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

Abstract

Filoviruses, including the 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 Å 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 the virion. A comparison of intracellular nucleocapsids with prior in-virion nucleocapsid structures reveals that the nucleocapsid further condenses vertically in the virion. The interfaces responsible for nucleocapsid assembly are highly conserved and offer targets for broadly effective antivirals.

Keywords: Ebola virus; Mononegavirales; antiviral; correlative light and electron microscopy; cryo-electron tomography; filovirus; focused-ion beam milling; immune suppressor; in situ structural biology; integrative modeling; nucleocapsid; nucleoprotein; phosphoprotein; subtomogram averaging; virus assembly; virus pathogenesis; virus structure.