶克隆方法，以评估<em&gt;在体外</em&gt; HIV-1 C亚型的Gag-MJ4嵌合病毒的复制能力

The protective effect of many HLA class I alleles on HIV-1 pathogenesis and disease progression is, in part, attributed to their ability to target ...

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.

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 ...

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 ...

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under controlled laboratory conditions. Ex vivo infection of human tissues with human immunodeficiency virus (HIV), among other viruses, has been successfully used to investigate early disease pathogenesis, as well as a platform to test the efficacy and toxicity of antiviral drugs. In the present protocol, we explain how to process and infect with HIV-1 tissue explants from human tonsils and cervical mucosae, and maintain them in culture on top of gelatin sponges at the liquid-air interface for about two weeks. This non-polarized culture setting maximizes access to nutrients in culture medium and oxygen, although progressive loss of tissue integrity and functional architectures remains its main limitation. This method allows monitoring HIV-1 replication and pathogenesis using several techniques, including immunoassays, qPCR, and flow cytometry. Of importance, the physiologic variability between tissue donors, as well as between explants from different areas of the same specimen, may significantly affect experimental results. To ensure result reproducibility, it is critical to use an adequate number of explants, technical replicates, and donor-matched control conditions to normalize the results of the experimental treatments when compiling data from multiple experiments (i.e., conducted using tissue from different donors) for statistical analysis.

Ex Vivo Infection of Human Lymphoid Tissue and Female Genital Mucosa with Human Immunodeficiency Virus 1 and Histoculture

Infection of human tissues with human immunodeficiency virus (HIV) ex vivo provides a valuable 3D model of virus pathogenesis. Here, we describe a protocol to process and infect tissue specimens from human tonsils and female genital mucosae with HIV-1 and maintain them in culture at the liquid-air interface.

体内人淋巴组织和女性生殖器粘膜与人体免疫机能丧失病毒1和 Histoculture 的感染

Histocultures allow studying intercellular interactions within human tissues, and they can be employed to model host-pathogen interactions under ...

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

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or fluorescence microscopy. An HIV-1 reporter virus (HIV-1 Gag-iCre) is generated by inserting Cre recombinase into the HIV-1 genome between the matrix and the capsid proteins of the Gag polyprotein. This results in a packaging of Cre recombinase into virus particles, which is then released into a target cell line stably expressing a Cre recombinase-activated red fluorescent protein (RFP) to GFP switch cassette. In the basal state, this cassette expresses RFP only. Following the delivery of Cre recombinase into the target cell, the RFP, flanked by loxP sites, excises, resulting in GFP expression. This assay can be used to test any inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems and has been used to identify a class of purinergic receptor antagonists as novel inhibitors of HIV-1 viral membrane fusion.

We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection systems.

高通量的高效液氧活化病毒膜融合检测 HIV-1 病毒膜融合抑制剂的研究

This assay is designed to specifically report on HIV-1 fusion via the expression of green fluorescent protein (GFP) detectable by flow cytometry or ...

CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites

Human immunodeficiency virus (HIV) integrates its proviral DNA non-randomly into the host cell genome at recurrent sites and genomic hotspots. Here we present a detailed protocol for the generation of novel in vitro models for HIV infection with chosen genomic integration sites using CRISPR-Cas9-based genome engineering technology. With this method, a reporter sequence of choice can be integrated into a targeted, chosen genomic locus, reflecting clinically relevant integration sites.
In the protocol, the design of an HIV-derived reporter and choosing of a target site and gRNA sequence are described. A targeting vector with homology arms is constructed and transfected into Jurkat T cells. The reporter sequence is targeted to the selected genomic site by homologous recombination facilitated by a Cas9-mediated double-strand break at the target site. Single-cell clones are generated and screened for targeting events by flow cytometry and PCR. Selected clones are then expanded, and correct targeting is verified by PCR, sequencing, and Southern blotting. Potential off-target events of CRISPR-Cas9-mediated genome engineering are analyzed.
By using this protocol, novel cell culture systems that model HIV infection at clinically relevant integration sites can be generated. Although the generation of single-cell clones and verification of correct reporter sequence integration is time-consuming, the resulting clonal lines are powerful tools to functionally analyze proviral integration site choice.

We present a genome engineering workflow for the generation of new in vitro models for HIV-1 infection that recapitulate proviral integration at selected genomic sites. Targeting of HIV-derived reporters is facilitated by CRISPR-Cas9-mediated, site-specific genome manipulation. Detailed protocols for single-cell clone generation, screening, and correct targeting verification are provided.

基于 crispr-cas9 的基因组工程, 用于生成 hiv-1 感染与选定的证明病毒整合点的 jurkat 报告模型

Human immunodeficiency virus (HIV) integrates its proviral DNA non-randomly into the host cell genome at recurrent sites and genomic hotspots. Here we ...