_Archives
Virology
J Arch Virol (1992) 123:193 207
© Springer-Verlag 1992 Printed in Austria
Human cytomegalovirus nuclear and cytoplasmic dense bodies B. Severi 1, M. P. Landini 2, G. Cenaechi ~, N. Zini 3, and N. M. Maraldi 3 1Istituto di Microscopia Elettronica Clinica and 2 Istituto di Microbiologia, UniversitA di Bologna, and 3Isfituto di Citomorfologia Normale e Patotogica del C.N.R., Istituto Rizzoli, Bologna, Italy Accepted July 8, 1991
Summary. One of the characteristic features of cytomegalovirus (CMV) replication is the formation of cytoplasmic dense bodies. Recent findings revealed similar structures also in the nuclei of CMV-infected cells. By transmission electron microscopy, immuno electronmicroscopy, and cytochemistry, we have studied the morphogenetic steps and macromolecular composition of both structures. Our results show that both structures contain DNA, RNA and viral antigenic proteins. Nuclear dense bodies are probably an expression of a stimulated cellular metabolism, while cytoplasmic dense bodies may represent the site where surplus cellular and viral molecules are stored before being eliminated. Introduction During the late phases of human cytomegalovirus (CMV) replication, electrondense structures varying in size, antigenically related but morphologically distinct from virions, are formed in the cytoplasm of infected cells. These structures are called cytoplasmic dense bodies (CDB) and represent one of the characteristic features of CMV replication [-8, 23, 24, 27]. Data in the literature suggest that CDB do not contain DNA and have surface antigens in common with virions [8, 10, 27]. Recently we determined the antigenic correlation between virions and CDB and found that CDB contain several host cell proteins in addition to some viral proteins (unpubl. data). These results suggest that CDB represent the final step of a cellular mechanism by which the cell eliminates macromolecules that have been overproduced. In fact, it is well known that infection of human fibroblasts by human CMV dramatically alters the mechanisms responsible for gene expression, leading to an increase in host cell DNA, RNA and protein syntheses [-12, 26, 28]. This overproduction of cellular material resembles ex novo synthesis of virus-specific components
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and gives rise to an abundance of viral and cellular macromolecules to be segregated or discharged by the cell. Furthermore, recent findings disclosed structures similar to CDB also in the nuclei of CMV infected cells [163. These organelles have been called nuclear dense bodies (NDB) to distinguish them from CDB. Their size, submicroscopic features, and localization resemble dense bodies (DB) or nuclear bodies (NB), widely described in the nuclei of various cell systems, in both normal and pathological conditions, including viral diseases [6]. The present study aimed to define the morphogenetic steps of N D B and CDB formation at different phases of the C M V replication cycle and to determine their macromolecular composition. In particular we tested for the presence of the most a b u n d a n t CMV-structural component of 65 k D a (p65) that, in a previous paper, we localized by immunoelectronmicroscopy on the CDB and N D B matrix [16], and the presence of D N A and R N A , by cytochemical methods.
Material and methods Viruses and cell cultures The Towne strain of CMV (originally obtained through the courtesy of M. Stinski, University of Iowa, USA) was used in our experiments. Human embryo fibroblasts were obtained from the Istituto Zooprofilattico, Brescia, Italy and were infected at a m.o.i, of 0.5 pfu/cells. Infected cells were used at different times after infection (from 1 to 10 days p.i.). For the immunoreaction with p65, a monoclonal antibody against CMV late nuclear protein (N. 9220, Biotech Research Labs., Inc., Rockville, MD) was employed.
Transmission electron microscopy Human embryo fibroblasts were processed for TEM at 24, 48, 96 h and 4 and 6 days after CMV infection. Each sample was fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, postfixed in 1% osmium tetroxide in veronal acetate buffer, dehydrated in graded alcohols and embedded in Epon or LR White resins. When required, thin sections were counterstained with uranyt acetate and/or lead citrate. Osmium postfixation was omitted for the samples to be processed for cytochemistry and immunoelectronmicroscopy.
Cytochemistry Regressive EDTA staining was carried out according to the procedure of Bernhard [43. Briefly, thin sections of Epon embedded infected fibroblasts were floated for 1 rain on 5% aqueous uranyl acetate at room temperature, washed with distilled water and treated with 0.2 M EDTA for 60 min. They were washed again with distilled water and contrasted for 1 min on standard lead citrate at room temperature. Controls were not treated with the EDTA solution. The HAPTA technique Epon embedded thin sections were hydrolyzed with a solution of HC1 5 N for 45 min, washed in distilled water and processed with a 1% phosphotungstic acid (PTA) solution in 80% ethanol, pH 3.5, for 2rain at 20 °C and again rinsed in distilled water. Controls were obtained omitting the HC1 step [13].
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HC1 extraction Epon embedded thin sections were hydrolyzed with a HC1 0.5 N solution for 25-45 min and washed in distilled water [1, 17, 19]. Control sections were incubated in distilled water adjusted to pH 2.0 for the same time and temperatures instead of the HC1 treatment. The DNase and RNase-gold techniques A monodisperse colloidal sol composed of gold particles with an average size of 15 nm was prepared according to Frens [11]. To a boiling aqueous solution of 100ml of 0.01% tetrachloroauric acid (Merck, Darmstadt, Federal Republic of Germany) were added 4 ml of a 1% aqueous solution of sodium citrate and the mixture allowed to boil for 5 rain. The solution was cooled to room temperature and adjusted to pH 9.0 for RNase and to pH 6 for DNase with 0.2 M KzCO 3. The enzyme-gold complexes were prepared essentially according to Bendayan [3]. The concentrations used were 0.0056 mg/ml for RNase A (type XII-A Sigma, St. Louis, MO) and 0.05 mg/ml for DNase I (type DN.EP, Sigma). Both complexes were centrifuged for 30 rain at 33.000 g at 4 °C. The sediments obtained from an 8 ml suspension were diluted with 0.150ml of 0.02M PBS (pH7.5 for RNase A and pH6.0 for DNase I) containing 0.5 mg/ml polyethylene glycol. Cytochemical labeling For RNase A incubation, the complex was generally used diluted up to 1 : 20 with PBS containing 0.5 mg/ml of polyethylene glycol and for DNase I up to t : 200. The grids were floated for 5 rain on a drop of PBS and incubated on a drop of the enzyme-colloidal gold complexes for 45 rain at room temperature. Following incubation, the sections were thoroughly washed with PBS, rinsed with distilled water, dried and stained with aqueous solutions of uranyl acetate and lead citrate. The specificity of the labeling was tested by the following control experiments. For RNase A: (a) incubation with enzyme-gold complex at non-optimal values of pH; (b) incubation with non-stabilized gold suspensions; (c) incubation with albumin-gold complex; (d) incubation with enzyme-gold complex to which the appropriate substrate was added, i.e. RNA 1 rag/rot (type IV, from calf liver, Sigma, St. Louis, MO); and (e) incubation with RNase inhibitors, like heparin, 0.5 mg/ml, or polyvinyl sulphate, 0.1 mg/ml. For DNase: (a) incubation with DNase-gold complex to which the appropriate substrate, DNA type I (from calf thymus, Sigma, St. Louis, MO) 1 mg/ml was added; (b) incubation with nonstabilized gold suspension; (c) incubation with an enzyme-gold complex prepared with DNase inactivated by heating at 100 °C for 10 rain.
Immunoelectronmicroscopy Post-embedding immunolabeling of infected fibroblasts was done by the protein-gold technique, according to Roth et al. [22]. For more details, see our previous paper on p65-69 and p28 intracetlular and intraviral localization [16].
Results
Conventional electron microscopy and p65 immunodetection A t 24 h p.i., cells revealed a clear i m m u n o g o l d positivity for p65 on occasional nuclear r o u n d i s h electrondense structures m a i n l y localized in c o n t a c t with hyp e r t r o p h i c nucleoli, often as if b u d d i n g f r o m t h e m (Fig. 1). In a previous p a p e r [16] we indicated these structures as nuclear dense bodies (NDB).
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l,ig. 1. p65 inmmnotabcting. 24 h p.i. Two nuclear dense masses positive lbr p65 (arrows), Onc sccms to be in the process of budding from the nucleolus, the second appears slightly separated from it. Bar: 0.3 gm Fig. 2. p65 immunolabeling. 96 h p.i. Three p65 positive NDB localized along the contours of the skein inclusion. Bar: 0.6 tam
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Between 48 and 96 h p.i., CDB were observed at different levels of organization and decorated with gold particles. Positive NDB, although few, were more numerous; they could be sparsely distributed in the nucleoplasm, in contact with nucleoli, or immersed in a slightly electrondense granular material which later turned into ringshaped or reticular structures filled with developing nucleocapsids ("skein"). Only some of the infected cells presented these NDB, but several of them could be observed inside the same nucleus. They did not always show identical morphological features: they could be quite similar to conventional CDB, or showed a looser texture and less regular contours. At 96 h p.i., NDB were slightly increased in number and were either sparse in the nucleoplasm or located in the skein in a circular fashion. The positivity towards p65 was always evident (Fig. 2). In the cytoplasm many lysosomes, mainly of the multivesicular body (MVB)-type were seen, while Golgi apparatuses were numerous and hypertrohpic. Many CDB were often enveloped by Golgi-derived vacuoles or saccules, or began to fuse with each other and with viral particles and MVB (Fig. 3). The same degree of positivity for p65 seen in NDB was also revealed in CDB (Fig. 4). Later on (4-6 days p.i.), both these viral-related cellular changes and the gold labeling were enhanced. Numerous large "black holes" [24], derived from the fusion between viral particles, CDB and MVB, were also evident and positive for p65 (Fig. 5). CDB rich in gold particles were often seen close to, or in contact with, nuclear membranes, generally in spatial relationship with the nuclear pores, with which they seemed to be connected by a thin thread of faint material. At tirnes, gold particles were seen to connect CDB and nuclear pores (Fig. 6). NDB were always found isolated or arranged in a circle on the skein inclusion which appeared filled with partially or fully organized capsids. At each time p.i., mock infected cells failed to show any specific gold labeling when p65 antibody was omitted. Anti p65 antibody was also assayed on uninfected human embryo fibroblasts both by immunofluorescence and immunoelectronmicroscopy. The reaction obtained with both techniques was negative (data not shown).
Cytochemistt3, Cytochemical staining and gold-labeling were carried out on CMV-infected cells at 5 days p.i. The results are summarized in Tables 1 and 2. EDTA technique Introduced by Bernhard [4], this technique is considered a regressive staining procedure in which the uranyl stain fixed on deoxyribonucleoproteins is bleached out by an EDTA solution. On the contrary, structures containing ribonucleoproteins retain the stain. With this procedure the nucleolus maintained a high contrast, mostly on the fibrillar zones. Equally stained were the clusters of interchromatin granules,
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Fig. 3. Conventional electron microscopy. 96 h p.i. CDB which are going to be phagocytosed by Golgi-derived cisternae (arrows). It is also illustrated the fusion between a CDB and a MVB (A). A mature CDB (arrowhead) is surrounded by two membranes. Bar: 0.8 ~tm Fig. 4. p65 immunolabeling. 96 h p.i. Two gold particle-labeled CDB are seen free in the cytoplasm. Bar: 0.6pm
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Fig. 5. p65 immunolabeling. 4-6 days p.i. p65 positive CDB and capsids are going to form a so called "black hole". Bar: 0.5 gm Fig. 6. p65 immunolabeling. 4-6 days p.i. A p65 labeled CDB which seems connected to a nuclear pore by a stream of gold particles; N nucleus. Bar: 0.3 ~tm
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Cytochemical reactions ~ with
Nucleolus Viral core NDB Skein Fteterochromatin Interchromatinic granules CDB
EDTA
HAPTA
HCI
+ + + +
+ +
+ + + + + + -~+ +
+ + + + + + +
+ + +
+ + +
-
+ + + +
**See Materials and methods
Table2. Cytochemical detection of RNA and DNAcontaining structures in CMV-infected cells (Tell structures
Cytochemical reactions ~' with
Nucleolus NDB Skein Heterochromatin CDB Ribosomes
RNase
DNase
+ + + + + + + +
+ + + + -
+ + + + +
~'See Materials and methods
N D B a n d C D B . M o s t o f the N D B a p p e a r e d as r e t i c u l a t e d s t r u c t u r e s w h e r e e l e c t r o n d e n s e a r e a s w e r e i n t e r m i n g l e d w i t h light z o n e s (Fig. 7 A). In the c o n t r o l , t h e y g e n e r a l l y a p p e a r e d u n i f o r m l y a n d d e n s e l y stained; the skein was m a d e u p o f a l o w e r e l e c t r o n d e n s e a n d tess c o m p a c t m a t e r i a l t h a n the c o n t r o l (Fig. 7 A), while C D B s h o w e d the s a m e e l e c t r o n d e n s i t y as the s a m p l e s n o t t r e a t e d with the regressive s t a i n i n g (Fig. 7 B). B o t h free a n d b o u n d r i b o s o m e s w e r e clearly s t a i n e d (Fig. 7 B). C h r o m a t i n a r e a s a n d viral c o r e s w e r e c o m p l e t e l y o r a l m o s t c o m p l e t e l y u n c o l o r e d (Fig. 7 A). HAPTA
technique
W h e n a tissue is h y d r o l y z e d b y HC1, p h o s p h o t u n g s t i c acid d o e s n o t stain D N A c o n t a i n i n g s u b s t a n c e s . U s i n g this t e c h n i q u e , we f o u n d t h a t the s a m e o r g a n e l l e s a n d s t r u c t u r e s s t a i n e d with E D T A , w e r e also s t a i n e d w i t h the H A P T A r e a c t i o n
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Fig. 7. EDTA technique. 5 days p.i. A A nucleus devoid of heterochromatin containing some stained NDB. The skein inclusion appears largely extracted. Bar: 1gm. B Some CDB showing a high contrast, as well as free and bound ribosomes do; N nucleus. Bar: 0.75 lain
in a similar way. Only the nucleolus and ribosomes appeared stained to a lesser _degree (Fig. 8 A, B). HC1 technique Treatment of sections of fixed tissues with 0.5N HC1 at 20°C for 15-60rain (Feulgen hydrolysis) produces aldehydes by the specific hydrolysis of DNA [1, 17, 19]. In our samples, HC1 strongly extracted DNA from the chromatin clumps and viral cores (Fig. 9 A). NDB and CDB appeared almost unstained (Fig. 9 A, B), as did black holes. The nucleolus showed no substantial changes compared with uninfected cells. Since their background substance was extracted by HCt, the cords forming the skein were seen to be almost completely composed of strands of viral capsids (Fig. 9 A), tess visible when other staining techniques were employed. We stress that nucleocapsids, NDB, CDB and black holes were not unstained in the sense that their content was not visible or sufficiently contrasted, but presented as bleached, i.e., with a translucent and light aspect, similar to hyalin. Only a thin rim of electrondense deposit could be seen at the periphery of NDB, CDB and black holes (Fig. 9 A, B). RNase-gold In the nucleus, the structures that bound the RNase were the nucleolus ( + + ) and the skein ( + ) , generally along the contours of the cords. NDB (Fig. 10 A)
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Fig. 8. H A P T A technique. 5 days p.i. A An infected cell whose nucleus clearly shows a skein inclusion with some contrasted and reticulated NDB. Heterochromatin appears unstained, while CDB are markedly stained (arrows); N nucleus. Bar: 2.7gin. B Both the nucleolus (arrow) and the skein (arrowheads) are of medium electrondensity, while NDB appear very electrondense. Bar: 0.9 gm Fig. 9. HC1 technique. 5 days p.i. A A bleached NDB (arrow) is seen close to the skein inclusion where numerous capsids appear similarly extracted. Bar: 0.5 lain. B This picture shows two bleached CDB, as well as are nucleocapsids along the skein cords. Bar: 1.5 gm
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Fig. 10. RNase-gold. 5 days p.i. A The positivity of the reaction is present on the NDB and skein matrix. Bar: 0.9 gm. B Numerous enveloped and unenveloped CDB are labeled with this technique. Bar: 0.9 gm Fig. 11. DNase-gold. 5 days p.i. A The positivity of the reaction in the nucleus is present on NDB, skein and heterochromatin. Note a gold labeled NDB contained in the nucleolus. Bar: 1.4 gm. B In the cytoplasm CDB and the black holes are both markedly labeled by the reaction. Bar: 1.5 gm
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were slightly marked, while viral cores were always unlabeled. In the cytoplasm the positivitiy was seen on the ribosomes and on CDB (Fig. 10 B). DNase-gold In the nucleus, a clear positivity was found on the heterochromatin areas, NDB and all over the skein, while the nucleolus appeared almost unlabeled. CDB were markedly covered with gold particles, as well as the "black holes" (Fig. 11). Control experiments (see Materials and methods) failed to show any deposition of gold particles either with DNase or with RNase. Discussion
NDB (DB or NB) are inclusions whose shape and composition seem to vary widely and whose biological significance is still obscure. The classification by Bouteille et al. [6] has been universally adopted to describe the different types of NB. in fact, five types of these structures have been reported in the literature, some of which are considered of nucleolar origin. A relationship between NDB and nucleolar fibrillar centers has also been suggested [9, 18]. In plant cells two types of NDB have been identified [31]: (a) nucleolus-associated bodies, in proximity or in contact with the nucteolus; (b) NDB free in the nuclear sap and of higher electrondensity than the first type. Both seem to contain proteins, RNA and DNA. In mammals they can be found both in normal cells and, more numerous and evident, in tumour cells, virus infected cells, inflammation and after immunological stimulation. For this reason they are considered an expression of a stimulated or metabolically active state (for a review of the literature, see [-14]). Though they seem to derive from the nucleolus by a budding process, their exact macromolecular content is still debated. Simar [25], after enzymatic digestion on frozen sections of plasma cells, suggested that NDB contained both RNA and DNA. Other conflicting results have been reported by other authors [15, 20, 30]. In HSV-infected cells, besides the appearance of NDB, a progressive destruction of nucleoli has been observed. These data, together with the demonstration of nucleolar 100 kDa proteins both in the nucleolus and in NDB, have been related to alterations in the synthesis and maturation of ribosomal RNA and to degradation of the host cell metabolism after infection [21]. On the basis of our results, CMV-induced NDB seem to correspond to the dense bodies and nuclear bodies (DB, NB) described above. This is confirmed by their submicroscopic features and by their position in the nuclei. They seem to bud from the nucleolus, as previously reported [29], and their biological behaviour and macromolecular composition are in agreement with many reported findings. The results obtained in this study suggest the following sequence of events regarding the origin and the role of NDB and CDB of human CMV-infected
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cells: p65 molecules are produced early in the cytoplasm, as already shown by Britt and Vugler [7]. During the viral cycle, p65 is produced in large amounts and part of it is collected and concentrated in the CDB (beginning 48 h p.i.). The majority of them are sequestered into Golgi-derived vesicles, others fuse with each other and with different cell structures forming the so called "black holes"; some reach the extracetlular space [24-]. A few CDB are located near or in contact with nuclear membranes where they appear to inject p65 molecules into the nucleus via nuclear pores. In the nucleus p65 is found in dense structures (NDB) that seem budding from nucleoli. Once the skein inclusion is formed, we observed the majority of NDB arranged in a circle along the contours of this structure and exhibiting a clear positivity for p65, DNA and RNA. The cytochemical demonstration of both DNA and RNA in the CDB is more difficult to interpret. However, since CDB appear to represent the sites of accumulation and retention of overproduced viral and cellular material, the nucleic acids in CDB could have the same meaning. Thus, traffic from the nucleus to the cytoplasm would account for both DNA and RNA inside CDB. Many literature reports deal with the passage of nuclear substances to the cytoplasm (for review of the literature, see [5]). The cytochemical results are in agreement and indicate that both NDB and CDB contain DNA as well as RNA. However, some conflicting results are obtained by the HAPTA reaction. In fact, only the structures that do not contain D N A should maintain their contrast with this stain. On the contrary, the bulk of our findings indicate that NDB, the skein and CDB contain some DNA, even if a certain reactivity for HAPTA was found. This contradiction may be only apparent assuming that the above mentioned structures contain substances other than DNA (ribonucleoproteins, p65, other viral and/or cell proteins, etc). This would account for the contrast found after HAPTA treatment. The conflicting results reported by some authors regarding the macromolecular composition of NDB, could depend on the particular cell model investigated, (i.e., in normal or pathological conditions, pharmacologically or hormonally activated cells, virus infected cells), or the different cytotypes employed. Some working hypotheses have been suggested by Vagner-Capodano et al. [30] on the role of NDB in cultured thyrotropin-stimulated thyroid cells: a) they are the site of RNA degradation; b) they are the organelles that ensure transport of molecules from the nucleolus to the cytoplasm; c) they represent stored immature ribosomal RNA sequestered by the nucleotus and waiting to be discarded; d) they are a storage form of ribosomal RNA. Antoine et al. I-2-] and Nistal etal. [18], maintain that new nucleolar structures are produced in the form of NB to supply the need for new ribosomes to cope with the increased proteinosynthetic activity of the cell. In conclusion, our results on CMV-infected cells suggest that new NDB appear to originate from the nucleolus of infected cells and are loaded with different molecules (p65, nucleic acids, etc.) related to their particular functional role in the nucleus. Due to their macromolecular content and their position on
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the skein inclusion (which is considered the site o f nucelocapsid assembly), it is conceivable that they are directly implicated in virus morphogenesis. Besides, we c a n n o t rule out that N D B are simplified or separated nucleolar structures whose task is to p r o d u c e ribosomes for viral use. M o r e o v e r , C D B seem to represent the site where surplus cellular, viral or virus-induced molecules are stored before being eliminated.
Acknowledgements We would like to thank Mr. W. Mantovani for the excellent technical assistance on the preparation of photographic material. This work was partially supported by CNR and Ministero della Pubblica Istruzione (40 and 60%) and Istituto Superiore di Sanit/t (AIDS Project).
References 1. Aldrich HC, Coleman SE (1986) Localization of nucleic acids in sections. In: Aldrich HC, Todd WJ (eds) Ultrastructure technique for microorganisms. Plenum, New York, pp 447-467 2. Antoine N, Thiry M, Goessens G (1989) Ultrastructural and cytochemicat studies on extranucleolar bodies in rat oocytes at the preovulatory follicle stage. Biol Cell 65: 6166 3. Bendayan M (1981) Ultrastructural localization of nucleic acids by the use of enzymegold complexes. J Histochem Cytochem 29:531-541 4. Bernhard W (1966) A new staining procedure for electron microscopical cytology. J Ultrastruct Res 27:250-265 5. Borer RA, Lehuer HM, Eppenherger HM, Nigg EA (1989) Major nucleolar proteins shuttle between nucleus and cytoplasm. Cell 56:379-390 6. Bouteille M, Kalifat SR, Delarue J (1967) Ultrastructural variations of nuclear bodies in human diseases. J Ultrastruct Res 19:474-486 7. Britt WG, Vugter L (1987) Structural and immunological characterization of the intracellular forms of an abundant 68.000 M2 human cytomegalovirus protein. J Gen Virol 68:1897-1907 8. Craighead JE, Kanich RE, Almeida JD (1972) Non viral microbodies with viral antigenicity produced in cytomegalovirus infected cells. J Virol 10:766-775 9. Dupuy-Coin AM, Kalifat SR, Bouteille M (1972) Nuclear bodies as proteinaceous structures containing ribonucteoproteins. J Ultrastruct Res 38:174-187 10. Forgani B, Schmidt NJ (I980) Humoral immune response to virions and dense bodies of human cytomegalovirus determined by enzyme immunofluorescence assay. J Med Virol 6:119-127 11. Frens G (1973) Controlled nucleation for the regulation of particle size in monodisperse gold suspension. Nature (Phys Sci) 241:20-22 12. Furukawa T, Fioretti A, Plotkin SA (1973) Growth characteristic of human fibroblasts with demonstration of protein synthesis early in viral replication. J Virol 11:991-997 13. Gautier A (1968) Mise en evidence, sur coupes, du complex DNA-histones par la technique "HAPTA". In: Bocciarelli DS (ed) Electron microscopy, vol 2. Tipografia Poliglotta Vaticana, Roma, pp 81-82 14. Ghadially FN (1982) Nuclear bodies. In: Ghadially FN (ed) Ultrastructural pathology of the cell and matrix, 2nd edn. Butterworth, London, pp 118-I21 15. Krishan A, Uzman BG, Hedley-White ET (1967) Nuclear bodies, a component of cell nuclei in hamster tissues and human tumours. J Ultrastruct Res 19:563-572
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16. Landini MP, Severi B, Furlini G, Badiali De Giorgi L (1987) Human cytomegalovirus structural components: intracellular and intraviral localization of p28 and p65-69 by immunoelectron microscopy. Virus Res 8:15-23 17. Leduc EH, Bernhard W (1961) Ultrastructural cytochemistry. J Biophys Biochem Cytol 10:437-455 18. Nistal M, Regadera J, Paniagua R, Rodriguez MC (1990) Nuclear bodies (Sphaeridia) in Sertoli cells of a man with Acquired Immunodeficiency Syndrome (AIDS) and testicular infection by cytomegalovirus. Ultrastruct Pathol 14:21-26 19. Pearse AGE (1968) Histochemistry theoretical and applied, vol 1, 3rd edn. Churchill, London 20. Puvioll-Dutilleul F, Pichard E, Sheldnick P, Amalric F, Puvion E (1985) Appearance of host-specific nucleolar proteins in intranuclear "dense bodies" following herpes simplex infection. Eur J Cell Biol 39:458-468 21. Pu¢ion-Dutitleul F, Pichard E (1986) Viral alkaline nuclease in intranuclear dense bodies induced by herpes simplex infection. Biol Cell 58:15-22 22. Roth J, Bendayan M, Orci L (1978) Ultrastructural localization of intracellular antigens by the use of protein-A gold complex. J Histochem Cytochem 26:1074-1081 23. Sarov I, Abady I (1975) The morphogenesis of human cytomegalovirus. Isolation and polypeptide composition of cytomegalovirions and dense bodies. Virology 66:464-473 24. Severi B, Landini MP, Govoni E (1988) Human cytomegalovirus morphogenesis: an ultrastructural study of the late cytoplasmic phases. Arch Virol 98:51-64 25. Simar LJ (1969) Ultrastructure et constitution des corps nucleaires dans les plasmocytes. Z Zellforsch 99:235-251 26. St Jeor SC, Albrecht TB, Funk FD, Rapp F (1974) Stimulation of host cell DNA synthesis of human cytomegalovirus. J Virol 13:353-362 27. Talbot P, Almeida JD (1977) Human cytomegalovirus: purification of enveloped virions and dense bodies. J Gen Virol 36:345-349 28. Tanaka S, Furukawa T, Plotkin SA (1975) Human cytomegalovirus stimulates host cell RNA synthesis. J Virol 15:65-73 29. Vagner-Capodano AM, Manchamp J, Stahl A, Lissitzky S (1980) Nucleolar budding and formation of nuclear bodies in cultured thyroid cells stimulated by Thyrotropin, Dibutyryl Cyclic AMP, and Prostaglandin E2. J Ultrastruct Res 70:37-51 30. Vagner-Capodano AM, Bouteille M, Stahl A, Lissitzky S (1982) Nucleolar ribonucleoprotein release into the nucleoptasm as nuclear bodies in cultured Thyrotropinstimulated thyroid cells: autoradiographic kinetics. J Ultrastruct Res 78:13-25 31. Williams LM, Charest PM, Fitgerald GJ, Lafontaine J-H (1985) A comparison of nuclease-gold and protease-gold complex labeling over the nucleolus and nuclear bodies of Pisum sativum root tip cells. Biol Cell 54:65-72 Authors' address: Dr. B. Severi, Istituto di Microscopia Elettronica Clinica, Policlinico S. Orsola 9, 1-40138 Bologna, Italy. Received January 29, 1991
Virology
J Arch Virol (1992) 123:193 207
© Springer-Verlag 1992 Printed in Austria
Human cytomegalovirus nuclear and cytoplasmic dense bodies B. Severi 1, M. P. Landini 2, G. Cenaechi ~, N. Zini 3, and N. M. Maraldi 3 1Istituto di Microscopia Elettronica Clinica and 2 Istituto di Microbiologia, UniversitA di Bologna, and 3Isfituto di Citomorfologia Normale e Patotogica del C.N.R., Istituto Rizzoli, Bologna, Italy Accepted July 8, 1991
Summary. One of the characteristic features of cytomegalovirus (CMV) replication is the formation of cytoplasmic dense bodies. Recent findings revealed similar structures also in the nuclei of CMV-infected cells. By transmission electron microscopy, immuno electronmicroscopy, and cytochemistry, we have studied the morphogenetic steps and macromolecular composition of both structures. Our results show that both structures contain DNA, RNA and viral antigenic proteins. Nuclear dense bodies are probably an expression of a stimulated cellular metabolism, while cytoplasmic dense bodies may represent the site where surplus cellular and viral molecules are stored before being eliminated. Introduction During the late phases of human cytomegalovirus (CMV) replication, electrondense structures varying in size, antigenically related but morphologically distinct from virions, are formed in the cytoplasm of infected cells. These structures are called cytoplasmic dense bodies (CDB) and represent one of the characteristic features of CMV replication [-8, 23, 24, 27]. Data in the literature suggest that CDB do not contain DNA and have surface antigens in common with virions [8, 10, 27]. Recently we determined the antigenic correlation between virions and CDB and found that CDB contain several host cell proteins in addition to some viral proteins (unpubl. data). These results suggest that CDB represent the final step of a cellular mechanism by which the cell eliminates macromolecules that have been overproduced. In fact, it is well known that infection of human fibroblasts by human CMV dramatically alters the mechanisms responsible for gene expression, leading to an increase in host cell DNA, RNA and protein syntheses [-12, 26, 28]. This overproduction of cellular material resembles ex novo synthesis of virus-specific components
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and gives rise to an abundance of viral and cellular macromolecules to be segregated or discharged by the cell. Furthermore, recent findings disclosed structures similar to CDB also in the nuclei of CMV infected cells [163. These organelles have been called nuclear dense bodies (NDB) to distinguish them from CDB. Their size, submicroscopic features, and localization resemble dense bodies (DB) or nuclear bodies (NB), widely described in the nuclei of various cell systems, in both normal and pathological conditions, including viral diseases [6]. The present study aimed to define the morphogenetic steps of N D B and CDB formation at different phases of the C M V replication cycle and to determine their macromolecular composition. In particular we tested for the presence of the most a b u n d a n t CMV-structural component of 65 k D a (p65) that, in a previous paper, we localized by immunoelectronmicroscopy on the CDB and N D B matrix [16], and the presence of D N A and R N A , by cytochemical methods.
Material and methods Viruses and cell cultures The Towne strain of CMV (originally obtained through the courtesy of M. Stinski, University of Iowa, USA) was used in our experiments. Human embryo fibroblasts were obtained from the Istituto Zooprofilattico, Brescia, Italy and were infected at a m.o.i, of 0.5 pfu/cells. Infected cells were used at different times after infection (from 1 to 10 days p.i.). For the immunoreaction with p65, a monoclonal antibody against CMV late nuclear protein (N. 9220, Biotech Research Labs., Inc., Rockville, MD) was employed.
Transmission electron microscopy Human embryo fibroblasts were processed for TEM at 24, 48, 96 h and 4 and 6 days after CMV infection. Each sample was fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, postfixed in 1% osmium tetroxide in veronal acetate buffer, dehydrated in graded alcohols and embedded in Epon or LR White resins. When required, thin sections were counterstained with uranyt acetate and/or lead citrate. Osmium postfixation was omitted for the samples to be processed for cytochemistry and immunoelectronmicroscopy.
Cytochemistry Regressive EDTA staining was carried out according to the procedure of Bernhard [43. Briefly, thin sections of Epon embedded infected fibroblasts were floated for 1 rain on 5% aqueous uranyl acetate at room temperature, washed with distilled water and treated with 0.2 M EDTA for 60 min. They were washed again with distilled water and contrasted for 1 min on standard lead citrate at room temperature. Controls were not treated with the EDTA solution. The HAPTA technique Epon embedded thin sections were hydrolyzed with a solution of HC1 5 N for 45 min, washed in distilled water and processed with a 1% phosphotungstic acid (PTA) solution in 80% ethanol, pH 3.5, for 2rain at 20 °C and again rinsed in distilled water. Controls were obtained omitting the HC1 step [13].
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HC1 extraction Epon embedded thin sections were hydrolyzed with a HC1 0.5 N solution for 25-45 min and washed in distilled water [1, 17, 19]. Control sections were incubated in distilled water adjusted to pH 2.0 for the same time and temperatures instead of the HC1 treatment. The DNase and RNase-gold techniques A monodisperse colloidal sol composed of gold particles with an average size of 15 nm was prepared according to Frens [11]. To a boiling aqueous solution of 100ml of 0.01% tetrachloroauric acid (Merck, Darmstadt, Federal Republic of Germany) were added 4 ml of a 1% aqueous solution of sodium citrate and the mixture allowed to boil for 5 rain. The solution was cooled to room temperature and adjusted to pH 9.0 for RNase and to pH 6 for DNase with 0.2 M KzCO 3. The enzyme-gold complexes were prepared essentially according to Bendayan [3]. The concentrations used were 0.0056 mg/ml for RNase A (type XII-A Sigma, St. Louis, MO) and 0.05 mg/ml for DNase I (type DN.EP, Sigma). Both complexes were centrifuged for 30 rain at 33.000 g at 4 °C. The sediments obtained from an 8 ml suspension were diluted with 0.150ml of 0.02M PBS (pH7.5 for RNase A and pH6.0 for DNase I) containing 0.5 mg/ml polyethylene glycol. Cytochemical labeling For RNase A incubation, the complex was generally used diluted up to 1 : 20 with PBS containing 0.5 mg/ml of polyethylene glycol and for DNase I up to t : 200. The grids were floated for 5 rain on a drop of PBS and incubated on a drop of the enzyme-colloidal gold complexes for 45 rain at room temperature. Following incubation, the sections were thoroughly washed with PBS, rinsed with distilled water, dried and stained with aqueous solutions of uranyl acetate and lead citrate. The specificity of the labeling was tested by the following control experiments. For RNase A: (a) incubation with enzyme-gold complex at non-optimal values of pH; (b) incubation with non-stabilized gold suspensions; (c) incubation with albumin-gold complex; (d) incubation with enzyme-gold complex to which the appropriate substrate was added, i.e. RNA 1 rag/rot (type IV, from calf liver, Sigma, St. Louis, MO); and (e) incubation with RNase inhibitors, like heparin, 0.5 mg/ml, or polyvinyl sulphate, 0.1 mg/ml. For DNase: (a) incubation with DNase-gold complex to which the appropriate substrate, DNA type I (from calf thymus, Sigma, St. Louis, MO) 1 mg/ml was added; (b) incubation with nonstabilized gold suspension; (c) incubation with an enzyme-gold complex prepared with DNase inactivated by heating at 100 °C for 10 rain.
Immunoelectronmicroscopy Post-embedding immunolabeling of infected fibroblasts was done by the protein-gold technique, according to Roth et al. [22]. For more details, see our previous paper on p65-69 and p28 intracetlular and intraviral localization [16].
Results
Conventional electron microscopy and p65 immunodetection A t 24 h p.i., cells revealed a clear i m m u n o g o l d positivity for p65 on occasional nuclear r o u n d i s h electrondense structures m a i n l y localized in c o n t a c t with hyp e r t r o p h i c nucleoli, often as if b u d d i n g f r o m t h e m (Fig. 1). In a previous p a p e r [16] we indicated these structures as nuclear dense bodies (NDB).
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l,ig. 1. p65 inmmnotabcting. 24 h p.i. Two nuclear dense masses positive lbr p65 (arrows), Onc sccms to be in the process of budding from the nucleolus, the second appears slightly separated from it. Bar: 0.3 gm Fig. 2. p65 immunolabeling. 96 h p.i. Three p65 positive NDB localized along the contours of the skein inclusion. Bar: 0.6 tam
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Between 48 and 96 h p.i., CDB were observed at different levels of organization and decorated with gold particles. Positive NDB, although few, were more numerous; they could be sparsely distributed in the nucleoplasm, in contact with nucleoli, or immersed in a slightly electrondense granular material which later turned into ringshaped or reticular structures filled with developing nucleocapsids ("skein"). Only some of the infected cells presented these NDB, but several of them could be observed inside the same nucleus. They did not always show identical morphological features: they could be quite similar to conventional CDB, or showed a looser texture and less regular contours. At 96 h p.i., NDB were slightly increased in number and were either sparse in the nucleoplasm or located in the skein in a circular fashion. The positivity towards p65 was always evident (Fig. 2). In the cytoplasm many lysosomes, mainly of the multivesicular body (MVB)-type were seen, while Golgi apparatuses were numerous and hypertrohpic. Many CDB were often enveloped by Golgi-derived vacuoles or saccules, or began to fuse with each other and with viral particles and MVB (Fig. 3). The same degree of positivity for p65 seen in NDB was also revealed in CDB (Fig. 4). Later on (4-6 days p.i.), both these viral-related cellular changes and the gold labeling were enhanced. Numerous large "black holes" [24], derived from the fusion between viral particles, CDB and MVB, were also evident and positive for p65 (Fig. 5). CDB rich in gold particles were often seen close to, or in contact with, nuclear membranes, generally in spatial relationship with the nuclear pores, with which they seemed to be connected by a thin thread of faint material. At tirnes, gold particles were seen to connect CDB and nuclear pores (Fig. 6). NDB were always found isolated or arranged in a circle on the skein inclusion which appeared filled with partially or fully organized capsids. At each time p.i., mock infected cells failed to show any specific gold labeling when p65 antibody was omitted. Anti p65 antibody was also assayed on uninfected human embryo fibroblasts both by immunofluorescence and immunoelectronmicroscopy. The reaction obtained with both techniques was negative (data not shown).
Cytochemistt3, Cytochemical staining and gold-labeling were carried out on CMV-infected cells at 5 days p.i. The results are summarized in Tables 1 and 2. EDTA technique Introduced by Bernhard [4], this technique is considered a regressive staining procedure in which the uranyl stain fixed on deoxyribonucleoproteins is bleached out by an EDTA solution. On the contrary, structures containing ribonucleoproteins retain the stain. With this procedure the nucleolus maintained a high contrast, mostly on the fibrillar zones. Equally stained were the clusters of interchromatin granules,
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Fig. 3. Conventional electron microscopy. 96 h p.i. CDB which are going to be phagocytosed by Golgi-derived cisternae (arrows). It is also illustrated the fusion between a CDB and a MVB (A). A mature CDB (arrowhead) is surrounded by two membranes. Bar: 0.8 ~tm Fig. 4. p65 immunolabeling. 96 h p.i. Two gold particle-labeled CDB are seen free in the cytoplasm. Bar: 0.6pm
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Fig. 5. p65 immunolabeling. 4-6 days p.i. p65 positive CDB and capsids are going to form a so called "black hole". Bar: 0.5 gm Fig. 6. p65 immunolabeling. 4-6 days p.i. A p65 labeled CDB which seems connected to a nuclear pore by a stream of gold particles; N nucleus. Bar: 0.3 ~tm
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Cytochemical reactions ~ with
Nucleolus Viral core NDB Skein Fteterochromatin Interchromatinic granules CDB
EDTA
HAPTA
HCI
+ + + +
+ +
+ + + + + + -~+ +
+ + + + + + +
+ + +
+ + +
-
+ + + +
**See Materials and methods
Table2. Cytochemical detection of RNA and DNAcontaining structures in CMV-infected cells (Tell structures
Cytochemical reactions ~' with
Nucleolus NDB Skein Heterochromatin CDB Ribosomes
RNase
DNase
+ + + + + + + +
+ + + + -
+ + + + +
~'See Materials and methods
N D B a n d C D B . M o s t o f the N D B a p p e a r e d as r e t i c u l a t e d s t r u c t u r e s w h e r e e l e c t r o n d e n s e a r e a s w e r e i n t e r m i n g l e d w i t h light z o n e s (Fig. 7 A). In the c o n t r o l , t h e y g e n e r a l l y a p p e a r e d u n i f o r m l y a n d d e n s e l y stained; the skein was m a d e u p o f a l o w e r e l e c t r o n d e n s e a n d tess c o m p a c t m a t e r i a l t h a n the c o n t r o l (Fig. 7 A), while C D B s h o w e d the s a m e e l e c t r o n d e n s i t y as the s a m p l e s n o t t r e a t e d with the regressive s t a i n i n g (Fig. 7 B). B o t h free a n d b o u n d r i b o s o m e s w e r e clearly s t a i n e d (Fig. 7 B). C h r o m a t i n a r e a s a n d viral c o r e s w e r e c o m p l e t e l y o r a l m o s t c o m p l e t e l y u n c o l o r e d (Fig. 7 A). HAPTA
technique
W h e n a tissue is h y d r o l y z e d b y HC1, p h o s p h o t u n g s t i c acid d o e s n o t stain D N A c o n t a i n i n g s u b s t a n c e s . U s i n g this t e c h n i q u e , we f o u n d t h a t the s a m e o r g a n e l l e s a n d s t r u c t u r e s s t a i n e d with E D T A , w e r e also s t a i n e d w i t h the H A P T A r e a c t i o n
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Fig. 7. EDTA technique. 5 days p.i. A A nucleus devoid of heterochromatin containing some stained NDB. The skein inclusion appears largely extracted. Bar: 1gm. B Some CDB showing a high contrast, as well as free and bound ribosomes do; N nucleus. Bar: 0.75 lain
in a similar way. Only the nucleolus and ribosomes appeared stained to a lesser _degree (Fig. 8 A, B). HC1 technique Treatment of sections of fixed tissues with 0.5N HC1 at 20°C for 15-60rain (Feulgen hydrolysis) produces aldehydes by the specific hydrolysis of DNA [1, 17, 19]. In our samples, HC1 strongly extracted DNA from the chromatin clumps and viral cores (Fig. 9 A). NDB and CDB appeared almost unstained (Fig. 9 A, B), as did black holes. The nucleolus showed no substantial changes compared with uninfected cells. Since their background substance was extracted by HCt, the cords forming the skein were seen to be almost completely composed of strands of viral capsids (Fig. 9 A), tess visible when other staining techniques were employed. We stress that nucleocapsids, NDB, CDB and black holes were not unstained in the sense that their content was not visible or sufficiently contrasted, but presented as bleached, i.e., with a translucent and light aspect, similar to hyalin. Only a thin rim of electrondense deposit could be seen at the periphery of NDB, CDB and black holes (Fig. 9 A, B). RNase-gold In the nucleus, the structures that bound the RNase were the nucleolus ( + + ) and the skein ( + ) , generally along the contours of the cords. NDB (Fig. 10 A)
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Fig. 8. H A P T A technique. 5 days p.i. A An infected cell whose nucleus clearly shows a skein inclusion with some contrasted and reticulated NDB. Heterochromatin appears unstained, while CDB are markedly stained (arrows); N nucleus. Bar: 2.7gin. B Both the nucleolus (arrow) and the skein (arrowheads) are of medium electrondensity, while NDB appear very electrondense. Bar: 0.9 gm Fig. 9. HC1 technique. 5 days p.i. A A bleached NDB (arrow) is seen close to the skein inclusion where numerous capsids appear similarly extracted. Bar: 0.5 lain. B This picture shows two bleached CDB, as well as are nucleocapsids along the skein cords. Bar: 1.5 gm
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Fig. 10. RNase-gold. 5 days p.i. A The positivity of the reaction is present on the NDB and skein matrix. Bar: 0.9 gm. B Numerous enveloped and unenveloped CDB are labeled with this technique. Bar: 0.9 gm Fig. 11. DNase-gold. 5 days p.i. A The positivity of the reaction in the nucleus is present on NDB, skein and heterochromatin. Note a gold labeled NDB contained in the nucleolus. Bar: 1.4 gm. B In the cytoplasm CDB and the black holes are both markedly labeled by the reaction. Bar: 1.5 gm
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were slightly marked, while viral cores were always unlabeled. In the cytoplasm the positivitiy was seen on the ribosomes and on CDB (Fig. 10 B). DNase-gold In the nucleus, a clear positivity was found on the heterochromatin areas, NDB and all over the skein, while the nucleolus appeared almost unlabeled. CDB were markedly covered with gold particles, as well as the "black holes" (Fig. 11). Control experiments (see Materials and methods) failed to show any deposition of gold particles either with DNase or with RNase. Discussion
NDB (DB or NB) are inclusions whose shape and composition seem to vary widely and whose biological significance is still obscure. The classification by Bouteille et al. [6] has been universally adopted to describe the different types of NB. in fact, five types of these structures have been reported in the literature, some of which are considered of nucleolar origin. A relationship between NDB and nucleolar fibrillar centers has also been suggested [9, 18]. In plant cells two types of NDB have been identified [31]: (a) nucleolus-associated bodies, in proximity or in contact with the nucteolus; (b) NDB free in the nuclear sap and of higher electrondensity than the first type. Both seem to contain proteins, RNA and DNA. In mammals they can be found both in normal cells and, more numerous and evident, in tumour cells, virus infected cells, inflammation and after immunological stimulation. For this reason they are considered an expression of a stimulated or metabolically active state (for a review of the literature, see [-14]). Though they seem to derive from the nucleolus by a budding process, their exact macromolecular content is still debated. Simar [25], after enzymatic digestion on frozen sections of plasma cells, suggested that NDB contained both RNA and DNA. Other conflicting results have been reported by other authors [15, 20, 30]. In HSV-infected cells, besides the appearance of NDB, a progressive destruction of nucleoli has been observed. These data, together with the demonstration of nucleolar 100 kDa proteins both in the nucleolus and in NDB, have been related to alterations in the synthesis and maturation of ribosomal RNA and to degradation of the host cell metabolism after infection [21]. On the basis of our results, CMV-induced NDB seem to correspond to the dense bodies and nuclear bodies (DB, NB) described above. This is confirmed by their submicroscopic features and by their position in the nuclei. They seem to bud from the nucleolus, as previously reported [29], and their biological behaviour and macromolecular composition are in agreement with many reported findings. The results obtained in this study suggest the following sequence of events regarding the origin and the role of NDB and CDB of human CMV-infected
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cells: p65 molecules are produced early in the cytoplasm, as already shown by Britt and Vugler [7]. During the viral cycle, p65 is produced in large amounts and part of it is collected and concentrated in the CDB (beginning 48 h p.i.). The majority of them are sequestered into Golgi-derived vesicles, others fuse with each other and with different cell structures forming the so called "black holes"; some reach the extracetlular space [24-]. A few CDB are located near or in contact with nuclear membranes where they appear to inject p65 molecules into the nucleus via nuclear pores. In the nucleus p65 is found in dense structures (NDB) that seem budding from nucleoli. Once the skein inclusion is formed, we observed the majority of NDB arranged in a circle along the contours of this structure and exhibiting a clear positivity for p65, DNA and RNA. The cytochemical demonstration of both DNA and RNA in the CDB is more difficult to interpret. However, since CDB appear to represent the sites of accumulation and retention of overproduced viral and cellular material, the nucleic acids in CDB could have the same meaning. Thus, traffic from the nucleus to the cytoplasm would account for both DNA and RNA inside CDB. Many literature reports deal with the passage of nuclear substances to the cytoplasm (for review of the literature, see [5]). The cytochemical results are in agreement and indicate that both NDB and CDB contain DNA as well as RNA. However, some conflicting results are obtained by the HAPTA reaction. In fact, only the structures that do not contain D N A should maintain their contrast with this stain. On the contrary, the bulk of our findings indicate that NDB, the skein and CDB contain some DNA, even if a certain reactivity for HAPTA was found. This contradiction may be only apparent assuming that the above mentioned structures contain substances other than DNA (ribonucleoproteins, p65, other viral and/or cell proteins, etc). This would account for the contrast found after HAPTA treatment. The conflicting results reported by some authors regarding the macromolecular composition of NDB, could depend on the particular cell model investigated, (i.e., in normal or pathological conditions, pharmacologically or hormonally activated cells, virus infected cells), or the different cytotypes employed. Some working hypotheses have been suggested by Vagner-Capodano et al. [30] on the role of NDB in cultured thyrotropin-stimulated thyroid cells: a) they are the site of RNA degradation; b) they are the organelles that ensure transport of molecules from the nucleolus to the cytoplasm; c) they represent stored immature ribosomal RNA sequestered by the nucleotus and waiting to be discarded; d) they are a storage form of ribosomal RNA. Antoine et al. I-2-] and Nistal etal. [18], maintain that new nucleolar structures are produced in the form of NB to supply the need for new ribosomes to cope with the increased proteinosynthetic activity of the cell. In conclusion, our results on CMV-infected cells suggest that new NDB appear to originate from the nucleolus of infected cells and are loaded with different molecules (p65, nucleic acids, etc.) related to their particular functional role in the nucleus. Due to their macromolecular content and their position on
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the skein inclusion (which is considered the site o f nucelocapsid assembly), it is conceivable that they are directly implicated in virus morphogenesis. Besides, we c a n n o t rule out that N D B are simplified or separated nucleolar structures whose task is to p r o d u c e ribosomes for viral use. M o r e o v e r , C D B seem to represent the site where surplus cellular, viral or virus-induced molecules are stored before being eliminated.
Acknowledgements We would like to thank Mr. W. Mantovani for the excellent technical assistance on the preparation of photographic material. This work was partially supported by CNR and Ministero della Pubblica Istruzione (40 and 60%) and Istituto Superiore di Sanit/t (AIDS Project).
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