In vitro assembly of Ebola virus nucleocapsidlike complex expressed in E. coli

Abstract

Ebola virus (EBOV) harbors an RNA genome encapsidated by nucleoprotein (NP) along with other viral proteins to form a nucleocapsid complex. Previous Cryoeletron tomography and biochemical studies have shown the helical structure of EBOV nucleocapsid at nanometer resolution and the first 450 amino-acid of NP (NPΔ451–739) alone is capable of forming a helical nucleocapsid-like complex (NLC). However, the structural basis for NP-NP interaction and the dynamic procedure of the nucleocapsid assembly is yet poorly understood. In this work, we, by using an E. coli expression system, captured a series of images of NPΔ451–739 conformers at different stages of NLC assembly by negative-stain electron microscopy, which allowed us to picture the dynamic procedure of EBOV nucleocapsid assembly. Along with further biochemical studies, we showed the assembly of NLC is salt-sensitive, and also established an indispensible role of RNA in this process. We propose the diverse modes of NLC elongation might be the key determinants shaping the plasticity of EBOV virions. Our findings provide a new model for characterizing the self-oligomerization of viral nucleoproteins and studying the dynamic assembly process of viral nucleocapsid in vitro.

References

[1] Albertini AA (2006) Crystal structure of the rabies virus nucleoprotein-RNA complex. Science 313:360–363

[2] Alvarez CP (2002) C-type lectins DC-SIGN and L-SIGN mediate cellular entry by Ebola virus in cis and in trans. J Virol 76:6841–6844

[3] Beniac DR (2012) The organisation of Ebola virus reveals a capacity for extensive, modular polyploidy. PLoS One 7:e29608

[4] Bharat TA (2012) Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography. Proc Natl Acad Sci USA 109:4275–4280

[5] Bhella D (2002) Significant differences in nucleocapsid morphology within the Paramyxoviridae. J Gen Virol 83:1831–1839

[6] Booth TF, Rabb MJ, Beniac DR (2013) How do filovirus filaments bend without breaking? Trends Microbiol 21:583–593

[7] Dong S (2015) Insight into the Ebola virus nucleocapsid assembly mechanism: crystal structure of Ebola virus nucleoprotein core domain at 1.8 A resolution. Protein Cell 6:351–362

[8] Dziubanska PJ (2014) The structure of the C-terminal domain of the Zaire ebolavirus nucleoprotein. Acta Crystallogr D Biol Crystallogr 70:2420–2429

[9] Egelman EH (2000) A robust algorithm for the reconstruction of helical filaments using single-particle methods. Ultramicroscopy 85:225–234

[10] Gao GF, Feng Y (2014) On the ground in Sierra Leone. Science 346:666

[11] Gong X (2016) Structural insights into the Niemann-Pick C1 (NPC1)-mediated cholesterol transfer and ebola infection. Cell 165:1467–1478

[12] Green TJ (2006) Structure of the vesicular stomatitis virus nucleoprotein-RNA complex. Science 313:357–360

[13] Huang Y (2002) The assembly of Ebola virus nucleocapsid requires virion-associated proteins 35 and 24 and posttranslational modification of nucleoprotein. Mol Cell 10:307–316

[14] Hunt CL (2011) The Tyro3 receptor kinase Axl enhances macropinocytosis of Zaire ebolavirus. J Virol 85:334–347

[15] Iseni F (1998) Characterization of rabies virus nucleocapsids and recombinant nucleocapsid-like structures. J Gen Virol 79(Pt 12):2909–2919

[16] Kirchdoerfer RN(2015) Assembly of the Ebola virus nucleoprotein from a chaperoned VP35 complex. Cell Rep 12:140–149

[17] Kuhn JH (2010) Proposal for a revised taxonomy of the family Filoviridae: classification, names of taxa and viruses, and virus abbreviations. Arch Virol 155:2083–2103

[18] Leung DW (2015) An intrinsically disordered peptide from Ebola virus VP35 controls viral RNA synthesis by modulating nucleoprotein-RNA interactions. Cell Rep 11:376–389

[19] Ludtke SJ, Baldwin PR, Chiu W (1999) EMAN: semiautomated software for high-resolution single-particle reconstructions. J Struct Biol 128:82–97

[20] Maclellan K (2007) The 24-angstrom structure of respiratory syncytial virus nucleocapsid protein-RNA decameric rings. J Virol 81:9519–9524

[21] Miller EH (2012) Ebola virus entry requires the host-programmol/ led recognition of an intracellular receptor. Embo J 31:1947–1960

[22] Mindell JA, Grigorieff N (2003) Accurate determination of local defocus and specimen tilt in electron microscopy. J Struct Biol 142:334–347

[23] Nanbo A (2010) Ebolavirus is internalized into host cells via macropinocytosis in a viral glycoprotein-dependent manner. PLoS Pathog 6:e1001121

[24] Noda T (2005) Nucleocapsid-like structures of Ebola virus reconstructed using electron tomography. J Vet Med Sci 67:325–328

[25] Noda T (2006) Assembly and budding of Ebolavirus. PLoS Pathog 2:e99

[26] Noda T (2010) Characterization of the Ebola virus nucleoprotein-RNA complex. J Gen Virol 91:1478–1483

[27] Ruigrok RW, Crepin T, Kolakofsky D (2011) Nucleoproteins and nucleocapsids of negative-strand RNA viruses. Curr Opin Microbiol 14:504–510

[28] Shaikh TR (2008) SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs. Nat Protoc 3:1941–1974

[29] Tawar RG (2009) Crystal structure of a nucleocapsid-like nucleoprotein-RNA complex of respiratory syncytial virus. Science 326:1279–1283

[30] Wang H (2015) Crystal structures of human TIM members: Ebolavirus entry-enhancing receptors. Chin Sci Bull. doi:10.1360/ N972015-01255 (in Chinese)

[31] Watanabe S, Noda T, Kawaoka Y (2006) Functional mapping of the nucleoprotein of Ebola virus. J Virol 80:3743–3751

[32] White JM, Schornberg KL (2012) A new player in the puzzle of filovirus entry. Nat Rev Microbiol 10:317–322

[33] Yabukarski F (2014) Structure of Nipah virus unassembled nucleoprotein in complex with its viral chaperone. Nat Struct Mol Biol 21:754–759

[34] Zhou H (2013) Structural perspective on the formation of ribonucleoprotein complex in negative-sense single-stranded RNA viruses. Trends Microbiol 21:475–484

RIGHTS & PERMISSIONS

2016 The Author(s) 2016. This article is published with open access at Springerlink.com and journal.hep.com.cn