Localization of adenovirus morphogenesis players, together with visualization of assembly intermediates and failed products, favor a model where assembly and packaging occur concurrently at the periphery of the replication center

Abstract

Adenovirus (AdV) morphogenesis is a complex process, many aspects of which remain unclear. In particular, it is not settled where in the nucleus assembly and packaging occur, and whether these processes occur in a sequential or a concerted manner. Here we use immunofluorescence and immunoelectron microscopy (immunoEM) to trace packaging factors and structural proteins at late times post infection by either wildtype virus or a delayed packaging mutant. We show that representatives of all assembly factors are present in the previously recognized peripheral replicative zone, which therefore is the AdV assembly factory. Assembly intermediates and abortive products observed in this region favor a concurrent assembly and packaging model comprising two pathways, one for capsid proteins and another one for core components. Only when both pathways are coupled by correct interaction between packaging proteins and the genome is the viral particle produced. Decoupling generates accumulation of empty capsids and unpackaged cores.

Fig 1. AdV genomes and packaging proteins are found together at the periphery of replication centers.

(A) Cartoon summarizing previous observations on the distribution of AdV DNAs in the replication centers. (B, D, E) Confocal immunofluorescence sections (~0.3 μm thick) showing the localization of the different factors in HEK 293 cells infected with Ad5 wt or Ad5/FC31, as indicated. MOI = 50, 36 hpi. (B) Label for viral DNA (BrdU). The bottom row shows higher magnification views of selected areas from the top row. Scale bars: 20 μm (top row), 10 μm (bottom row). (C) Quantification of BrdU label. The plot depicts the percentage of infected cells (as ascertained by GFP expression) showing BrdU label. (D) Double labeling for BrdU (magenta) and DBP (green) in cells infected with Ad5/FC31. Bars: 10 μm (left panel), 5 μm (right panel). (E) Double labeling for BrdU (magenta) and L1 52/55 kDa (green). Dotted rectangles indicate the areas shown at higher magnification in the bottom row for each virus. Bars: 3 μm. In B and D, dashed white contours indicate the periphery of infected cells (assessed by GFP expression).

Fig 2. Localization of newly synthesized viral DNA in infected cells.

Viral replication centers labeled with anti-BrdU in HEK293 cells infected with Ad5 wt (A-D) or Ad5/FC31 (E-H). (A, E) Low magnification views of replication centers with the DAS (electron-clear) highlighted in green and the PRZ in magenta. (B, F) Zoom of areas highlighted by white rectangles in A and E. (C, G) Details of other PRZs showing BrdU signal on bundles, indicating that they contain viral DNA. (D, H) EOGs produced by Ad5 wt and Ad5/FC31 respectively. Scale bars: 1 μm (A and E); 0.4 μm (B and F); 200 nm (C and G) and 100 nm (D and H). Nucleus (N); cytoplasm (C); DNA bundles (b); nucleoplasm (np). Arrows indicate viral particles with BrdU signal. Unless stated otherwise, all EM images correspond to 48 hpi at MOI = 50. (I) Quantification of BrdU label in FS samples. The number of gold particles (gp) per unit area (μm²) is shown for three different nuclear regions: PRZ, DAS, rest of nucleus (other). Label in control, uninfected cells is also shown.

Fig 3. Localization of protein L1 52/55 kDa by immunoelectron microscopy.

HEK 293 cells infected with Ad5 wt (A-D) or Ad5/FC31 (E-H) labeled for L1 52/55 kDa. (A, E) General view of replication centers. DAS area highlighted in green and PRZ in magenta. Arrows indicate the presence of L1 52/55 kDa in EOGs. (B, F) Viral particles. (C, G) Electron-opaque grains. (D, H) Arch-shaped labels in the nucleoplasm close to the PRZs. Nucleoplasm (np); protein crystal (pc); DNA bundle (b). Scale bars: A and E 200 nm; B and F 100 nm; C-D and G-H 50 nm. (I) Quantification of gold labels for L1 52/55k in the different nuclear regions in the infected cell. SB refers to speckled bodies, introduced later on in the text.

Fig 4. Presence of core protein VII in the putative AdV assembly zone.

Replication centers labeled for protein VII in HEK293 cells infected with Ad5 wt (A-C) or Ad5/FC31 (D-F). Sections treated with DNase before immunolabeling to unmask VII epitopes. (B, E) Zoom of square areas in A, D. Green area: DAS; magenta area: PRZ. White arrows indicate signal in viral particles; black arrows indicate signal in DNA bundles. Nucleoplasm (np); nucleus (N); cytoplasm (C); chromatin (ch); DNA bundle (b); lobes (lo). Scale bars: A and D 1 μm; B, C, E and F 0.2 μm. (G) Quantification of label for protein VII in FS samples.

Fig 5. Presence of capsid protein (fiber) in the putative AdV assembly zone.

HEK 293 cells infected with Ad5 wt (A, B, E) or Ad5/FC31 (C, D, F) labeled for fiber. (A, C) Protein crystals. (B, D) Viral particles. (E, F) Replication centers. Green area: DAS. Magenta area: PRZ. Cytoplasm (C); nucleoplasm (np); chromatin (ch); phagocytic vacuoles (ph); DNA bundles (b). Open and closed arrows indicate signal in electron-opaque grains and DNA bundles respectively. Scale bars: 200 nm. (G) Quantification of label for fiber in FS samples.

Fig 6. AdV assembly site and possible sequence of events.

Selected details from the PRZ region in freeze-substituted HEK293 cells infected with Ad5/FC31 show where and how AdV capsid and core meet. (A) EOGs (arrows) arise from DNA bundles (b). Section labeled for L1 52/55 kDa. (B) A partially formed capsid engulfs a protrusion in a DNA bundle (white circle). (C) Possible sequence of events to assemble a viral particle. White circles indicate the specific detail the text refers to. In the early stage for capsids, capsid caps are assembled in the nucleoplasm (np). These caps contain L1 52/55 kDa, which will act as a Velcro to bind to other L1 52/55 kDa molecules or other packaging elements in the core. In the early stage for core 1 and 2, small condensations appear at the periphery of the DNA bundle (b), also containing L1 52/55 kDa. In the intermediate stage 1, the capsid caps bind to these condensations through L1 52/55kDa, and capsids grow around the coalescing core (intermediate stage 2). In the late stage 1, the capsid is almost complete and the core is still connected to the DNA bundle. Finally (late stage 2), the viral particle is sealed and separated from the bundle. Sections labeled against L1 52/55 kDa or BrdU, as indicated. The scale bars represent 100 nm.

Fig 7. Failed core assembly products and schematics for the proposed AdV assembly pathway.

(A–D) Cells infected with Ad5/FC31 (MOI = 5) and embedded in Epon. (A) Loose speckled bodies (white dotted circles) adjacent to the PRZ (magenta contour) at 48 hpi. The insets show zooms into a speckled body (dotted black square) and viral particles (black square). (B) Compact speckled bodies adjacent to the PRZ at 56 hpi. (C-D) Speckled bodies found at 36 and 56 hpi respectively. (E) A nucleolus in a cell infected with Ad5 wt at 48 hpi is shown for comparison. (F and G) Speckled bodies in freeze-substituted cells labeled for core protein VII, without (F) or with (G) DNase treatment. Nucleus (N); cytoplasm (C); virus particles (vp); lobe (lo); protein crystal (pc); phagocytic vacuoles (ph). Scale bars: A 700 nm; B 1.5 μm; C-E 500 nm; F, G 300 nm. (H) Quantitative comparison of polypeptide VII label in SBs with or without DNase treatment of the sections. (I) Schematics of an infected nucleus summarizing the localization of assembly factors in the AdV-induced structures discussed in this work. Only when interactions between packaging proteins and the viral genome are correctly coordinated, virions are assembled in the PRZ (J). Defective interactions give rise to dead end products: empty capsids and EOGs/SBs.