Ultrastructural studies on the morphogenesis of the Densonucleosis virus (Parvovirus)

VIROLOGY

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517-531 (1976)

Ultrastructural

Studies

on the Morphogenesis Virus (Parvovirus)

SIMON GARZON

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of the Densonucleosis

EDOUARD KURSTAK’

Laboratoire de Virologie Cornparke, Dtfpartement de Microbiologic et Immunologic, Facultk de MPdecine, UniversitP de Montreal, MontrPal, P. Q., Canada Accepted December 30,1975 Morphogenesis of Densonucleosis virus (DNV), a Parvovirus, has been studied in its natural host, the larvae of Galleria mellonella. This infection is characterized by the formation of dense, DNA-positive inclusions in the hypertrophied nuclei of the infected cells. This type of lesion is observed in the majority of tissues, with the exception of the midgut. The first ultrastructural changes consist of the increase in the number of free ribosomes and of the formation of “microbody-like” structures arising from the accumulation of small, round bodies of 17-20 nm inside the vesicles. In the nucleus, the nucleolus undergoes hypertrophy which is accompanied by the segregation of its fibrillar and granular components. The development of the granular portion coincides with the synthesis of the double-stranded DNA of the replicative form inside the virogenic stroma. As the infection progresses, the granular portion of the nucleolus regresses, in favor of the fibrillar portion. On the other hand, DNV seems to stimulate the formation of intranuclear bodies associated with the virogenic stroma. The virions are assembled inside the virogenic stroma and may be in relation with the nucleolus and the intranuclear bodies. Each particle encapsidates a single-strand of the replicative form, giving rise to two distinct populations of mature virions. The replication of DNV shows certain similarities with that of other Parvoviruses, particularly of the AAV and H-l. INTRODUCTION

Densonucleosis virus (DNV) is a small, nonenveloped DNA virus, classified as a Parvovirus (Wildy, 1971, 1973). DNV exhibits physicochemical characteristics similar to those of the animal Parvoviruses (Hoggan, 1971; Kurstak, 1972; Mayor and Kurstak, 1974). However, the host for DNV is the larvae of the Lepidoptera Galleria mellonella. The virions of the DNV have a diameter of 20-24 nm and contain a single-stranded DNA with a molecular weight of 1.6 to 2.0 x lo6 daltons (Kurstak et al., 1973). The virus seems to be made up of three structural proteins with molecular weights of 49,000, 58,500, and 69,000 daltons (Tijssen et al., 1976). From these observations it ’ To whom reprint

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Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved,

would appear that the viral genome would theoretically code only for these three proteins, unless these proteins contain common sequences. On the other hand, preliminary studies indicated the existence of two populations of DNV particles, each containing complementary single-stranded DNA chains (Kurstak et al., 1971,1973). A similar situation also exists in the case of the adenoassociated-viruses (MV) where two populations of virions have been isolated (Berns and Adler, 1972; Mayor and Kurstak, 1974). The DNV, however, differs biologically from the AAV by its autonomous replication. It should be noted that the replication of several Parvoviruses seems to depend on the replicative state of the host’s cells. In fact, it was demonstrated that the H-l virus, the rat Kilham 517

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virus (RV), and the minute virus of the mouse (MVM) preferentially infect cells in their S-phase of replication (Hampton, 1970; Tennant and Hand, 1970; Tattersall, 1972; Rhode, 1974). The replication of the DNV in its natural host G. mellonella is not well characterized at the ultrastructural level. Preliminary electron microscopic studies confirmed only the viral nature of the dense nuclear inclusions induced by the infection and the presence of viral particles (Vago et al., 1966; Kurstak et al., 1968). In the present study, the morphogenesis of DNV in the cells of infected larvae of G. mellonella was investigated and compared to that of the other members of the Parvovirus group.

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aqueous solution of uranyl acetate, are washed, and are treated with 0.2 M ethylene diamine tetraacetic acid, pH 7.2, for 45 min. After another washing, the preparations are stained with lead citrate for 1 min and examined with the electron microscope. RESULTS

Spectrum of virulence. The present ultrastructural study revealed the characteristic lesions caused by DNV in the nucleus of the cells of various tissues in the larvae of G. mellonella. Thus, the adipose tissue, trachea, silk glands, hypodermis, hemolymph, and the posterior and anterior gut constitute the primary foci of infection, detectable from the first days of the disease. Later, similar lesions can be MATERIALS AND METHODS observed in the salivary glands, dermal The L4-L5 larvae of G. mellonella ob- glands, moulting glands (or Verson’s tained from different laboratories are in- glands), wing buds, nervous ganglia, and jected ip with 0.01 ml of a purified suspen- in cells of the tunica externa and the intersion of DNV (0.1 mg/ml). The larvae are stitial cells of the gonads (Fig. 1). Infection then kept in an incubator at 30”. At 2-hr of the key organs by the DNV inhibits the intervals during the first 24 hr of the infec- moulting and the metamorphosis of the tion and daily thereafter, different tissues larvae. However, DNV has so far not been detected in the nuclei of the midgut cells. are harvested and prefixed with a mixture Morphogenesis . The first ultrastrucof 0.4% paraformaldehyde and 1% glutaraldehyde in 0.067 M phosphate buffer, pH tural changes of the infected cells have 7.4, for 2 hr at 4” (Karnovsky, 1965). After been observed 6-12 hr after the infection, washing overnight at 4” with the phos- both in the cytoplasm and nucleus. In the cytoplasm one can observe: (i) the phate buffer supplemented with 0.2 M sucrose, the tissues are postfixed for 1 hr regression of the endoplasmic reticulum with 2% osmic acid in the same buffer. and the appearance of a large number of Some of the samples are kept without post- dispersed ribosomes; (ii) the swelling and fixation. After dehydratation, the tissues degeneration of various organelles, such are embedded in Epon 812 or Spin-r’s mix- as the mitochondria and the lysosomes ture @purr, 1969). Thin sections are pre- (Fig. 2a); and (iii) the gradual developpared with an LKB ultramicrotome and ment of new structures due to the progresare contrasted with 1% uranyl acetate and sive accumulation of small, round partilead citrate (Reynolds, 19631,prior to their cles of 17-20 nm of diameter inside the examination with a Philips 300 electron vesicles (Fig. 2). These particles form paracrystalline arrays (Fig. 2b) and within microscope. Regressive staining with EDTA (Bern- the same structure several plans of cryshard, 19681, which differentiated ribonu- tallization can be observed (Fig. 2c and d). The number of these structures increases cleoproteins from deoxyribonucleoproteins at the ultrastructural level, was also used rapidly during the first 2 days of infection, in this study. For this purpose, the sec- particularly in the hemocytes, adipose tistions of tissues which have been fixed only sue, and silk glands. Simultaneously, in the nucleus, the nuwith paraformaldehyde-glutaraldehyde prior to embedding in Spurr’s or Epon 812 cleolus undergoes a rapid hypertrophy, acresins, are placed for 1 min in a 5% companied by the segregation of the pars

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FIG. 1. Spectrum of virulence and polytropic nature of DNV. Nuclear lesions (nuclear hypertrophy and virogenic stroma) in the cells of various tissues atier 5 days of infection: (a) moulting gland (x 3000); (b) epithelium of posterior gut ( x 6000); (c) epithelium of anterior gut (X 5000); (d) epithelial sheet of gonad ( x 6000). Bar = 2.0 pm,

fibrosa and the pars granulosa. The nucleolar hypertrophy is very important in some cells (Fig. 3). The segregation of the two nucleolar components is complete with one or several strongly colored zones of fibrillar material and to one or several

networks of granular material (Figs. 3 and 4). The granular portion is well amplified when the virogenic stroma appears in the center of the nucleus in close relation with the nucleolus (Fig. 4). The cellular chromatin and the nucleolus are pushed to the

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EVIG. 2. Cells of the silk glands and adipose tissue 6-24 hr aRer infection with DNV. (a) Changes in the mitt ochondria of a silk gland cell: swelling, vacuolization, loss of cristae, and accumulation of small partic :les array (W ( x 48,600); (b) higher magnification showing the format #ion (+) condensing into a paracrystalline and arrangement of a paracrystalline array ( x 79,000); (c) and (d) same changes in a cell of adipose tissue . In this cell the cytoplasm shows several “microbody-like” structures. Different plans of crystallization can I be recc Bgnized (c: x 18,000; d: x 51,600). Bar = 0.2 pm.

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FIG. 3. Nucleus of a hypodermal cell after 12 hr of infection. Note the hypertrophy and segregation nucleolus. f, Fibrillar component; g, granular component; nu, nucleolus (X 24,000). Bar = 0.5 pm.

periphery of the nucleus by the invading stroma. Dense fibrous material is accumulated in the virogenic stroma from which mature viral particles emerge. On the other hand, inside the virogenic stroma the presence of one or several round-shaped intranuclear bodies can be observed. These intranuclear bodies of variable diameter are composed of winding filaments with no limiting membrane (Figs. 5 and 6a). Regressive staining with EDTA produces a “bleaching” of the virogenic stroma, viral particles, and of the cellular chromatin which lose their usual contrast. However, the intra and perichromatidic granules, the ribosomes, the nucleolus, and the intranuclear bodies retain their normal contrast. The nucleolus clearly shows the segregation of its fibrillar and granular components (Fig. 5b). In many infected nuclei we observed, between the nucleolus and the virogenic stroma, an accumulation of empty viral particles as well as mature virions. Some virions could also be seen inside the network of the granular portion of the nucleo-

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lus (Fig. 5a and 6b). As the virogenic stroma develops, the granular portion of the nucleolus decreases gradually until its complete disappearance whereas the fibrillar portion keeps increasing (Fig. 7 and 8). The virogenic stroma invades the whole nucleus and the continued synthesis of viral material leads to a progressive nuclear hypertrophy. The nucleolus which is already reduced to its fibrillar portion disintegrates completely. Mature virions assembled in the virogenic stroma are grouped together in islets, some of which are in relation with an intranuclear body (Fig. 6a). Gradually the viral inclusions are joined together to replace the virogenic stroma (Fig. 9). The production of virions per cell is considerable, and at the last stage of the infection the viral mass ruptures the nuclear membrane at several points which allows the passage of the virions to the cytoplasm. Paracrystalline viral inclusions can then be observed in the cytoplasm and nucleus (Fig. 9b). At that time, the cytoplasm is considerably reduced due to the hypertrophy of the nucleus.

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FIG. 4. Hypodermal cells after 24 hr of infection. Two of these cells present a virogenic stroma (vs) which is in close relation with the nucleolus. The latter shows hypertrophy and segregation of its two components. The cytoplasm is rich in free ribosomes (r) and shows the swelling of the endoplasmic reticulum (er) and mitochondria (m). The third cell which presents no signs of infection has a compact and uniformly dense nucleolus. ch, Cellular chromatin (X 16,100). Bar = 1.0 pm.

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FIN:. 5. Nuclei of cells after 48 hr of infection. (a) The virogenic stroma (vs) increases and pushes the chror natin and nucleolus (fragmented in this cell) to the periphery. In the center of the virogenic Strom: aan intra nuclear body is visible (p). Inside and around the virogenic stroma, mature virions (v) are released (X 18,5010). (b) Regressive staining with EDTA: the granular (g) and fibrillar (f) components of the nuclec IlUS, the il ntranuclear body (b) and the ribosomes (r) retain their contrast. The virogenic stroma (replica .tive form) and the mature virions are completely “bleached” (X 18,800). Bar = 0.5 pm.

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FIG. 6. Hypodermal cells after 72 hr of infection: (a) Several intranuclear bodies ( t>o) are enclc)sed in the vi1-ogenic stroma, some of them localized between the virogenic stroma and the mature virions gl in isle ‘ts (Do) (X 22,000); (b) some mature DNV particles (v) seem to be present inside (+) and a the gr anular portion (g) of the nucleolus (nu) (X 42,700). Bar = 0.5 pm.

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FIG. 7. Hypodermal cells after 4 days of infection. The viral inclusion occupies almost the entire nlucleus. (a ) Mature virions (v) are grouped together between the nucleolus (nu) and the virogenic stroma I:vs) (X 14 ,500); (b) the virions are assembled inside the virogenic stroma and grouped into islets. The nu cleolus component (X 10,500). Bar = 1.0 1urn. P” shed to the periphery of the nucleus is reduced to the fibrillar DISCUSSION

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This study demonstrates the polytr epic nature of the DNV and describes the It is P**incipal stages of its replication.

clear that under the experimental Iconditions used in this study it is rather dij Micult to interpret kinetically the data obt #ained from static in viuo observations in asynchronous cells.

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FIG. 8. Different stages in the evolution of the morphology of the nucleolus during the replicative cycl e of DNV. ( a, b, and c) After 24 hr of infection: the granular portion (g) is well developed and clearly separz tted from tkie fibrillar portion (f~ (a: x 19,300; b: x 19,500; c: x 16,400). Cd) After 72 hr: the granular porl tion regress #es while the fibrillar portion increases (X 12,000). (e) After 4 days: complete regression of the granuli 3.r portion. The nucleolus, pushed to the periphery of the nucleus by the virogenic stroma, consist .s of only th e fibrillar portion ( x 11,200). (0 After 5-6 days: lysis of the residual nucleolus (fibrillar portion) ’ (x 16,500) Note the evolution of the virogenic stroma in parallel. Bar = 0.5 km.

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FIG. 9. Hypodermal cells alter 6 days of infection. (a) The virogenic replaced by plaques of virus particles (v) (X 19,500). (b) The membrane of production of virions is ruptured, allowing the passage of the virions paracrystalline formations of DNV in the nucleus (~1 (X 26,000). Bar =

I. Polytropic nature. Earlier studies described the characteristic nuclear lesions caused by the DNV in the cells of several tissues which constitute the primary foci of

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stroma (dense) is progressively the nucleus hypertrophied by the to the cytoplasm c-t). Note the 0.5 pm.

the infection (Amargier et al., 1965; Garzon and Kurstak, 1968; Kurstak et al., 1968a,b; Kurstak, 1972). The present study identifies the secondary foci of infection in

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several other tissues. It is evident from these results that with the exception of the cells of the midgut, where the pH may be too high, all other tissues of the host are susceptible to the DNV infection. 2. Mode of replication. After a latent period of 2-4 hr (Kurstak et al., 1970), which corresponds to the early stages in the viral cycle, the first ultrastructural changes occur in the cytoplasm and in the nucleus. These changes are characteristic for DNV-infected cells and consist of the accumulations of free ribosomes, the appearance of new cytoplasmic structures, and the hypertrophy and segregation of the nucleolus. The formation of new intracytoplasmic structures seems to be caused by the accumulation and crystallization of ribosomes inside vesicles which may arise from the degeneration of mitochondria. Such crystallization of ribosomes has been observed earlier in chick embryonic cells under hypothermic conditions (Byers, 1967) and also in infection with cowpea mosaic virus (van der Scheer and Groenewegen, 1974). According to Byers, crystallization of ribosomes within interphase cells must be preceded by their release from polyribosomes. On the other hand, during mitosis there is an inhibition of new protein synthesis which may produce many free ribosomes in the cytoplasm, making them available for crystallization during telophase. One might speculate that DNV infection causes the degranulation of the rough endoplasmic reticulum which provides many free ribosomes for crystallization. It was always considered that the nucleolus undergoes lysis at the beginning of the infection with DNV (Vago et al., 1966). According to the results of the present study, however, the nucleolus does not disappear at the onset of the infection by DNV but undergoes hypertrophy and segregation of its fibrillar and granular components. The presence and the structure of the nucleolus, as well as the evolution of its structure during different stages of infection have been examined in the present study by regressive staining with EDTA. The influence of intranuclear viruses on

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the nucleolus during viral infections has been reviewed recently (Heine, 1974). Herpes and adenoviruses were observed to induce segregation of nucleolar material (Sirtori and Basisio-Bestetti, 1967; Matsui and Bernhard, 1967; Philips and Raskas, 1972). Henry et al. (19721, Al-Lami et al. (1968), and Singer and Toolan (1975) reported abnormal changes in the nucleoli of infected cells after infection with the parvoviruses AAV and H-l. A study with a variety of chemically unrelated substances capable of causing nucleolar segregation has lead to the following conclusion: nucleolar segregation is a specific lesion which corresponds to an interference in the nucleolar RNA synthesis. On the other hand, natural segregation in Newts @eddy and Svoboda, 1972) and other stimuli for nucleolar segregation, like cycloheximide or amino sugars, cannot be related to a direct action on RNA synthesis because of their as yet undefined mode of action (Simard et al., 1974). Philips and Raskas (1972) suggested that adenovirus type 2, which inhibits the formation of ribosomal RNA (Raskas et al., 1970) and causes nucleolar segregation, may do so by interfering with normal processing of 45 S RNA rather than by blocking its synthesis. In this respect, it is also noteworthy that Fong et al. (1970) reported abnormalities in the processing of 45 S rRNA in NB cells 12 hr after infection with H-l. This infection with H-l was also observed to cause a fragmentation of the fibrous part of the nucleolus (Singer and Toolan, 1975). Actinomycin D inhibits the nucleolar RNA synthesis but does not inhibit synthesis of H-l hemagglutinating factor (Rhode, 1973), indicating strongly that nucleoli are relatively unimportant in the development of the H-l virus. It is difficult to interpret the hypertrophy of the nucleolus. In general, a hypertrophy of the nucleolus corresponds to an increase in the synthesis of nucleolar RNA and of the high molecular-weight ribosomal precursors (Koulish and Kleinfeld, 1964; Steele et al., 1965). The hypertrophy of the nucleolus observed in DNV-infected cells might represent the early stage of the segregation. However, it is conceivable

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that the hypertrophy of the nucleolus reflects the starvation conditions of the larvae (infected larvae stop feeding themselves), which can also lead to the increase of the nucleolus (Stenram, 1963). The findings of the present study and the above data obtained from the literature could suggest that the redistribution of the nucleolar components subsequent to DNV infection might represent the morphologic manifestations of an action of the virus at some unknown level of nucleic acid metabolism in the host cell. The mechanism by which these morphological changes are induced in the nucleolus of infected cells is still unknown but is presently under investigation. One might speculate that the segregation of the nucleolus and the release of ribosomes from polysomes are not unrelated processes. The intranuclear bodies observed in DNV-infected cells show a close association with the virogenic stroma. They give a staining with EDTA which corresponds to that of ribonucleoproteins. The formation of such structures has been also observed in other viral infections. In certain cases, these structures could correspond to the site of protein synthesis and the early foci of viral production (Oyanagi et al., 1970; Bouteille et al., 1974). In the present study the virogenic stroma was shown to contain the viral antigens and double-stranded DNA (replicative form), whereas the mature particles contained single-stranded DNA as revealed by staining with acridine orange (Kurstak, 1972). Since both complementary strands are separately encapsidated, unwinding of the double-stranded DNA is thus necessary for the assembly of mature virions (Kurstak et al., 1973). Preliminary investigations on multiple viral infection of a single cell show that in doubly infected cells, DNV present the same morphological changes as described in the present work. However, a reduction in the level of production of the virus has been noted (Kurstak et al., 1972; Kurstak and Garzon, 1975). It was demonstrated by Morris (1971) using autoradiography in light microscopy that cytoplasmic proteins migrate towards

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the nucleus in DNV-infected cells. More complete studies, employing high resolution autoradiography and histochemical techniques as well as immunoenzymatical methods (Kurstak et al., 1975) using monospecific antisera directed against each of the structural proteins of DNV, are presently in progress in this laboratory. The results of these studies should further elucidate the site and cycle of replication of this Parvovirus. ACKNOWLEDGMENTS The authors wish to express their appreciation to Dr. P. A. Onji, from Wakayama Medical University for his collaboration. Supported by Grants No. MA-2385 from the Medical Research Council of Canada, and No. A-3746 from the National Research Council of Canada. REFERENCES AL-LAMI, F., LEDINKO, N., and TOOLAN, H. (1969). Electron microscope study of human NB and SMH cells infected with the parvovirus H-I: Involvement ofthe nucleolus. J. Gen. Viral. 5,485-492. AMARGIER, A., VAGO, C., and MEYNADIER, G. (1965). Etude histopathologique dun nouveau type de virose mise en evidence chez le Lepidoptere Galleria mellonella. Arch. Ges. Virusforsch. 15, 659-667. BERNHARD, W. (1968). Une etude de coloration regressive a l’usage de la microscopic electronique. C.R. Acad. Sci. 267, 2170-2173. BERNS, K. I., and ADLER, S. (19721. Separation of two types of adeno-associated virus particles containing complementary polynucleotide chains. J. Viral. 9, 394-396. BOUTEILLE, M., LAVAL, M., and DUPUY-COIN, A. M. (19741. Localization of nuclear functions as revealed by ultrastructural autoradiography and cytochemistry. In “The Cell Nucleus” (H. Busch, ed.), Vol. 1, 51-60. Academic Press, New York. BYERS, B. (1967). Structure and formation of ribosome crystals in hypothermic chick embryo cells. J. Mol. Biol. 26, 155-167. FONG, C. K. Y., TOOLAN, H. W., and HOPKINS, M. S. (1970). Effect of H-l virus infection on RNA synthesis in NB cells. Proc. Sot. Exp. Biol. Med. 135, 585-588. GARZON, S., and KURSTAK, E. (19681. Infection des cellules des gonades et du systeme nerveux de Galleria mellonella par le virus de la densonucleose. Natural&e Canad. 95, 1125-1129. HAMPTON, E. G. (19701. H-l virus growth in synchronized rat embryo cells. Canad. J. Microbial. 16, 266-268. HEINE, U. I. (19741. Intranuclear viruses. In “The

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