Research seminars Beyond the Helix: Unprecedented Features of the Filovirus Nucleocapsid Revealed by in situ Cryo-Electron Tomography

Filoviruses, including the Ebola and Marburg viruses, cause hemorrhagic fevers with up to 90% lethality. Antibody treatments have been approved, but thus far, they have been for just one species. Therefore, developing antiviral drugs active against a range of filoviruses that can access immune-privileged sites is a high priority. To meet this objective, we must understand the critical and conserved nucleocapsid structure indispensable for viral replication. Until recently, it was impossible to reveal protein structures directly inside virus-infected cells, except for very thin peripheral parts of cells or budded virus particles. Therefore, we had no structural insights into how the virus replicates and how nucleocapsids assemble in infected cells before budding and release. The viral nucleocapsid is assembled by polymerizing 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 the 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 and the C-terminal domain of this outer layer of NP provide the constant width between intracellular nucleocapsid bundles and likely function 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 among all pathogenic filoviruses and offer targets for broadly effective antivirals.