Figure 1.
Characterisation of the course of infection in cultured cells.
(A) Quantitative Western Blot showing the relative amounts of major viral structural proteins NP and VP40 released into cell culture supernatant during the indicated time periods. (B) Correlation of morphology and specific infectivity of MARV particles released into cell culture supernatant during the indicated time periods. Infectivity in each supernatant was determined in a TCID50 assay and normalized for NP amounts in each supernatant to determine the specific infectivity (light grey areas). Relative amounts of filamentous virus (grey bars) and spherical virus (black bars) in each supernatant were determined by EM of thin sections. (C) Quantification of MARV-infected cell morphology over a 4-day infection period. HUH-7 cells were infected, fixed at the indicated time points, embedded in their in situ orientation and cell morphology was determined by EM of thin sections in a systematic random sampling manner. (D) Electron micrograph showing the intact morphology of an infected cell at day 1 p.i.. (E) 6 nm digital slice of a tomogram showing the periphery of an intact cell and the filamentous morphology of the viruses associated with the cell profile. (F) Quantification of morphology of cell-associated MARV particles over a 4-day infection period. Quantification was done as in C. (G) Vesiculated morphology of an infected cell at day 4 p.i.. (H) 8 nm digital slice of a tomogram showing the periphery of a vesiculated cell and the spherical morphology of two viruses and several cell-derived vesicles associated with the cell profile.
Table 1.
Correlation of virus and cell morphology during the time course of MARV infection.
Figure 2.
The different steps of MARV budding and release.
(A-F) Left panels show digital slices of tomograms of MARV-infected cells at 1 day p.i.. Right panels are the corresponding schematic 3D representations of the viral NC (blue) and the cytoplasm and surrounding membrane (yellow). The different steps of MARV budding and release are (A) Intracellular NC in the cytoplasm. (B) Intracellular NC associated on one side along its whole length with the PM of a filopodia-like membrane protrusion. (C) Viral NC with its tip inserted in a budding structure and associated with the PM along its whole length on one side. (D, E) Viral NCs completely inserted into filamentous budding structures. In (E), a bud neck-like membrane shape is seen at the rear of the budding structure. (F) Filamentous virus with a membrane distortion at one end. Note that all NCs have the same length. Different contrast of NCs in individual images results from differences in electron-dense stain access during sample preparation. (G) 8 nm digital slice of a tomogram of a MARV-infected cell at 1 day p.i.. Coloured overlay is the surface representation of viral NC (blue) and 3D surface rendering of surrounding membrane and cytoplasm (yellow). It shows a filamentous budding structure with the viral NC fully inserted into the budding site and several released virions. 3D surface representation is shifted relative to the tomogram slice for improved visualisation. See also Video S1.
Table 2.
3D analysis of virus shape and NC length at different steps of filamentous MARV budding.
Figure 3.
Membrane distortions are found at the rear end of filamentous MARV particles.
MARV infected cells were embedded and sectioned in their in situ orientation and the morphology of filamentous viruses was examined in electron tomograms of 300 nm thick sections. (A, B) Digital slices of tomograms showing typical examples of (A) distorted ends and (B) intact, round ends of filamentous viruses. Scale bars 100 nm. (C) Quantification of the distance of distorted and round virus ends from the nearest cellular membrane. Distances were measured in 3D tomograms and assigned to one of three distance classes >250 nm, 50–250 nm, <50 nm. At a distance <50 nm from the nearest cell membrane, most virus ends are distorted. At a distance >250 nm away from the nearest cell membrane the majority of virus ends is intact and round.
Figure 4.
Release and spherical morphology of MARV particles after prolonged infection.
(A) 8 nm digital slice of a tomogram taken from a MARV-infected cell at 4 days p.i.. Coloured overlay is the surface representation of viral NCs (blue) and 3D surface rendering of cytoplasm and membranes (yellow). Several spherical viruses and a six-shaped particle near the surface of a vesiculated cell and surrounded by cell-derived vesicles and debris are shown. 3D surface representation is shifted relative to the tomogram slice for improved visualisation. See also Video S2. (B) Left panel is a digital slice of a tomogram taken from a MARV-infected cell at 4 days p.i. showing the round shape of a virus and three cross sections of the viral NC. Right panel is the corresponding schematic 3D representation of the viral NC (blue) and the viral membrane (yellow). Note that the 3D reconstruction reveals that the viral NC is kinked but continuous.
Figure 5.
Steps of filamentous virus budding and release.
(A) An individual viral NC in the cytoplasm of an infected cell. (B) The viral NC is delivered in full-length form to the PM, with which it associates along one side for its entire length. (C) Slow initiation of NC envelopment at one end of the NC at the PM. (D) Envelopment proceeds in a fast process along the length of the NC until the nascent virion protrudes from the membrane, remaining attached at only one end. (E) Scission of the virus particle from the PM leaves a local membrane instability at the rear end of the virus particle. For details see Discussion. The model is based on 3D tomographic reconstructions of MARV-infected cell sections; Membrane and cytoplasm are depicted in yellow, NCs are blue. Single and triple arrows depict slow and fast processes, respectively.