Structure and assembly of the Ebola virus nucleocapsid

Abstract

Ebola and Marburg viruses are filoviruses: filamentous, enveloped viruses that cause hemorrhagic fever1. Filoviruses are within the order Mononegavirales2 which also includes rabies virus, measles virus, and respiratory syncytial virus. Mononegaviruses have non-segmented, single-stranded negative-sense RNA genomes that are encapsidated by nucleoprotein (NP) and other viral proteins to form a helical nucleocapsid (NC). NC acts as a scaffold for virus assembly and as a template for genome transcription and replication. Insights into NP-NP interactions have been derived from structural studies of oligomerized, RNA-encapsidating NP3–6 and cryo-electron microscopy (cryo-EM) of NC7–12 or NC-like structures11–13. There have been no high-resolution reconstructions of complete mononegavirus NCs. Here, we have applied cryo-electron tomography and subtomogram averaging to determine the structure of Ebola virus NC within intact viruses and recombinant NC-like assemblies. These structures reveal the identity and arrangement of the NC components, and suggest that the formation of an extended alpha-helix from the disordered C-terminal region of NP-core links NP oligomerization, NC condensation, RNA encapsidation, and accessory protein recruitment.

Electron Cryo-microscopy Structure of Ebola Virus Nucleoprotein Reveals a Mechanism for Nucleocapsid-like Assembly

“…Wan and co-workers recently described an eNP helical assembly using a NC-like complex with a construct corresponding to eNP-3 in our study (Wan et al, 2017) (Figure S6). In that structure, all eNPs are arranged in the same orientation to form a helical structure.…”

“…In our structure, there are significant differences with respect to asymmetric unit composition and tube diameters. Binding of ssRNA can potentially drive incorporation of helix α23 into α22 helix, forming an extended helix in the previous structure (Wan et al, 2017). Helical extension can then induce additional conformational changes that lead to clamping of the RNA to the binding pocket (Figure 5C and Figure S6).…”

“…Lower resolution structures were obtained with RNA, where RNA appears to bind to the outer face of the helical NC-like assembly (Wan et al, 2017). This observation would be similar to the NC-like structures of paramxyoviruses showing ssRNA binding to the exterior face (Tawar et al, 2009), which can provide access for the viral polymerase during viral RNA synthesis.…”

“…Nucleocapsid-like assemblies from VLPs were generated by Wan et al in the presence of eVP24 and eVP35, but only eVP24 was seen in the density map (Wan et al, 2017). Although eVP35 could not be docked into the density, eVP24 was assigned to bind eNPs asymmetrically into two distinct sites on eNP.…”

“…The impacts of these structural observations were tested in biochemical and functional studies, including ebola virus (EBOV) minigenome (MG) assay that reports on the impact of viral RNA synthesis. Additionally, we compare our structural observations with the recently solved eNP N-terminus (residues 1–450) assembly at 6.6 Å and virus-like particle (VLP) structure of at sub-nanometer resolution by electron cryo-tomography (Wan et al, 2017). Taken together, these studies offer a framework towards understanding Ebola NC architecture and remodeling of the NC-assembly during viral RNA replication.…”