Figure 1.
Mimivirus membrane organization and membrane assembly.
A. 0.8 nm slice from STEM tomography of a mature intracellular Mimivirus particle. B. Magnified representation of the region delineated in (A). The layers comprising the Mimivirus virion are: 1: DNA core; 2: Core wall; 3: Inner membrane; 4: A layer previously proposed to represent an outer membrane [27]; 5: Inner capsid shell; 6: Outer capsid shell; 7: Fibers (Movie S1). C. Density plot of the various layers along the line drawn in (B). Digits correspond to those depicted in B. D, E. Confocal images of a 0.5 µm z-section (D) and a projection composed of 11 0.5 µm z-sections (E) showing the Mimivirus factory in the cytoplasm of an 8 hours PI cell revealing viral DNA (DAPI, blue) surrounded by assembling viruses (anti-fibril antibody, red). F. TEM of a factory (VF) in an 8-hour PI cell showing icosahedral capsids at the factory periphery. G. Magnification of the delineated region in (F), depicting viral assembly zones overlying the TEM image. Three distinct zones are proposed: viral replication, membrane assembly, and capsid assembly zones. H, I. 7.5 nm digital slice derived from STEM tomography of 280 nm-thick section (H) and 3-dimensional surface rendering (I) reveal that the membrane assembly region consists of an elaborate membrane network (Movie S2). Scale bars: 200 nm in A, H; 20 nm in B; 5 µm in D, E; 2 µm in F and 500 nm in G.
Figure 2.
Cisternae and vesicles apposing viral factories at the onset of capsid assembly.
A. 10 nm digital slice derived from a 280 nm-thick STEM tomogram of a 7.5-hour PI viral factory. The slice reveal cisternae (arrows) and clusters of ∼70 nm vesicles (arrowheads) at close vicinity to the viral factory (VF). B. Magnification of the delineated regions in (A). C. A different digital slice from the STEM tomogram shown in (A), with 3-dimensional surface rendering of vesicles; numbers correspond to the regions delineated in (A) and (B). The insets depict magnified 3-dimensional surface rendering of the vesicles from region 2 at two different angles, demonstrating the spherical structure of the vesicles (Movie S3). D. 10 nm digital slice from a different 280 nm-thick STEM tomogram of a 7.5-hour PI viral factory, showing membrane structures surrounded by multiple vesicles near viral factories (Movie S4). The inset demonstrates that membrane structures apposing the factories are studded with ribosomes (black arrows). E, F. Two STEM tomogram slices of an 8-hour PI viral factory that are derived from serial sections and are located 480 nm apart. These tomograms reveal that as Mimivirus progeny are assembled, host cisternae are excluded from the membrane assembly zone (Movies S5, S6). Scale bars: 200 nm in A, D; 100 nm in B; 250 nm in E.
Figure 3.
Mimivirus infection induces synthesis of host ER.
A. polyphaga cells were infected at 10 MOI (multiplicity of infection), fixed at the indicated PI time points, and stained with phalloidin-488 actin probe (green) to trace cell border and DAPI (blue). A. Cells stained with mouse anti-protein disulfide isomerase (red), a soluble protein residing in the ER lumen. B. Cells stained with KDEL retention peptide that specifically labels RER membrane proteins. In both staining experiments, ‘*’ indicate non-infected cells, revealing that Mimivirus infection results in a massive redistribution of RER markers. N: nucleous; VF: viral factory. Due to the large amount of dsDNA within VFs relative to that present in cell nuclei, the nuclei are not visible at DAPI exposure levels required to detect VFs. Scale bars: 15 µm in A; 5 µm in B.
Figure 4.
Multivesicular bodies and open sheets in the membrane assembly zone.
A. 10 nm digital slice derived from a 280 nm STEM tomogram of an 8-hour PI viral factory (VF). B, C. Two magnified 10 nm slices of the region delineated in A that are 20 nm apart in the z-axis. D, E. Top and side views, respectively, of a 3-dimensional surface rendering representation derived from the tomogram shown in A–C, highlighting the multi-vesicle nature of the body and the vesicular connectivity within these bodies. F–H. 3-D surface rendering from a STEM tomogram of a different 8-hour PI VF, revealing a cluster of open membrane sheets (arrowheads in F) connected to membrane tubules (arrowheads and arrow in g), and to 70 nm vesicles (arrowheads and arrows in H). See Movies S7, S8. Scale bars: 200 nm in A; 100 nm in B–H.
Figure 5.
Assembly of icosahedral capsids on top of membrane sheets.
A, B. 10 nm digital slices 40 nm apart derived from 320 nm STEM tomogram of 8-hour PI VFs, revealing formation of angular structures by a layer lying on top of open membrane sheets (arrow) that are surrounded by vesicles (arrowheads). C, D. Two angles of 3-dimensional surface rendering presentations derived from panels A and B, showing an early assembling capsid. Membrane sheets and vesicles (arrowheads) are depicted in blue and newly generated angular structures are indicated in yellow (arrows). E, F. Two 10 nm digital slices from a later assembly stage, revealing the progression of angular structures (arrows) into truncated icosahedral morphologies and open membrane sheets still connected to vesicles (arrowheads). G, H. Two different angles of 3-dimensional surface rendering presentations of an assembling icosahedral capsid (yellow; arrows) on top of a membrane layer (blue; arrowheads). The corresponding movies are S10 and S11 for A–D, and Movie S12 for E–H. Scale bars: 100 nm.
Figure 6.
Mimivirus L425 protein acts as an icosahedral capsid scaffolding protein.
A–D. 8-hours PI cells were cryo-preserved and exposed to rabbit anti-L425 antibodies, followed by exposure to gold-conjugated goat anti-rabbit antibody, revealing that pre-assembled capsids (arrows) as well as fully assembled icosahedral capsids (arrowheads) include L425. E–G. Infected cells processed by the immuno-TEM Tokuyasu method. As is the case for cryo-preserved specimens, anti-L425 antibodies specifically label the external layer of the open sheet membrane. Scale bars: 500 nm in A, D and E; 200 nm in B–C; 100 nm in F–G.
Figure 7.
Trimming of open-sheet membrane surplus.
A, B. Two 10 nm digital slices derived from a 320 nm-thick STEM tomogram from the periphery of an 8-hour PI Mimivirus VF, revealing that the inner viral membrane underlying an icosahedral capsid layer (arrowheads) is connected to external membrane sheets (arrows). C. 3-D surface rendering view of icosahedral capsids (arrowheads; yellow) and inner membrane (blue) that are still connected to external open membrane sheets (arrows; blue). D, E. 10 nm digital slice (D) and a 3-dimensional surface rendering view (E) of fully assembled Mimivirus virions in which excess external membrane is trimmed (arrow in E), leaving a ∼20 nm DNA entry portal. See Movies S13, S14. Scale bars: 100 nm.
Figure 8.
Model of Mimivirus membrane and capsid assembly.
A. Host cisternae, from which ∼70 nm vesicles bud out, are recruited to early (7.5-hour PI) viral factories. B. Zoom-in of multiple ∼70 nm vesicles reaching membrane assembly zone. C. Vesicles fuse into multivesicular bodies. D. Multivesicular bodies generated at 8-hour PI rupture to form large open membrane sheets that act as precursors for the inner Mimivirus membrane. The open sheet is expanding by fusing with additional incoming vesicles. E. An icosahedral vertex is generated on top of the open membrane sheets through the assembly of structural Mimivirus capsid proteins in a process involving the L425 protein. F. Upon assembly of the capsid, the inner membrane layer is shaped into icosahedral morphology. G, H. Membrane overhangs consisting of the open membrane sheets are suggested to prevent premature closure of icosahedral capsids, thus enabling formation of a DNA-encapsidating portal [28]. I. As multiple Mimivirus capsid progeny are generated, large host cisternae that act as viral lipid reservoir are excluded from the membrane assembly zone, thus highlighting the need for small, ∼70 nm vesicles capable of reaching this zone, to enable continuous viral assembly. The model underscores the notion that Mimivirus factories represent ‘production lines’ where all stages of viral generation occur simultaneously. Gray: viral DNA; Blue: membrane network; Yellow: capsids.