Conceptualizaciones y reflexiones sobre la Acción Colectiva de Alto Riesgo

Doucas et al (1996) have shown that at least two early transcribed viral proteins namely, the E4 ORF3 (llkd protein) and the E1B 55kd protein, are targeted to PML nuclear bodies, which in turn, undergo reorganisation into novel structures which the authors referred to as tracks. These structures had an identical appearance to the thread-like structures which were shown to contain pVID/Vni and the 23kd protease late in infection.

Interestingly, prolonged infection of HeLa cells with HSV-1 results in the localisation of PML, and another nuclear autoantigen SP100, to translucent patches of fine granular

material, and viral capsids, at the nuclear border (Puvion-Dutilleul et al., 1995c). These authors also determined that HSV-1 infection induces the redistribution of at least some of the components of interchromatin granule-associated zones and coiled bodies, in particular U1 snRNA, U2 snRNA and P80-coilin, to translucent patches. The translucent patches are detected only at the late stage of nuclear transformation, and contain a high concentration of capsid proteins (and also viral capsids), which suggested a possible role for these structures in the assembly of capsid proteins into capsid shells. Direct comparisons were made with the adenovirus induced clear amorphous inclusions, which the authors noted did not contain redistributed P80-coilin and snRNAs.

It was thought possible that 23kd protease may play an active role within clear amorphous inclusions in the inactivation of cellular proteins relocated to these intranuclear structures. A SWISSPROT findpatterns database search of all human proteins containing the consensus cleavage sequences for the Ad2 23kd protease ((M,L,I)xGxG and (M,L,I)xGGx, with zero mismatches) revealed that PML, P80-coilin, SP100, fibrillarin and U1 snRNP proteins all had potential cleavage sites for the viral enzyme. For PML, the potential cleavage sites are 679-LWGPG and the C-terminal 869-LAGRG, Likewise, P80-coilin has two potential cleavage sites which are 61-LEGGL and 512-MRGRG. Polyclonal antiserum and monoclonal antibody cell culture supernatant generated against P80-coilin were generously provided by Dr.C. Lyons and Dr.A.I. Lamond from the University of Dundee. Cell culture supernatant containing Mab 5E10, which specifically recognises PML protein (Stuurman et al., 1992) was kindly donated by Professor Roel Van Driel (University of Amsterdam).

The distribution of both PML and P80-coilin within non-infected HeLa cells was examined using the BIO-RAD MRC-600 laser scanning confocal imaging system (figure 3.56). Both proteins were immunolocalised to specific foci within non-infected nuclei, with partial colocalisation observed within most cells examined. This was not surprising as Schul et al

(1996) have described trios of coiled bodies, PML bodies and cleavage bodies (not examined here) which partially overlap within nucleoplasmic domains.

The distribution of both proteins late in infection (28 h.p.i) was markedly different (figure 3.57), with the track-like staining patterns of PML described by Doucas et al (1996) easily identified. P80-coilin was likewise shown to be redistributed, but was not shown to colocalise with PML.

Figure 3.56: Immunofluorescent staining of PML and P80-coilin within non-infected

HeLa cells. Part A: BIO-RAD MRC-600 laser scanning confocal image of PML (green) and P80-coilin (red) partial colocalisation within non-infected cells. Part B: Immunolocalisation of PML (top right) and P80- coilin (bottom right) using the Nikon-microphot light microscope. Mab 5E10 (anti-PML) and polyclonal antiserum 204/4 (anti-P80-coilin) were immunodetected using FITC and Texas-red conjugated anti-mouse and anti-rabbit IgG respectively (section 2.8.2). Both parts xlOO magnification (oil immersion).

Figure 3.57: The distribution of PML and P80-coilin within Ad2 infected HeLa cells (28 h.p.i). BIO-RAD MRC-600 laser scanning confocal image of PML (part A) and P80-coilin (part B). The

merge of both images is shown in part C with PML showing as red and P80-coilin as green (channel leads reversed). The same antibody solutions were used as outlined in figure 3.56. Viewed at xlOO magnification (oil immersion).

Surprisingly, PML and pVIII were also found to be differentially localised within infected cell nuclei at 28 h.p.i (figure 3.58). Puvion-Dutilleul et al (1995b) detected PML within clear amorphous inclusions at 17 h.p.i, and it may be that PML inactivation during the subsequent later phases of infection has resulted in the protein (or cleaved products of the protein) being relocated from these structures.

Figure 3.58: Immunolocalisation of PML and pVIII within Ad2 infected HeLa cells (28

h.p.i). BIO-RAD MRC-600 laser scanning confocal image of PML (part A) and pVIII (part B). The merge of both images is shown in part C with PML shown as green and pVIII as red. Viewed at xlOO magnification (oil immersion).

Figure 3.59: Immunolocalisation of the 23kd protease and P80-coilin within Ad2 infected HeLa cells (28 h.p.i). BIO-RAD MRC-600 laser scanning confocal image of the 23kd protease (detected using OA10b3)(part A) and P80-coilin (part B). The merge of both images is shown in part C with the 23kd protease shown as green and P80-coilin as red. Viewed at xlOO magnification (oil immersion).

These authors also noted that P80-coilin was not localised to the clear amorphous inclusions during adenovirus infection. However, some colocalisation of the 23kd protease and P80- coilin was detected in this study (figure 3.59). Colocalisation appeared to occur at specific foci which contain the viral protease. It is possible that both proteins are localised to different intranuclear structures, which may overlap during the late stages of nuclear transformation. Rebelo et al (1996) have shown that coiled bodies are disassembled during adenovirus infection into microfoci, which they have determined using electron microscopy to be electron dense microbodies which contain both P80-coilin and fibrillarin. It is unclear whether these dense microbodies are the same electron dense nuclear structures which have been found in this study to be in close proximity to the clear amorphous inclusions at the nuclear periphery (figure 3.55). These electron dense nuclear bodies are discussed in more detail in section 3.3.6.

PML and P80-coilin were not detected in 4% formaldehyde or 2% paraformaldehyde/0.05% glutaraldehyde fixed (10 minutes) non-infected or infected HeLa cells. The post-embedding technique for immunocytochemistry which was used throughout this study has also been found to be unsuitable for immunodetection of P80-coilin in other studies (Dr.Carol Lyons, University of Dundee, pers.comm.). The pre-embedding technique, with immunolabelling of P80-coilin prior to infiltration of hydrophilic acrylic resins such as Lowicryl K4M, was found to be more suitable. Likewise, Stuurman et al (1992) used the pre-embedding technique to determine the intranuclear distribution of PML within permeabilised T24 and HeLa cells.