Intracellular Ebola Virus nucleocapsid assembly revealed by in situ cryo-electron tomography.

Filoviruses, including Ebola and Marburg viruses, cause hemorrhagic fevers with up to 90% lethality. The viral nucleocapsid is assembled by polymerization of the nucleoprotein (NP) along the viral genome, together with the viral proteins VP24 and VP35. We employed cryo-electron tomography of cells transfected with viral proteins and infected with model Ebola virus, to illuminate assembly intermediates as well as a 9[A] map of the complete intracellular assembly. This structure reveals a previously unresolved, third, and outer layer of NP complexed with VP35. The intrinsically-disordered-region together with the C-terminal domain of this outer layer of NP provides the constant-width between intracellular nucleocapsid bundles and likely functions as a flexible tether to the viral matrix protein in virion. A comparison of intracellular nucleocapsid with prior in-virion nucleocapsid structures reveals the nucleocapsid further condenses vertically in-virion. The interfaces responsible for nucleocapsid assembly are highly conserved and offer targets for broadly effective antivirals.