The in situ Structural Approach to Reveal the Filovirus Budding Mechanism

Ebola virus (EBOV) and related filoviruses cause severe hemorrhagic fever with high mortality [1]. The nucleocapsid (NC) of EBOV lies at the virion core and contains elements required for replication in host cells. NCs assemble in inclusion bodies (replication factories) before transport to the plasma membrane from which new virions bud. Understanding the structure and conserved principles involved in NC assembly and budding is critical to developing antiviral treatments. Cryo-electron tomography combined with cryo-fluorescence microscopy and focused ion beam (FIB) milling (cryo-FIB-ET) allows direct protein structure determination inside cells [2,3]. where viral proteins are assembled. Using cryo-FIB-ET, we visualized EBOV NC assembly inside viral inclusion bodies in cells expressing VP24, VP35 and NP or in EBOV-infected cells (Fig.1). We have identified various NC intermediates and fully assembled NC inside viral inclusion bodies and revealed their spatial arrangement during their assembly. Using subtomogram averaging, we obtained the first in-cell structures of NC (Fig.2). Surprisingly, in-cell NC has a distinct confirmation from NC previously published in virus particles [4] (in-virion NC), suggesting that NC undergoes conformational changes upon binding to virus matrix protein and incorporation into the virion. Comparisons of in-cell NC and in-virion NC suggest that the C-terminal half of the nucleoprotein (NP), which forms part of the NC by packaging viral genetic material, protrudes from the core NP-RNA helix to form a region of the NC exterior that has the most remarkable structural variability. Our study reveals protein structural arrangements that occur during NC assembly and incorporation into virions and increases our understanding of this critical step in the life cycle of filoviruses and other enveloped viruses. NC assembly in EBOV-infected cells. Cryo-electron tomogram slice of EBOVΔVP30-infected, VP30-expressing Vero cells 24 hours post-infection. Loosely-coiled NP-like structures fill viral inclusion bodies and fully assembled NCs are emerging. Intracellular NC structure and arrangements revealed by cryo-FIB-ET. (A) Our intracellular NC structure obtained HEK293T cells expressing VP24, VP35, and NP. (B) Slice (20 nm-thick) from a representative tomogram showing EBOV NC assembly in cells. (C) Previous cryoEM of NP N-term-RNA complex [5] (green) filling the core of our intracellular NC (grey). (D) Average is mapped back to the 3D volume. (E) A bivariate histogram showing relative angle and distance between intracellular NC particles sampled every 4 nm reveals that intracellular NC frequently forms bundles with a regular center-to-center distance of peak 64 nm.