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Crystalline arrangement of a picornavirus infecting Drosophila melanogaster cell culture schneider cell line No. 2
Micron, Voi. 13, No. 1, pp. 23-33, 1982 Printed in Great Britain
0047-7206/82/010023-11503.00/0 Pergamon Press Ltd.
CRYSTALLINE A R R A N G E M E N T OF A PICORNAVIRUS INFECTING DROSOPHILA MELANOGASTER CELL CULTURE SCHNEIDER CELL LINE No. 2 JOSEPH SECKBACH* a n d MARTIN KESSEL Department of Membrane and Ultrastructure Research, Hebrew University, Hadassah Medical School, Jerusalem, Israel 91000
(Received 14 May 1981; received for publication 3 June 1981) Al~traet--Drosophila melanogaster embryonic Schneider cell line No. 2 cultured in standard Schneider's medium revealed aggregated particles of picornavirus (PCV) clusters located almost exclusively in cytoplasmic vesicles. The PCV particle is about 25 nm in diameter and aggregates are found in graduate degrees of crystallinity. The PCV aggregates are visible as small or larger membrane bound clusters (ca. 2.2 t~m in diameter). This limiting membrane is sometimes shown to be associated with cytoplasmic membranes such as endoplasmic reticulum. Membranes are also observed sandwiching single rows of PCV particles (viral lamellae). Membrane thickness is about 7 nm and is found in various locations associated with the particles. In general the degree of the crystallinity extends from scattered free particles to well ordered crystalline arrays. In this communication we propose a possible stepwise model in the formation of the PCV crystals. Index key words: Drosophila, picornaviruses, crystalline vesicle, electron microscopy.
INTRODUCTION It is well known that Drosophila melanogaster harbors different viruses detected both in cell lines and in tissues of wild type flies (Gateff, 1978; Haars et al., 1980; Plus et al., 1975, Plus, 1978, 1980; Teninges et al., 1979, 1979a). A high percentage of cells from the above D. melanogaster sources have been found to be infected with viruses (Gateff, 1978; Plus, 1978) that vary in shape, e.g., from a spherical or round particle to an icosahedron with surface projections, and size ranges from 25 to 72 nm in diameter (Gateff, 1978; Plus et al., 1975, Plus, 1978; Teninges, 1979a). The occurrence of infected viral particles may evolve accidentally in the cell line or may pre-exist in flies in a non-pathogenic form. It has been suggested that fetal bovine serum used in culture media may induce pathogenicity through either an agent present in the serum which acts upon an intracellular masked viral state and contaminates the culture or that the virus is actually present in the serum (Plus, 1978). Among these viruses of D. melanogaster are the three endemic types of picornaviruses, DPV,
DAV, and DCV (Plus et al., 1975; Teninges et al., 1979). This report is concerned with picornavirus particles and their crystalline formation in a D. melanogaster cell line. These particles appear to be identical to DPV and DAV strains of Drosophila picornaviruses (Plus, personal communication). MATERIALS AND METHODS The original embryonic cell cultures of D. melanogaster Schneider Cell line No. 2 were obtained from Dr. R. Sederoff, the Univelsity of Oregon. The cell lines were cultured in modified Schneider medium at 24°C (Sederoff and Clynes, 1974). Fixation of the samples and electron microscopic observation was according to the following standard procedures. Cells were fixed in 2 ~ glutaraldehyde in 0. I M phosphate buffer, pH 6.7 on ice at 4°C for l hr; postfixed with 1 OsO4 (phosphate buffer on ice for lhr). Dehydration was in a series of increasing concentrations of EtOH. Sections were post stained with lead hydroxide and uranyl acetate. Embedded blocks were sectioned with a LKB ultratome-III and observed either in a Philips EM 300 or EM 400 operating at 60 or 80 kV. Tilting series
*Dr. Seckbach's present address: School for Overseas Students, Hebrew University, Mount Scopus, Jerusalem, Israel, 91905.
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Joseph Seckbach and Martin Kessel
were recorded using the Philips eucentric goniometer stage mounted on the EM-400. Optical diffractograms were recorded directly from the electron microscope negative using a 2 mW He Laser source. RESULTS Figure 1 depicts a triangular shaped cell of D. melanogaster which exhibits several membrane-bound vesicles (0.35-1.0/zm in diameter) containing loosely packed picornavirus particles in a non-crystalline arrangement. This central cell (Fig. 1) is bordered by a typical neighboring cell showing abundant rough endoplasmic reticulum (ER), while the lack of crystalline bodies near the cell periphery is noticeable. Both this cell and the central cell are limited by a double membrane system which encloses picornavirus (PCV) particles in single rows. More viral particles appear sandwiched by the external membrane of the vesicle containing cell. A similar segment of a single row of viruses resembling a necklace is seen located between the cell membrane and a membrane from lysed adjacent cellular contents. This sandwich phenomenon might occur as a result of cell lysis and the free particles being trapped between the cell and the lysed membranes. Besides the nine viral containing bodies, we see in the central cell, the cytoplasm contains mitochondria, lysosome-like vesicles and ribosomes. Figure 2 shows a higher magnification of some of the viral vesicles seen in Fig. 1. Each of the viral vesicles has a diameter of about 1 tzm and we observe a small, fine granular area designated viroplasm. The cytoplasm surrounding the aggregates contains particles of similar size and shape to those within the vesicles in addition to free ribosomes. Although the viral clusters are comprised mainly of irregularly arranged closely packed particles in one ot the vesicles we observe short rows of inter-connected viruses showing initial crystallinity. The same vesicle also shows segments of membranes associated with the particles. Figure 3 shows part of a Drosophila cell containing a large multi-lobed viral vesicle (4.35 × 5.85 tzm) packed with picornaviruses. There is as yet little evidence of extensive crystallinity except in small patches dispersed within the cluster. These patches appear to be more concentrated in a localized region. Adjacent to the PCV cluster is a conspicuous nucleus and its nucleolus. Lytic fragments of an extra-
cellular neighboring region (lower left) exhibit attachment of a row of virus particles to the intact cell membrane similar to that seen in Fig. 1. Packing of the multilobed-viral body is considered as mainly close packed and noncrystalline in appearance with, however, early evidence of initial steps of crystallization. At higher magnification (Fig. 4) it is possible to detect the following viral configurations: helical or zig zag ribbons, patches of hexagonally arrayed virus particles and non-crystalline aggregates; as well as short segments of membranes with the viruses clearly attached on both sides, or sandwiched between a double membrane. A different view of the organization of intravesicular PCV particles is demonstrated in Fig. 5. This micrograph shows more extensive crystallinity within the large PCV vesicle, viral lamellae and myelin-like bodies. Vesicles also contain areas of viroplasm which are scattered throughout, and embedded amongst, the viral particles. In addition to the dominant hexagonal packing of viral particles some areas of square packing are also present. The vesicle membrane to which viruses are attached exhibits areas of bulging. Figure 6 shows a higher magnification of an area from Fig. 5. This section leads to an impression that crystallization has begun from a number of different foci resulting in many small crystalline patches which appear to change orientation as crystal growth is forced to stop due to lack of physical space. The striated appearance of some of the particle rows within the vesicle showing a periodicity of roughly 15 nm, seem to be a consequence of the change of direction within the plane of section of a row of viral particles thus presenting a different view as if by tilting. Some bundles of PCV particle rows may also take on a parallel curved appearance. In addition, small areas of hexagonal arrays are also evident. In the change from face to striated view, some of the particles exhibit a ring structure, i.e. having an electron transparent center surrounded by a darker periphery. The highly developed crystalline vesicle in Fig. 7 is bordered by one to three layers of viral lamellae which also show several discontinuities. This conspicuous vesicle which extends to 44 ~m in diameter, contains closely packed PCV particles and shows several areas of highly organized membrane associated viruses. A number of classes of viral aggregates are present
Pieornavirus Crystalline in Drosophila Cell Line
25
Fig. 1. The central Drosophila cell contains membranes bound vesicles (VV), each containing loosely packed picornavirai particles. Pairs of membranous systems enclosing a single row of viruses, i.e. viral lamellae (VL) border both this cell and the upper neighbouring cell. This adjacent cell possesses rough endoplasmic reticulum (ER) membranes but in the area shown lacks viral vesicles. A 'necklace' composed of a short segment of a row of single particles within a double membrane complex (VL) is attached to the lower border of the cell and is in contact with the lysed cellular contents (arrows) M, mitochondria; Ly, lysosome-like vesicle, x 24,450; bar= 1 t~m.
within the tightly packed central non-crystalline region. There are single m e m b r a n e - P C V particle segments with a helical appearance (l) there are viral lameilae (2); two distinct highly ordered crystalline areas are, respectively m e m b r a n e associated (3) and membrane-free (4). Optical diffraction patterns from the marked areas in the micrograph (Fig. 8) show a clear hexagonal lattice of the non-lamellar region (Fig. 8c) and a less clear indication of this lattice in the lamellar region (Fig. 8b). In the latter diffraction pattern the spacing between the lamellae is
clearly shown by a pair of strong reflections. At this higher magnification (Fig. 8) the membrane associated crystalline viruses are seen to have a discontinuous appearance which appears to be the result of the orientation of the viral crystal to the plane of the section. The notion that the different views of virus organization seen in Fig. 8 arise from different planes of section is indeed verified in the tilting pair of micrographs presented in Figs. 9(a) and 9(b) in which the transformation from a viral lamellar appearance to a non-lamellar crystalline array
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Joseph Seckbach and Martin Kessel
Fig. 2. A higher magnification of the central area from the previous cell showing details of the viral packing in the membrane limited vesicles. Irregular segments of membrane bundles (Mb) and finely granulated areas of viroplasm (Vp) are scattered between the picornavirus vesicles. The cytoplasm surrounding these vesicles contains ribosomes and particles similar in size and shape to those enclosed inside the clusters. Short, straight and curved rows of interconnected viral particles are visible within the viral vesicle on the right of the micrograph. The vesicle displays the following features in addition: (a) the membrane enveloping the viral vesicle (arrow); (b) small area of hexagonally arranged viruses (double arrows) and (c) loose membrane fragments dispersed within the vesicle, x 60,350; bar= 1 t~m. and vice versa is clearly seen. The non-lamellar crystalline array therefore appears to result from a section parallel to and between the lamellae, whereas the very small change in appearance of areas lying close to the tilt axis shows that the lamellae extend throughout the plane of section. Figure 10 shows a viral vesicle which is almost totally crystalline and is comprised almost exc!usively of parallel rows of viruses. Not all the viruses are membrane bound and in some locations there appear to be more than one pair of membranes. In such cases the membranes are twisted braid-like a r o u n d each other and are usually shorter in length than membranes with viruses attached. The fact that this single section presents three different views of the viral rows within an apparent close packed arrangement shows once again that the three dimensional arrangement of the crystal is comprised of crystalline elements in different orientation to each other.
DISCUSSION The presence of the viral particles described in this study have been established purely on morphological grounds, particle shape, size, membrane-virus association, loci of crystallization and comparison of these characteristics with published data. Drosophila picornaviruses are more 'specialized' to insects than other insect viruses (Teninges et al., 1979) and have been found ubiquitously (Plus et al., 1975). The characteristics of Drosophila melanogaster picornaviruses are: R N A content, size of the particle 20-30nm in diameter, resistance to pH3 and lipid solvents, and intracytoplasmic localization (Plus et al., 1975, 1976). In Figs. 5, 7, 8, 9 and 10 there is evidence of a strong m e m b r a n e viral particle association in which two membranes enclose a row o f virus particles (viral lamellae) a step which may possibly be involved in the formation of the virus crystals. Similar patterns of two membranes sandwiching a
Picornavirus Crystalline in Drosophila Coil Line
Figs. 3 and 4. Figure legends on p. 29.
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Fig. 5. Part of a vesicle containing numerous crystalline areas as well as short segments of viral lamellae (VL). A bulging area of the vesicle is noticeable (Bu). Fingerprint-like areas of granulated viroplasm (Vp) are dispersed within the vesicle. In addition the dominant hexagonal packing, an area of square packing of virus particles is also present (Sq). My, myelin-like figure, x 31,350; bar= l#m. Fig. 6. A higher magnification of an area from Fig. 5 emphasizes the initial crystallization from a number of different foci. The small crystalline patches appear to change orientation as crystal growth is forced to stop due to lack of physical space. The striated appearance of the particle rows, showing a periodicity of roughly 15 nm, seems to be a consequence of the change within the plane of section of a row of particles thus presenting a different view as if by tilting. Some rows appear as parallel and curved hexagonal arrays. Some particles exhibit an electron transparent central core surrounded by an electron dense periphery (R). x 51,300; bar=0.5 ~m.
single row of viruses have been observed in other viral systems, e.g., in Simian virus 40 (Oshiro et al., 1967) a n d in Theiler's murine encephalomyelitis virus ( F r i e d m a n n a n d Lipton, 1980). In our present observations, all degrees of the virus aggregation have been observed within vesicles within the cytoplasma, while other virallike particles in Drosophila cell cultures crystallized within the nucleus (Gateff, 1978 ; Seckbach, unpublished data).
Our electron micrographs exhibit several appearances of viral particles within virus containing vesicles: a m o r p h o u s packing; paracrystalline packing; association of PCV with m e m b r a n e s ; (Figs. 2, 3, 5, 7 and 10) and well ordered hexagonal crystallinity. It seems to us that the m e m b r a n e system has a role to play in the formation of the crystal. We therefore suggest the following stepwise PCV crystallization: viruses become attached to membranes in
Picornavirus Crystalline in Drosophila Coil Line
Fig. 7. A highly developed crystalline vesicle is bordered by one to three layers of viral lamellae which show several discontinuities. This vesicle contains closely packed picornaviruses and possesses several groups of highly organized intravesicular, membrane bound viruses. (1) single membrane-viral particle association with a helical appearance, (2) the viral lamellae (VL) in the peripheral areas showing double membrane loaded w,th single rows of particles, (3) membrane associated viruses and (4) membrane-free crystalline ordered viruses. × 31,350; bar= I/~m.
Legends to page 27
Fig. 3. A central multilobed membrane-bound vesicle packed with picornaviruses is adjacent to a conspicuous nucleus (N) and its nucleolus (Nu). Lytic fragments in an extracellular neighbouring region (arrow) exhibit attachment of a row of viral particles to the intact cell membrane (VL) and also of viruses grouped into small crystalloids (C). × 20,250; bar= 1 /~m. Fig. 4. A higher magnification view of the central zone in the multilobed vesicle shown in Fig. 3 demonstrates the following: (a) short membrane segments with viruses attached on both sides or sandwiched between a double membrane (VL); (b) helical appearance of the membrane associated viruses (h) and (c) small hexagonal arrays of particles (hx) scattered amongst the loosely packed viruses, x 62,700; bar= 0.5 t~m.
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Joseph Seckbach and Martin Kessel
Figs. 8 (a) -(c). A higher magnification of an area from Fig. 7 showing optical diffraction patterns from lamellar (8b) and non-lamellar (8c) regions of the PCV crystal. The hexagonal packing of the nonlamellar region is very clearly seen both in the micrograph and the optical pattern. In the lamellar region the most dominant feature of both the micrograph and the diffraction pattern is the pair of strong reflections which arise from the interlamellar spacing, x 66,700; bar=0.5 p.m.
a helical fashion; the virus associated membranes aggregate to form a stack of several layers or virus lamellae, enclosing a row of viral particles between each pair of membranes; membranevirus association forms concentric layers of crystalline arrays; the final stage is the highly organized crystalline body with perhaps an eventual disintegration of the membranes. Most noticeable are helical arrangements of virus particles attached to short fragments of membrane (Figs. 3, 4 and 7). These structures resemble the zig-zag patterns of helical ribosomes in non-membrane bound ribosomal crystals observed in the cytoplasm of prokaryotic and eukaryotic ceils. For example similar helices were observed in growing cells of E. coli in the presence of vinblastine sulfate (Kingsbury et al., 1970). However, the PCV particles in the present study differ basically from ribosomal crystalline units observed in tetrameric sheets in lizard oocytes (Unwin and Taddei, 1977) and the hexagonally packed ribosome helices in Entarnoeba (Lake and Slayter, 1972). In these
crystal-membrane complexes of ribosomal aggregates, views in transverse section show two layers of endoplasmic reticulum enclosing double rows of ribosomes (Hubert, 1977; Unwin, 1979; Unwin and Taddei, 1977), whereas in our case a single row of virus particles is sandwiched between two membranes (Figs. 5, 7, 8, 9 and 10). The unusual ribosomal lamellar inclusions observed by Weise et al. (t980) observed in lymphocytes appear similar to our observations in that a single row of particles is sandwiched between two sheets of lamellae. However, the inclusions are not enclosed within vesicles and we have never observed the tubular arrangement of the virus lameilae as seen in the lymphocytes. The crystalline arrays of viruses described are not unique to D. melanogaster since other virus crystals have also been shown to be arranged as hexagonal or square arrays such as crystals of artichoke mottle crinkle virus (Russo et al., 1968). The wheat curl mite Aceria tulipae also exhibits a fully crystalline hexagonal pattern of brome mosaic virus whose particle size is similar
Picornavirus Crystalline in Drosophila Cell Line
Fig. 9 (a) and (b). Figure legends on p. 33.
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Fig. 10. This vesicle exhibits various types of highly ordered arrays of picornaviruses which mostly appear to be line in parallel rows. These rows are interspersed with twisted bundles of braid-like and nonviral containing membranes.
to PCV (Paliwal, t972). These observations suggest that the crystallization of insect viruses is a c o m m o n a n d related process. I n summary, we may conclude that there is an existence of a gradual assembly process of PCV aggregates m a t u r i n g into the virus crystal and that the PCV particles most closely resemble the DPV a n d D P A strains both belonging systematically to the class Picornaviridae.
Acknowledgements--The authors wish to express their appreciation to Dr. Ira S. Hammerman for initiation of this research while at Bar-Ilan University. We wish to thank Mrs. J. Hanania and Mrs. S. Kitov for their able technical assistance. Special thanks are due to Dr.
Nadine Plus for her valuable remarks and evaluations concerning the observations presented here prior to publication. This investigation was supported by the Israel-U.S. Binational Research Foundation. REFERENCES Friedmann, A. and Lipton, H. L., 1980. Replication of Theiler's murine encephalomyelitis viruses in BHK 21 Cells: an electron microscopic study. Virology, 101: 389-398. Gateff, E., 1978.The genetics and epigenetics of neoplasms in Drosophila. Biol. Rev., 53: 123-168. Haars, R., Zehtgraf, H., Gateff, E., and Bautz, F. A., 1980. Evidence for endogenous reovirus-like particles in a tissue culture cell line from Drosophila melanogaster. Virology, 101 : 124-130.
Picornavirus Crystalline in Drosophila Cell Line Hubert, J., 1977. Effets d'un sejour a une temperature de 6°C sur l'ultrastructure des ovaires du lezard lacerta vivipara Jacquin. Archs Anat. microsc., 66: 37-52. Kingsbury, E. W., Nauman, R. K., Morgan, R. S., and Voelz, H., 1970. Structure and function of ribosomal helices in Escherichia coli. In: Societ6 de Microscopie Electronique, Paris. lmprim6 en France. p. 69. Lake, J. A. and Slayter, H. S., 1972. Three-dimensional structure of the cromatoid body helix of Entamoeba invadens. J. tool. Biol., 66: 271-282. Oshiro, L. S., Rose, H. M., Morgan, C., and Hsu, K. C., 1967. Electron microscopic study of the development of Simian virus 40 by use of ferritin-labeled antibodies. J. Virol., 1: 384-399. Paliwal, Y. C., 1972. Brome mosaic virus infection in the wheat curl mite Aceria tulipae, a nonvector of the virus. J. Invert. Path. 20: 288-302. Plus, N., 1980. Further studies on the origin of the endogenous viruses of Drosophila melanogaster cell lines. In: Invertebrate Systems In Vitro, Kurstak, Maramorosch and Dubendorfer (eds). Elsevier, North-Holland Biomedical Press, 436-439. Plus, N., 1978. Endogenous viruses of Drosophila melanogaster cell lines: Their frequency, identification and origin. In Vitro, 14: 1015-1021. Plus, N. G., Croizier, Veyrunes, C., and David, J., 1976. A comparison of buoyant density and polypeptides of Drosophila P, C and A viruses, lntervirology, 7: 346-350.
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Plus, N. G., Croizier, Jousset, F.-X., and David, J., 1975. Picornaviruses of laboratory and wild Drosophila melanogaster Geographical distribution and serotypic composition. Arms Microbiol. (Inst. Pasteur) 126A: 107-117. Russo, M., Martelli, G. P., and Quacquarelli, A., 1968. Studies on the agent of artichoke mottled crinkle. IV. lntracellular localization of the virus. Virology, 34: 679-693. Sederoff, R. and Clynes, R., 1974. A modified medium for culture of Drosophila cells. Drosophila Inf. Service Newsletter, 51 : 153-155. Teninges, D., Ohanessian, A., Richard-Molard, C., and Contamine, d., 1979a. Contamination and persistent infection of Drosophila cell lines by reovirus type particles. In Vitro, 15: 425-428. Teninges, D., Ohanessian, A., Richard-Molard, C., and Contamine, C., 1979. Isolation and biological properties of Drosophila X virus. J. gen. Virol. 42: 241-254. Unwin, P. N. T. and Taddei, C., 1977. Packing of ribosomes in crystals from the lizard Lacerta sicula. J. mol. Biol., 114: 491-506. Unwin, P. N. T., 1979. Attachment of ribosome crystals to intracellular membranes. J. mol. Biol., 132: 69-84. Weise, R. W., Rozek, R., and Palutke, M., 1980. Unusual ribosomal lamellar inclusions in lymphocytes. Micron, 11: 127-135.
Legends to page 31 Figs. 9. (a) and 9 (b) are from a tilt series of micrographs recorded at +45 ° and - 4 5 ° and clearly show a number of changes occurring in the appearance of non-attached and membrane attached picornaviruses. In regions where the membranes and their associated viruses lie parallel to the tilt axis in Fig. 9 (a) the same region in Fig. 9 (b) now appears as a non-lamellar viral array. Similarly in non-lamellar areas in Fig. 9 (a), in Fig. 9 (b) the viral lamellar now appear in the parallel configuration. Viral lamellae lying close to the tilt axis hardly change their appearance at all. x 66,700; bar=0.5 t~m.
0047-7206/82/010023-11503.00/0 Pergamon Press Ltd.
CRYSTALLINE A R R A N G E M E N T OF A PICORNAVIRUS INFECTING DROSOPHILA MELANOGASTER CELL CULTURE SCHNEIDER CELL LINE No. 2 JOSEPH SECKBACH* a n d MARTIN KESSEL Department of Membrane and Ultrastructure Research, Hebrew University, Hadassah Medical School, Jerusalem, Israel 91000
(Received 14 May 1981; received for publication 3 June 1981) Al~traet--Drosophila melanogaster embryonic Schneider cell line No. 2 cultured in standard Schneider's medium revealed aggregated particles of picornavirus (PCV) clusters located almost exclusively in cytoplasmic vesicles. The PCV particle is about 25 nm in diameter and aggregates are found in graduate degrees of crystallinity. The PCV aggregates are visible as small or larger membrane bound clusters (ca. 2.2 t~m in diameter). This limiting membrane is sometimes shown to be associated with cytoplasmic membranes such as endoplasmic reticulum. Membranes are also observed sandwiching single rows of PCV particles (viral lamellae). Membrane thickness is about 7 nm and is found in various locations associated with the particles. In general the degree of the crystallinity extends from scattered free particles to well ordered crystalline arrays. In this communication we propose a possible stepwise model in the formation of the PCV crystals. Index key words: Drosophila, picornaviruses, crystalline vesicle, electron microscopy.
INTRODUCTION It is well known that Drosophila melanogaster harbors different viruses detected both in cell lines and in tissues of wild type flies (Gateff, 1978; Haars et al., 1980; Plus et al., 1975, Plus, 1978, 1980; Teninges et al., 1979, 1979a). A high percentage of cells from the above D. melanogaster sources have been found to be infected with viruses (Gateff, 1978; Plus, 1978) that vary in shape, e.g., from a spherical or round particle to an icosahedron with surface projections, and size ranges from 25 to 72 nm in diameter (Gateff, 1978; Plus et al., 1975, Plus, 1978; Teninges, 1979a). The occurrence of infected viral particles may evolve accidentally in the cell line or may pre-exist in flies in a non-pathogenic form. It has been suggested that fetal bovine serum used in culture media may induce pathogenicity through either an agent present in the serum which acts upon an intracellular masked viral state and contaminates the culture or that the virus is actually present in the serum (Plus, 1978). Among these viruses of D. melanogaster are the three endemic types of picornaviruses, DPV,
DAV, and DCV (Plus et al., 1975; Teninges et al., 1979). This report is concerned with picornavirus particles and their crystalline formation in a D. melanogaster cell line. These particles appear to be identical to DPV and DAV strains of Drosophila picornaviruses (Plus, personal communication). MATERIALS AND METHODS The original embryonic cell cultures of D. melanogaster Schneider Cell line No. 2 were obtained from Dr. R. Sederoff, the Univelsity of Oregon. The cell lines were cultured in modified Schneider medium at 24°C (Sederoff and Clynes, 1974). Fixation of the samples and electron microscopic observation was according to the following standard procedures. Cells were fixed in 2 ~ glutaraldehyde in 0. I M phosphate buffer, pH 6.7 on ice at 4°C for l hr; postfixed with 1 OsO4 (phosphate buffer on ice for lhr). Dehydration was in a series of increasing concentrations of EtOH. Sections were post stained with lead hydroxide and uranyl acetate. Embedded blocks were sectioned with a LKB ultratome-III and observed either in a Philips EM 300 or EM 400 operating at 60 or 80 kV. Tilting series
*Dr. Seckbach's present address: School for Overseas Students, Hebrew University, Mount Scopus, Jerusalem, Israel, 91905.
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Joseph Seckbach and Martin Kessel
were recorded using the Philips eucentric goniometer stage mounted on the EM-400. Optical diffractograms were recorded directly from the electron microscope negative using a 2 mW He Laser source. RESULTS Figure 1 depicts a triangular shaped cell of D. melanogaster which exhibits several membrane-bound vesicles (0.35-1.0/zm in diameter) containing loosely packed picornavirus particles in a non-crystalline arrangement. This central cell (Fig. 1) is bordered by a typical neighboring cell showing abundant rough endoplasmic reticulum (ER), while the lack of crystalline bodies near the cell periphery is noticeable. Both this cell and the central cell are limited by a double membrane system which encloses picornavirus (PCV) particles in single rows. More viral particles appear sandwiched by the external membrane of the vesicle containing cell. A similar segment of a single row of viruses resembling a necklace is seen located between the cell membrane and a membrane from lysed adjacent cellular contents. This sandwich phenomenon might occur as a result of cell lysis and the free particles being trapped between the cell and the lysed membranes. Besides the nine viral containing bodies, we see in the central cell, the cytoplasm contains mitochondria, lysosome-like vesicles and ribosomes. Figure 2 shows a higher magnification of some of the viral vesicles seen in Fig. 1. Each of the viral vesicles has a diameter of about 1 tzm and we observe a small, fine granular area designated viroplasm. The cytoplasm surrounding the aggregates contains particles of similar size and shape to those within the vesicles in addition to free ribosomes. Although the viral clusters are comprised mainly of irregularly arranged closely packed particles in one ot the vesicles we observe short rows of inter-connected viruses showing initial crystallinity. The same vesicle also shows segments of membranes associated with the particles. Figure 3 shows part of a Drosophila cell containing a large multi-lobed viral vesicle (4.35 × 5.85 tzm) packed with picornaviruses. There is as yet little evidence of extensive crystallinity except in small patches dispersed within the cluster. These patches appear to be more concentrated in a localized region. Adjacent to the PCV cluster is a conspicuous nucleus and its nucleolus. Lytic fragments of an extra-
cellular neighboring region (lower left) exhibit attachment of a row of virus particles to the intact cell membrane similar to that seen in Fig. 1. Packing of the multilobed-viral body is considered as mainly close packed and noncrystalline in appearance with, however, early evidence of initial steps of crystallization. At higher magnification (Fig. 4) it is possible to detect the following viral configurations: helical or zig zag ribbons, patches of hexagonally arrayed virus particles and non-crystalline aggregates; as well as short segments of membranes with the viruses clearly attached on both sides, or sandwiched between a double membrane. A different view of the organization of intravesicular PCV particles is demonstrated in Fig. 5. This micrograph shows more extensive crystallinity within the large PCV vesicle, viral lamellae and myelin-like bodies. Vesicles also contain areas of viroplasm which are scattered throughout, and embedded amongst, the viral particles. In addition to the dominant hexagonal packing of viral particles some areas of square packing are also present. The vesicle membrane to which viruses are attached exhibits areas of bulging. Figure 6 shows a higher magnification of an area from Fig. 5. This section leads to an impression that crystallization has begun from a number of different foci resulting in many small crystalline patches which appear to change orientation as crystal growth is forced to stop due to lack of physical space. The striated appearance of some of the particle rows within the vesicle showing a periodicity of roughly 15 nm, seem to be a consequence of the change of direction within the plane of section of a row of viral particles thus presenting a different view as if by tilting. Some bundles of PCV particle rows may also take on a parallel curved appearance. In addition, small areas of hexagonal arrays are also evident. In the change from face to striated view, some of the particles exhibit a ring structure, i.e. having an electron transparent center surrounded by a darker periphery. The highly developed crystalline vesicle in Fig. 7 is bordered by one to three layers of viral lamellae which also show several discontinuities. This conspicuous vesicle which extends to 44 ~m in diameter, contains closely packed PCV particles and shows several areas of highly organized membrane associated viruses. A number of classes of viral aggregates are present
Pieornavirus Crystalline in Drosophila Cell Line
25
Fig. 1. The central Drosophila cell contains membranes bound vesicles (VV), each containing loosely packed picornavirai particles. Pairs of membranous systems enclosing a single row of viruses, i.e. viral lamellae (VL) border both this cell and the upper neighbouring cell. This adjacent cell possesses rough endoplasmic reticulum (ER) membranes but in the area shown lacks viral vesicles. A 'necklace' composed of a short segment of a row of single particles within a double membrane complex (VL) is attached to the lower border of the cell and is in contact with the lysed cellular contents (arrows) M, mitochondria; Ly, lysosome-like vesicle, x 24,450; bar= 1 t~m.
within the tightly packed central non-crystalline region. There are single m e m b r a n e - P C V particle segments with a helical appearance (l) there are viral lameilae (2); two distinct highly ordered crystalline areas are, respectively m e m b r a n e associated (3) and membrane-free (4). Optical diffraction patterns from the marked areas in the micrograph (Fig. 8) show a clear hexagonal lattice of the non-lamellar region (Fig. 8c) and a less clear indication of this lattice in the lamellar region (Fig. 8b). In the latter diffraction pattern the spacing between the lamellae is
clearly shown by a pair of strong reflections. At this higher magnification (Fig. 8) the membrane associated crystalline viruses are seen to have a discontinuous appearance which appears to be the result of the orientation of the viral crystal to the plane of the section. The notion that the different views of virus organization seen in Fig. 8 arise from different planes of section is indeed verified in the tilting pair of micrographs presented in Figs. 9(a) and 9(b) in which the transformation from a viral lamellar appearance to a non-lamellar crystalline array
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Joseph Seckbach and Martin Kessel
Fig. 2. A higher magnification of the central area from the previous cell showing details of the viral packing in the membrane limited vesicles. Irregular segments of membrane bundles (Mb) and finely granulated areas of viroplasm (Vp) are scattered between the picornavirus vesicles. The cytoplasm surrounding these vesicles contains ribosomes and particles similar in size and shape to those enclosed inside the clusters. Short, straight and curved rows of interconnected viral particles are visible within the viral vesicle on the right of the micrograph. The vesicle displays the following features in addition: (a) the membrane enveloping the viral vesicle (arrow); (b) small area of hexagonally arranged viruses (double arrows) and (c) loose membrane fragments dispersed within the vesicle, x 60,350; bar= 1 t~m. and vice versa is clearly seen. The non-lamellar crystalline array therefore appears to result from a section parallel to and between the lamellae, whereas the very small change in appearance of areas lying close to the tilt axis shows that the lamellae extend throughout the plane of section. Figure 10 shows a viral vesicle which is almost totally crystalline and is comprised almost exc!usively of parallel rows of viruses. Not all the viruses are membrane bound and in some locations there appear to be more than one pair of membranes. In such cases the membranes are twisted braid-like a r o u n d each other and are usually shorter in length than membranes with viruses attached. The fact that this single section presents three different views of the viral rows within an apparent close packed arrangement shows once again that the three dimensional arrangement of the crystal is comprised of crystalline elements in different orientation to each other.
DISCUSSION The presence of the viral particles described in this study have been established purely on morphological grounds, particle shape, size, membrane-virus association, loci of crystallization and comparison of these characteristics with published data. Drosophila picornaviruses are more 'specialized' to insects than other insect viruses (Teninges et al., 1979) and have been found ubiquitously (Plus et al., 1975). The characteristics of Drosophila melanogaster picornaviruses are: R N A content, size of the particle 20-30nm in diameter, resistance to pH3 and lipid solvents, and intracytoplasmic localization (Plus et al., 1975, 1976). In Figs. 5, 7, 8, 9 and 10 there is evidence of a strong m e m b r a n e viral particle association in which two membranes enclose a row o f virus particles (viral lamellae) a step which may possibly be involved in the formation of the virus crystals. Similar patterns of two membranes sandwiching a
Picornavirus Crystalline in Drosophila Coil Line
Figs. 3 and 4. Figure legends on p. 29.
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Joseph Seckbach and Martin Kessel
Fig. 5. Part of a vesicle containing numerous crystalline areas as well as short segments of viral lamellae (VL). A bulging area of the vesicle is noticeable (Bu). Fingerprint-like areas of granulated viroplasm (Vp) are dispersed within the vesicle. In addition the dominant hexagonal packing, an area of square packing of virus particles is also present (Sq). My, myelin-like figure, x 31,350; bar= l#m. Fig. 6. A higher magnification of an area from Fig. 5 emphasizes the initial crystallization from a number of different foci. The small crystalline patches appear to change orientation as crystal growth is forced to stop due to lack of physical space. The striated appearance of the particle rows, showing a periodicity of roughly 15 nm, seems to be a consequence of the change within the plane of section of a row of particles thus presenting a different view as if by tilting. Some rows appear as parallel and curved hexagonal arrays. Some particles exhibit an electron transparent central core surrounded by an electron dense periphery (R). x 51,300; bar=0.5 ~m.
single row of viruses have been observed in other viral systems, e.g., in Simian virus 40 (Oshiro et al., 1967) a n d in Theiler's murine encephalomyelitis virus ( F r i e d m a n n a n d Lipton, 1980). In our present observations, all degrees of the virus aggregation have been observed within vesicles within the cytoplasma, while other virallike particles in Drosophila cell cultures crystallized within the nucleus (Gateff, 1978 ; Seckbach, unpublished data).
Our electron micrographs exhibit several appearances of viral particles within virus containing vesicles: a m o r p h o u s packing; paracrystalline packing; association of PCV with m e m b r a n e s ; (Figs. 2, 3, 5, 7 and 10) and well ordered hexagonal crystallinity. It seems to us that the m e m b r a n e system has a role to play in the formation of the crystal. We therefore suggest the following stepwise PCV crystallization: viruses become attached to membranes in
Picornavirus Crystalline in Drosophila Coil Line
Fig. 7. A highly developed crystalline vesicle is bordered by one to three layers of viral lamellae which show several discontinuities. This vesicle contains closely packed picornaviruses and possesses several groups of highly organized intravesicular, membrane bound viruses. (1) single membrane-viral particle association with a helical appearance, (2) the viral lamellae (VL) in the peripheral areas showing double membrane loaded w,th single rows of particles, (3) membrane associated viruses and (4) membrane-free crystalline ordered viruses. × 31,350; bar= I/~m.
Legends to page 27
Fig. 3. A central multilobed membrane-bound vesicle packed with picornaviruses is adjacent to a conspicuous nucleus (N) and its nucleolus (Nu). Lytic fragments in an extracellular neighbouring region (arrow) exhibit attachment of a row of viral particles to the intact cell membrane (VL) and also of viruses grouped into small crystalloids (C). × 20,250; bar= 1 /~m. Fig. 4. A higher magnification view of the central zone in the multilobed vesicle shown in Fig. 3 demonstrates the following: (a) short membrane segments with viruses attached on both sides or sandwiched between a double membrane (VL); (b) helical appearance of the membrane associated viruses (h) and (c) small hexagonal arrays of particles (hx) scattered amongst the loosely packed viruses, x 62,700; bar= 0.5 t~m.
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Joseph Seckbach and Martin Kessel
Figs. 8 (a) -(c). A higher magnification of an area from Fig. 7 showing optical diffraction patterns from lamellar (8b) and non-lamellar (8c) regions of the PCV crystal. The hexagonal packing of the nonlamellar region is very clearly seen both in the micrograph and the optical pattern. In the lamellar region the most dominant feature of both the micrograph and the diffraction pattern is the pair of strong reflections which arise from the interlamellar spacing, x 66,700; bar=0.5 p.m.
a helical fashion; the virus associated membranes aggregate to form a stack of several layers or virus lamellae, enclosing a row of viral particles between each pair of membranes; membranevirus association forms concentric layers of crystalline arrays; the final stage is the highly organized crystalline body with perhaps an eventual disintegration of the membranes. Most noticeable are helical arrangements of virus particles attached to short fragments of membrane (Figs. 3, 4 and 7). These structures resemble the zig-zag patterns of helical ribosomes in non-membrane bound ribosomal crystals observed in the cytoplasm of prokaryotic and eukaryotic ceils. For example similar helices were observed in growing cells of E. coli in the presence of vinblastine sulfate (Kingsbury et al., 1970). However, the PCV particles in the present study differ basically from ribosomal crystalline units observed in tetrameric sheets in lizard oocytes (Unwin and Taddei, 1977) and the hexagonally packed ribosome helices in Entarnoeba (Lake and Slayter, 1972). In these
crystal-membrane complexes of ribosomal aggregates, views in transverse section show two layers of endoplasmic reticulum enclosing double rows of ribosomes (Hubert, 1977; Unwin, 1979; Unwin and Taddei, 1977), whereas in our case a single row of virus particles is sandwiched between two membranes (Figs. 5, 7, 8, 9 and 10). The unusual ribosomal lamellar inclusions observed by Weise et al. (t980) observed in lymphocytes appear similar to our observations in that a single row of particles is sandwiched between two sheets of lamellae. However, the inclusions are not enclosed within vesicles and we have never observed the tubular arrangement of the virus lameilae as seen in the lymphocytes. The crystalline arrays of viruses described are not unique to D. melanogaster since other virus crystals have also been shown to be arranged as hexagonal or square arrays such as crystals of artichoke mottle crinkle virus (Russo et al., 1968). The wheat curl mite Aceria tulipae also exhibits a fully crystalline hexagonal pattern of brome mosaic virus whose particle size is similar
Picornavirus Crystalline in Drosophila Cell Line
Fig. 9 (a) and (b). Figure legends on p. 33.
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Fig. 10. This vesicle exhibits various types of highly ordered arrays of picornaviruses which mostly appear to be line in parallel rows. These rows are interspersed with twisted bundles of braid-like and nonviral containing membranes.
to PCV (Paliwal, t972). These observations suggest that the crystallization of insect viruses is a c o m m o n a n d related process. I n summary, we may conclude that there is an existence of a gradual assembly process of PCV aggregates m a t u r i n g into the virus crystal and that the PCV particles most closely resemble the DPV a n d D P A strains both belonging systematically to the class Picornaviridae.
Acknowledgements--The authors wish to express their appreciation to Dr. Ira S. Hammerman for initiation of this research while at Bar-Ilan University. We wish to thank Mrs. J. Hanania and Mrs. S. Kitov for their able technical assistance. Special thanks are due to Dr.
Nadine Plus for her valuable remarks and evaluations concerning the observations presented here prior to publication. This investigation was supported by the Israel-U.S. Binational Research Foundation. REFERENCES Friedmann, A. and Lipton, H. L., 1980. Replication of Theiler's murine encephalomyelitis viruses in BHK 21 Cells: an electron microscopic study. Virology, 101: 389-398. Gateff, E., 1978.The genetics and epigenetics of neoplasms in Drosophila. Biol. Rev., 53: 123-168. Haars, R., Zehtgraf, H., Gateff, E., and Bautz, F. A., 1980. Evidence for endogenous reovirus-like particles in a tissue culture cell line from Drosophila melanogaster. Virology, 101 : 124-130.
Picornavirus Crystalline in Drosophila Cell Line Hubert, J., 1977. Effets d'un sejour a une temperature de 6°C sur l'ultrastructure des ovaires du lezard lacerta vivipara Jacquin. Archs Anat. microsc., 66: 37-52. Kingsbury, E. W., Nauman, R. K., Morgan, R. S., and Voelz, H., 1970. Structure and function of ribosomal helices in Escherichia coli. In: Societ6 de Microscopie Electronique, Paris. lmprim6 en France. p. 69. Lake, J. A. and Slayter, H. S., 1972. Three-dimensional structure of the cromatoid body helix of Entamoeba invadens. J. tool. Biol., 66: 271-282. Oshiro, L. S., Rose, H. M., Morgan, C., and Hsu, K. C., 1967. Electron microscopic study of the development of Simian virus 40 by use of ferritin-labeled antibodies. J. Virol., 1: 384-399. Paliwal, Y. C., 1972. Brome mosaic virus infection in the wheat curl mite Aceria tulipae, a nonvector of the virus. J. Invert. Path. 20: 288-302. Plus, N., 1980. Further studies on the origin of the endogenous viruses of Drosophila melanogaster cell lines. In: Invertebrate Systems In Vitro, Kurstak, Maramorosch and Dubendorfer (eds). Elsevier, North-Holland Biomedical Press, 436-439. Plus, N., 1978. Endogenous viruses of Drosophila melanogaster cell lines: Their frequency, identification and origin. In Vitro, 14: 1015-1021. Plus, N. G., Croizier, Veyrunes, C., and David, J., 1976. A comparison of buoyant density and polypeptides of Drosophila P, C and A viruses, lntervirology, 7: 346-350.
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Plus, N. G., Croizier, Jousset, F.-X., and David, J., 1975. Picornaviruses of laboratory and wild Drosophila melanogaster Geographical distribution and serotypic composition. Arms Microbiol. (Inst. Pasteur) 126A: 107-117. Russo, M., Martelli, G. P., and Quacquarelli, A., 1968. Studies on the agent of artichoke mottled crinkle. IV. lntracellular localization of the virus. Virology, 34: 679-693. Sederoff, R. and Clynes, R., 1974. A modified medium for culture of Drosophila cells. Drosophila Inf. Service Newsletter, 51 : 153-155. Teninges, D., Ohanessian, A., Richard-Molard, C., and Contamine, d., 1979a. Contamination and persistent infection of Drosophila cell lines by reovirus type particles. In Vitro, 15: 425-428. Teninges, D., Ohanessian, A., Richard-Molard, C., and Contamine, C., 1979. Isolation and biological properties of Drosophila X virus. J. gen. Virol. 42: 241-254. Unwin, P. N. T. and Taddei, C., 1977. Packing of ribosomes in crystals from the lizard Lacerta sicula. J. mol. Biol., 114: 491-506. Unwin, P. N. T., 1979. Attachment of ribosome crystals to intracellular membranes. J. mol. Biol., 132: 69-84. Weise, R. W., Rozek, R., and Palutke, M., 1980. Unusual ribosomal lamellar inclusions in lymphocytes. Micron, 11: 127-135.
Legends to page 31 Figs. 9. (a) and 9 (b) are from a tilt series of micrographs recorded at +45 ° and - 4 5 ° and clearly show a number of changes occurring in the appearance of non-attached and membrane attached picornaviruses. In regions where the membranes and their associated viruses lie parallel to the tilt axis in Fig. 9 (a) the same region in Fig. 9 (b) now appears as a non-lamellar viral array. Similarly in non-lamellar areas in Fig. 9 (a), in Fig. 9 (b) the viral lamellar now appear in the parallel configuration. Viral lamellae lying close to the tilt axis hardly change their appearance at all. x 66,700; bar=0.5 t~m.