Fig 1.
Viral entry into target cells is initiated after attachment to the plasma membrane and is accomplished by macropinocytosis (1). During maturation of macropinocytic vesicles into LE/Lys, the viral envelope fuses with the LE/Lys membrane, and the NC is released into the cytosol (2), where it serves as a template for the primary transcription of capped viral mRNAs (3). Translation of viral proteins (4) occurs at free ribosomes, with the exception of GP, which is translated at the rough endoplasmatic reticulum (ER/RER). GP is transported through the classical secretory pathway via the Golgi apparatus to the plasma membrane. The matrix protein VP40 associates with membrane vesicles and is transported to the plasma membrane to form the viral envelope together with GP and cellular lipids (10). Expression of NC proteins (NP, VP35, VP30, VP24, and L) leads to the formation of IBs (5) where secondary transcription (6), genome replication (7), and NC assembly are organized (8). NCs are transported directionally from IBs to budding sites through actin polymerization-driven transport mediated by actin tail formation at one end (9). Envelopment of NCs by membranes containing VP40 and GP occurs at the plasma membrane where budding of filoviruses take place (11). Figure was created with BioRender.com. IB, inclusion body; LE/Lys, late endosomes/lysosomes; RER, rough endoplasmatic reticulum; RNP, ribonucleoprotein complex.
Fig 2.
Structure of Ebola virus nucleocapsids [15].
(A) NPNTD monomer (amino acid 1–450) derived from subtomogram averaging of NPNTD helices (see B) with the aligned crystal structure in cyan and the putative RNA binding site in yellow. An arrow indicates the N-terminal arm region with the N-terminal helix. (B) The left-handed NPNTD helix bound to an RNA chain (yellow). (C) Model of the EBOV NC derived from Cryo-ET and subtomogram averaging. Left: NC structure of EBOV. Subunits of 1 rung are shown in dark or light gray. One subunit is highlighted in pink. Middle: The NPNTD subunits are highlighted in cyan and blue, the structures highlighted in orange, and purple are NPCTD protrusions, 2 VP24 molecules and VP35. The bound RNA is highlighted in yellow. Right: side view of the asymmetric protomer showing 2 molecules of NPNTD (cyan and blue), 2 molecules of VP24 (orange and purple), and unassigned densities most likely corresponding to NPCTD and VP35 (light gray). (D) Structure of recombinant EBOV NC obtained from virus-like particles composed of NP, VP35, VP24, and VP40. Top: longitudinal presentation of the helix; bottom: cross-section of the helix. The boomerang-shaped protrusions are highlighted in purple, and small protrusions are highlighted in orange. The inner layer composed of NPNTD is shown in gray. Figures are modified from Wan and colleagues [15]. Figure was created with BioRender.com. Cryo-ET, cryo-electron tomography; NC, nucleocapsid.
Fig 3.
(A) Current working model of IB organization. Transcription and genome replication occur inside IBs. It is possible that the 2 activities are separated in different subcompartments which, however, remains to be investigated. Possible subcompartmentalization of IBs might result from liquid–liquid phase separation based on RNA and protein concentrations. Regulation of viral transcription is partially based on the phosphorylation status of VP30, which modulates interactions with the polymerase cofactor VP35 and the polymerase L. VP30 phosphorylation is regulated by host cell kinases and phosphatases whose localization and availability might be interdependent. Increasing levels of NC proteins and possibly the recruitment of VP40 and/or host cell factors into IBs is thought to trigger the assembly of NCs. (B) Model of RNPs condensation to NCs in IBs. Electron micrographs from Noda and colleagues [20] show thin-walled helices upon NP expression and thick-walled helices upon coexpression of NP+VP35+VP24. The thin-walled helices detected inside IBs may represent RNPs that are active in transcription or replication or may represent intermediates of assembly. (C) Left picture: Electron micrograph from Noda and colleagues [20] of an IB (left) upon coexpression of EBOV NP, VP35, and VP24 showing cross-sectioned NCLS. Right picture: Electron micrograph from Kolesnikova and colleagues [14] of an IB from a MARV-infected cell with cross-sectioned NCs. Blue arrowheads indicate condensed thick-walled NCs at the periphery of the IB. Figure was created with BioRender.com. IB, inclusion body; NC, nucleocapsid; NCLS, NC-like structure; RNP, ribonucleoprotein complex.
Fig 4.
Transport of nucleocapsids from inclusion bodies to budding sites.
Transport-competent NCs composed of all the NC proteins are formed inside IBs. The involvement of different host cell factors is required for the actin-dependent transport of NCs (gray box). Outside the IBs, actin tails are formed at one end of the NC in the cytosol, which drive their transport. After reaching the plasma membrane, budding of filoviruses occurs mainly at filopodia, in which myosin 10 may support the transport of NCs along parallel actin filaments. Figure was created with BioRender.com. IB, inclusion body; NC, nucleocapsid.