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Pathogenesis of oncogenic simian adenoviruses
© 1970by AcademicPress,Inc. 344
J. ULTRASTRUCTURERESEARCH30, 344-353 (1970)
Pathogenesis of Oncogenic Simian Adenoviruses VII. The Origin of Annulate Lamellae in LLC-MK2 Cells Infected with SV301,2 LEONARD P. MERKOW, ~ MALCOLM SLIFKIN, 3 MATIAS PARDO, AND NORBERT P. RAPOZA4
Departmentsof Experimental Pathology andMicrobiology, William H.Singer Memorial Research Institute of the Allegheny General Hospital, Pittsburgh, Pennsylvania 15212, and Department of Microbiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 Received June 9, 1969 LLC-MK2 cells were grown as monolayers, infected with the oncogenic simian adenovirus 30 (SV30), and harvested 1-192 hours post infection (p.i.). As early as 1 hour p.i., increased numbers of annulate lamellae were observed within the cytoplasm and nucleus of infected L L C - M K 2 cells. The cytoplasmic annulate lamellae were usually in close proximity to the nucleus and frequently contiguous with the outer nuclear envelope. Intertubular connections between parallel lamellae, electron dense particles consistent with ribosomes, and crystalloid bodies in contact with annulate lamellae were observed. The increased number of annulate lamellae over that observed in control cultures may be either a cellular response concomitant with virus replication or a secondary manifestation of viral toxicity. The formation of annulate lamellae appears to occur by way of at least two processes in SV30-associated cells. The presence of i n t r a n u c l e a r (12, 13, 24-26, 29, 30) a n d / o r i n t r a c y t o p l a s m i c a n n u l a t e lamellae has been described in m a n y n o r m a l (5,15-18, 49, 52, 56, 58, 59), altered (2, 42, 43), p r e n e o p l a s t i c (46, 47), o r neoplastic (4, 6, 8, 21, 28, 38, 39-41, 47, 48, 51, 55) h u m a n or a n i m a l cells. Since a n n u l a t e lamellae have been considered a specialized f o r m of e n d o p l a s m i c reticulum (3, 8, 10, 39) a n d quite frequently are contiguous with the latter, their m e c h a n i s m of f o r m a t i o n is of considerable interest. M o r e recently, n u m e r ous annulate lamellae have been observed in viral i n d u c e d neoplastic cells in vivo (45), p r e s u m a b l y noninfected cell lines in vitro (9, 10, 36, 37) a n d virus infected cell 1 Presented in part at the Fourth European Regional Conference on Electron Microscopy, Rome, Italy, September, 1968. 2 This investigation was supported in part by Grant AI-02535 from the National Institutes of Health. 3 Adjunct Assistant Professor at Pennsylvania State University. 4 Present address: Division of Biological Research, G. D. Searle and Company, Chicago, Illinois 60680.
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lines (7, 11, 34, 35). I n c r e a s e d n u m b e r s of a n n u l a t e lamellae have been e n c o u n t e r e d in L L C - M K 2 cells infected with the oncogenic simian a d e n o v i r u s 30 (SV30). This r e p o r t describes the origin a n d f o r m a t i o n of a n n u l a t e lamellae in L L C - M K 2 cells infected with SV30 in vitro. This process is c o m p a r e d with the m e c h a n i s m of f o r m a tion of a n n u l a t e lamellae in cells i n d u c e d b y the oncogenic simian a d e n o v i r u s 20 in
vivo (44). M A T E R I A L S A N D METHODS Simian adenovirus 30 obtained from a purified strain described by Hoffert, Bates, and Cheever (19) was used throughout this investigation. Its origin has been previously described (54). The cell line LLC-MK2, a derivative of rhesus monkey kidney, was grown in basal medium of Eagle (BME) containing 5 % calf serum and 1% glutamine. These cells were obtained from the American Type Culture Collection and two commercial sources (Flow Laboratories, Rockville, Maryland, and Microbiological Associates, Bethesda, Maryland). Two percent agamma calf serum was used in all cell cultures when virus was propagated or titrated. Virus stocks used in these investigations were prepared in the LLC-MK2 cell line of African green monkey kidney cells containing SV5 and SV40 antisera. These stocks were demonstrated to be free of SV40 virus and adeno-associated viruses by procedures previously described (54). Virus stocks were purified by centrifuging frozen and thawed specimens in the Spinco Model L ultracentrifuge. Preparative density gradient centrifugation with cesium chloride was used as a further step in purification (54). Electron microscopy. L L C - M K 2 cells were grown as monolayers in 32-ounce prescription bottles. These monolayers containing 108 to 2 x 108 cells were infected with SV30 at an input of 2 TCID~0 per cell. Cultures inoculated with BME alone served as controls and were processed in an identical manner. A t 1, 3, 6, 9, 24, 48, 72, 120, and 192 hours post infection (p.i.), L L C - M K 2 cells were dislodged from the glass surface with a rubber policeman and centrifuged in tubes at 2000 rpm in a Servall centrifuge for 2 minutes. The supernatant was decanted, and the cell pellet was immediately fixed with 1% phosphate-buffered osmium tetroxide at p H 7.4. The 1-2 ml of packed cells obtained from each of 2 bottles was then sequentially resuspended through a series of ethanols and centrifuged as above; the supernatants were decanted. The resultant dehydrated pellet of cells was treated with propylene oxide and then embedded in Epon 812. One-micron sections were prepared and stained with methylene blue. Masses of L L C - M K 2 cells were easily identifiable in these sections with the light microscope. Ultrathin sections displaying silver or gold interference colors were obtained with a Porter-Blum MT-1 or MT-2 ultramicrotome equipped with a glass knife. These ultrathin sections were stained with a 5 % solution of uranyl acetate followed by lead citrate. All ultrathin sections were then examined with a Philips 100B or EM300 electron microscope at 60 kV. RESULTS A s early as 1 h o u r p. i. a n d at all subsequent times, increased n u m b e r s of a n n u l a t e lamellae were o b s e r v e d within the c y t o p l a s m a n d nucleus of n u m e r o u s L L C - M K 2 cells i n o c u l a t e d in vitro with SV30 (Figs. 1-8). This increased n u m b e r of a n n u l a t e
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lamellae was most evident at low magnification, and frequently in a survey view one or more infected cells showed several individual stacks of annulate lamellae (Fig. 1). Cytoplasmic annulate lamellae were often in close proximity to the nucleus and frequently appeared intimately associated with the nuclear envelope (Figs. 1, 3-6). The number of double membranes or lamellae vary from 1 to 115 (Fig. 7). This organelle in a longitudinal plane of section displayed the characteristic periodic constrictions in register with its adjacent parallel membranes (Figs. 1-7). Perpendicular intertubular connections between parallel lamellae (Figs. 2 and 4), as well as an increased density of the matrix surrounding each lamella were observed (Figs. 2-4). Individual 150 A or smaller electron dense particles were unattached (free between lamellae) or attached to lamellae (Fig. 3). Annulate lamellae occasionally appeared simultaneously in several planes of section (Figs. 1 and 7). A lateral margin or end of annulate lamellae usually in the longitudinal plane of section was frequently contiguous with strands of smooth or rough endoplasmic reticulum (Figs. 1, 6, and 7). An en face plane of section revealed the annuli to consist of numerous microcylinders (subannuli). At an increased magnification, the annulate pores displayed an octagonal symmetry. The lateral margins of annulate lamellae frequently displayed bulbous dilatation (Figs. 1, 2, and 6). Clusters of scalloped lipid bodies with a variable affinity for osmium were often adjacent to and/or contiguous with annulate lamellae (Figs. 2, 4, and 6). Rarely, a honeycomb crystalloid body was in contact with annulate lamellae (Fig. 2). The presence of input intracellular virus particles was noted between 1 and 10 hours p.i. (Figs. 1 and 2). Nuclear projections were observed in infected cells and individual annulate lamellae rarely encircled these projections. In a fortuitous plane of section, direct continuity between stacks of annulate lamellae and the outer nuclear membrane was observed (Figs. 3, 5, and 6). Usually a stack of contiguous annulate lamellae was parallel to the nuclear envelope (Figs. 3 and 4). However, occasionally these attached annulate lamellae appeared either in an oblique (Fig. 5) or perpendicular (Fig. 6) plane of section to the nuclear envelope. The presence of intranuclear annulate lamellae was infrequently observed (Figs. 7 and 8). This type of annulate lamellae was similar in morphology to those observed in the cytoplasm and often was in contact with either the internal nuclear membrane and/or FIG. 1. LLC-MK2 cell 2 hours p.i. with SV30. Four stacks of annulate lamellae (A1) are in close proximity to the nucleus (N). The largest stack shows dilated vesicular (V) lateral ends (arrows). Two annulate lamellae are sectioned in a tangential plane and display pores. Virus particles (Vp, arrows) are adjacent to or within dilated cisternae. This and all subsequent photographs represent osmium-fixed, Epon-embedded material stained with uranyl acetate and lead citrate, x 12,000. FIG. 2. LLC-MK2 cell 3 hours p.i. The annulate lamellae (AI) display interlamellar granular material and tubular connections (arrows). Three lamellae are continuous with an egg-shaped, membranelimited crystalline body (arrows). The lateral ends of lamellae are dilated and contain ribosomes on their outer surface. Several scalloped lipid bodies (Lb) are in close proxmity or in contact with the annulate lamellae. A virus particle (Vp, arrow) is present, x 46,000.
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peripheral nuclear chromatin (Fig. 7). Organelles such as endoplasmic reticulum, the Golgi complex, and vacuoles did not differ considerably from those in control LLCM K 2 cell cultures. However, virus-infected cells did display the cytopathology associated with adenovirus infection (Fig. 8) whereas control cells did not. At 12 hours or later, intranuclear virus particles were first apparent. By 18 hours, crystals of newly synthesized SV30 were observed. The infrequent presence of annulate lamellae was observed in control L L C - M K 2 cells.
DISCUSSION Infection by SV30 in L L C - M K 2 cells provided an experimental model to study the formation of annulate lamellae in somatic mammalian cells. In these in vitro infected cells, annulate lamellae appear to form by way of a sequential delamination of individual lamellae on either side of the nuclear envelope. Annulate lamellae formed by this process are similar to that described by Harrison for seagull adrenal cortical cells (17). Therefore, origin of annulate lamellae may occur by way of a template or replicative process with delamination (shedding) as the mechanism of separation from the nuclear envelope. The mechanism regulating either template "availability" or delamination is unknown. However, since annulate lamellae were seen (although infrequently) in control L L C - M K 2 cells, it is tempting to speculate that in this instance the virus might act by augmenting the translation or transcription of focal portions of the host cell genome. In contrast, annulate lamellae appear to develop by way of a slightly different and potentially more complex mechanism in SV20 or SV30 induced neoplastic cells in vivo (44, 45). In the in vivo model, an association between annulate lamellae, endoplasmic reticulum, and the external membrane of the nuclear envelope appears to exist. Subsequent to in vivo infection with SV20 (44) portions of cisternal smooth endoplasmic reticulum adjacent to the nucleus are arrayed so as to suggest formation of periodic constrictions in register. As this occurs, interlamellar (intertubular) connections appear between these focally constricted cisternae of endoplasmic reticulum FIG. 3. LLC-MK2 cell 9 hours p.i. Continuity exists between this annulate lamellae (Al)and the outer nuclear envelope. Some interlamellar granularity is present between the 6 lamellae. Several 150 ~ dense particles are either free or between or attached to lamellae (arrows) and comparable to ribosomes attached to the external nuclear envelope (arrows). x 50,000. FIG. 4. LLC-MK2 cell 3 hours p.i. A stack of three lamellae are adjacent to the nucleus (N). Note the similar morphology of the nuclear envelope subjacent to the most proximal portion of the annulate lamellae (AI, between arrows). The interlamellar granularity and tubular connections are present (arrows). x 29,000. Fit. 5. LLC-MK2 cell 3 hours p.i. Annulate lamellae (Al) are not parallel to the nuclear envelope. Three lamellae are in continuity with the outer nuclear envelope. Each lamella makes its own contact with the nucleus (N). x 25,000.
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(44: Figs. 11-14). This mechanism is similar to the process of coalescence of vesicles derived and budded off from the nuclear membrane in alligator and Necturus oocytes as described by Harrison (17) and Kessel (28), respectively. Therefore, annulate lamellae appear to form by way of at least two processes in cells containing simian adenovirus virions. Fong et al. (14) and Tyrrell et al. (57) did not report the presence of annulate lamellae in simian adenovirus (SV15, SV17) infected primary monkey kidney cells. This could be due to the oncogenicity of SV30. Furthermore, the use of a continuous cell line, as opposed to a primary cell line (14, 57) may play a role in the incidence of this organelle. K i m and Boatman (34) suggested that annulate lamellae may be involved in the synthesis of viral material since this organelle was observed by them only in rubellainfected L L C - M K 2 cells. Our observation of annulate lamellae in uninfected LLCM K 2 cells could be attributed to our extensive ultrastructural examination of control cells. Cytoplasmic crystals have been reported in cells infected with human adenovirus (50) and rubella virus (34). Continuity between these crystals and annulate lamellae has been reported (34) and observed in the present investigation. The presence of lipid bodies in close proximity to annulate lamellae has been reported (34, 52). Similarly, lipid bodies were observed in our experimental model and may play some role in the function(s) of annulate lamellae. The presence of annulate lamellae has been reported in conditions of altered protein synthesis (20, 22, 23, 36); accordingly, this organelle has been implicated as having some role in protein synthesis (34, 52, 56). For this reason, it is conceivable that their increased number and size contribute to the formation of viral protein. The nonparticulate interlamellar ribonucleic acid (1, 27, 48, 53) as well as ribosomes associated with lamellae (29-33) could possibly result in increased cytoplasmic synthesis of protein which is subsequently transported into the nucleus for the completion of virion "coating." Some annulate lamellae displayed attached or interlamellar free ribosomes in infected cells. These ribosomes were quite similar to the 150 A or F~6. 6. An LLC-MK2 cell 3 hours p.i. A large nucleus (N) displays peripheral condensed chromatin. The annulate lamellae (Al) on the left is contiguous with and perpendicular to the nuclear envelope. The 3 lamellae on the right are contiguous with and perpendicular to one dilated cisterna of rough endoplasmic reticulum (Rer). Lipid bodies (Lb) are present, x 30,000. FI6. 7. LLC-MK2 cell 1 hour p.i. Two cytoplasmic annulate lamellae (Al) are near the nucleus. The larger group consists of approximately 115 lamellae in the longitudinal, tangential, and en face planes of section. Some lamellae are contiguous with endoplasmic reticulum (arrows). An intranuclear annulate lamella (lal) is juxtaposed to the internal nuclear membrane. × 12,000. FI~. 8. An LLC-MK2 cell 120 hours p.i. Within this degenerating cell there are intranuclear annulate lamellae (Ial). Adjacent to the degenerating nucleolus (Ne) and nuclear inclusions (Ni) there is a translucent region containing several virus particles (Vp, arrows), x 15,000.
ANNULATE LAMELLAE IN SV30=INFECI'ED LLC-MK2 CELLS
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smaller granules associated with fibrils within annulate lamellae and nucleoli (32, 33) in gonadal cells of several lower species. Since it has been suggested that annulate lamellae act as a nuclear "controlling" mechanism in the cytoplasm (32, 33), viral genome could potentiate this process. On the other hand, the viral-nuclear D N A interaction or the toxicity of the virus per se (34) may mediate the production of increased numbers of annulate lamellae. Similarly, the toxic effects of certain chemicals appear to "induce" the formation of this organelle (20, 22, 23, 36, 46). Therefore, annulate lamellae may be associated with virus replication, viral target cell interaction, or both. Annulate lamellae probably have multiple functions, and these may differ depending upon the cell type. Since this organelle is, in fact, a constituent of many different types of cells, it appears to be quite ubiquitous.
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AFZELIUS,B. A., Z. Zellforsch. Mikroskop. Anat. 45, 660 (1957). ANCLA, M. and DEBRU×, J., Obstet. Gyneeol. 26, 23 (1965). BARER, R. S., JOSEPH, S. and MEEK, G. A., Proc. Roy. Soc. B152, 353 (1960). BINGGELI,M. F., J. Biophys. Biochem. Cytol. 5, 143 (1959). CAVAZOS,F., GREEN, J. A., HALL, D. G. and LUCAS, F. V., Am. J. Obstet. Gynecol. 99, 833 (1967). CHAMBERS,V. C. and WEISER, R. S., J. Cell Biol. 21, 133 (1964). CO~RIYGTON,D. and VOGT, P. K., J. Virol. 1, 400 (1967). ELLIOT,R. L. and ARHELCER, R., Arch. Pathol. 81, 2130 (1966). EPSTEIN, M. A., J. Biophys. Biochem. Cytol. 3, 567 (1957). - - - - ibid. 10, 153 (1961). EPSTEIN,M. A. and ACHONG, B. G., J, Natl. Cancer Inst. 34, 241 (1965). EVERINGHAM,J. W., J. Cell Biol. 37, 540 (1968). - - - - ibid. 37, 551 (1968). FONt, C. K. Y., 13ENSCrt, K. G. and HSIUNG, G. D., Virology 35, 297 (1968). FRASCA,J. M., AUERBACH,0., PARKS,V. R. and STOECKENIUS,W., ExptL Mol. Pathol. 6, 261 (1967). GROSS,B. G., or. Ultrastruct. Res. 14, 64 (1966). HARRISON,G. A., or. Ultrastruct. Res. 14, 158 (1966). HERTIG,A. T. and ADAMS, E. C., J. Cell Biol. 34, 647 (1967). HOFFERT,W. R., BATES, M. E. and CHEERER,F. S., Am. J. Hyg. 68, 15 (1958). HRUBAN,Z., SWIFT, H., DUNN, F. W. and LEwis, D. E., Lab. Invest. 14, 70 (1965). HRUBAN,Z., SWIFT, H. and RECHCIOL,M., or. Natl. Cancer Inst. 35, 459 (1965). HRImAN, Z., SWIFT, H. and SLESERS,A., Lab. Invest. 14, 1652 (1965). -Cancer Res. 25, 708 (1965). Hsu, W. S., Z. Zellforsch. Mikroskop. Anat. 58, 17 (1962). - - - - ibid. 58, 660 (1963). - - - - ibid. 82, 376 (1967).
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J. ULTRASTRUCTURERESEARCH30, 344-353 (1970)
Pathogenesis of Oncogenic Simian Adenoviruses VII. The Origin of Annulate Lamellae in LLC-MK2 Cells Infected with SV301,2 LEONARD P. MERKOW, ~ MALCOLM SLIFKIN, 3 MATIAS PARDO, AND NORBERT P. RAPOZA4
Departmentsof Experimental Pathology andMicrobiology, William H.Singer Memorial Research Institute of the Allegheny General Hospital, Pittsburgh, Pennsylvania 15212, and Department of Microbiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15213 Received June 9, 1969 LLC-MK2 cells were grown as monolayers, infected with the oncogenic simian adenovirus 30 (SV30), and harvested 1-192 hours post infection (p.i.). As early as 1 hour p.i., increased numbers of annulate lamellae were observed within the cytoplasm and nucleus of infected L L C - M K 2 cells. The cytoplasmic annulate lamellae were usually in close proximity to the nucleus and frequently contiguous with the outer nuclear envelope. Intertubular connections between parallel lamellae, electron dense particles consistent with ribosomes, and crystalloid bodies in contact with annulate lamellae were observed. The increased number of annulate lamellae over that observed in control cultures may be either a cellular response concomitant with virus replication or a secondary manifestation of viral toxicity. The formation of annulate lamellae appears to occur by way of at least two processes in SV30-associated cells. The presence of i n t r a n u c l e a r (12, 13, 24-26, 29, 30) a n d / o r i n t r a c y t o p l a s m i c a n n u l a t e lamellae has been described in m a n y n o r m a l (5,15-18, 49, 52, 56, 58, 59), altered (2, 42, 43), p r e n e o p l a s t i c (46, 47), o r neoplastic (4, 6, 8, 21, 28, 38, 39-41, 47, 48, 51, 55) h u m a n or a n i m a l cells. Since a n n u l a t e lamellae have been considered a specialized f o r m of e n d o p l a s m i c reticulum (3, 8, 10, 39) a n d quite frequently are contiguous with the latter, their m e c h a n i s m of f o r m a t i o n is of considerable interest. M o r e recently, n u m e r ous annulate lamellae have been observed in viral i n d u c e d neoplastic cells in vivo (45), p r e s u m a b l y noninfected cell lines in vitro (9, 10, 36, 37) a n d virus infected cell 1 Presented in part at the Fourth European Regional Conference on Electron Microscopy, Rome, Italy, September, 1968. 2 This investigation was supported in part by Grant AI-02535 from the National Institutes of Health. 3 Adjunct Assistant Professor at Pennsylvania State University. 4 Present address: Division of Biological Research, G. D. Searle and Company, Chicago, Illinois 60680.
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lines (7, 11, 34, 35). I n c r e a s e d n u m b e r s of a n n u l a t e lamellae have been e n c o u n t e r e d in L L C - M K 2 cells infected with the oncogenic simian a d e n o v i r u s 30 (SV30). This r e p o r t describes the origin a n d f o r m a t i o n of a n n u l a t e lamellae in L L C - M K 2 cells infected with SV30 in vitro. This process is c o m p a r e d with the m e c h a n i s m of f o r m a tion of a n n u l a t e lamellae in cells i n d u c e d b y the oncogenic simian a d e n o v i r u s 20 in
vivo (44). M A T E R I A L S A N D METHODS Simian adenovirus 30 obtained from a purified strain described by Hoffert, Bates, and Cheever (19) was used throughout this investigation. Its origin has been previously described (54). The cell line LLC-MK2, a derivative of rhesus monkey kidney, was grown in basal medium of Eagle (BME) containing 5 % calf serum and 1% glutamine. These cells were obtained from the American Type Culture Collection and two commercial sources (Flow Laboratories, Rockville, Maryland, and Microbiological Associates, Bethesda, Maryland). Two percent agamma calf serum was used in all cell cultures when virus was propagated or titrated. Virus stocks used in these investigations were prepared in the LLC-MK2 cell line of African green monkey kidney cells containing SV5 and SV40 antisera. These stocks were demonstrated to be free of SV40 virus and adeno-associated viruses by procedures previously described (54). Virus stocks were purified by centrifuging frozen and thawed specimens in the Spinco Model L ultracentrifuge. Preparative density gradient centrifugation with cesium chloride was used as a further step in purification (54). Electron microscopy. L L C - M K 2 cells were grown as monolayers in 32-ounce prescription bottles. These monolayers containing 108 to 2 x 108 cells were infected with SV30 at an input of 2 TCID~0 per cell. Cultures inoculated with BME alone served as controls and were processed in an identical manner. A t 1, 3, 6, 9, 24, 48, 72, 120, and 192 hours post infection (p.i.), L L C - M K 2 cells were dislodged from the glass surface with a rubber policeman and centrifuged in tubes at 2000 rpm in a Servall centrifuge for 2 minutes. The supernatant was decanted, and the cell pellet was immediately fixed with 1% phosphate-buffered osmium tetroxide at p H 7.4. The 1-2 ml of packed cells obtained from each of 2 bottles was then sequentially resuspended through a series of ethanols and centrifuged as above; the supernatants were decanted. The resultant dehydrated pellet of cells was treated with propylene oxide and then embedded in Epon 812. One-micron sections were prepared and stained with methylene blue. Masses of L L C - M K 2 cells were easily identifiable in these sections with the light microscope. Ultrathin sections displaying silver or gold interference colors were obtained with a Porter-Blum MT-1 or MT-2 ultramicrotome equipped with a glass knife. These ultrathin sections were stained with a 5 % solution of uranyl acetate followed by lead citrate. All ultrathin sections were then examined with a Philips 100B or EM300 electron microscope at 60 kV. RESULTS A s early as 1 h o u r p. i. a n d at all subsequent times, increased n u m b e r s of a n n u l a t e lamellae were o b s e r v e d within the c y t o p l a s m a n d nucleus of n u m e r o u s L L C - M K 2 cells i n o c u l a t e d in vitro with SV30 (Figs. 1-8). This increased n u m b e r of a n n u l a t e
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lamellae was most evident at low magnification, and frequently in a survey view one or more infected cells showed several individual stacks of annulate lamellae (Fig. 1). Cytoplasmic annulate lamellae were often in close proximity to the nucleus and frequently appeared intimately associated with the nuclear envelope (Figs. 1, 3-6). The number of double membranes or lamellae vary from 1 to 115 (Fig. 7). This organelle in a longitudinal plane of section displayed the characteristic periodic constrictions in register with its adjacent parallel membranes (Figs. 1-7). Perpendicular intertubular connections between parallel lamellae (Figs. 2 and 4), as well as an increased density of the matrix surrounding each lamella were observed (Figs. 2-4). Individual 150 A or smaller electron dense particles were unattached (free between lamellae) or attached to lamellae (Fig. 3). Annulate lamellae occasionally appeared simultaneously in several planes of section (Figs. 1 and 7). A lateral margin or end of annulate lamellae usually in the longitudinal plane of section was frequently contiguous with strands of smooth or rough endoplasmic reticulum (Figs. 1, 6, and 7). An en face plane of section revealed the annuli to consist of numerous microcylinders (subannuli). At an increased magnification, the annulate pores displayed an octagonal symmetry. The lateral margins of annulate lamellae frequently displayed bulbous dilatation (Figs. 1, 2, and 6). Clusters of scalloped lipid bodies with a variable affinity for osmium were often adjacent to and/or contiguous with annulate lamellae (Figs. 2, 4, and 6). Rarely, a honeycomb crystalloid body was in contact with annulate lamellae (Fig. 2). The presence of input intracellular virus particles was noted between 1 and 10 hours p.i. (Figs. 1 and 2). Nuclear projections were observed in infected cells and individual annulate lamellae rarely encircled these projections. In a fortuitous plane of section, direct continuity between stacks of annulate lamellae and the outer nuclear membrane was observed (Figs. 3, 5, and 6). Usually a stack of contiguous annulate lamellae was parallel to the nuclear envelope (Figs. 3 and 4). However, occasionally these attached annulate lamellae appeared either in an oblique (Fig. 5) or perpendicular (Fig. 6) plane of section to the nuclear envelope. The presence of intranuclear annulate lamellae was infrequently observed (Figs. 7 and 8). This type of annulate lamellae was similar in morphology to those observed in the cytoplasm and often was in contact with either the internal nuclear membrane and/or FIG. 1. LLC-MK2 cell 2 hours p.i. with SV30. Four stacks of annulate lamellae (A1) are in close proximity to the nucleus (N). The largest stack shows dilated vesicular (V) lateral ends (arrows). Two annulate lamellae are sectioned in a tangential plane and display pores. Virus particles (Vp, arrows) are adjacent to or within dilated cisternae. This and all subsequent photographs represent osmium-fixed, Epon-embedded material stained with uranyl acetate and lead citrate, x 12,000. FIG. 2. LLC-MK2 cell 3 hours p.i. The annulate lamellae (AI) display interlamellar granular material and tubular connections (arrows). Three lamellae are continuous with an egg-shaped, membranelimited crystalline body (arrows). The lateral ends of lamellae are dilated and contain ribosomes on their outer surface. Several scalloped lipid bodies (Lb) are in close proxmity or in contact with the annulate lamellae. A virus particle (Vp, arrow) is present, x 46,000.
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348
M~RKOW ST AL.
peripheral nuclear chromatin (Fig. 7). Organelles such as endoplasmic reticulum, the Golgi complex, and vacuoles did not differ considerably from those in control LLCM K 2 cell cultures. However, virus-infected cells did display the cytopathology associated with adenovirus infection (Fig. 8) whereas control cells did not. At 12 hours or later, intranuclear virus particles were first apparent. By 18 hours, crystals of newly synthesized SV30 were observed. The infrequent presence of annulate lamellae was observed in control L L C - M K 2 cells.
DISCUSSION Infection by SV30 in L L C - M K 2 cells provided an experimental model to study the formation of annulate lamellae in somatic mammalian cells. In these in vitro infected cells, annulate lamellae appear to form by way of a sequential delamination of individual lamellae on either side of the nuclear envelope. Annulate lamellae formed by this process are similar to that described by Harrison for seagull adrenal cortical cells (17). Therefore, origin of annulate lamellae may occur by way of a template or replicative process with delamination (shedding) as the mechanism of separation from the nuclear envelope. The mechanism regulating either template "availability" or delamination is unknown. However, since annulate lamellae were seen (although infrequently) in control L L C - M K 2 cells, it is tempting to speculate that in this instance the virus might act by augmenting the translation or transcription of focal portions of the host cell genome. In contrast, annulate lamellae appear to develop by way of a slightly different and potentially more complex mechanism in SV20 or SV30 induced neoplastic cells in vivo (44, 45). In the in vivo model, an association between annulate lamellae, endoplasmic reticulum, and the external membrane of the nuclear envelope appears to exist. Subsequent to in vivo infection with SV20 (44) portions of cisternal smooth endoplasmic reticulum adjacent to the nucleus are arrayed so as to suggest formation of periodic constrictions in register. As this occurs, interlamellar (intertubular) connections appear between these focally constricted cisternae of endoplasmic reticulum FIG. 3. LLC-MK2 cell 9 hours p.i. Continuity exists between this annulate lamellae (Al)and the outer nuclear envelope. Some interlamellar granularity is present between the 6 lamellae. Several 150 ~ dense particles are either free or between or attached to lamellae (arrows) and comparable to ribosomes attached to the external nuclear envelope (arrows). x 50,000. FIG. 4. LLC-MK2 cell 3 hours p.i. A stack of three lamellae are adjacent to the nucleus (N). Note the similar morphology of the nuclear envelope subjacent to the most proximal portion of the annulate lamellae (AI, between arrows). The interlamellar granularity and tubular connections are present (arrows). x 29,000. Fit. 5. LLC-MK2 cell 3 hours p.i. Annulate lamellae (Al) are not parallel to the nuclear envelope. Three lamellae are in continuity with the outer nuclear envelope. Each lamella makes its own contact with the nucleus (N). x 25,000.
w~
~Z
350
MERKOW ET AL.
(44: Figs. 11-14). This mechanism is similar to the process of coalescence of vesicles derived and budded off from the nuclear membrane in alligator and Necturus oocytes as described by Harrison (17) and Kessel (28), respectively. Therefore, annulate lamellae appear to form by way of at least two processes in cells containing simian adenovirus virions. Fong et al. (14) and Tyrrell et al. (57) did not report the presence of annulate lamellae in simian adenovirus (SV15, SV17) infected primary monkey kidney cells. This could be due to the oncogenicity of SV30. Furthermore, the use of a continuous cell line, as opposed to a primary cell line (14, 57) may play a role in the incidence of this organelle. K i m and Boatman (34) suggested that annulate lamellae may be involved in the synthesis of viral material since this organelle was observed by them only in rubellainfected L L C - M K 2 cells. Our observation of annulate lamellae in uninfected LLCM K 2 cells could be attributed to our extensive ultrastructural examination of control cells. Cytoplasmic crystals have been reported in cells infected with human adenovirus (50) and rubella virus (34). Continuity between these crystals and annulate lamellae has been reported (34) and observed in the present investigation. The presence of lipid bodies in close proximity to annulate lamellae has been reported (34, 52). Similarly, lipid bodies were observed in our experimental model and may play some role in the function(s) of annulate lamellae. The presence of annulate lamellae has been reported in conditions of altered protein synthesis (20, 22, 23, 36); accordingly, this organelle has been implicated as having some role in protein synthesis (34, 52, 56). For this reason, it is conceivable that their increased number and size contribute to the formation of viral protein. The nonparticulate interlamellar ribonucleic acid (1, 27, 48, 53) as well as ribosomes associated with lamellae (29-33) could possibly result in increased cytoplasmic synthesis of protein which is subsequently transported into the nucleus for the completion of virion "coating." Some annulate lamellae displayed attached or interlamellar free ribosomes in infected cells. These ribosomes were quite similar to the 150 A or F~6. 6. An LLC-MK2 cell 3 hours p.i. A large nucleus (N) displays peripheral condensed chromatin. The annulate lamellae (Al) on the left is contiguous with and perpendicular to the nuclear envelope. The 3 lamellae on the right are contiguous with and perpendicular to one dilated cisterna of rough endoplasmic reticulum (Rer). Lipid bodies (Lb) are present, x 30,000. FI6. 7. LLC-MK2 cell 1 hour p.i. Two cytoplasmic annulate lamellae (Al) are near the nucleus. The larger group consists of approximately 115 lamellae in the longitudinal, tangential, and en face planes of section. Some lamellae are contiguous with endoplasmic reticulum (arrows). An intranuclear annulate lamella (lal) is juxtaposed to the internal nuclear membrane. × 12,000. FI~. 8. An LLC-MK2 cell 120 hours p.i. Within this degenerating cell there are intranuclear annulate lamellae (Ial). Adjacent to the degenerating nucleolus (Ne) and nuclear inclusions (Ni) there is a translucent region containing several virus particles (Vp, arrows), x 15,000.
ANNULATE LAMELLAE IN SV30=INFECI'ED LLC-MK2 CELLS
23
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smaller granules associated with fibrils within annulate lamellae and nucleoli (32, 33) in gonadal cells of several lower species. Since it has been suggested that annulate lamellae act as a nuclear "controlling" mechanism in the cytoplasm (32, 33), viral genome could potentiate this process. On the other hand, the viral-nuclear D N A interaction or the toxicity of the virus per se (34) may mediate the production of increased numbers of annulate lamellae. Similarly, the toxic effects of certain chemicals appear to "induce" the formation of this organelle (20, 22, 23, 36, 46). Therefore, annulate lamellae may be associated with virus replication, viral target cell interaction, or both. Annulate lamellae probably have multiple functions, and these may differ depending upon the cell type. Since this organelle is, in fact, a constituent of many different types of cells, it appears to be quite ubiquitous.
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