由腺病毒感染正常细胞分离病毒复制车厢富集亚核分数
Summary
We provide a novel strategy to isolate viral replication compartments (RC) from adenovirus (Ad)-infected human cells. This approach represents a cell-free system that can help to elucidate the molecular mechanisms regulating viral genome replication and expression as well as regulation of viral-host interactions established at the RC.
Abstract
During infection of human cells by adenovirus (Ad), the host cell nucleus is dramatically reorganized, leading to formation of nuclear microenvironments through the recruitment of viral and cellular proteins to sites occupied by the viral genome. These sites, called replication compartments (RC), can be considered viral-induced nuclear domains where the viral genome is localized and viral and cellular proteins that participate in replication, transcription and post-transcriptional processing are recruited. Moreover, cellular proteins involved in the antiviral response, such as tumor suppressor proteins, DNA damage response (DDR) components and innate immune response factors are also co-opted to RC. Although RC seem to play a crucial role to promote an efficient and productive replication cycle, a detailed analysis of their composition and associated activities has not been made. To facilitate the study of adenoviral RC and potentially those from other DNA viruses that replicate in the cell nucleus, we adapted a simple procedure based on velocity gradients to isolate Ad RC and established a cell-free system amenable to conduct morphological, functional and compositional studies of these virus-induced subnuclear structures, as well as to study their impact on host-cell interactions.
Introduction
腺病毒包含复制在被感染细胞核的双链DNA基因组。当病毒DNA进入细胞核，它局部化相邻的PML核机构1。随着病毒的早期基因的表达，核架构大幅改组，诱导形成病毒性微环境，被称为病毒复制车厢（RC）2。由于腺病毒（广告）RC是网站，病毒基因组复制和病毒晚期基因表达发生，他们提供了一个环境，招募所有参与这些过程中必要的病毒和细胞的因素。有趣的是，各种细胞蛋白负责细胞抗病毒反应，如DNA损伤反应的，先天免疫应答和抑制肿瘤是增选到这些病毒网站2。因此，广告RC可以考虑监管中心之一，促进高效病毒复制，同时伴随调节蜂窝的抗病毒反应，表明这些结构是关键的病毒 – 宿主细胞相互作用的理解。尽管如此，钢筋混凝土形成的分子机制，其组合物和相关活动是知之甚少。
腺病毒的RC，以及RC从复制在核中的其他DNA病毒不相关联的膜，而相比之下，细胞质的RC 3。此外，这些病毒诱导的结构是可能的蛋白质和核酸的待全部由。 RC形成在感染的细胞的RNA病毒（通常被称为病毒工厂）已被分离，同时它们的细胞质定位的和膜结合的状态，这促进了对它们的详细的形态，功能和生化特征4的优势。
据我们所知，核病毒的RC没有被隔离，这可能是由于所述的核内米的核结构和不存在的复杂性embranes这将有利于他们的孤立。他们的研究一直依靠而不是在免疫荧光显微镜，荧光原位杂交和透射电子显微镜。然而，尽管固有的隔离亚核结构复杂，其它核领域，如核仁和Cajal间机构一直在5,6前隔离。因为核仁和RC都是由蛋白质和核酸的，并且具有0.5之间的直径 – 5微米，我们推测，RC也应该适合于隔离。因此，为了更精确地表征相关联的RC的分子组成和功能，我们建立了一种新方法，以分离富含RC亚核级分。为此，我们制备使用速度梯度和蔗糖垫类似程序用于分离核仁7或其他核结构域6子核级分，并建立了无细胞系统，使分子组合物的研究和有关的活动RC。因此这种技术应该提前的病毒 – 宿主细胞相互作用的理解，并表示一个强大的工具，也应有助于RC的详细分析从该复制在核中的其他病毒和诱导形成类似的尺寸的复制隔间的那些形成在腺病毒感染的细胞，如，疱疹病毒，乳头状瘤病毒或多瘤病毒。
Protocol
Representative Results
Discussion
In order to elucidate the molecular mechanisms that govern regulation of cellular activities by viral infection understanding the composition and activities associated with RC would be instrumental. Therefore, to make a detailed analysis of RC, we established a cell-free system that takes advantage of the size and biochemical composition of these virus-induced structures, to isolate subnuclear fractions enriched with RC using a simple procedure that relies on velocity gradients with sucrose cushions. Critical steps of th…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported by grants from CONACyT-SEP (SEP-2008-84582; CB-2011-01-168497) and Promep-SEP for R.A.G.; P.H. received a scholarship from CONACyT (447442).
Materials
| DMEM | Gibco | 12100-046 | Warm in 37 ºC water bath before use |
| Fetal Bovine Serum | Gibco | 12484-028 | |
| Sucrose, Ultra Pure | Research Organics | 0928S | Prepare a 2.55 M stock solution and store at 4 ºC |
| Dounce homogenizer | Kontess Glass Company | 884900-0000 | |
| Branson 1800 Ultrasonic Bath | Branson | Z769533 SIGMA | Turn on 15 min before use. |
| Peroxidase AffiniPure F(ab')₂ Fragment Goat Anti-Mouse IgG (H+L) | Jackson ImmunoResearch | 115-036-003 | Use at a 1:10,000 dilution in PBS/0.03% non-fat milk |
| Goat anti-Mouse IgG1 Secondary Antibody, Alexa Fluor 488 conjugate | Life Technologies | A-21121 | Use at a 1:2,000 dilution in PBS |
| Silane-Prep Slides | Sigma | S4651-72EA | Open in a laminar flow cabinet |
| SuperSignal West Pico Chemiluminescent Substrate | Pierce ThermoScientific | 34080 |
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