The eukaryotic cell nucleus performs a series of tasks including replication and packaging of DNA and transcription, splicing, polyadenylation, and transport of RNA. The nucleus is organised into a series of structural domains of which the nucleolus is best characterised. Much of the nuclear interior is occupied by chromatin (in various degrees of condensation) and interchromatin regions that contain several characteristic structures, including perichromatin fibrils and granules, interchromatin granules, and other nuclear bodies including coiled bodies (Brasch and Ochs, 1992; Chaly and Chen, 1993; Puvion-Dutilleul and Puvion, 1995; Bridge and Pettersson, 1995). In this study, mock infected cells, and non infected cells, were examined to form a basis for comparison with nuclei of infected cells. By conventional EM, the nuclei of mock infected HeLa cells exhibited few distinctive features (figures 3.35 and 3.36). The nucleoplasm was quite homogenous in appearance with condensed chromatin, nucleoli and nuclear bodies detected. It was beyond the scope of this study to accurately identify specific structures within non-infected HeLa cell nuclei (for instance coiled bodies), however, based on comparisons with micrographs presented in publications by Brasch and Ochs (1992) and Chaly and Chen (1993), it appears likely that
Figure 3.35: Electron micrograph of a mock-infected HeLa cell. Mock-infected HeLa cells were
fixed with 2% paraformaldehyde/ 0.05% glutaraldehyde (pH 7.4) for one hour then dehydrated and embedded in epon as described in section 2.8. Thin sections were contrasted with uranyl acetate/lead citrate then viewed at 80kv. The nucleolus (Nu), regions containing condensed chromatin (CC), nuclear membrane (NM), cytoplasm (C), and mitochondria (M) are easily identified. Automatic exposure at x5700 magnification.
Figure 3.36: Intranuclear domains of the non-infected HeLa cell. Higher magnification (x25000) view of the nucleus of a HeLa cell from the same thin-section as that shown in figure 3.35. Nucleoli (Nu), condensed chromatin (CC), and possible nuclear bodies (Nb). Automatic exposure.
many of the domains shown in figures 3.35 and 3.36 are genuine and are not artefacts of fixation methods, or dehydration and/or embedding in hydrophobic resin.
Nuclei of adenovirus infected cells progressively accumulate new induced structures as a consequence of viral replication. Due to the low multiplicity of infection (10 p.f.u/cell) used during the course of this study, some of these structures were detected, and are highlighted in figures 3.37 and 3.38.The earliest ultrastructural changes detected are small, irregularly shaped masses of thin fibrils (Puvion-Dutilleul and Puvion, 1995) that rapidly increase in size and become pleomorphic, appearing as crescents, rings and spheres (figure 3.37). These fibrillar inclusions contain DBP-ssDNA (ssDNA accumulation sites are discussed in more detail in section 3.3.4) and are present in the fibrillogranular network of the peripheral replicative zone, which appears to be the main site of viral replication and transcription. At late stages of nuclear transformation, DBP-ssDNA accumulation sites are located at the periphery of the viral genome storage sites (which are frequently associated with crystalline arrays of virus particles), and become smaller and more homogenous in shape, appearing as globular inclusions rather than crescents and rings (figure 3.38). Viral genome storage sites are typically detected within nuclei after 20 h.p.i (Bridge and Pettersson, 1995), and it has been suggested that DNA from these compartments may be incorporated into virions. Structures which are thought to be viral genome storage sites (VGSS) are highlighted in figures 3.37 and 3.38, although it should be noted that the identification of these structures is based only on comparisons with data presented by Chaly and Chen (1993) and Puvion- Dutilleul and Puvion (1995). However, a viral genome storage site identified by Puvion- Dutilleul et al (1995b) is shown in figure 3.53 (page 163).
Clusters of interchromatin granules are more discernible as infection progresses (figure 3.37) and have been shown to contain snRNP proteins, regardless of the stage of infection (Puvion-Dutilleul et al., 1994). Electron microscopic studies of non-infected HeLa cells (Puvion-Dutilleul et al., 1994; Rebelo et al., 1996) have shown that snRNPs are present in clusters of interchromatin granules, interchromatin granule-associated zones, coiled bodies and perichromatin fibrils. It was not possible to accurately identify these structures in this study (figures 3.35 and 3.36), although in late-phase adenovirus infected cells the interchromatin granules and less commonly observed compact rings (not shown) were easily identified after comparing data presented here, and that provided by Chaly and Chen (1993)
Figure 3.37: The nucleus of an adenovirus infected cell (I). Adenovirus infected HeLa cells (28
h.p.i) were fixed with 4% formaldehyde (pH 7.4) for 10 minutes then dehydrated, embedded, and contrasted as described in section 2.8. Thin-sections were viewed at 80kv. Residual nucleolus (rNu), ssDNA-accumulation sites (fibrillar inclusions; FI), interchromatin granules (IG),viral genome storage site (possible)(VGSS), clear amorphous inclusions (CAI), condensed chromatin (CC), and virions (V). Automatic exposure at x9100 magnification.
Figure 3.38: The nucleus of an adenovirus infected cell (II). Ad2 infected HeLa cells (28 h.p.i),
were fixed with 2% paraformaldehyde/ 0.05% glutaraldehyde (pH 7.4) for one hour then dehydrated, embedded, and contrasted as outlined in section 2.8. Crystalline arrays of virions (V), randomly distributed virions (rV), condensed chromatin (CC), viral genome storage site (possible)(VGSS), and fibrillar inclusions (FI), viewed at 80kv, automatic exposure at x7100 magnification.
and Puvion-Dutilleul et al (1994). The latter authors have detected spliceosome components, viral RNA and poly(A)+ RNA within the fibrillogranular network and clusters of interchromatin granules and suggest that these could be the sites of active splicing and/or post-splicing events. Pulse-chase labelling studies undertaken by the same authors (Puvion- Dutilleul et al., 1992 and 1994) have also indicated that clusters of interchromatin granules might contribute to some sorting of viral RNA molecules before their transport to the cytoplasm.
During the course of an adenovirus infection nucleolar proteins (B23, NOR90, Poll, and NuFl; Bridge and Pettersson, 1995) and nucleolus-associated proteins such as fibrillarin (Puvion-Dutilleul and Christensen, 1993) and P80-coilin (Rebelo et al., 1996) are redistributed, some of which are later detected within and/or near ssDNA accumulation sites. Data is consistent with idea that adenovirus infection disrupts the organisation of the nucleolus (figure 3.37), with viral replication being associated with pseudonucleoli.
Many of the cellular and virus-induced structures emphasised within the micrographs presented so far, have been tentatively compared with published data from studies which have been primarily focused on the elucidation of structure-function relationships of specific cellular compartments involved in the replicative cycle of the virus. Comparisons are made difficult for several reasons. Firstly, other studies have described the formation of several types of nucleoplasmic inclusions that differ in size, morphology, and in some instances, whether they incorporated [3H]uridine, [3H] thymidine, or neither. Since these studies used different cell types, Ad strains, stages of infection (most studies have focused on events occurring from 1 to 20 h.p.i), and inclusion body nomenclatures, it is difficult to compare the results directly.
Comparisons were made more difficult when different methods of fixation (in particular if cryo-fixation was used instead of chemical fixation), or different types of embedding media were utilised (in most studies hydrophilic acrylic resins or hydrophobic epoxy resins were used, although in a few cases cryo-electron and embedment-free section electron microscopy techniques were employed), and whether or not thin sections were contrasted, or post-fixed with osmium tetroxide. Even during the course of this study there were observed differences in the apparent structure of clear amorphous inclusions (figure 3.37), which appeared to be
dependant on the chemical fixative used, and/or the duration time of fixation. This is discussed in detail below.