Summary

呼吸系统同步病毒病毒特定核胶囊的生成和组装

Published: July 27, 2021
doi:

Summary

为了深入分析呼吸道同步病毒 (RSV) RNA 合成,我们报告了利用伴生磷蛋白 (P) 共同表达无RNA核蛋白 (N0)的协议,用于病毒特定核胶囊 (NCs) 的后续 体外 组装。

Abstract

使用正宗的RNA模板对于推进病毒RNA合成的基本知识至关重要,该知识可以指导机械发现和病毒学的检测发展。非受教负感 (NNS) RNA 病毒(如呼吸道同步病毒 (RSV))的 RNA 模板不是单单 RNA 分子,而是核蛋白 (N) 封装核糖核蛋白复合物。尽管正宗RNA模板很重要,但这种核糖核蛋白复合物的生成和组装仍然复杂,需要深入阐明。主要的挑战是,过度表达的RSV N非具体地与细胞RNA结合,形成随机核胶囊状粒子(NCLPs)。在这里,我们建立了一个协议,通过与伴郎磷蛋白 (P) 共同表达 N,然后用 RNA 寡头与 RSV 特异性 RNA 序列组装 N0来获得无RNA N (N0),以获得病毒特异性核胶囊 (NCs)。该协议展示了如何克服在准备这个传统上具有挑战性的病毒核糖蛋白复合物的困难。

Introduction

非隔离性负感(NNS)RNA病毒包括许多重要的人类病原体,如狂犬病、埃博拉和呼吸道同步病毒(RSV)1、2。RSV是导致全球幼儿和老年人患支气管炎和肺炎等呼吸系统疾病的主要原因。目前,没有有效的疫苗或抗病毒疗法可用于预防或治疗RSV4。作为生命周期的一部分,RSV基因组作为RSV RNA依赖RNA聚合酶复制的模板,以产生抗原体,而抗基因组又充当生成后代基因组的模板。基因组和抗基因组RNA都完全被核蛋白(N)封装,形成核胶囊(NCs)3。由于NC是RSV聚合酶复制和转录的模板,因此适当的NC组装对于聚合酶获得RNA合成5的模板至关重要。有趣的是,根据NNS病毒聚合酶的结构分析,假设几个N蛋白暂时脱离NC,允许聚合酶进入,并在RNA合成6、7、8、9、10、11、12后重新绑定RNA。

目前,RSV RNA聚合检测已建立使用纯化RSV聚合酶短裸RNA模板13,14。然而,RSV聚合酶的活动没有达到最佳水平,正如RSV聚合酶在使用裸RNA模板时产生的非加工和流产产品中观察到的。缺乏带有病毒特异性RNA的NC是进一步机械化地理解RSV RNA合成的主要障碍。因此,使用正宗的RNA模板成为推进RSV RNA合成基础知识的关键需要。来自RSV和其他NNS RNA病毒的核胶囊状粒子(NCLPs)的已知结构显示,NCLP中的RNA要么是随机细胞RNA,要么是平均病毒基因组RNA15、16、17、18、19。总之,主要障碍是,当N在主机单元中表达过度时,N与细胞RNA结合,形成NCLP。

为了克服这一障碍,我们建立了一个协议,首先获得无RNA(N0),并组装N0 与正宗的病毒基因组RNA到NCLP20。该协议的原则是获得大量的重组RNA无N(N0)通过共同表达N与伴郎,RSV磷蛋白(PNTD)的N终端域。纯化的 N0P 可以通过添加 RSV 特异性 RNA 寡头来刺激并组装成 NCLP,在组装过程中,伴郎 PNTD 在添加 RNA 寡头后会被移位。

在这里,我们详细说明了 RSV 特定 RNA 特定 NC 的生成和组装协议。在此协议中,我们描述了分子克隆、蛋白质制备、 体外 组装和复杂组件的验证。我们强调克隆策略,为分子克隆的蛋白质共表达生成双柠檬结构。在蛋白质制备方面,我们描述了细胞培养、蛋白质提取和蛋白质复合物的纯化过程。然后,我们讨论RSV RNA特定NC的 体外 组装方法。最后,我们使用大小排除色谱 (SEC) 和负污渍电子显微镜 (EM) 来描述和可视化组装的 NCLP。

Protocol

1. 分子克隆 注:利气独立克隆(LIC)用于制造RSV双语气联表达构造质粒。LIC是一种在20世纪90年代初开发的方法,它利用T4DNA聚合酶的3′-5’Exo活性来制造载体和DNA插入21,22之间的互补性悬垂。构造使用 2BT-10 矢量 DNA 进行,该 DNA 由他在开放读取帧 (ORF) (图 1)N 端的标记组成。 使用 SSPI ?…

Representative Results

无RNA N0P蛋白的纯化通过此协议,可以获得大规模可溶性异质 RSV N0P 复合体。P蛋白的N和N终端部分的全长与大肠杆菌中的N蛋白的10倍共同表达。N0P 使用钴柱、离子交换和大小排除色谱进行纯化。N0P 包含全长 N 端和 N 端 P,但不包含基于紫外线吸收 A260 /A 280比率20 (图4)的蜂窝RNA。 <p class="jove_…

Discussion

已知的核胶囊状粒子(NCLP)结构的非隔离负感(NNS)RNA病毒表明,组装的NCLP是复杂的N与宿主细胞RNA时,过度表达在细菌或真核表达系统15,16,17,18,19。先前的研究已经尝试通过各种方法获得RNA免费N,如RNA酶A消化,高盐洗涤,或调整不同的pH缓冲器,以去除非特异性细胞…

Disclosures

The authors have nothing to disclose.

Acknowledgements

埃默里梁实验室的研究项目由美国国家普通医学科学研究院 (NIGMS)、国家卫生研究院 (NIH) 提供,奖励编号为 R01GM130950,以及埃默里大学医学院的研究启动基金。作者感谢梁实验室的成员给予的帮助和支持和批判性讨论。

Materials

Agarose SIgma A9539-500G making construct using LIC method
Amicon Ultra-15 Centrifugal Filter Unit Millipore UFC901024 concentrate the protein sample
Ampicillin sodium GOLD BIOTECHNOLOGY 5118.111317A antibiotic for cell culture
AseI NEB R0526S making construct using LIC method
Cobalt (High Density) Agarose Beads Gold Bio H-310-500 For purification of His-tag protein
Corning LSE Digital Dry Bath Heater CORNING 6885-DB Heate the sample
dCTP Invitrogen 10217016 making construct using LIC method
dGTP Invitrogen 10218014 making construct using LIC method
Glycerol Sigma G5516-4L making solution
HEPES Sigma H3375-100G making solution
HiTrap Q HP Sigma GE29-0513-25 Protein purification
Imidazole Sigma I5513-100G making solution
IPTG (Isopropyl-beta-D-thiogalactopyranoside) GOLD BIOTECHNOLOGY 1116.071717A induce the expression of protein
Microcentrifuge Tubes VWR 47730-598 for PCR
Misonix Sonicator XL2020 Ultrasonic Liquid Processor SpectraLab MSX-XL-2020 sonicator for lysing cell
Negative stain grids Electron Microscopy Sciences CF400-Cu-TH For making negative stain grids
New Brunswick Innova 44/44R eppendorf M1282-0000 Shaker for culturing the cell
Nonidet P 40 Substitute Sigma 74385-1L making solution
OneTaq DNA Polymerase NEB M0480L PCR
QIAquick Gel Extraction Kit QIAGEN 28706 Purify DNA
SSPI-HF NEB R3132S making construct using LIC method
Superose 6 Increase 10/300 GL Sigma GE29-0915-96 Protein purification
T4 DNA polymerase Sigma 70099-3 making construct using LIC method
Thermo Scientific Sorvall RC 6 Plus Centrifuge Fisher Scientific 36-101-0816 Centrifuge, highest speed 20,000 rpm
Trizma hydrochloride Sigma T3253-250G making solution
Uranyl Formate Electron Microscopy Sciences 22451 making negative stain solution

References

  1. Whelan, S. P., Barr, J. N., Wertz, G. W. Transcription and replication of nonsegmented negative-strand RNA viruses. Current Topics in Microbiology and Immunology. 283, 61-119 (2004).
  2. Lamb, R. A., Knipe, D. M., Howley, P. M. . Fields virology. , (2013).
  3. Collins, P. L., Fearns, R., Graham, B. S. Respiratory syncytial virus: virology, reverse genetics, and pathogenesis of disease. Current Topics in Microbiology and Immunology. 372, 3-38 (2013).
  4. Fearns, R., Deval, J. New antiviral approaches for respiratory syncytial virus and other mononegaviruses: Inhibiting the RNA polymerase. Antiviral Research. 134, 63-76 (2016).
  5. Grosfeld, H., Hill, M. G., Collins, P. L. RNA replication by respiratory syncytial virus (RSV) is directed by the N, P, and L proteins; transcription also occurs under these conditions but requires RSV superinfection for efficient synthesis of full-length mRNA. Journal of Virology. 69 (9), 5677-5686 (1995).
  6. Pan, J., et al. Structure of the human metapneumovirus polymerase phosphoprotein complex. Nature. 577 (7789), 275-279 (2020).
  7. Horwitz, J. A., Jenni, S., Harrison, S. C., Whelan, S. P. J. Structure of a rabies virus polymerase complex from electron cryo-microscopy. Proceedings of the National Academy of Sciences of the United States of America. 117 (4), 2099-2107 (2020).
  8. Cao, D., et al. Cryo-EM structure of the respiratory syncytial virus RNA polymerase. Nature Communications. 11 (1), 368 (2020).
  9. Abdella, R., Aggarwal, M., Okura, T., Lamb, R. A., He, Y. Structure of a paramyxovirus polymerase complex reveals a unique methyltransferase-CTD conformation. Proceedings of the National Academy of Sciences of the United States of America. 117 (9), 4931-4941 (2020).
  10. Gilman, M. S. A., et al. Structure of the Respiratory Syncytial Virus Polymerase Complex. Cell. 179 (1), 193-204 (2019).
  11. Liang, B., et al. Structure of the L Protein of Vesicular Stomatitis Virus from Electron Cryomicroscopy. Cell. 162 (2), 314-327 (2015).
  12. Jenni, S., et al. Structure of the Vesicular Stomatitis Virus L Protein in Complex with Its Phosphoprotein Cofactor. Cell Reports. 30 (1), 53-60 (2020).
  13. Renner, M., et al. Nucleocapsid assembly in pneumoviruses is regulated by conformational switching of the N protein. Elife. 5, 12627 (2016).
  14. Cox, R. M., Plemper, R. K. Structure and organization of paramyxovirus particles. Current Opinion in Virology. 24, 105-114 (2017).
  15. Desfosses, A., et al. Self-organization of the vesicular stomatitis virus nucleocapsid into a bullet shape. Nature Communications. 4, 1429 (2013).
  16. Green, T. J., et al. Common mechanism for RNA encapsidation by negative-strand RNA viruses. Journal of Virology. 88 (7), 3766-3775 (2014).
  17. Jamin, M., Yabukarski, F. Nonsegmented Negative-Sense RNA Viruses-Structural Data Bring New Insights Into Nucleocapsid Assembly. Advances in Virus Research. 97, 143-185 (2017).
  18. Wan, W., et al. Structure and assembly of the Ebola virus nucleocapsid. Nature. 551 (7680), 394-397 (2017).
  19. Mendes, A., Kuhn, R. J. Alphavirus Nucleocapsid Packaging and Assembly. Viruses. 10 (3), (2018).
  20. Gao, Y., et al. In vitro trackable assembly of RNA-specific nucleocapsids of the respiratory syncytial virus. Journal of Biological Chemistry. 295 (3), 883-895 (2020).
  21. Aslanidis, C., de Jong, P. J. Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Research. 18 (20), 6069-6074 (1990).
  22. Li, C., Evans, R. M. Ligation independent cloning irrespective of restriction site compatibility. Nucleic Acids Research. 25 (20), 4165-4166 (1997).
  23. Hanahan, D. Studies on transformation of Escherichia coli with plasmids. Journal of Molecular Biology. 166 (4), 557-580 (1983).
  24. Green, R., Rogers, E. J. Transformation of chemically competent E. coli. Methods in Enzymology. 529, 329-336 (2013).
  25. De Carlo, S., Harris, J. R. Negative staining and cryo-negative staining of macromolecules and viruses for TEM. Micron. 42 (2), 117-131 (2011).
  26. Ohi, M., Li, Y., Cheng, Y., Walz, T. Negative Staining and Image Classification – Powerful Tools in Modern Electron Microscopy. Biological Procedures Online. 6, 23-34 (2004).
  27. Aebi, U., Pollard, T. D. A glow discharge unit to render electron microscope grids and other surfaces hydrophilic. Journal of Electron Microscopy Technique. 7 (1), 29-33 (1987).
  28. Green, T. J., et al. Access to RNA encapsidated in the nucleocapsid of vesicular stomatitis virus. Journal of Virology. 85 (6), 2714-2722 (2011).
  29. Alvarez Paggi, D., et al. A conformational switch balances viral RNA accessibility and protection in a nucleocapsid ring model. Archives of Biochemistry and Biophysics. 671, 77-86 (2019).
  30. Green, T. J., Zhang, X., Wertz, G. W., Luo, M. Structure of the vesicular stomatitis virus nucleoprotein-RNA complex. Science. 313 (5785), 357-360 (2006).
  31. Tawar, R. G., et al. Crystal structure of a nucleocapsid-like nucleoprotein-RNA complex of respiratory syncytial virus. Science. 326 (5957), 1279-1283 (2009).
  32. Galloux, M., et al. Characterization of a viral phosphoprotein binding site on the surface of the respiratory syncytial nucleoprotein. Journal of Virology. 86 (16), 8375-8387 (2012).
  33. Milles, S., et al. Self-Assembly of Measles Virus Nucleocapsid-like Particles: Kinetics and RNA Sequence Dependence. Angewandte Chemie Interntional Edition English. 55 (32), 9356-9360 (2016).
  34. Desfosses, A., et al. Assembly and cryo-EM structures of RNA-specific measles virus nucleocapsids provide mechanistic insight into paramyxoviral replication. Proceedings of the National Academy of Sciences of the United States of America. 116 (10), 4256-4264 (2019).

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Cite This Article
Gao, Y., Ogilvie, C., Raghavan, A., Von Hoffmann, C., Liang, B. Generation and Assembly of Virus-Specific Nucleocapsids of the Respiratory Syncytial Virus. J. Vis. Exp. (173), e62010, doi:10.3791/62010 (2021).

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