Name: assembly and purification of prototype foamy virus intasomes
Uploaded: 2018-03-19T15:00:00.000+00:00
Duration: 10 min 20 s
Description: Watch this Scientific Journal Video about Assembly and Purification of Prototype Foamy Virus Intasomes at JoVE.com

这项工作得到了 NIH AI099854 和 AI126742 的支持。

1. vDNA 的退火
<ol><li>组合1X 十缓冲器 (10X 十库存: 100 毫米三盐酸, pH 值 8.0, 1 米氯化钠, 10 毫米 EDTA), 10 µM 寡聚 1 (5 ' ATTGTCATGGAATTTTGTATATTGAGTGGCGCCCGAACAG 3 ', 100 µM 股票) 和10µM 寡聚 2 (5 ' CTGTTCGGGCGCCACTCAATATACAAAATTCCATGACA 3 ', 100 µM 股票) 在最后容量1.5 毫升.整除25µL 六十0.2 毫升聚合酶链反应 (PCR) 管。 注: 根据应用的不同, 低聚糖可以用显影或标签在 5 '-末端的寡聚2或作为内部氨基 t 在基13从 5 '-端的寡聚1。在退火前, 应采用高效液相色谱 (HPLC) 纯化改性寡聚物。我们发现, 5 '-端 Cy3 或 Cy5 荧光标签的寡2降低 intasome 组装效率10倍。内部荧光标签不会降低装配效率。</li>
	<li>退火使用 thermocycler 与以下时间和温度: 1 周期在94.0 ˚C 3 分钟, 99 个周期在 (94.0 ˚C 1 分钟, 93.6 ˚C 1 分钟) 减少每周期0.8 ˚C 的温度 (最后一个周期是14.8 ˚C 1 分钟, 14.4 ˚C 1 分钟), 并存储在4.0 ˚C。</li>
	<li>将所有六十管的退火反应结合起来。负载500µL 到两个0.5 毫升 3 kDa 分子量截止 (截留分子量) ultracentrifugal 过滤器浓度单位。将退火后的 vDNA 通过离心离心在室温下 1.4万 x g 10 分钟。 注: retentate 体积应为每浓度单位50µL。</li>
	<li>丢弃流。将剩余退火 vDNA 的250µL 添加到每个过滤器单元。重复旋转并丢弃流。缓冲交换成 TE 缓冲器 (10 毫米三盐酸, pH 值 8.0, 1 毫米 EDTA) 洗涤三次与450µL TE。</li>
	<li>反转过滤单元, 并在 RT 1000 x g 处旋转2分钟。每个过滤器单元的最终 retentate 体积应为 ~ 50 µL. 将 retentates 和转移到1.5 毫升螺钉盖管。</li>
	<li>测量 vDNA 的 260 nm 紫外线 (UV) 吸收量。使用此值计算最终的摩尔 DNA 浓度。此 vDNA 的消光系数 (ε260) 为622803厘米-1 M-1 , 其分子量为 23972 Da. 存储在-20 ˚C。</li>
</ol>2. Intasome 总成
<ol><li>如果可能, 在带有可变恒温器的小房间中执行 intasome 组件。调整恒温器到大约18-22 ˚C。 注: 按照我们的经验, intasome 装配在4˚C 是低效的。</li>
	<li>用搅拌板上的搅拌条, 在2升烧杯中制备 1 l 的透析缓冲器 (20 毫米双三丙烷, pH 7.5, 200 毫米氯化钠, 25 毫米 dithiothreitol (), 2 µM ZnCl2)。平衡18-22 ˚C 房间的缓冲器, 至少6小时。</li>
	<li>组装 intasomes, 结合50毫米双三丙烷, pH 7.5, 500 毫米氯化钠, 120 µM PFV, 和50µM vDNA 在总容积150µL。 注意: PFV 在核酸酶活动 7, 8 中应该没有污染.如果 PFV 浓度太稀, 不能容纳150µL 体积内的120µM, 则可以将最终体积增加到200µL。这一步骤的关键因素是保持摩尔比的 vDNA (2.4: 1) 和有足够的复杂, 以可视化的色谱。</li>
	<li>用双蒸馏水 (ddH2O) 清洁10厘米长片10毫米 6-8 kDa 截留分子量透析油管和夹, 或最高纯度水。夹紧油管的一端。 注: 透析油管应小心处理, 以防止任何可能的污染, 从实验室的空间。</li>
	<li>使15毫升透析油管冲洗缓冲器 (50 毫米双三丙烷, pH 7.5, 500 毫米氯化钠)。用1-2 毫升冲洗缓冲液冲洗三次透析油管内。用吸管和薄凝胶加载尖端尽可能多地去除冲洗缓冲器。</li>
	<li>如有必要, 使用干净的剃刀刀片或手术刀切开透析油管, 使薄的凝胶加载尖端可以到达油管的末端。</li>
	<li>将 intasome 组件从步骤2.3 转移到透析油管, 用吸管和薄凝胶加载尖端。夹住透析油管的开端, 尽量减少油管内引入的空气量。将油管放入透析缓冲器。</li>
	<li>透析隔夜 (16-20 小时), 温和搅拌, 使油管旋转, 但不是在漩涡。确保透析油管被淹没和移动。大约一小时后, 观察管内的可见沉淀。 注: Cy3 或 Cy5 荧光标记 vDNA, 沉淀更明显。这对于两端标记和内部标记的荧光 vDNA 是正确的。</li>
</ol>3. Intasome 增溶
<ol><li>准备两个宽口径的吸管提示, 从2-4 毫米从200µL 尖端, 用新剃刀或手术刀刀片在干净的表面。 注意: 这些提示将在步骤3.3 和3.5 中使用。</li>
	<li>从透析缓冲液中取出透析油管。为了防止稀释的 intasome 样品与透析缓冲, 可能留在油管末端附近的剪辑, 使用微与小吸管提示, 以消除任何过剩的透析缓冲。从透析油管的一端取出夹子。</li>
	<li>从步骤3.1 中使用宽口径吸管尖端将样品包括透析油管内的沉淀到冰上的1.5 毫升管上。注意回收材料的总容积;总成交量一般为140µL。</li>
	<li>样品与沉淀是在200毫米氯化钠;添加适量的一股5米氯化钠溶液, 将盐增加到最终浓度为320毫米的氯化钠。例如, 对于150µL 的示例, 请添加3.9 µL 5 M 氯化钠和1.1 µL ddH2O 作为最终卷155µL. 将冰桶与样品一起转到4˚C 冷室。</li>
	<li>使用宽口径的吸管尖从步骤3.1 到并用重悬和溶解沉淀吹打。每20分钟重复至少1小时, 观察到大部分沉淀物都应该进入溶液中。 注: 吹打到并用重悬的沉淀可以停止时, 明显的沉淀不再明显减少时间点之间。当 vDNA 未标记或在内部标记时, 根据目视检查, 沉淀减少了90%。在 Cy5 端标记为 vDNA 的情况下, 沉淀仅减少 20%;多数沉淀与荧光末端标记 vDNA 不会溶解。有效可溶性的沉淀量是可变的, 应进行经验主义的测定。</li>
</ol>4. Intasome 纯化
<ol><li>准备250毫升的尺寸排除色谱 (秒) 运行缓冲器 (20 毫米双三丙烷, pH 7.5, 320 毫米氯化钠, 10% 甘油)。无菌过滤器的缓冲区与一个0.2 µm 过滤器单元和存储在4˚C。 注: 所有净化步骤都在4˚C 的冷室进行。</li>
	<li>平衡交联琼脂糖秒柱 (直径 = 10 毫米; 长度 = 300 毫米; 床容积 = 24 毫升; 样品体积 = 25-500 µL; 最大压力 = 1.5 MPa; 排除限制 = 4 x 107 Da; 在5和 5000 kDa 之间分离分子权重, 请参阅垫子表材料), SEC 以0.4 毫升/分钟的流速运行缓冲区。 注: 如果能够有效地将 300 kDa 从 44 kDa (如 Superose 12 10/300 GL 或 Superose 6 增加) 中分离出来, 这种净化可以适应不同大小的排除柱。Superose 12 10/300 GL 的分辨率较低, 导致峰值重叠。相反, Superose 6 的增加有更高的分辨率, 并提供更好的分离。</li>
	<li>离心 intasome 样品在离心在 1.4万 x g 在4˚C 的10分钟, 以颗粒任何剩余的沉淀。小心移除上清。载入上清至200µL 注射回路 (可容纳 200 ul 的油管)。将该示例应用于 SEC 列。 注意: 较小的负载量允许 SEC 更大的分辨率。</li>
	<li>洗脱与25毫升秒运行缓冲器和收集95个分数270µL. 观察 SEC 色谱显示三个 280/260 峰值 ( 图1)。 注: 第一个应为骨料 (~ 9.0-11.5 毫升洗脱), 其次是 PFV 聚丙烯 intasome 峰值 (~ 12.0-14.75 毫升洗脱) 和 PFV 在单体与自由 vDNA 峰值 (~ 15.0-18.0 毫升)。</li>
</ol>5. 集成链转移分析、分数选择和存储
<ol><li>将每个 intasome 峰值分数和 50 ng 3 kb 超螺旋质粒 DNA (库存浓度为50µL/µL) 结合在反应缓冲器中 (10 毫米双三丙烷), pH 值 7.5, 110 毫米氯化钠, 5 毫米 MgSO4, 4 µM ZnCl2, 10 毫米) 在最后的反应容量15µL。孵育37˚C 5 分钟。</li>
	<li>包括一个没有添加 PFV intasome 的负控制。停止与0.75 µL 蛋白酶 K (20 毫克/毫升库存溶液) 和0.75 µL SDS (10% 库存溶液) 的反应。孵化在55˚C 1 小时, 根据需要在-20 ˚C 存储样本, 供以后分析。 注: 此化验是对整合活动的定性评估。intasome 摩尔浓度的每一个分数是不确定之前, 该化验。积分链转移分析中包含的分数可以储存在冰上 (包括隔夜储存), 直到整合活动被确认和选择的分数是 aliquoted。这里我们使用 pMP2, 一个 pUC19 导数9。我们还成功地使用了 5.4 kb 质粒 pcDNA 3.1。任何能被解析为松弛圆、线性和超螺旋形式的质粒都可以作为整合的目标。</li>
	<li>120毫升1% 重量每卷 (w/v) 琼脂糖凝胶在1X 泰式缓冲 (40 毫米三乙酸, 1 毫米 EDTA) 与0.1 微克/毫升溴溴 (EtBr, 10 毫克/毫升库存解决方案)10。融化琼脂糖溶液, 倒入15厘米 x 10 厘米凝胶铸造托盘。插入一个15井梳子与5毫米宽, 0.75 毫米厚实的井。 注: 较窄的井比1.5 毫米厚井产生更好的带分辨率。</li>
	<li>允许凝胶在室温下凝固。将凝胶浸泡在1X 泰中, 0.1 微克/毫升 EtBr。</li>
	<li>从步骤5.2 中添加3µL 50% 甘油到每个集成反应。将整个反应体积加载到凝胶上。将1µL 10 kb DNA 大小标记 (150 ng/µL 库存浓度) 加到样品两侧的车道上;换句话说, DNA 大小标记应该侧面的样本车道。</li>
	<li>在外部车道, 装载6µL 橙 g 染料 (0.25% 橙 g, 30% 甘油)。在恒定电压下运行凝胶, 10 伏/厘米 (100 v) 在室温下为1小时, 或直到橙色 G 染料前面到达凝胶的末端。</li>
	<li>立即图像的琼脂糖凝胶与荧光扫描仪设置, 以检测 EtBr (532 nm 励磁, 610 nm 发射过滤器)。如果使用了荧光标记 vDNAs, 也图像以适当的荧光设置。 注: 例如, 要检测 Cy5 标记的 DNA, 图像使用 633 nm 激发和 670 nm 发射过滤器。协调一致的产品 (CI, 线性 dna) 应该运行真实的大小在 ~ 3 kb, 反应超螺旋质粒 (SC) 应该运行更快 (~ 2 kb), 和半现场集成产品 (HSI, 放松循环 DNA) 应该运行慢 (~ 3.5 kb)。反应 vDNA 应该运行真实的大小在 40 bp。</li>
	<li>使用图像软件7量化每个车道中的带区。计算每个车道中的 DNA 分数, 即 CI、HSI 或 SC;此计算中不包括 vDNA。 注: 积分效率, 或转换为线性产品的 SC 分数, 是通过将协调集成产品 (ci) 的像素体积除以三波段 (HSI + SC) 的总像素量来计算的。如果 intasomes 被荧光标记, 则 HSI 和 CI 产品也可能被荧光量化。</li>
	<li>选择具有最一致的集成活动的分数。 注意: 根据我们的经验, 峰值协同整合活动与蛋白质峰值相一致, 确定为 tetrameric PFV intasomes。测量每个分数的280 , 并计算最终的 intasome 浓度。这里描述的 PFV 的聚丙烯280是908339厘米-1M-1 , 分子量为 225.5 kDa。浓度应遵循与 SEC 色谱峰和链转移试验相同的模式。</li>
	<li>将每一个分数分布在5µL 整除数中, 并在液氮中凝固。商店整除数在-80 ˚C。为存储选择的分数通常介于 200-600 nM 之间。</li>
</ol>

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逆转录病毒的 multimeric 与病毒基因组 DNA 进行整合, 并继续病毒生命周期。每 intasome 单体的数量可以是 tetramers、octamers 或可能更高的顺序多聚体11, 12, 13, 14.PFV intasomes 是聚丙烯重组的双链 DNA 寡聚物, 模仿病毒基因组 dna 结束3.这些 intasomes 已用于结构和动态研究3</s...

将病毒基因组集成到宿主基因组是所有逆转录1生命周期中的一个强制性步骤。病毒酶整合酶 (in) 催化病毒 DNA 基因组的每一端的共价键连接到宿主 DNA。在细胞感染期间, 它是一个整合整合的预集成复合体的一部分。重组复合双链 DNA 寡聚物模拟病毒 DNA 末端也可以进行集成到目标 DNA体外2。一个共同的综合试验体外利用超螺旋质粒作为靶 DNA。将两种病毒 DNA 寡聚物 (vDNA) 与质粒整合在一起会产生线性乘积, 并被称为协同集成 (图 2A)。集成化验体外也可能产生的产品只有一个 vDNA 共价键加入到目标质粒, 导致一个松弛的循环。这种半站点集成产品似乎是检测体外的一个伪品。
重组和 vDNA 可能执行集成在体外, 但它们不是理想的试剂, 研究的动力学或结构的集成络合物时, 单体将模糊相关的可视化。纯集成复合物, 或 "intasomes", 是动态单分子分析或结构研究所必需的。PFV 和 vDNAs 可以通过透析从一个相对较高的盐浓度缓冲, 以较低的盐浓度3,4。在透析过程中, 形成沉淀形式。这种沉淀从透析中除去, 盐浓度增加。盐浓度越高, 含 PFV intasomes 的沉淀 solubilizes。然后用尺寸排除色谱 (SEC) 纯化 intasomes。重组原型泡沫病毒 (PFV) 已被证明存在作为单体在溶液中的浓度高达225µM5。SEC 分离有效地分离 PFV intasomes (225.5 kDa), 其中包括 PFV 的聚丙烯和二 vDNAs, 从单体 PFV 在 (44.4 kDa) 和自由 vDNA (24.0 kDa)。PFV intasomes 可能会被冻结, 并在-80 ˚C 至少保存六月的集成活动。
重组 PFV intasomes 也可能被修改, 包括在氨基酸替代或截断突变或 vDNAs 标记为显影或生物素4,6。纯化后的 PFV intasomes 能很容易地整合成超螺旋质粒靶 DNA体外。intasomes 的散装生化积分化验可以测试突变, 抑制剂, 或其他化学添加剂的影响。生物素化 intasomes 可以用来探测与核酸或蛋白质的亲和性。PFV intasomes 在室温下功能, 允许用磁镊进行单分子显微分析, 测量两个 vDNA 端或全内反射荧光之间的连接时间, 以可视化目标的 intasome 搜索。DNA6。此外, PFV intasomes 是第一个结构上具有显著影响的逆转录病毒集成领域3。

<table><tbody><tr><td>DNA Oligomers</td><td>IDT</td><td>N/A</td><td>Custom DNA Oligos</td></tr><tr><td>Tris Ultra Pure</td><td>Gojira Fine Chemicals</td><td>UTS1003</td><td></td></tr><tr><td>NaCl</td><td>P212121</td><td>RP-S23020</td><td></td></tr><tr><td>UltraPure EDTA</td><td>Invitrogen/Gibco</td><td>15575</td><td></td></tr><tr><td>Amicon Ultra 0.5 mL centrifugal filters</td><td>Sigma-Aldrich</td><td>Z677094-24EA</td><td>3 kDa MWCO</td></tr><tr><td>DTT</td><td>P212121</td><td>SV-DTT</td><td></td></tr><tr><td>BIS-TRIS propane,&gt;=99.0% (titration)</td><td>Sigma-Aldrich</td><td>B6755-500G</td><td></td></tr><tr><td>ZnCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>208086</td><td></td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Amresco</td><td>0662</td><td></td></tr><tr><td>Glycerol</td><td>Thermo Fisher Scientific</td><td>G37-20</td><td></td></tr><tr><td>Gel-loading tips, 1 - 200 &amp;mu;L</td><td>Corning</td><td>CLS4853-400EA</td><td></td></tr><tr><td>Razor blade; Single-edged; 100/Pk.; Pack of 100</td><td>Fisher Scientific</td><td>12-640</td><td></td></tr><tr><td>Sterile Disposable Filter Units with PES Membrane &gt; 250mL</td><td>Thermo Fisher Scientific</td><td>568-0020</td><td></td></tr><tr><td>Dialysis tubing clips</td><td>Spectrum Labs</td><td>132734</td><td></td></tr><tr><td>6-8 kDa 10 mm Dialysis Tubing</td><td>Spectrum Medical</td><td>132645</td><td></td></tr><tr><td>Superose 6 10/300 GL</td><td>GE Healthcare Life Sciences</td><td>17517201</td><td></td></tr><tr><td>Hi-Res Standard Agarose</td><td>AGTC Bioproducts</td><td>AG500D1</td><td></td></tr><tr><td>Ethidium bromide</td><td>Thermo Fisher Scientific</td><td>BP1302</td><td></td></tr><tr><td>Orange G</td><td>Fisher Scientific</td><td>0-267</td><td></td></tr><tr><td>Hyladder 10kb, 500 lanes</td><td>Denville Scientific</td><td>CB4225-4</td><td></td></tr></tbody></table>

assembly and purification of prototype foamy virus intasomes

反转录病毒生命周期的一个定义特征和必要步骤是将基因组整合到宿主基因组中。所有逆转录编码一个整合酶 (in) 酶, 催化共价连接病毒的宿主 DNA, 这被称为链转移。集成可以建模体外与重组逆转录病毒和 DNA 寡聚物模仿病毒基因组的两端。为了更仔细地重述发生体内的集成反应, 在减少的盐浓度缓冲液中, 通过透析在重组和合成寡聚物中组装出集成络合物。集成复合体称为 intasome, 可通过尺寸排除色谱法纯化。在原型泡沫病毒 (PFV) 的情况下, intasome 是一个聚丙烯和两个 DNA 寡聚物, 是容易分离的单体和游离寡聚 dna。PFV intasomes 的整合效率可以在各种实验条件下进行测定, 以更好地了解逆转录病毒集成的动力学和机理。

PFV intasomes 由重组和 vDNA 组装而成。程序集后, intasomes 由 SEC (图 1) 进行纯化。用超螺旋 DNA 靶和琼脂糖凝胶电泳 (图2) 测定各分数的积分活性。这种凝胶是成像与荧光扫描仪设置, 以检测 EtBr (和荧光, 如果使用荧光标记 vDNA)。使用图像分析软件, 可以使用带像素卷来计算集成效率 (图 3)。积分效率最高的分数...

Watch this Scientific Journal Video about Assembly and Purification of Prototype Foamy Virus Intasomes at JoVE.com

Assembly and Purification of Prototype Foamy Virus Intasomes

重组逆转录病毒整合酶和 dna 寡聚物模仿病毒 dna 末端可以形成一个酶活跃的复杂称为 intasome。Intasomes 可用于生物化学、结构和动力学研究。本协议详细介绍了如何组装和纯化原型泡沫病毒 intasomes。

泡沫病毒 Intasomes 原型的组装与纯化

A defining feature and necessary step of the retrovirus life cycle is the integration of the viral genome into the host genome. All retroviruses encode an ...

assembly-and-purification-of-prototype-foamy-virus-intasomes

Research

JoVE Journal

Immunology and Infection

5.4K 次观看.  The Ohio State University College of Medicine. 重组逆转录病毒整合酶和 dna 寡聚物模仿病毒 dna 末端可以形成一个酶活跃的复杂称为 intasome。Intasomes 可用于生物化学、结构和动力学研究。本协议详细介绍了如何组装和纯化原型泡沫病毒 intasomes。

视频: Assembly and Purification of Prototype Foamy Virus Intasomes

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal phase during establishment of infection in a given host. Consequently, study of encapsidation deciphers the information regarding the know-how of the mechanism regulating virus assembly to form infectious virions. Such information is vital in formulating novel methods of curbing virus spread and disease control. Virus encapsidation can be studied in vivo and in vitro. Genome encapsidation in vivo is a highly regulated selective process involving macromolecular interactions and subcellular compartmentalization. Therefore, study leading to dissect events encompassing virus encapsidation in vivo would provide basic knowledge to understand how viruses proliferate and assemble. Recently in vitro encapsidation has been exploited for the research in the area of biomedical imaging and therapeutic applications. Non-enveloped plant viruses stand far ahead in the venture of in vitro encapsidation of the negatively charged foreign material. Brome mosaic virus (BMV), a non-enveloped multicomponent RNA virus pathogenic to plants, has been used as a model system for studying genome packaging in vivo and in vitro. For encapsidation assays in Nicotiana benthamiana plants, Agrobacterium -mediated transient expression, refer to as agroinfiltration, is an efficient and robust technique for the synchronized delivery and expression of multiple components to the same cell. In this approach, a suspension of Agrobacterium tumefaciens cells carrying binary plasmid vectors carrying cDNAs of desiredviral mRNAs is infiltrated into the intercellular space withina leaf using nothing more sophisticated than a 1 ml disposable syringe (without needle). This process results in the transfer of DNA insert into plant cells; the T-DNA insert remains transiently in the nucleus and is then transcribed by the host polymerase II, leading to the transient expression. The resulting mRNA transcript (capped and polyadenylated) is then exported to the cytoplasm for translation. After approximately 24 to 48 hours of incubation,sections of infiltrated leaves can be sampled for microscopyor biochemical analyses. Agroinfiltration permits large numbers (hundreds to thousands) of cells to be transfected simultaneously. For in vitro encapsidation, purified virions of BMV are dissociated into capsid protein by dialyzing against dissociation buffer containing calcium chloride followed by removal of RNA and un-dissociated virions by centrifugation. Genome depleted capsid protein subunits are then reassembled with desired viral genome components or non-viral components such as indocyanine dye.

Simple and Robust in vivo and in vitro Approach for Studying Virus Assembly

A simple, efficient and robust way to synchronize the delivery of multiple viral components to plant cells via Agrobacterium-mediated transient expression is described. This approach is amenable for studying replication, encapsidation followed by in vitro reassembly of non-viral components into genome depleted optical viral ghosts suitable for biomedical applications.

简单而强大在体内和在体外方法研究病毒大会

In viruses with positive-sense RNA genomes pathogenic to humans, animals and plants, progeny encapsidation into mature and stable virions is a cardinal ...

科研

免疫与感染

Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States alone, an average of 41,400 deaths and 1.86 million hospitalizations are caused by influenza virus infection each year 1. Point mutations in the polymerase basic protein 2 subunit (PB2) have been linked to the adaptation of the viral infection in humans 2. Findings from such studies have revealed the biological significance of PB2 as a virulence factor, thus highlighting its potential as an antiviral drug target.
The structural genomics program put forth by the National Institute of Allergy and Infectious Disease (NIAID) provides funding to Emerald Bio and three other Pacific Northwest institutions that together make up the Seattle Structural Genomics Center for Infectious Disease (SSGCID). The SSGCID is dedicated to providing the scientific community with three-dimensional protein structures of NIAID category A-C pathogens. Making such structural information available to the scientific community serves to accelerate structure-based drug design.
Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for new lead discovery by targeting a pathway or an entire protein family. Emerald Bio has developed a high-throughput, multi-target parallel processing pipeline (MTPP) for gene-to-structure determination to support the consortium. Here we describe the protocols used to determine the structure of the PB2 subunit from four different influenza A strains.

Structure-based drug design plays an important role in drug development. Pursuing multiple targets in parallel greatly increases the chance of success for lead discovery. The following article highlights how the Seattle Structural Genomics Center for Infectious Disease utilizes a multi-target approach for gene-to-structure determination of the PB2 influenza A subunit.

流感聚合酶PB2亚基基因结构测定多目标并行处理方法

Pandemic outbreaks of highly virulent influenza strains can cause widespread morbidity and mortality in human populations worldwide. In the United States ...

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum (ER) resident proteins implicated in homotypic fusion of the ER. We detail here a method for purifying a glutathione S-transferase (GST) and poly-histidine tagged Drosophila atlastin by two rounds of affinity chromatography. Studying fusion reactions in vitro requires purified fusion proteins to be inserted into a lipid bilayer. Liposomes are ideal model membranes, as lipid composition and size may be adjusted. To this end, we describe a reconstitution method by detergent removal for Drosophila atlastin into preformed liposomes. While several reconstitution methods are available, reconstitution by detergent removal has several advantages that make it suitable for atlastins and other similar proteins. The advantage of this method includes a high reconstitution yield and correct orientation of the reconstituted protein. This method can be extended to other membrane proteins and for other applications that require proteoliposomes. Additionally, we describe a FRET based lipid mixing assay of proteoliposomes used as a measurement of membrane fusion.

Detergent-assisted Reconstitution of Recombinant Drosophila Atlastin into Liposomes for Lipid-mixing Assays

Biological membrane fusion is catalyzed by specialized fusion proteins. Measuring the fusogenic properties of proteins can be achieved by lipid mixing assays. We present a method for purifying recombinant Drosophila atlastin, a protein that mediates homotypic fusion of the ER, reconstituting it to preformed liposomes, and testing for fusion capacity.

洗涤剂辅助重组重组果蝇阿拉他素到脂质混合测定脂质体

Membrane fusion is a crucial process in the eukaryotic cell. Specialized proteins are necessary to catalyze fusion. Atlastins are endoplasmic reticulum ...

生物化学

Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and assay development in virology. The RNA template of nonsegmented negative-sense (NNS) RNA viruses, such as the respiratory syncytial virus (RSV), is not an RNA molecule alone but rather a nucleoprotein (N) encapsidated ribonucleoprotein complex. Despite the importance of the authentic RNA template, the generation and assembly of such a ribonucleoprotein complex remain sophisticated and require in-depth elucidation. The main challenge is that the overexpressed RSV N binds non-specifically to cellular RNAs to form random nucleocapsid-like particles (NCLPs). Here, we established a protocol to obtain RNA-free N (N0) first by co-expressing N with a chaperone phosphoprotein (P), then assembling N0 with RNA oligos with the RSV-specific RNA sequence to obtain virus-specific nucleocapsids (NCs). This protocol shows how to overcome the difficulty in the preparation of this traditionally challenging viral ribonucleoprotein complex.

For in-depth mechanistic analysis of the respiratory syncytial virus (RSV) RNA synthesis, we report a protocol of utilizing the chaperone phosphoprotein (P) for coexpression of the RNA-free nucleoprotein (N0) for subsequent in vitro assembly of the virus-specific nucleocapsids (NCs).

呼吸系统同步病毒病毒特定核胶囊的生成和组装

The use of an authentic RNA template is critical to advance the fundamental knowledge of viral RNA synthesis that can guide both mechanistic discovery and ...

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus (IAV), virus fusion with the endosomal membrane as well as the subsequent disassembly of the viral capsid, called uncoating, is governed by the ionic conditions inside endocytic vesicles. The early steps in the virus life cycle are hard to study because endosomes cannot be directly accessed experimentally, creating the need for an in vitro approach. Here, we describe a method based on velocity gradient centrifugation of purified virions through a two-layer glycerol gradient, which enables analysis of the IAV core and its stability. The gradient contains a non-ionic detergent (NP-40) in its lower layer to remove the viral membrane by solubilization as the virus sediments toward the bottom. At neutral pH, viral cores are pelleted as stable structures. The major core components, matrix protein (M1) and the viral ribonucleoproteins (vRNPs), can be clearly identified in the pellet fraction by SDS-PAGE. Decreasing the pH to 6.0 or lower in the bottom layer selectively removes M1 from the pellet followed by release of vRNPs at more acidic conditions. Viral protein bands on Coomassie-stained gels can be subjected to densitometric quantification to monitor intermediate states of IAV disassembly. Besides pH, other factors that influence viral core stability can be assessed, such as salt concentration and putative viral uncoating factors, simply by modifying the detergent-containing glycerol layer accordingly. Taken together, the presented technique allows highly reproducible and quantitative analysis of viral uncoating in vitro. It can be applied to other enveloped viruses that undergo complex uncoating processes.

In Vitro Disassembly of Influenza A Virus Capsids by Gradient Centrifugation

Disassembly of influenza A virus cores during virus entry into host cells is a multistep process. We describe an in vitro method to analyze the early stages of viral uncoating. In this approach, velocity gradient centrifugation is used to biochemically dissect the steps that initiate uncoating under defined conditions.

在甲型流感病毒的衣壳的体外拆卸梯度离心

Acid-triggered molecular processes closely control cell entry of many viruses that enter through the endocytic system. In the case of influenza A virus ...

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-&#197;-10 &#197;. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.

Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy.

推进液体和冰中病毒组件的高分辨率成像

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It ...

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 ...

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell deformation, division, migration, and adhesion. However, studying the dynamics and structure of the actin network in vivo is complicated by the biochemical and genetic regulation within live cells. To build a minimal model devoid of intracellular biochemical regulation, actin is encapsulated inside giant unilamellar vesicles (GUVs, also called liposomes). The biomimetic liposomes are cell-sized and facilitate a quantitative insight into the mechanical and dynamical properties of the cytoskeleton network, opening a viable route for bottom-up synthetic biology. To generate liposomes for encapsulation, the inverted emulsion method (also referred to as the emulsion transfer method) is utilized, which is one of the most successful techniques for encapsulating complex solutions into liposomes to prepare various cell-mimicking systems. With this method, a mixture of proteins of interest is added to the inner buffer, which is later emulsified in a phospholipid-containing mineral oil solution to form monolayer lipid droplets. The desired liposomes are generated from monolayer lipid droplets crossing a lipid/oil-water interface. This method enables the encapsulation of concentrated actin polymers into the liposomes with desired lipid components, paving the way for in vitro reconstitution of a biomimicking cytoskeleton network.

In Vitro Reconstitution of the Actin Cytoskeleton Inside Giant Unilamellar Vesicles

In this manuscript, we demonstrate the experimental techniques to encapsulate the F-actin cytoskeleton into giant unilamellar lipid vesicles (also called liposomes), and the method to form a cortex-biomimicking F-actin layer at the inner leaflet of the liposome membrane.

体外 重组巨型单层囊泡内的肌动蛋白细胞骨架

The actin cytoskeleton, the principal mechanical machinery in the cell, mediates numerous essential physical cellular activities, including cell ...

生物工程

Sample Preparation for Single Virion Atomic Force Microscopy and Super-resolution Fluorescence Imaging

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule imaging assays which allows adhesion of single virions to glass surfaces with specificity. This preparation is based on grafting the surface of the glass with a mixture of PLL-g-PEG and PLL-g-PEG-Biotin, adding a layer of avidin, and finally creating virion anchors through attachment of biotinylated virus specific antibodies. We have applied this technique across a range of experiments including atomic force microscopy (AFM) and super-resolution fluorescence imaging. This sample preparation method results in a control adhesion of the virions to the surface.

The attachment of virions to a surface is a requirement for single virion imaging by Super-resolution fluorescence imaging or atomic force microscopy (AFM). Here we demonstrate a sample preparation method for controlled adhesion of virions to glass surfaces suitable for use in AFM and super-resolution fluorescence imaging.

单病毒原子力显微镜和超分辨率荧光成像的样品制备

Immobilization of virions to glass surfaces is a critical step in single virion imaging. Here we present a technique&#160;adopted from single molecule ...

生物学

Expression and Purification of Virus-like Particles for Vaccination

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased biosafety and stability over use of live pathogens. Non-infectious and self-assembling, VLPs are used to present structural proteins as immunogens, bypassing the need for live pathogens or recombinant viral vectors for antigen delivery. In this article, we demonstrate the different stages of VLP design and development for future applications in preclinical animal testing. The procedure includes the following stages: selection of antigen, expression of antigen in cell line of choice, purification of VLPs/SVPs, and quantification for antigen dosing. We demonstrate use of both mammalian and insect cell lines for expression of our antigens and demonstrate how methodologies differ in yield. The methodology presented may apply to a variety of pathogens and can be achieved by substituting the antigens with immunogenic structural proteins of the user&#39;s microorganism of interest. VLPs and SVPs assist with antigen characterization and selection of the best vaccine candidates.

Here, we present a protocol for synthesizing virus-like particles using either baculovirus or mammalian expression systems, and ultracentrifugation purification. This highly customizable approach is used to identify viral antigens as vaccine targets in a safe and flexible manner.

表达和疫苗病毒样颗粒的纯化

Virus-like particles (VLPs) and subviral particles (SVPs) are an alternative approach to viral vaccine design that offers the advantages of increased ...

泡沫病毒 Intasomes 原型的组装与纯化

摘要

探索更多视频

此视频中的章节

简单而强大在体内和在体外方法研究病毒大会

流感聚合酶PB2亚基基因结构测定多目标并行处理方法

洗涤剂辅助重组重组果蝇阿拉他素到脂质混合测定脂质体

呼吸系统同步病毒病毒特定核胶囊的生成和组装

在甲型流感病毒的衣壳的体外拆卸梯度离心

推进液体和冰中病毒组件的高分辨率成像

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

体外重组巨型单层囊泡内的肌动蛋白细胞骨架

单病毒原子力显微镜和超分辨率荧光成像的样品制备

表达和疫苗病毒样颗粒的纯化

泡沫病毒 Intasomes 原型的组装与纯化

摘要

探索更多视频

此视频中的章节

简单而强大在体内和在体外方法研究病毒大会

流感聚合酶PB2亚基基因结构测定多目标并行处理方法

洗涤剂辅助重组重组果蝇阿拉他素到脂质混合测定脂质体

呼吸系统同步病毒病毒特定核胶囊的生成和组装

在甲型流感病毒的衣壳的体外拆卸梯度离心

推进液体和冰中病毒组件的高分辨率成像

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

体外 重组巨型单层囊泡内的肌动蛋白细胞骨架

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