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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作者没有什么可透露的。

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3,5,13,14。
PFV intasomes 的组装发生在透析过程中, 从相对较高的盐浓度缓冲到较低的盐浓度。这导致形成一个沉淀, 其中包括 PFV intasomes。intasomes 通过增加盐浓度可溶性。PFV 在225µM5的浓度单体。然后从单体和自由 vDNA 中分离出复合物。我们修改了 PFV intasomes 的纯化, 将甘油在 SEC 缓冲区6中。添加甘油允许 PFV intasomes 在不丢失活动的情况下被冻结以供将来使用 (图 4)。
该协议有几个关键的特点。虽然蛋白质纯化通常是在低温下进行的, 但我们发现, PFV intasome 的组装效率在4˚C 是低效的。相反, 大会透析应发生在18-22 ˚C 的范围内。使用重组 PFV 在无污染细菌核酸酶 7 8 中也很重要.最后, vDNA 的比例是装配的一个关键因素。如果蛋白质浓度的起始低于这里的推荐, vDNA 浓度必须按比例降低。虽然可以组装 PFV intasomes 在较低的浓度, 配合物必须在足够高的浓度, 以检测在 SEC 分离。
此方法可用于未标记或标记为 vDNA 的 PFV intasomes。我们发现, vDNA 可以有效地标记为荧光或生物素6。其他小分子标签也可能是可能的。在 Cy5 荧光的情况下, 我们发现标有 vDNA 的末端降低了聚集 intasomes 的收率, 峰值浓度降低了10倍。但是, 内部 Cy5 标记为 vDNA 的产量与未标记的 vDNA 有等价的收益率。此装配方法也可用于4中 PFV 的点突变或截断突变。由于 PFV 在临床上对 HIV-1 中的链转移抑制剂 (那里玩) 药物有抑制作用, 因此可以使用这些 PFV intasomes 研究 INSTIs 3 的机制.此外, 在 PFV 的保守残留物中, HIV-1 的一些那里玩抗性突变发生在, 允许研究与工程 PFV intasomes 的耐药性。

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&#160;</td>
			<td>P212121</td>
			<td>SV-DTT</td>
			<td></td>
		</tr>
		<tr>
			<td>BIS-TRIS propane,&#62;=99.0% (titration)</td>
			<td>Sigma-Aldrich</td>
			<td>B6755-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>ZnCl2</td>
			<td>Sigma-Aldrich</td>
			<td>208086</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4</td>
			<td>Amresco</td>
			<td>0662</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>G37-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel-loading tips, 1 - 200 &#956;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 &#62; 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为快照冻结后使用。冻结分数保留集成活动 (图 4), 允许对组装的 intasomes 进行长期存储。任何标签分子及其在 vDNA 中的位置都可能影响 intasome 组件的效率 (图 5)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57453/57453fig1.jpg"> 图 1: 大小排除色谱.PFV intasome 组件与琼脂糖秒列分离。本专栏允许分离聚合体 (1), PFV intasome 由 PFV 的聚丙烯和两个 vDNA (2) 和单体 PFV (3) 组成。这个例子包括 vDNA 与内部 Cy5 荧光标签检测到 650 nm 激发。y 轴表示光学密度 (OD) 在毫吸光度单位 (茂)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57453/57453fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57453/57453fig2.jpg"> 图 2: SEC 分数的集成检测.(A) 集成链转移反应示意图。左, 卡通的 PFV intasome 包括聚丙烯 (蓝色圆圈) 和两个 vDNA (粗黑线), 和目标质粒 (薄黑线)。不绘制目标质粒的 supercoils (SC)。中心, 一个半站点集成 (HSI) 产品的卡通, 当一个 vDNA 已经共价键加入到目标质粒。加入反应引入了一个尼克, 放松了 supercoils。正确的, 一个协同集成 (CI) 产品的卡通, 两个 vDNAs 已经加入到目标 DNA。这种集成产品导致了一个线性 DNA 与 vDNAs 在末端。(B) 超螺旋质粒 DNA 添加到每秒分数 #49-55。在孵化后, 通过1% 去蛋白琼脂糖凝胶电泳 EtBr, 将其整合反应分离出来。阴性对照为质粒 DNA, 无蛋白质 (T)。DNA 大小标记显示为 kb。超螺旋质粒 (SC)、协同集成产品 (CI)、半现场集成产品 (HSI) 和 vDNA。(C) 扫描琼脂糖凝胶的存在 Cy5。只有 vDNA、CI 和 HSI 产品是可见的。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57453/57453fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57453/57453fig3v2.jpg"> 图 3: 对集成检测的定量.扫描并量化图 2中的琼脂糖凝胶 (A) EtBr 和 (B) Cy5 的存在。(A) 为 EtBr 计算、HSI、CI 和 SC 波段像素卷使用凝胶分析软件获得。通过将协同集成产品 (ci) 的像素体积除以所有三波段 (HSI + SC) 的总像素体积, 计算出集成效率。例如, 分数 #49 带的 EtBr 通道中的像素值为: 110565.2 SC、25152.56 CI 和 6313.04 HSI。分数 #49 的总 DNA 像素值是这些值的总和, 142030.8。积分效率为25152.56 除以总 DNA 值142030.8 等于0.18 的 CI 像素体积。(B) Cy5 CI 波段像素卷被绘制为任意单位。峰值积分活动的分数分别 aliquoted 和冻结。在此示例中, 选中了分数 #50-53。整除数与液氮冷冻, 储存在-80 ˚C, 供将来使用。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57453/57453fig4.jpg"> 图 4: 冻结对 intasome 活动的影响.其中一半是用液氮冷冻,-80 ˚C 1 小时, 然后在冰上慢慢解冻。另一半的 SEC 分数被保存在冰冷的房间的冰, 而前半部分被冻结和解冻。Intasomes 测试的活动, 没有 (-) 和 (+) 冷冻/解冻。集成效率的测量如上文所述。(A) EtBr 染色琼脂糖凝胶的集成反应产品。超螺旋质粒 (SC)、协同集成产品 (CI)、半现场集成产品 (HSI) 和反应 vDNA。DNA 大小标记显示为 kb。(B) 从 EtBr 图像中定量化集成效率。计算在图 3图例中进行了说明。冻结和解冻后的一体化活动没有损失。5项独立实验的平均集成效率为 2 intasome 准备。误差线指示标准偏差。配对 t 检验分析产生了一个双尾 P = 0.011, 表明冻结/解冻可能有轻微增强的整合活动。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57453/57453fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/57453/57453fig5.jpg"> 图 5: 标签分子和位置影响 intasome 组件.PFV intasome 组件包括未标记的 vDNAs (红色), 末端标记在寡聚2与 Cy5 (蓝色), 内部标记在寡1与 Cy5 (黑色), 或末端标记在寡糖2与生物素 (橙色)。所有程序集均与琼脂糖秒列分离。y 轴表示茂中的外径。图谱是每个 intasome 类型至少2个独立组件的代表。末端贴标降低了 PFV intasomes Cy5 10 倍和生物素1.8 倍的产量。在高盐 resolubilization (步骤 3.5) 后, 端 Cy5 和生物素组件明显增加沉淀, 导致尺寸排除柱上的材料明显丢失。vDNA 的内部标记与 Cy5 荧光导致 intasomes 的相等的产量作为未标记的 vDNA。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57453/57453fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

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

Nanyang Technological University

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

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

流感病毒从感染细胞的染色质的核糖核蛋白复合物的亲和纯化

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

病原液泡的分离纯化<em&gt;军团</em&gt;感染吞噬细胞

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Rescue of Recombinant Newcastle Disease Virus from cDNA

Newcastle disease virus (NDV), the prototype member of the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented, negative-sense, single-stranded, enveloped RNA virus (Figure 1) with potential applications as a vector for vaccination and treatment of human diseases. In-depth exploration of these applications has only become possible after the establishment of reverse genetics techniques to rescue recombinant viruses from plasmids encoding their complete genomes as cDNA2-5. Viral cDNA can be conveniently modified in vitro by using standard cloning procedures to alter the genotype of the virus and/or to include new transcriptional units. Rescue of such genetically modified viruses provides a valuable tool to understand factors affecting multiple stages of infection, as well as allows for the development and improvement of vectors for the expression and delivery of antigens for vaccination and therapy. Here we describe a protocol for the rescue of recombinant NDVs.

Newcastle disease virus (NDV) has been extensively studied in the last few years in order to develop new vectors for vaccination and therapy, among others. These studies have been possible due to techniques to rescue recombinant virus from cDNA, such as those we describe here.

从救援的cDNA重组新城疫病毒

Newcastle disease virus (NDV), the prototype member of  the Avulavirus genus of the family Paramyxoviridae1, is a non-segmented,  negative-sense, ...

Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

分离，鉴定和小鼠胸腺上皮细胞的分离纯化

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple ...

Neutrophil Isolation and Analysis to Determine their Role in Lymphoma Cell Sensitivity to Therapeutic Agents

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of inflammation. They are key players in the innate immune system and play major roles in cancer biology. Neutrophils have been proposed as key mediators of malignant transformation, tumor progression, angiogenesis and in the modulation of the antitumor immunity; through their release of soluble factors or their interaction with tumor cells. To characterize the specific functions of neutrophils, a fast and reliable method is coveted for in vitro isolation of neutrophils from human blood. Here, a density gradient separation method is demonstrated to isolate neutrophils as well as mononuclear cells from the blood. The procedure consists of layering the density gradient solution such as Ficoll carefully above the diluted blood obtained from patients diagnosed with chronic lymphocytic leukemia (CLL), followed by centrifugation, isolation of mononuclear layer, separation of neutrophils from RBCsby dextran then lysis of residual erythrocytes. This method has been shown to isolate neutrophils &#8805; 90 % pure. To mimic the tumor microenvironment, 3-dimensional (3D) experiments were performed using basement membrane matrix such as Matrigel. Given the short half-life of neutrophils in vitro, 3D experiments with fresh human neutrophils cannot be performed. For this reason promyelocytic HL60 cells are differentiated along the granulocytic pathway using the differentiation inducers dimethyl sulfoxide (DMSO) and retinoic acid (RA). The aim of our experiments is to study the role of neutrophils on the sensitivity of lymphoma cells to anti-lymphoma agents. However these methods can be generalized to study the interactions of neutrophils or neutrophil-like cells with a large range of cell types in different situations.

Neutrophils are the most abundant type of white blood cells. The isolation of neutrophils from human blood by density gradient separation method and the differentiation of human promyelocytic (HL60) cells along the granulocytic pathway are described here; to test their role on sensitivity of lymphoma cells to anti-lymphoma agents.

中性粒细胞分离和分析，以确定他们的淋巴瘤细胞的敏感性对治疗药物的作用

Neutrophils are the most abundant (40% to 75%) type of white blood cells and among the first inflammatory cells to migrate towards the site of ...

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 ...

Rescue and Characterization of Recombinant Virus from a New World Zika Virus Infectious Clone

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral evolution. Here we describe the construction of an infectious clone for Zika virus (ZIKV), which is currently causing an explosive outbreak in the Americas. To prevent toxicity to bacteria that is commonly observed with flavivirus-derived plasmids, we generated a two-plasmid system which separates the genome at the NS1 gene and is more stable than full-length constructs that could not be successfully recovered without mutations. After digestion and ligation to join the two fragments, full-length viral RNA can be generated by in vitro transcription with T7 RNA polymerase. Following electroporation of transcribed RNA into cells, virus was recovered that exhibited similar in vitro growth kinetics and in vivo virulence and infection phenotypes in mice and mosquitoes, respectively.

This protocol describes the recovery of infectious Zika virus from a two-plasmid infectious cDNA clone.

来自新世界Zika病毒感染性克隆的重组病毒的救援和表征

Infectious cDNA clones allow for genetic manipulation of a virus, thus facilitating work on vaccines, pathogenesis, replication, transmission and viral ...

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 ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

快速、安全、简便的人工床边核酸提取法检测全血标本中的病毒

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...