The investigators thank all the volunteers in Zambia who participated in this study and all the staff at the Zambia Emory HIV Research Project in Lusaka who made this study possible. The investigators would like to thank Jon Allen, Smita Chavan, and Mackenzie Hurlston for technical assistance and sample management. We would also like to thank Dr. Mark Brockman for his discussions and generous donation of the GXR25 cells.
This study was funded by R01 AI64060 and R37 AI51231 (EH) and the International AIDS Vaccine Initiative. This work was made possible in part by the generous support of the American people through the United States Agency for International Development (USAID).&#160;The contents are the responsibility of the study authors and do not necessarily reflect the views of USAID or the United States Government. This work also was supported, in part, by the Virology Core at the Emory Center for AIDS Research (Grant P30 AI050409). DC and JP were supported in part by Action Cycling Fellowships. This work was supported in part by the Yerkes National Primate Research Center base grant (2P51RR000165-51). This project was also funded in part by the National Center for Research Resources P51RR165 and is currently supported by the Office of Research Infrastructure Programs/OD P51OD11132.

1，扩增HIV-1的gag基因从受感染，冰冻血浆<ol><li>提取病毒RNA从140微升使用提取试剂盒解冻的HIV-1感染的血浆。 <ol><li>如果可行后提取RNA作为解冻病毒RNA产生最佳的放大效果，立即进行cDNA合成。如果可能的话，建立的PCR master mix首轮DNA扩增和储存在4℃之前，病毒RNA提取。 </li></ol></li><li>从RNA反转录的cDNA，并使用逆转录酶，并在单步RT-PCR的热稳定DNA聚合酶扩增的第一轮的DNA产物。 <ol><li>取的RNA进行的-80℃下冷冻（如果冷冻是必要的），并简单地在室温下解冻，然后放置在冷块。转移5μl的RNA的含有45μl反应混合液每个PCR管中，得到的50微升的最终体积。立即将剩余的RNA样品到-80℃的冰箱中。 </li><li>酶是经过dded到第一轮PCR主混合物（ 表1A），等份加入45μl到每个薄壁PCR扩增管用于所需反应的数量。请务必使用PCR管与个别上限，而不是剥夺上限，以确保样本之间的最小交叉污染。将PCR管在寒冷的块，以保护对温度敏感的逆转录酶和RNA模板。注:样品应一式三份，以充分品尝病毒准种运行。同样重要的是，利用具有高保真DNA聚合酶酶的产物，以减少PCR引入的错误掺入。请参考试剂列表中推荐的产品。 </li><li>后的RNA模板被添加到所有的反应管，转移PCR管，以热循环器使用表1B中描述的循环仪程序扩增。注:本扩增的产品将在第二轮巢式PCR使用。 </li></ol></li><li>执行嵌套SECOND轮PCR扩增后，使用1μl的第一轮PCR扩增（1.2）作为DNA模板。 <ol><li>等分试样49微升二轮PCR主混合物（ 表2A）到每个薄壁PCR管中进行期望的反应的数量。传递来自第一轮PCR扩增到每个反应管加入1μl得到的50微升的最终体积。注意:这将作为模板DNA用于第二轮扩增。同样重要的是，利用DNA聚合酶具有很高的保真度和校对能力，以便最小化的PCR引入的错误掺入。请参考试剂列表中推荐的产品。 </li><li>转让第二轮PCR反应混合物，以热循环和表2B描述运行程序。注意:该程序结束后，本反应的产物将在琼脂糖凝胶上运行，以确认生产产物。 </li></ol></li><li>添加3微升的5X样染料5＆＃181;微升50微升第二轮反应体积中，并在120伏上含有UV-DNA荧光染色在1％琼脂糖TAE凝胶上运行，直到带被解决。可视化在蓝色光照明1.6 kb的条带（ 见图1A）。 </li><li>运行包含的DNA染色1％琼脂糖凝胶TAE，其余45μl反应，并切除适当的波段用干净的刀片。 <ol><li>从用凝胶纯化试剂盒凝胶切片中提取DNA，洗脱在无核酸酶的水，结合每个个体三个正反应，并且在-20℃下以供后续使用冷冻产品。 </li></ol></li></ol> 2，准备堵嘴扩增子的克隆通过必要的限购站点简介<ol><li>扩增的长末端重复序列（LTR）的MJ4质粒/ 5&#39;非编码区部分（ 见表3）。 </li><li>可视化1.3 kb的PCR产物在照明，消费积极扩增，凝胶净化，冷冻如前上述（1.4,1.5， 见图1B）。 </li><li>创建通过"剪接重叠延伸"稠MJ4 LTR- 的gag扩增PCR（ 见表4）。 </li><li>可视化，消费税，净化，并冻结了3.2 kb的扩增子在1.4,1.5（参见图1C）。 </li></ol> 3，克隆扩增GAG基因导入MJ4，C ​​亚型，感染性分子克隆<ol><li>消化1.5微克MJ4质粒和1.5μg的纯化LTR- 的gag PCR产物与在50℃下（ 参见表5A）的BclI位（甲基化敏感）限制性内切酶进行1.5小时的。 </li><li>加入1μlNgoMIV到消化反应并在37℃下反应1小时。 </li><li>用于克隆添加5×上样染料，以限制酶消化反应而慢慢electrophorese上含有100伏形象化，附加的DNA凝胶染料1-2 hr的1％琼脂糖TAE凝胶的总体积，并纯化表示频带如先前降序进项税（参见图2）。 </li><li> 1插入到载体比例:用纯化LTR- 插科打诨插入和MJ4载体DNA在3准备连接反应。过夜孵育连接反应在4℃下（ 见表5B）。 </li><li>转化JM109化学感受态细菌用连接产物并涂布在LB琼脂平板上以100微克/毫升氨苄青霉素和生长在30℃下为22 +小时。 <ol><li>解冻JM109在冰上感受态细胞15分钟。标注的1.5 ml离心管和冰浴。 </li><li>等分50-100微升JM109细胞的预冷的1.5 ml离心管中。添加2.5-5微升连接反应到JM109细胞，轻弹管轻轻混合，并立即返回到冰。孵育感受态细胞用连接产物在冰上30分钟。 </li><li>热休克含有JM109感受态细胞，并在45秒的42℃的热块的连接反应，并返回到冰上至少3分钟的1.5 ml离心管中。 </li><li>加入50微升SOC培养基中，以每个微量离心管和板的整个转化反应到补充有氨苄青霉素的100微克/毫升室温的LB-琼脂平板上。 </li><li>转板至30℃培养箱中培养，并留下了20小时，或直至菌落明显形成。 </li></ol></li><li>挑分离的菌落并生长在4毫升的LB与100微克/毫升氨苄青霉素，于30℃为22 +小时。 </li><li>降速的文化，在3200×g离心15分钟，倒出汤汁，并提取质粒DNA。 </li><li>为了证实克隆保真度，切带NgoMIV高致病性禽流感限制性内切酶小量DNA的双消化在37℃2小时。在1％琼脂糖TAE凝胶分析（ 见表5C）。 </li><li>序列使用下面的引物每种质粒的LTR-GAG插入区域:GagInnerF1，GagF2，修订版1，Rev3型，和GagR6（ 表6），以确认序列同一性。 </li><li>比较与人口序列DER获得克隆序列从最初的纯化的扩增子ived从1.5.1。注:以最终评估两个独立的克隆的复制能力，以确保任何体外复制的表型不是由于在克隆过程中引入主链的错误是很重要的。 </li></ol>四代复制能力的Gag-MJ4嵌合病毒和滴定<ol><li>所描述的王子等人 39比1 FUGENE高清:通过转1.5微克的嵌合MJ4小量提取质粒DNA导入使用4 293T细胞产生复制能力的病毒。 </li><li>下面记载在王子等人 39滴度收获病毒储存于TZM-BL细胞。 <ol><li>板TZM-BL细胞感染病毒前24小时，以便有翌日30-40％汇合单层细胞。这通常可以通过在24孔板中加入5×10 4个细胞，每孔800微升的总体积来实现。 </li><li>加入细胞的井后，轻轻移动24孔板前进和后退，然后一边以有效地分配单元到另一边。从未漩涡 - 这将导致细胞积聚在板的中心。 </li><li>翌日，在DMEM中制备1％FBS;这将用于稀释的DEAE-葡聚糖和稀释的病毒种群。股票DEAE葡聚糖是10毫克/毫升或125X，和80微克/毫升或1个是期望的终浓度。 </li><li>取出病毒原液可以从-80℃冷冻并置于测试在摇床上以在室温下解冻。 </li><li>使用多道移液器，连续稀释病毒储存在一个圆底组织培养处理96孔板盖。这是一个3倍的稀释方案。 见表7的稀释方案。 </li><li>使用真空吸引器，确保不打扰单层细胞接种TZM-BL 24孔板取出介质。一次只能从一个24孔板中取出介质，使细胞做不能干。 </li><li>在DMEM混合物中添加150μl的1倍的DEAE-葡聚糖+ 1％FBS的用1000微升吸管每孔中。不要重复使用吸管，因为这破坏了单层。 </li><li>加入150μl每种病毒稀释至适当的井。添加病毒到孔的中心和漩涡轻轻跨细胞单层均匀分布的病毒。培养感染的细胞2小时在组织培养培养箱中于37℃和5％CO 2。 </li><li> 2小时温育后，在DMEM中加入0.5毫升10％FBS至每孔，并返回板到培养箱另外48小时。 </li><li> 48小时后，细胞应该是100％汇合。因为MJ4是具有复制能力的病毒，会有一些细胞死亡，但是这是可以预料的。 </li><li>从各取出介质好，加入400微升/孔固定溶液（ 见表8A配方）。在固定的时间只有一个板块。添加使用1000微升吸管固定的解决方案，并让坐5分钟室温。 </li><li>除去固定液，用PBS冲洗3次与钙 /镁离子 。使用喷瓶轻轻的PBS添加到井的一侧，以避免破坏单层。在第三次洗涤后，吸干板干上吸收纸。 </li><li>加入400微升/孔的染色溶液（ 见表8B为配方）到24孔板的每个孔中，并在37℃下进行至少2小时。较长的孵育时间是可以接受的，但温育时间应保持滴定实验之间保持一致。 </li><li> 2个洗净，用PBS与钙 /镁离子和得分感染，或加PBS中，并储存在4℃下得分后。板可保持在4℃下进行长达四天，只要该单层保持水合。 </li><li>得分感染，使用永久性标记来划分的24孔板的孔中插入象限。算上所有的蓝色细胞的视场范围内，使用200X的总放大倍率，曾经在每四个象限的阱。 </li><li>计算每微升感染单位如下:[（＃蓝色细胞/ 4）×67] /（微升加病毒）=国际单位/微升。参考表8C为在微升病毒加入到各孔的体积。平均多口井在一起;这些数字应该是准确滴定比较相似。 </li></ol></li></ol> 5，准备文化传媒和GXR25细胞繁殖的体外复制检测<ol><li>准备完全RPMI（cRPMI）为GXR25细胞的繁殖。注:这是文化GXR25细胞的复制计划实验至少4个月前极为重要的（ 见表9）。 </li><li>分裂细胞1时10每周两次，并维持细胞密度细胞/ ml之间的1×10 5〜2×10 6。 </li></ol> 6， 在加格-MJ4嵌合体的GXR25（CEM-CCR5-GFP）的体外复制细胞<ol><li>在我以前的日子nfection，分裂细胞的浓度之间的2-3×10 5个细胞/ ml，使它们在对感染的天数增殖。 </li><li>在感染当天，从-80删除病毒的股票℃的冰柜和解冻。计算从每个股票需要病毒的基础上从TZM-BL细胞测定中的国际单位/微升滴度的体积，以便感染5×10 5个GXR25细胞以0.05的感染复数，使用下式: 病毒量感染（微升）= 1μL/ X IU×0.05点¯x500000 注:我们选择0.05的教学语言，因为这会导致对数生长期为所有我们所测试的病毒。它，以便确定最佳的MOI捕获数增殖的病毒进行试验进行初步的实验是非常重要的。 </li><li>稀释病毒cRPMI到100微升的体积（为了达到0.05 MOI），并添加到一个V形底组织培养的良好待遇96孔板。注:同时包括positi已经（MJ4 WT感染）和一负（模拟感染的细胞）控制每一组的复制实验。设置板，使得每隔一列是空白的，以限制孔之间的交叉污染。阳性对照是必要的，因为这将在后面为标准用于标准化复制的斜坡，这是实验重复的复制率的措施。 </li><li>算使用自动细胞计数器GXR25细胞。计算需要的总的所有感染（5×10 5×＃感染）的细胞数。分装至所需体积到50ml锥形管中并离心以沉淀细胞。 25％以上的感染性总是需要计算。 </li><li>小心吸媒体和重悬在cRPMI在5×10 5细胞/ 100微升的浓度。 </li><li>吸取细胞进入无菌槽调匀。使用多道移液器，添加100μl的细胞进入96孔板包含经稀释的病毒的各孔中。弥点¯x彻底。 </li><li>加入2微升的聚凝胺的5毫克/毫升（100倍）的溶液，以每孔中并彻底混合细胞。 </li><li>孵育于37℃在组织培养箱中，用5％CO 2下3小时。 </li><li>为了洗感染的细胞，离心分离96孔V型底板，以沉淀细胞。然后小心地取出150μl的培养基，并用新鲜的150微升cRPMI的更换而不干扰细胞沉淀。重复2次以上（包括离心分离），以充分地洗涤细胞。 </li><li>最后的离心和加入150微升cRPMI后，重悬细胞沉淀用多道移液器，并从各孔中分别以800微升cRPMI在24孔组织的阱加上整个细胞/病毒混合物（约200微升）培养板。 </li><li>地方板在5％CO 2的组织培养箱中于37℃。 </li><li>每两天，从文化的表面除去100微升上清良好，转移到96孔U汉字板和存储冷冻在-80°C，直到运行病毒定量检测。注:在96孔板中的上清液仔细安排将允许培养物上清液中的病毒粒子的定量通过放射标记的逆转录酶（RT）下测定多通道移液器转移。每块板应包含以标准化的实时检测读数板间的变化，从感染的标准样品。 </li><li>后取出，每100微升的样品，拆分单元​​格1:通过充分重悬细胞2和去除一半的剩余量（450微升）的。通过加入550微升新鲜cRPMI到每个孔中恢复原始体积（1ml）中。 </li></ol>逆转录酶的7分析（RT）在细胞培养上清液中改编自奥斯特洛夫斯基等 40协议。 <ol><li>建立生物安全罩在BSL-3设施与放射性材料的使用。 <ol><li>发生在胡胡吸水纸为吸引研发和取代吸气指定的放射性使用。注:所有放射性废物必须按照安全规定妥善处置。 </li></ol></li><li>加1-2微升[α-33 P] dTTP的10毫居里/毫升和4微升的1M二硫苏糖醇（DTT）以每次1毫升一份RT主混合物（ 参见表10和的Ostrowski 等人 40）。 </li><li>精心搭配RT预混液，DTT和[α-33 P] dTTP与每次1.5 ml离心管1000微升吸并转移到一个无菌的低谷。 </li><li>分配25μl的标记RT的混合进96孔的薄壁PCR板各孔中。注:包括阳性对照（MJ4 WT感染）和阴性对照（模拟感染）的空间。 </li><li>加入5μl的每种上清液含有该RT主混合物的PCR板。 </li><li>密封PCR板用胶粘剂电化铝盖，并在热循环孵育2小时，在37℃。为安全起见，RINSE枪头与Amphyl与处置在小容器中含有的提示Amphyl浪费，这将在后面的放射性废物进行处置。 </li><li>温育后，用200μl的多通道移液器使在箔覆盖的小孔。注意:如果枪头触及液体好，移动到下一列或行前更换提示。 </li><li>混合样品的5倍，并转移5μl的各样品的DE-81上的纸张。空气干燥10分钟。 </li><li>洗涤印迹5倍用1×SSC（氯化钠，柠檬酸钠），然后2倍与90％乙醇中，在每次洗涤5分钟。风干。 <ol><li>放置在一个单独的洗涤容器，每一个印迹，如夹层储物盒，补充足够的洗涤缓冲液（1×SSC或90％乙醇），以支付印迹，振摇5分钟。 </li><li>倒掉洗涤缓冲液成单独的容器中，然后重复。注意:第3次洗涤被认为是放射性的，应妥善处理。最后2清洗液会定期处理。 </li></ol></li><li>一旦干燥，车efully包装印迹的保鲜膜，并暴露在phosphoscreen在一个密封的纸盒在室温下过夜。 </li><li>分析phosphoscreens与phosphorimager和量化放射性成绩单。 </li><li>周围绘制使用OptiQuant软件的每个放射性信号的圆。图中的数字光单元（DLU）值来生成复制曲线。 </li><li>为了生成复制能力分数，DLU值分别记录10 -转化和使用第2天，第4和6的时间点，计算斜率。复制斜坡，然后由WT MJ4的斜率，以产生复制能力的分数分频。重要的是根据从同一RT板得到降低批内变异性的MJ4 WT值正常化。 </li></ol>

The authors have nothing to disclose.

由于长度和这个协议的技术性质，有几个步骤，这对嵌合的Gag-MJ4质粒均构建成功以及对病毒复制的能力的定量的关键。虽然限制性内切酶克隆的策略引进国外GAG基因导入MJ4本协议所列拥有超过以前使用重组为基础的方法具有许多优点，该协议可以是技术上的挑战，如果不采取关键步骤精确。 首先，这是绝对必要的使用已在主管菌株缺乏DCM和大坝的DNA甲基化酶生成MJ4质?...

确定主机和影响HIV-1发病机理和疾病进展是至关重要的合理的疫苗设计的病毒特征。细胞免疫应答是HIV-1感染人体免疫反应的重要组成部分。细胞毒性T淋巴细胞（CTL）所必需的急性病毒血症的初始控制，并允许主机建立一个稳定的状态（设定点）的病毒载量1,2。这些效应细胞的实验耗尽导致的病毒控制3,4的损失。尽管这样，逃避突变发生在病毒基因组的颠覆CTL识别病毒感染的细胞5-9的范围内。 某些HLA等位基因已低病毒载量和较慢的疾病进展，包括B * 57，B * 27和B * 81 10-15关联。的HLA I类等位基因的保护益处部分可以归因于它们的目标基因组的功能约束区域，例如Gag的事实并选择该减少病毒在体外 16-21复制的能力逃逸突变。虽然从细胞免疫系统逃逸，是在选择中的HLA I类等位基因的上下文中的病毒有益的，这些突变的影响可以具有用于发送时主机差动后果与HLA-错配的个体22,23。因此，了解病毒复制能力传输的HLA相关逃逸突变的影响，以促进我们的早期HIV-1发病机制的理解将是非常重要的。 尽管已经取得了很大的进展，以识别和描述与具体的HLA-I类等位基因24-29相关个人逃逸突变的适应度的缺陷，自然产生的HIV-1毒株的HLA相关基因多态性的独特而复杂的脚印，而产生的人类白细胞抗原可能不同的免疫遗传背景的30介导的免疫压力。在APrevious分析，格普费特等 。结果显示，从88急性感染赞比亚得到的传输加格序列的HLA相关的基因突变的积累与设定点病毒载量的31减少有关。这个建议的有害逃逸突变的传输，特别是在加格，对HLA不匹配的收件人提供临床益处，并且可能是由于减毒病毒复制。展望未来，就必须研究如何复杂的自然产生的菌株中的Gag基因多态性的组合协同工作来定义传播病毒的特性，例如复制能力，以及如何早期复制可能反过来影响HIV-1的临床指标和晚期发病。 布罗克曼等人首次证实GAG-Pro序列的过程中慢性期感染和病毒载量在这两个亚型C和B感染隔离复制能力之间的联系32-35。实验方法在这些研究中提出，虽然适当的审查由慢性感染个体的序列的体外复制能力，有几个技术上的警告和限制，使学习在C亚型HIV-1的复制能力急性感染的个体困难。此方法依赖于种群基于PCR扩增的序列引入B亚型NL4-3原病毒，这是来自部分来自LAV的重组，实验室适于病毒原液36。病毒的产生是完成通过共转染在CEM基于T细胞系37与PCR产物的消化Δ-GAG-亲 NL4-3的DNA。这种方法需要病毒在一段周的向外生长到几个月，这可能歪斜回收的病毒原液的性质有关的病毒准种的体内 ，并因此改变的体外复制能力的测定。这种方法Iš更适合于研究慢性感染的个体，它有效地用于病毒选择具有最高的复制能力，并在那里从大量的慢性感染个体的克隆许多不同的病毒变异体是相当劳力密集的，因此是不可行的。然而，急性感染个体中，有一般一到两个变体存在，并且因此，可以消除通过在体外选择压力偏斜所回收的病毒原液的性质的风险，使得在体外复制能力的更精确的评估。其次，这个方法需要重组C亚型的gag-Pro序列成B亚型来自骨干，并且可以引入骨干不相容偏压入分析。由于这些限制，大量的序列必须以克服引入的任何可能存在的偏差进行分析。 在这里，我们描述了一个另类的实验Appro公司ACH适合学习从C亚型急性感染个体的序列。我们用限制性内切酶克隆策略，引进源自HIV-1 C亚型感染者急性感染时间点进入C亚型前病毒的骨干，MJ4 gag基因。使用MJ4的为其中克隆的gag基因的共同骨架为C亚型的序列的分析至关重要。 MJ4从初级分离38演变而来，因此也不太可能引入偏倚由于骨干网和gag基因的亚型不兼容。另外，使用酶和限制性克隆的方法允许在原病毒构建体直接转染到293T细胞中，并在一个克隆的病毒原液的恢复相同的克隆的gag序列。 下面介绍的方法是一种高通量的方法来评估C亚型的复制能力得出的Gag-MJ4嵌合病毒。转染到293T细胞中是直接的和病毒的回收率只需要三天。 体外复制能力的测定在由布罗克曼等人创建的相同的CEM-CCR5的基于T细胞系37中 ，除了使用所必需的成功复制重要协议的修改的C亚型MJ4嵌合病毒。使用合适的T细胞系，而不是外周血单个核细胞的允许大量的C亚型MJ4-嵌合病毒对以高测定再现性测试。最后，使用放射性标记的逆转录酶测定法对病毒的上清液中定量更具成本比使用市售的p24 ELISA试剂盒有效。这也给了一个更高的动态范围，这是用于检测相同的检测中均不佳，高病毒复制和用于检测菌株之间的细微差别复制很重要。 总之，这里提出的方法允许对从HIV-1 C亚型来源的插科打诨序列的复制能力的深入研究急性赞比亚感染者，并作为书面，也可以扩展到其他学习C亚型病毒感染的人群。观察高度变异在不同的Gag菌株之间的复制能力。此外，我们能够显示发送加格的复制能力和设定点的病毒载量的CD4 +下降之间以及与统计协会在3年的时间39。这样的结果凸显学习如何传播的病毒特征，比如复制能力的重要性，与宿主免疫系统在感染早期，影响发病机制相互作用，将整体开发有效的疫苗干预以及治疗。

<table border="0" cellpadding="0" cellspacing="0" width="1024">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:444px;">Name of the Reagent</td>
			<td style="width:195px;">Company</td>
			<td style="width:133px;">Catalogue number</td>
			<td style="width:252px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GOF: 5&#39; ATTTGACTAGCGGAGGCTAGAA 3&#39;</td>
			<td>IDT DNA</td>
			<td>Custom Oligo</td>
			<td>25nmol, standard desalt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VifOR: 5&#39; TTCTACGGAGACTCCATGACCC 3&#39;</td>
			<td>IDT DNA</td>
			<td>Custom Oligo</td>
			<td>25nmol, standard desalt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GagInnerF1: 
			5&#39; AGGCTAGAAGGAGAGAGATG 3&#39;</td>
			<td>IDT DNA</td>
			<td>Custom Oligo</td>
			<td>25nmol, standard desalt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BclIDegRev2: 
			5&#39; AGTATTTGATCATAYTGYYTYACTTTR 3&#39;</td>
			<td>IDT DNA</td>
			<td>Custom Oligo</td>
			<td>25nmol, standard desalt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MJ4For1b: 5&#39; CGAAATCGGCAAAATCCC 3&#39;</td>
			<td>IDT DNA</td>
			<td>Custom Oligo</td>
			<td>25nmol, standard desalt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MJ4Rev: 5&#39; CCCATCTCTCTCCTTCTAGC 3&#39;</td>
			<td>IDT DNA</td>
			<td>Custom Oligo</td>
			<td>25nmol, standard desalt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BclIRev: 5&#39; TCTATAAGTATTTGATCATACTGTCTT 3&#39;</td>
			<td>IDT DNA</td>
			<td>Custom Oligo</td>
			<td>25nmol, standard desalt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GagF2: 5&#39; GGGACATCAAGCAGCCAT 3&#39;</td>
			<td>IDT DNA</td>
			<td>Custom Oligo</td>
			<td>25nmol, standard desalt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">For3: 5&#39; CTAGGAAAAAGGGCTGTTGGAAATG 3&#39;</td>
			<td>IDT DNA</td>
			<td>Custom Oligo</td>
			<td>25nmol, standard desalt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GagR6: 5&#39; CTGTATCATCTGCTCCTG 3&#39;</td>
			<td>IDT DNA</td>
			<td>Custom Oligo</td>
			<td>25nmol, standard desalt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rev3: 5&#39; GACAGGGCTATACATTCTTACTAT 3&#39;</td>
			<td>IDT DNA</td>
			<td>Custom Oligo</td>
			<td>25nmol, standard desalt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rev1: 5&#39; AATTTTTCCAGCTCCCTGCTTGCCCA 3&#39;</td>
			<td>IDT DNA</td>
			<td>Custom Oligo</td>
			<td>25nmol, standard desalt</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CoolRack PCR 96 XT</td>
			<td>Biocision</td>
			<td>BCS-529</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CoolRack M15</td>
			<td>Biocision</td>
			<td>BCS-125</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nuclease free water</td>
			<td>Fisher</td>
			<td>SH30538FS</td>
			<td>Manufactured by Hyclone</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAamp Viral RNA Mini Kit</td>
			<td>Qiagen</td>
			<td style="width:133px;">52906</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Simport PCR 8 Strip Tubes, Blue (Flat Cap)</td>
			<td>Daigger</td>
			<td>EF3647BX</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SuperScript III one-step RT-PCR system</td>
			<td>Life Technologies/Invitrogen</td>
			<td>12574035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phusion Hot-start II DNA polymerase</td>
			<td>Fisher</td>
			<td>F-549L&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCR Nucleotide Mix</td>
			<td>Roche</td>
			<td>4638956001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose, high gel strength</td>
			<td>Fisher</td>
			<td>50-213-128</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TAE 10X</td>
			<td>Life Technologies/Invitrogen</td>
			<td>AM9869</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Promega 1kb DNA ladder</td>
			<td>Fisher</td>
			<td>PRG5711</td>
			<td>Manufactured by Promega</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sybr Safe DNA Gel Stain, 10000x</td>
			<td>Life Technologies/Invitrogen</td>
			<td>S33102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wizard SV Gel and PCR Clean-Up System</td>
			<td>Promega</td>
			<td>A9282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blades, single-edged</td>
			<td>Fisher</td>
			<td>12-640</td>
			<td>Manufactured by Surgical Design</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermocycler, PTC-200</td>
			<td>MJ Research</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Microbiology &#38; Cloning reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LB Agar, Miller</td>
			<td>Fisher</td>
			<td>BP1425-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LB Broth, Lennox</td>
			<td>Fisher</td>
			<td>BP1427-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile 100mm x 15mm polystyrene petri dishes</td>
			<td>Fisher</td>
			<td>08-757-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ampicillin sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>A9518-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 14ml Polypropylene round-bottom tubes</td>
			<td>BD Biosciences</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NgoMIV restriction endonuclease</td>
			<td>New England BioLabs</td>
			<td>R0564L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BclI restriction endonuclease</td>
			<td>New England BioLabs</td>
			<td>R0160L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HpaI restriction endonuclease</td>
			<td>New England BioLabs</td>
			<td>R0105L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">T4 DNA Ligase, 5U/&#956;L</td>
			<td>Roche</td>
			<td>10799009001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">JM109 competent cells, &#62;10^8 cfu/&#956;g&#160;</td>
			<td>Promega</td>
			<td>L2001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PureYield plasmid miniprep system</td>
			<td>Promega&#160;</td>
			<td>A1222</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Safe Imager 2.0 Blue Light Transilluminator</td>
			<td>Invitrogen</td>
			<td>G6600</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microfuge 18 centrifuge</td>
			<td>Beckman Coulter</td>
			<td>367160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Amphyl cleaner/disinfectant</td>
			<td>Fisher</td>
			<td>22-030-394</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fugene HD, 1 mL</td>
			<td>VWR</td>
			<td>PAE2311</td>
			<td>Manufactured by Promega</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hexadimethrine bromide (Polybrene)</td>
			<td>Sigma-Aldrich</td>
			<td>H9268-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Costar Plates, 6-well, flat</td>
			<td>Fisher</td>
			<td>07-200-83</td>
			<td>Manufactured by Corning Life</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Costar Plates, 24-well, flat</td>
			<td>Fisher</td>
			<td>07-200-84</td>
			<td>Manufactured by Corning Life</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Costar Plates, 96-well, round</td>
			<td>Fisher</td>
			<td>07-200-95</td>
			<td>Manufactured by Corning Life</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flasks, Corning filter top/canted neck, 75 cm^2</td>
			<td>Fisher</td>
			<td>10-126-37</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Flasks, Corning filter top/canted neck, 150 cm^2</td>
			<td>Fisher</td>
			<td>10-126-34</td>
			<td>Manufactured by Corning Life</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conical Tubes, 50ml, blue cap</td>
			<td>Fisher</td>
			<td>14-432-22</td>
			<td>Manufactured by BD Biosciences</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Conical Tubes, 15ml, blue cap</td>
			<td>Fisher</td>
			<td>14-959-70C&#160;&#160;</td>
			<td>Manufactured by BD Biosciences</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA</td>
			<td>Fisher</td>
			<td>MT25052CI</td>
			<td>Manufactured by Mediatech</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI, 500 ml</td>
			<td>Life Technologies/Invitrogen</td>
			<td>11875-119</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM, 500 ml</td>
			<td>Life Technologies/Invitrogen</td>
			<td>11965-118</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin/Streptomycin/Glutamine, 100X</td>
			<td>Life Technologies/Invitrogen</td>
			<td>10378-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS with magnesium and calcium, 500ml</td>
			<td>Life Technologies/Invitrogen</td>
			<td>14040-133</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS without magnesium and calcium</td>
			<td>Life Technologies/Invitrogen</td>
			<td>20012-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sarstedt tubes, assorted colors</td>
			<td>Sarstedt</td>
			<td>72.694.996</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reservoir Trays for Multichannel, 55ml</td>
			<td>Fisher</td>
			<td>13-681-501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DEAE-Dextran</td>
			<td>Fisher</td>
			<td>NC9691007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning 96 well clear V bottom tissue culture treated microplate</td>
			<td>Fisher</td>
			<td>07-200-96</td>
			<td>Manufactured by Corning Life</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES, 1M Buffer Solution</td>
			<td>Life Technologies/Invitrogen</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS, Defined, 500 ml</td>
			<td>Fisher</td>
			<td>SH30070 03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">X-gal</td>
			<td>VWR</td>
			<td>PAV3941</td>
			<td>Manufactured by Promega</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutaraldehyde, Grade II, 25% in H2O</td>
			<td>Sigma-Aldrich</td>
			<td>G6257-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1M Magnesium chloride solution</td>
			<td>Sigma-Aldrich</td>
			<td>M1028-100ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Formaldehyde solution, for molecular biology, 36.5%</td>
			<td>Sigma-Aldrich</td>
			<td>F8775-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium hexacyanoferrate(II) trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>P9387-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium hexacyanoferrate(III)</td>
			<td>Sigma-Aldrich</td>
			<td>P8131-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Allegra X15-R centrifuge</td>
			<td>Beckman Coulter</td>
			<td>392932</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TC10 automated cell counter</td>
			<td>Bio-Rad</td>
			<td>1450001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VistaVision inverted microscope</td>
			<td>VWR</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reverse-Transcriptase Quantification Assay reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dTTP, [&#945;-33P]- 3000Ci/mmol, 10mCi/ml, 1 mCi</td>
			<td>Perkin-Elmer</td>
			<td>NEG605H001MC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1M Tris-Cl, pH 8.0</td>
			<td>Life Technologies/Invitrogen</td>
			<td>15568025</td>
			<td>Must be adjusted to pH 7.8 with KOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2M potassium chloride (KCl)</td>
			<td>Life Technologies/Invitrogen</td>
			<td>AM9640G</td>
			<td>Adjust to 1M solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.5M EDTA</td>
			<td>Life Technologies/Invitrogen</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nonidet P40</td>
			<td>Roche</td>
			<td style="width:133px;">11333941103</td>
			<td style="width:252px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Polyadenylic acid (Poly rA) potassium salt&#160;</td>
			<td>Midland Reagent Co.</td>
			<td>P-3001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oligo d(T) primer&#160;</td>
			<td>Life Technologies/Invitrogen</td>
			<td>18418-012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>43815-1G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SR, Super Resolution Phosphor Screen, Small</td>
			<td>Perkin-Elmer</td>
			<td>7001485</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning Costar Thermowell 96 well plate model (M) Polycarbonate</td>
			<td>Fisher</td>
			<td>07-200-245</td>
			<td>Manufactured by Corning Life</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corning 96 Well Microplate Aluminum Sealing Tape, Nonsterile</td>
			<td>Fisher</td>
			<td>07-200-684</td>
			<td>Manufactured by Corning Life</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DE-81 anion exchange paper</td>
			<td>Whatman</td>
			<td>3658-915</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trisodium citrate dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S1804-1KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Fisher</td>
			<td>S671-3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Autoradiography cassette</td>
			<td>Fisher</td>
			<td>FB-CA-810</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cyclone storage phoshpor screen</td>
			<td>Packard</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

为了正确地执行该协议，该协议创建能够组装功能齐全的，传染性的Gag-MJ4嵌合体的前病毒质粒，伟大必须小心，以生成适当的PCR扩增子。确定PCR是否已经生成的适当大小的gag扩增子是关键的。产品应在图1A中所示的大约1700碱基对的扩增子的100个碱基对（bp）。这个片段的确切长度将取决于所研究的gag基因的不同而不同。接着，MJ4分子克隆的5&#39;长末端重复序列（LTR）部?...

Watch this Scientific Journal Video about A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses at JoVE.com

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 ...

a-restriction-enzyme-based-cloning-method-to-assess-vitro-replication

Research

JoVE Journal

Immunology and Infection

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

15.5K 次观看.  Emory University. 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.

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

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

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 ...

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

Plasmodium falciparum, the causative agent of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important health problems worldwide, being responsible for a total of 4 million deaths annually1. Due to their extensive overlap in developing regions, especially Sub-Saharan Africa, co-infections with malaria and HIV-1 are common, but the interplay between the two diseases is poorly understood. Epidemiological reports have suggested that malarial infection transiently enhances HIV-1 replication and increases HIV-1 viral load in co-infected individuals2,3. Because this viremia stays high for several weeks after treatment with antimalarials, this phenomenon could have an impact on disease progression and transmission.
The cellular immunological mechanisms behind these observations have been studied only scarcely. The few in vitro studies investigating the impact of malaria on HIV-1 have demonstrated that exposure to soluble malarial antigens can increase HIV-1 infection and reactivation in immune cells. However, these studies used whole cell extracts of P. falciparum schizont stage parasites and peripheral blood mononuclear cells (PBMC), making it hard to decipher which malarial component(s) was responsible for the observed effects and what the target host cells were4,5. Recent work has demonstrated that exposure of immature monocyte-derived dendritic cells to the malarial pigment hemozoin increased their ability to transfer HIV-1 to CD4+ T cells6,7, but that it decreased HIV-1 infection of macrophages8. To shed light on this complex process, a systematic analysis of the interactions between the malaria parasite and HIV-1 in different relevant human primary cell populations is critically needed.
Several techniques for investigating the impact of HIV-1 on the phagocytosis of micro-organisms and the effect of such pathogens on HIV-1 replication have been described. We here present a method to investigate the effects of P. falciparum-infected erythrocytes on the replication of HIV-1 in human primary monocyte-derived macrophages. The impact of parasite exposure on HIV-1 transcriptional/translational events is monitored by using single cycle pseudotyped viruses in which a luciferase reporter gene has replaced the Env gene while the effect on the quantity of virus released by the infected macrophages is determined by measuring the HIV-1 capsid protein p24 by ELISA in cell supernatants.

An In vitro Co-infection Model to Study Plasmodium falciparum-HIV-1 Interactions in Human Primary Monocyte-derived Immune Cells

We have developed an in vitro malaria-HIV-1 co-infection model to study the impact of Plasmodium falciparum on the HIV-1 replicative cycle in human primary monocyte-derived macrophages. This versatile system can easily be adapted to other primary cell types susceptible to HIV-1 infection.

一个<em&gt;在体外</em&gt;共感染模型研究<em&gt;恶性疟原虫</em人原发性单核细胞源性的免疫细胞-HIV-1相互作用

Plasmodium falciparum, the causative agent  of the deadliest form of malaria, and human immunodeficiency virus type-1 (HIV-1) are among the most important ...

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The apical and basolateral surfaces of airway epithelial cells demonstrate directional responses to pathogen exposure in vivo. Thus, ideal in vitro models for examining cellular responses to respiratory pathogens polarize, forming apical and basolateral surfaces. One such model is differentiated normal human bronchial epithelial cells (NHBE). However, this system requires lung tissue samples, expertise isolating and culturing epithelial cells from tissue, and time to generate an air-liquid interface culture.
Calu-3 cells, derived from a human bronchial adenocarcinoma, are an alternative model for examining the response of proximal airway epithelial cells to respiratory insult1, pharmacological compounds2-6, and bacterial7-9 and viral pathogens, including influenza virus, rhinovirus and severe acute respiratory syndrome - associated coronavirus10-14. Recently, we demonstrated that Calu-3 cells are susceptible to respiratory syncytial virus (RSV) infection in a manner consistent with NHBE15,16 . Here, we detail the establishment of a polarized, liquid-covered culture (LCC) of Calu-3 cells, focusing on the technical details of growing and culturing Calu-3 cells, maintaining cells that have been cultured into LCC, and we present the method for performing respiratory virus infection of polarized Calu-3 cells.
To consistently obtain polarized Calu-3 LCC, Calu-3 cells must be carefully subcultured before culturing in Transwell inserts. Calu-3 monolayer cultures should remain below 90% confluence, should be subcultured fewer than 10 times from frozen stock, and should regularly be supplied with fresh medium. Once cultured in Transwells, Calu-3 LCC must be handled with care. Irregular media changes and mechanical or physical disruption of the cell layers or plates negatively impact polarization for several hours or days. Polarization is monitored by evaluating trans-epithelial electrical resistance (TEER) and is verified by evaluating the passive equilibration of sodium fluorescein between the apical and basolateral compartments17,18 . Once TEER plateaus at or above 1,000 &Omega;&times;cm2, Calu-3 LCC are ready to use to examine cellular responses to respiratory pathogens.

Establishing a Liquid-covered Culture of Polarized Human Airway Epithelial Calu-3 Cells to Study Host Cell Response to Respiratory Pathogens In vitro

The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

建立的液体覆盖的文化偏光人类呼吸道上皮的CALU-3细胞研究呼吸系统病原宿主细胞的反应<em在体外</em

The  apical and basolateral surfaces of airway epithelial cells demonstrate  directional responses to pathogen exposure in  vivo. Thus, ideal in vitro ...

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 ...

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.

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 ...

Intranasal Administration of Recombinant Influenza Vaccines in Chimeric Mouse Models to Study Mucosal Immunity

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the physiological mechanisms that mediate immune responses to vaccines perhaps due to the overall success of vaccination, which has reduced interest into the molecular and physiological mechanisms of vaccine immunity. However, several important human pathogens including influenza virus still pose a challenge for vaccination, and may benefit from immune-based strategies. 
Although influenza reverse genetics has been successfully applied to the generation of live-attenuated influenza vaccines (LAIVs), the addition of molecular tools in vaccine preparations such as tracer components to follow up the kinetics of vaccination in vivo, has not been addressed. In addition, the recent generation of mouse models that allow specific depletion of leukocytes during kinetic studies has opened a window of opportunity to understand the basic immune mechanisms underlying vaccine-elicited protection. Here, we describe how the combination of reverse genetics and chimeric mouse models may help to provide new insights into how vaccines work at physiological and molecular levels, using as example a recombinant, cold-adapted, live-attenuated influenza vaccine (LAIV). We utilized laboratory-generated LAIVs harboring cell tracers as well as competitive bone marrow chimeras (BMCs) to determine the early kinetics of vaccine immunity and the main physiological mechanisms responsible for the initiation of vaccine-specific adaptive immunity. In addition, we show how this technique may facilitate gene function studies in single animals during immune responses to vaccines. We propose that this technique can be applied to improve current prophylactic strategies against pathogens for which urgent medical countermeasures are needed, for example influenza, HIV, Plasmodium, and hemorrhagic fever viruses such as Ebola virus.

There is an overall lack of knowledge about how vaccines work. Here we propose the combined use of reverse genetics and bone marrow chimeric mice to gain insight into the early host immune responses to vaccines with a special focus on dendritic cells and T cell immunity.

重组流感疫苗的嵌合体小鼠模型鼻腔给药研究黏膜免疫

Vaccines are one of the greatest achievements of mankind, and have saved millions of lives over the last century. Paradoxically, little is known about the ...

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 ...

SILAC Based Proteomic Characterization of Exosomes from HIV-1 Infected Cells

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can characterize thousands of proteins at a time. These powerful techniques allow us to have a systemic understanding of cellular changes, especially when cells are subjected to various stimuli, such as infections, stresses, and specific test conditions. Even with recent developments, analyzing the exosomal proteome is time-consuming and often involves complex methodologies. In addition, the resultant large dataset often needs robust and streamlined analysis in order for researchers to perform further downstream studies. Here, we describe a SILAC-based protocol for characterizing the exosomal proteome when cells are infected with HIV-1. The method is based on simple isotope labeling, isolation of exosomes from differentially labeled cells, and mass spectrometry analysis. This is followed by detailed data mining and bioinformatics analysis of the proteomic hits. The resultant datasets and candidates are easy to understand and often offer a wealth of information that is useful for downstream analysis. This protocol is applicable to other subcellular compartments and a wide range of test conditions.

Here, we describe a quantitative proteomics method using the technique of stable isotope labeling by amino acids in cell culture (SILAC) to analyze the effects of HIV-1 infection on host exosomal proteomes. This protocol can be easily adapted to cells under different stress or infection conditions.

从HIV-1感染细胞的外来体基于SILAC蛋白质组学表征

Proteomics is the large-scale analysis of proteins. Proteomic techniques, such as liquid chromatography tandem mass spectroscopy (LC-MS/MS), can ...

Fluorescence-based Neuraminidase Inhibition Assay to Assess the Susceptibility of Influenza Viruses to The Neuraminidase Inhibitor Class of Antivirals

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against currently circulating strains. In addition to their use in treating seasonal influenza, the NA inhibitors have been stockpiled by a number of countries for use in the event of a pandemic. It is therefore important to monitor the susceptibility of circulating influenza viruses to this class of antivirals. There are different types of assays that can be used to assess the susceptibility of influenza viruses to the NA inhibitors, but the enzyme inhibition assays using either a fluorescent substrate or a chemiluminescent substrate are the most widely used and recommended. This protocol describes the use of a fluorescence-based assay to assess influenza virus susceptibility to NA inhibitors. The assay is based on the NA enzyme cleaving the 2&#8242;-(4-Methylumbelliferyl)-&#945;-D-N-acetylneuraminic acid (MUNANA) substrate to release the fluorescent product 4-methylumbelliferone (4-MU). Therefore, the inhibitory effect of an NA inhibitor on the influenza virus NA is determined based on the concentration of the NA inhibitor that is required to reduce 50% of the NA activity, given as an IC50 value.

We describe the use of a phenotypic fluorescence-based neuraminidase inhibition assay to assess the susceptibility of influenza A and B viruses to the neuraminidase inhibitor class of antivirals.

荧光为基础的神经氨酸酶抑制测定流感病毒的易感性评估抗病毒药的神经氨酸酶抑制剂类

The neuraminidase (NA) inhibitors are the only class of antivirals approved for the treatment and prophylaxis of influenza that are effective against ...