病毒 DNA 的纯化及相关病毒和细胞蛋白的鉴定
Summary
该协议的目的是专门标记和有选择地分离病毒 DNA 从受感染的细胞, 以表征病毒基因组相关的蛋白质。
Abstract
该协议的目的是分离单纯疱疹病毒1型 (HSV-1) DNA 从感染的细胞, 以确定相关的病毒和细胞蛋白的质谱。尽管与病毒基因组相互作用的蛋白质在决定感染的结果方面起着重要作用, 但对病毒基因组相关蛋白的综合分析在以前是不可行的。在这里, 我们演示了一种方法, 能够直接纯化 HSV-1 基因组从受感染的细胞。复制病毒 DNA 有选择性地用含有炔烃功能基团的修饰核苷酸标记。然后通过铜 (I)-催化叠氮化-炔烃加或单击反应, 通过生物素叠氮化物的共价键, 对标记的 DNA 进行具体和不可逆转的标记。生物素标记的 DNA 是纯化亲和包覆的珠子和相关的蛋白质是洗和鉴定的质谱。这种方法使 HSV-1 复制叉或整个基因组在复杂的生物环境中有选择的目标和隔离。此外, 这种方法的适应将允许调查 herpesviral 感染的各个方面, 以及检查其他 DNA 病毒的基因组。
Introduction
病毒具有执行基本功能的能力有限, 因此取决于宿主因素, 以促进感染的关键方面, 包括病毒基因表达、复制、修复、重组和运输。这些寄主因素的活动通常由病毒编码的蛋白质增强。此外, 病毒必须避免对病毒感染的细胞反应进行检测和干扰。因此, 病毒宿主交互决定感染的结果。最重要的是了解病毒如何改变细胞环境, 以适应细胞的机器, 以促进病毒的过程。特别感兴趣的是确定哪些因素和过程对病毒基因组在整个感染周期中的作用。
单纯疱疹病毒1型 (HSV-1) 是一种双链 DNA 病毒, 感染了相当一部分人的人口。在感染的第一个小时内, 病毒基因组进入细胞核, 病毒基因表达的有序级联随后与病毒 DNA (vDNA) 复制1进行协调。在细胞核中, 基因组受后生调节, 经过修复和重组, 并被包装成衣, 这样, 第一个子代病毒在不到六小时内生产。在整个感染过程中, 对病毒基因组相关蛋白的综合评价将为研究病毒基因组过程的分子细节打下基础, 并将提供对病毒和细胞因子的洞察参与感染的各个阶段。
以前的方法调查病毒感染的寄主因素包括亲和纯化病毒蛋白的相关细胞蛋白的分析2,3,4,5,6,7,8,9. 这些检测方法有助于识别宿主抗病毒应答中涉及的细胞因子, 以及病毒染色质修饰、基因表达和 DNA 修复。然而, 很难确定相互作用是否依赖于与 vDNA 的关联, 蛋白质组学只提供对作为特定病毒因素的功能而发生的相互作用的洞察。染色质沉淀 (芯片) 已被用来确定特定的病毒和细胞蛋白绑定到病毒基因组10,11,12,13,14, 15和荧光原位杂交 (fish) 结合免疫细胞化学技术, 使 colocalize 与 vDNA16,17,18的单元格因子的可视化19,20. 这些化验结果可以进行时空分析。然而, 限制包括对高度特异的抗体的需要, 有限的灵敏度, 以及对病毒宿主相互作用之前的洞察力的需要。因此, 我们开发了一种基于 iPOND (在新生的 DNA 上分离蛋白质)21和 aniPOND (加速本机 iPOND)22的方法, 以有选择地标记和纯化感染细胞的 vDNA, 以无偏见地识别病毒基因组。相关蛋白的质谱。iPOND 对细胞复制叉动力学的研究起到了很重要的作用。
对于被感染细胞的病毒基因组的选择性纯化, 复制 vDNA 被标记为乙炔修饰的苷、5-乙炔-2´-脱氧 (教育) 或 5-乙炔-2´-脱氧 () (图 1), 其次是共价键结合到生物素叠氮化通过点击化学促进单步纯化病毒基因组和相关蛋白的亲和涂层珠子 (图 2B)。重要的是, 感染是在固定细胞中进行的, 它们不从事细胞 DNA 复制, 从而能够对 vDNA 进行特定的标记。此外, HSV-1 感染导致细胞周期阻滞和抑制细胞 DNA 复制23,24。病毒可在感染前 prelabeled, 用于分析与传入病毒基因组相关的蛋白质 (图 1A), 或在 DNA 复制过程中标记, 用于分析与新合成的 vDNA 相关的蛋白质 (图 1B)25. 此外, 脉冲追踪分析可用于研究与病毒复制叉 (图 1C)26相关的蛋白质的性质。此外, 乙炔修饰的 vDNA 可以共价键共轭到一个荧光的空间研究蛋白质动力学 (图 2A和图 3)。成像允许直接可视化的 vDNA, 是一个免费的方法来验证 vDNA-蛋白质相互作用, 并可以适应跟踪病毒基因组在整个感染。我们预计, 这些方法可以进一步修改, 以研究任何方面的 herpesviral 感染, 包括潜伏期和重新激活, 并研究其他 DNA 病毒。此外, 用 5-乙炔苷 (EU) 进行标记可以分析 RNA 病毒基因组。
Protocol
1. 细胞培养、病毒感染和标记 ( 图 1 ) 以下协议涉及使用病毒。有关安全处理病毒和其他生物制剂的问题, 请参阅您的机构和 #39 的生物安全协议。该议定书得到了匹兹堡大学机构审查委员会的批准. 处理一个汇合的 150 cm 2 组织培养 MRC-5 细胞的烧瓶, 并将细胞转移到 600 cm 2 组织培养板中, 其中含有100毫升 DMEM 加 10% FBS。孵育?…
Representative Results
使用单击化学从细胞纯化 DNA 首先是通过 iPOND 方法21完成的。iPOND 的目的是纯化细胞复制叉, 用于鉴定相关的蛋白质。我们已经适应了这一技术, 专门研究 vDNA 蛋白相互作用期间感染。使用 vDNA 标记病毒基因组的方法 (图 1) 和同步感染的操作, 允许选择性地隔离和调查不同的种群。HSV-1 DNA 被贴上 (图 1), 以促进荧…
Discussion
该协议包括多个步骤, 如果不小心, 可能会导致蛋白质的产量和细胞 DNA 的污染显著降低。至关重要的是, 固定细胞用于所有的实验, 以确保细胞 DNA 没有标记和纯化。这可以证实, 由于缺乏细胞 dna 聚合在蛋白质样本, 因为 HSV-1 不利用细胞 dna 聚合酶的基因组合成。在标记和核收获步骤期间, 样品应错开, 以确保每一个处理平等。在纯化步骤中, 应注意不要过度移地操纵细胞核。这可能导致过早的原子?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
我们感谢汉娜在准备这份手稿方面的帮助。这项工作得到了 NIH 补助金 R01AI030612 的支持。
Materials
| MRC-5 cells | ATCC | CCL-171 | |
| Fetal Bovine Serum (FBS) | Gibco | 26140-179 | |
| Dulbecco's Modified Eagle Medium (DMEM) | Gibco | 12800-082 | substituted with 10% FBS, 2 mM L-glutamine, 12 mM (for growth in flasks) or 30 mM (for growth in dishes) sodium-bicarbinate |
| 600 cm2 tissue culture dish | Thermo Fisher Scientific | 166508 | |
| Tris Buffered Saline (TBS), pH 7.4 | 137 mM NaCl, 5 mM KCl, 491 mM MgCl, 680 mM CaCl, 25.1 mM Tricine | ||
| HSV-1 stock | stocks with titers greater than 1×109 PFU/mL work best | ||
| Sephadex G-25 column (PD-10 Desalting Column) | GE Healthcare | 17085101 | |
| Dimethyl sulfoxide (DMSO) | Fisher Scientific | D128-1 | |
| 5´-Ethynyl-2´-deoxycytidine (EdC) | Sigma-Aldrich | T511307 | Dissolve in DMSO to prepare 40 mM stock, aliquot, and store at -20 °C |
| 2´-deoxycytidine (deoxyC) | Sigma-Aldrich | D3897 | Dissolve in water to prepare 40 mM stock, aliquot, and store at -20 °C |
| Nuclear Extraction Buffer (NEB) | prepare fresh (20 mM Hepes pH 7.2, 50 mM NaCl, 3 mM MgCl2, 300 mM Sucrose, 0.5% Igepal) | ||
| Cell scraper | Bellco glass | 7731-22000 | Autoclave before use |
| Trypan blue solution | Sigma-Aldrich | T8154 | |
| PBS, pH 7.2 (10x) | 1.37 M NaCl, 27 mM KCl, 100 mM Na2HPO4, 18 mM KH2PO4 (dilute to 1x in sterile water before use) | ||
| Copper (II) sulfate pentahydrate (CuSO4-5H2O) | Fisher Scientific | C489 | Prepare 100 mM stock and store at 4 °C for up to 1 month |
| (+) Sodium L-ascorbate | Sigma-Aldrich | A4034 | Freshly prepare 100 mM stock and store on ice until use |
| Biotin azide | Invitrogen | B10184 | Prepare 10 mM stock in DMSO, aliquot, and store at -20 °C for up to 1 year |
| Click Reaction Mix | prepare immediately before use by adding reagents in the indicated order (10 mL: 8.8 mL 1x PBS, 200 mL 100 mM CuSO4, 25mL 10 mM Biotin Azide, 1 mL 100 mM sodium ascorbate) | ||
| cOmplete Protease Inhibitor Cocktail | Roche | 11697498001 | Dissolve in 1 mL water to prepare 50x stock, can store at 4 °C for up to 1 week, or directly add 1 pill to 50 mL buffer |
| freezing buffer | prepare fresh (7 mL 100% glycerol, 3 mL NEB, 200 μL 50x protease inhibitor) | ||
| Buffer B1 | prepare fresh (25 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl pH 8, 1% Igepal, 1x protease inhibitor) | ||
| Buffer B2 | prepare fresh (150 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl pH 8, 0.5% Igepal, 1x protease inhibitor) | ||
| Buffer B3 | prepare fresh (150 mM NaCl, 2 mM EDTA, 50 mM Tris-HCl pH 8, 1x protease inhibitor) | ||
| Vibra Cell Ultra Sonic Processer equipped with a 3 mm microtip probe | Sonics | VCX 130 | |
| Cell strainer | Falcon | 352360 | |
| Dynabeads MyOne Streptavidin T1 | Life Technologies | 65601 | |
| DynaMag-2 Magnet | Life Technologies | 12321D | |
| Mini-Tube Rotator | Fisher Scientific | 260750F | |
| 2X Laemmli sample buffer | Mix 400 mg of SDS, 2 mL 100% glycerol, 1.25 mL of 1 M Tris (pH 6.8), and 10 mg of bromophenol blue in 8 mL water. Store at 4 °C for up to 6 months. Before use add 1 M DTT to a final concentration of 200 mM. | ||
| cap locks for 1.5 mL tube | Fisher Scientific | NC9679153 | |
| Standard western blotting reagents | |||
| NOVEX Colloidal Blue Staining Kit | Invitrogen | LC6025 | |
| 12-well tissue culture dish | Corning | 3513 | |
| Coverslips | Fisher Scientific | 12-545-100 | Autoclave before use |
| Microscope slides | Fisher Scientific | 12-552-5 | |
| 16% paraformaldehyde | Electron Microscopy Sciences | 15710 | dilute to 3.7% in 1x PBS before use |
| Bovine serum albumin (BSA) | Fisher Scientific | BP1605 | prepare 3% in 1x PBS |
| Permeabilization buffer (0.5% Triton-X 100) | Sigma-Aldrich | T8787 | prepare 0.5% Triton-X 100 in 1x PBS |
| Click reaction cocktail – Click-iT EdU Alexa Fluor 488 Imaging Kit | Molecular Probes | C10337 | prepare according to manufactorer's protocol |
| Hoechst 33342 | Life Technologies | H1399 | prepare 10 mg/mL in water, can store at 4 °C for up to 1 year |
| mouse anti-ICP8 primary antibody | Abcam | ab20194 | Use a 1:200 dilution in 1x PBS |
| mouse anti-UL42 primary antibody (2H4) | Abcam | ab19311 | Use a 1:200 dilution in 1x PBS |
| Goat anti-mouse 594-conjugated secondary antibody | Life Technologies | a11005 | Use a 1:500 dilution in 1x PBS |
| Immu-mount | Thermo Fisher Scientific | 9990402 | |
| 2x SDS-bicarb solution | 2% SDS, 200 mM NaHCO3 | ||
| Phenol:chloroform:isoamyl alcohol | mix 25:24:1 at least 1 day before use, store at 4 °C in the dark | ||
| chloroform:isoamyl alcohol | mix 24:1 at least 1 day before use, store at room temperature in the dark | ||
| 10x Tris EDTA (TE), pH 8.0 | 100 mM Tris, 10 mM EDTA (dilute to 1x before use) | ||
| Qiaquick PCR purification kit | Qiagen | 28104 | |
| 3 M sodium acetate pH 5.2 | |||
| Qubit 2.0 Fluorometer | Invitrogen | Q32866 | |
| Qubit dsDNA HS Assay Kit | Invitrogen | Q32851 | |
| Qubit assay tubes | Invitrogen | Q32856 | |
| Fluorescence microscope equipped with imaging software | |||
| Microcentrifuge for 1.5 mL tubes | |||
| Tabletop centrifuge for 15 and 50 mL tubes | |||
| Cell culture incubator | |||
| Biosafety cabinet | |||
| Heat blocks | 65 °C and 95 °C |
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Citazione di questo articolo
Dembowski, J. A., Deluca, N. A. Purification of Viral DNA for the Identification of Associated Viral and Cellular Proteins. J. Vis. Exp. (126), e56374, doi:10.3791/56374 (2017).