- Email: [email protected]
Electron microscopic study of the development of herpesvirus saimiri
VIROLOGY
69, 810-815 (1976)
Electron
Microscopic
ADAM FRIEDMANN, Department
of Microbiology,
Study of the Development JOE E. COWARD,
College ofPhysicians
AND
of Herpesvirus
COUNCILMAN
and Surgeons, Columbia 10032
University,
Saimiri
MORGAN New York, New York
Accepted October 22, 1975
Electron microscopic study of herpesvirus saimiri in thin sections of infected OMK and Vero cells showed the apparent intranuclear envelopment of capsids by membranes of vesicles. Clusters of filaments were also encountered. Numerous unenveloped intracytoplasmic capsids were observed in cells devoid of nuclear changes, raising the possibility that differentiation can occur within the cytoplasm.
Herpesvirus saimiri (HVS), a viral agent isolated from the squirrel monkey (Saimiri sciureus), fails to produce any apparent disease in its natural host, However, when experimentally inoculated into or the owl monkey (Aotus triuirgutus) marmosets (Sanguinus Oedipus), HVS induces an acute malignant lymphoma (7). It has been previously reported that HVS, as in the case of other herpesviruses, replicates in the nuclei of infected cells (4). In addition, a series of tubules and coiled membranes have been reported in African green monkey kidney cells (Vera) showing advanced cytopathic effects (CPE) following infection with HVS (9). The present report describes a number of unusual developmental characteristics of this agent. In particular, while differentiation of many virions appears to take place within the nucleus, unenveloped capsids are also numerous in the cytoplasm and unique tubular structures form within the nuclei. The purpose of this report is to describe the morphologic events following infection of owl monkey kidney cells (OMK) and Vero cells with HVS and to suggest the possibility that differentiation of capsids may occur in the cytoplasm as well as the nucleus . Herpesvirus saimiri was generously supplied by Dr. L. V. Melendez. The owl monkey kidney cells (OMK) and African green monkey cells (Vera), kindly pro810 Copyright 0 1976by Academic Press, Inc. All rights of reproduction in any form reserved.
vided by Dr. Melendez, were grown in Eagle’s minimum essential medium (MEM) supplemented with 10% fetal calf serum. Sparse monolayers of cells were infected with 10-l dilution of stock virus. After incubation for 5 days, the cells were scraped into MEM and exposed to sonic vibration for 5 min. Cellular debris was removed by low speed centrifugation for 10 min, and 0.3 ml of the supernatant was used to infect cell cultures for electron microscopy. The cultures were harvested at sequential intervals after the appearance of cytopathic effects: 3-6 days postinfection with OMK cells and sll days postinfection with Vero cells. The cells were fixed in 1% glutaraldehyde for 1 hr, washed with Sorenson’s buffer, postfixed for 30 min in 1% dehydrated, and osmium tetroxide, embedded in epoxy resin (Epon 812). Thin sections stained with uranyl acetate and lead citrate were examined in a Phillips 200 electron microscope. Accumulation of nonenveloped capsids in the cytoplasm. During the late stages of
HVS development (10 days postinfection) large numbers of capsids accumulate in the cytoplasm of approximately 25% of the Vero cells. The capsids were usually found in clusters scattered through the cytoplasm (Fig. 1) often in close proximity to vacuoles, which were numerous at this stage of infection. It is remarkable that in many cells containing large numbers of
SHORT
intracytoplasmic capsids the nuclei were entirely normal in appearance and did not contain capsids or exhibit the margination of nuclear chromatin so characteristic of herpesvirus infection (Fig. 2). The fact that such capsids were repeatedly observed in cells which did not appear to be fused makes it unlikely that these unenveloped capsids gained access to the cytoplasm by fusion with adjacent infected cells. Occasional capsids were encountered in process of envelopment by cytoplasmic vacuolar membranes. Fewer capsids were found in the cytoplasm of infected OMK cells than in Vero cells. Intranuclear envelopment of HVS. In the majority of infected cells, capsids appeared to be assembled and enveloped within the nucleus. Heine et al. (1971) reported that HVS matures within the nucleus. Our observations confirm these findings and, in addition, indicate that capsid occurs during passage envelopment through membranes of intranuclear vesicular structures (Figs. 3 and 4). The frequency with which such vesicles were encountered inside the nucleus and the lack of any apparent continuity with the cytoplasm of the numerous cells examined argue against the possibility that they represent cross-sectioned cytoplasmic invaginations. Surprisingly, virus in process of envelopment by the peripheral nuclear membrane, an event so common in the case of other herpes-type viruses, was not encountered in these studies. The intranuclear envelopment of capsids as contrasted with the usual mode of envelopment during passage from the nucleus to the cytoplasm has been reported in FIG. 1. FIG. 2. aggregation x 44,000. FIG. 3. x 100,000. FIG. 4. x 75,000. FIG. 5. FIG. 6. FIG. 7. x 40,000. FIG. 8.
811
COMMUNICATIONS
the case of Herpesvirus saimiri (4) and more recently in association with Herpesvirus sylvilagus (5). Similar observations have been made in studies of the replicative cycle of another oncogenic virus, Herpesvirus ateles (Coward, Friedmann, and Morgan, unpublished). Apparently envelopment accompanies passage of the virus into intranuclear vacoules. Zntranuclear structures in HVS-infected cells. It was previously reported that the
nuclei of HVS-infected Vero cells contain filaments (4) and tubules (9). Such intranuclear structures were also observed in the present study. The arrangement of the filaments in both OMK and Vero cells assumed one of two forms. The first was composed of aggregates of fine fibrils (Fig. 51, while the second, which has not been described previously, consisted of linear arrays of granular material (Fig. 6). Such filaments were present in lO-12% of infected OMK and Vero cells. Tubules composed of repeating subunits (Fig. 7) were also found in OMK- and Vero-infected cells. In addition, collections of irregular, convoluted membranes were encountered but only in Vero cells (Fig. 8). The latter arrangement was uncommon being observed in no more than 1% of the cells. Intranuclear tubular and filamentous structures have been observed in association with a variety of herpesviruses. Such structures have been considered “bizarre” since no temporal or spatial relationship could be established between them and the differentiating virus. Curiously they are most frequently encountered in the nuclei of cells infected with oncogenic herpesviruses (1-3, 5, 9-12). It is tempting to pos-
HVS capsids in the cytoplasm of a Vero cell 10 days postinfection. x 36,000. Virus in the cytoplasm of a cell, the nucleus of which is devoid of capsids and does not exhibit or margination of chromatin. Nonspecific hyaline bodies are evident at the lower margin. Capsids
in process of envelopment
Capsids at differing
within
the nucleus
of an OMK
stages of passage into vesicles of a Vero-cell
cell 5 days postinfection.
nucleus
10 days postinfection.
An intranuclear aggregate of fine filaments in an OMK cell 5 days postinfection. x 65,000. A granular filament in the nucleus of an OMK cell 5 days postinfection. x 45,000. An intranuclear tubule composed of repeating subunits in an OMK cell 6 days postinfection. Irregular
membranous
structures
in the nucleus of a Vero cell 10 days postinfection.
x 50,000.
812
SHORT
COMMUNICATIONS
FIGURES 1 AND 2
SHORT
COMMUNICATIONS
FIGURES
3 AND 4
813
814
SHORT
COMMUNICATIONS
FIGURES 5-8
SHORT
COMMUNICATIONS
Mate that they may be the counterpart of polyheads, polysheaths, or polytaiis which accumulate in bacteria infected with amber and t.s mutants of T-even bacteriophages (6). The appearance of nonenveloped capsids in the cytoplasm of cells devoid of nuclear changes suggests that HVS capsids may be actually assembled in the cytoplasm. Should this be confirmed, it will be the first instance of a herpes-type virus differentiating in the cytoplasm. ACKNOWLEDGMENTS This work was supported by United States Public Health Grants AI-06814 from the National Institute of Allergy and Infectious Diseases and International Research Fellowship Award Number FO, TW0188701, National Institutes of Health (A.F.).
3. 4. 5. 6.
7.
8.
9. 10.
REFERENCES 1. COUCH, E. F., and NAHMIAS, J. Viral. 3,228-232 (1969). 2. EPSTEIN, M. A., ACHONG, B. G., CHURCHILL, A.
11. 12.
815
E., and BIGGS, P. M., J. Nat. Cancer Inst. 41, 805811 (1968). FAWCE~, D. W., J. Biophys. Biochem. Cytol. 2, 725-742 (1956). HEINE, U., ABLASHI, D. V., and ARMSTRONG, G. R., Cancer Res. 31, 10191029 (1971). HEINE, LJ., and HINZE, H. C., Cancer Res. 32, 1340-1350 (1972). KELLENBERGER, E., EISERLING, F. A., and BOY DE LA TOUR, E., J. Ultrastruct. Res. 21, 335 360 (1968). MELENDEZ, L. V., HUNT, R. D., DANIEL, M. D., GARCIA, F. G., and FRASER, C. E. O., Lab. Anim. Care 19, 378-386 (1969). MORGAN, D. G., EPSTEIN, M. A., ACHONG, B. G., and MELENDEZ, L. V., Nature (London) 228, 170-172 (1970). MORGAN, D. G., ACHONG, B. G., and EPSTEIN, M. A., Brit. J. Cancer 27, 434-444 (1973). MURPHY, F. A., HARRISON, A. K., and WHITFIELD, S. G., Arch. Ges. Virusforsch. 21, 463 468 (1967). NII, S., MORGAN, C., and ROSE, H. M., J. Viral. 2, 517-536 (1968). WATRACH, A. M., Virology 18, 324-327 (1962).
69, 810-815 (1976)
Electron
Microscopic
ADAM FRIEDMANN, Department
of Microbiology,
Study of the Development JOE E. COWARD,
College ofPhysicians
AND
of Herpesvirus
COUNCILMAN
and Surgeons, Columbia 10032
University,
Saimiri
MORGAN New York, New York
Accepted October 22, 1975
Electron microscopic study of herpesvirus saimiri in thin sections of infected OMK and Vero cells showed the apparent intranuclear envelopment of capsids by membranes of vesicles. Clusters of filaments were also encountered. Numerous unenveloped intracytoplasmic capsids were observed in cells devoid of nuclear changes, raising the possibility that differentiation can occur within the cytoplasm.
Herpesvirus saimiri (HVS), a viral agent isolated from the squirrel monkey (Saimiri sciureus), fails to produce any apparent disease in its natural host, However, when experimentally inoculated into or the owl monkey (Aotus triuirgutus) marmosets (Sanguinus Oedipus), HVS induces an acute malignant lymphoma (7). It has been previously reported that HVS, as in the case of other herpesviruses, replicates in the nuclei of infected cells (4). In addition, a series of tubules and coiled membranes have been reported in African green monkey kidney cells (Vera) showing advanced cytopathic effects (CPE) following infection with HVS (9). The present report describes a number of unusual developmental characteristics of this agent. In particular, while differentiation of many virions appears to take place within the nucleus, unenveloped capsids are also numerous in the cytoplasm and unique tubular structures form within the nuclei. The purpose of this report is to describe the morphologic events following infection of owl monkey kidney cells (OMK) and Vero cells with HVS and to suggest the possibility that differentiation of capsids may occur in the cytoplasm as well as the nucleus . Herpesvirus saimiri was generously supplied by Dr. L. V. Melendez. The owl monkey kidney cells (OMK) and African green monkey cells (Vera), kindly pro810 Copyright 0 1976by Academic Press, Inc. All rights of reproduction in any form reserved.
vided by Dr. Melendez, were grown in Eagle’s minimum essential medium (MEM) supplemented with 10% fetal calf serum. Sparse monolayers of cells were infected with 10-l dilution of stock virus. After incubation for 5 days, the cells were scraped into MEM and exposed to sonic vibration for 5 min. Cellular debris was removed by low speed centrifugation for 10 min, and 0.3 ml of the supernatant was used to infect cell cultures for electron microscopy. The cultures were harvested at sequential intervals after the appearance of cytopathic effects: 3-6 days postinfection with OMK cells and sll days postinfection with Vero cells. The cells were fixed in 1% glutaraldehyde for 1 hr, washed with Sorenson’s buffer, postfixed for 30 min in 1% dehydrated, and osmium tetroxide, embedded in epoxy resin (Epon 812). Thin sections stained with uranyl acetate and lead citrate were examined in a Phillips 200 electron microscope. Accumulation of nonenveloped capsids in the cytoplasm. During the late stages of
HVS development (10 days postinfection) large numbers of capsids accumulate in the cytoplasm of approximately 25% of the Vero cells. The capsids were usually found in clusters scattered through the cytoplasm (Fig. 1) often in close proximity to vacuoles, which were numerous at this stage of infection. It is remarkable that in many cells containing large numbers of
SHORT
intracytoplasmic capsids the nuclei were entirely normal in appearance and did not contain capsids or exhibit the margination of nuclear chromatin so characteristic of herpesvirus infection (Fig. 2). The fact that such capsids were repeatedly observed in cells which did not appear to be fused makes it unlikely that these unenveloped capsids gained access to the cytoplasm by fusion with adjacent infected cells. Occasional capsids were encountered in process of envelopment by cytoplasmic vacuolar membranes. Fewer capsids were found in the cytoplasm of infected OMK cells than in Vero cells. Intranuclear envelopment of HVS. In the majority of infected cells, capsids appeared to be assembled and enveloped within the nucleus. Heine et al. (1971) reported that HVS matures within the nucleus. Our observations confirm these findings and, in addition, indicate that capsid occurs during passage envelopment through membranes of intranuclear vesicular structures (Figs. 3 and 4). The frequency with which such vesicles were encountered inside the nucleus and the lack of any apparent continuity with the cytoplasm of the numerous cells examined argue against the possibility that they represent cross-sectioned cytoplasmic invaginations. Surprisingly, virus in process of envelopment by the peripheral nuclear membrane, an event so common in the case of other herpes-type viruses, was not encountered in these studies. The intranuclear envelopment of capsids as contrasted with the usual mode of envelopment during passage from the nucleus to the cytoplasm has been reported in FIG. 1. FIG. 2. aggregation x 44,000. FIG. 3. x 100,000. FIG. 4. x 75,000. FIG. 5. FIG. 6. FIG. 7. x 40,000. FIG. 8.
811
COMMUNICATIONS
the case of Herpesvirus saimiri (4) and more recently in association with Herpesvirus sylvilagus (5). Similar observations have been made in studies of the replicative cycle of another oncogenic virus, Herpesvirus ateles (Coward, Friedmann, and Morgan, unpublished). Apparently envelopment accompanies passage of the virus into intranuclear vacoules. Zntranuclear structures in HVS-infected cells. It was previously reported that the
nuclei of HVS-infected Vero cells contain filaments (4) and tubules (9). Such intranuclear structures were also observed in the present study. The arrangement of the filaments in both OMK and Vero cells assumed one of two forms. The first was composed of aggregates of fine fibrils (Fig. 51, while the second, which has not been described previously, consisted of linear arrays of granular material (Fig. 6). Such filaments were present in lO-12% of infected OMK and Vero cells. Tubules composed of repeating subunits (Fig. 7) were also found in OMK- and Vero-infected cells. In addition, collections of irregular, convoluted membranes were encountered but only in Vero cells (Fig. 8). The latter arrangement was uncommon being observed in no more than 1% of the cells. Intranuclear tubular and filamentous structures have been observed in association with a variety of herpesviruses. Such structures have been considered “bizarre” since no temporal or spatial relationship could be established between them and the differentiating virus. Curiously they are most frequently encountered in the nuclei of cells infected with oncogenic herpesviruses (1-3, 5, 9-12). It is tempting to pos-
HVS capsids in the cytoplasm of a Vero cell 10 days postinfection. x 36,000. Virus in the cytoplasm of a cell, the nucleus of which is devoid of capsids and does not exhibit or margination of chromatin. Nonspecific hyaline bodies are evident at the lower margin. Capsids
in process of envelopment
Capsids at differing
within
the nucleus
of an OMK
stages of passage into vesicles of a Vero-cell
cell 5 days postinfection.
nucleus
10 days postinfection.
An intranuclear aggregate of fine filaments in an OMK cell 5 days postinfection. x 65,000. A granular filament in the nucleus of an OMK cell 5 days postinfection. x 45,000. An intranuclear tubule composed of repeating subunits in an OMK cell 6 days postinfection. Irregular
membranous
structures
in the nucleus of a Vero cell 10 days postinfection.
x 50,000.
812
SHORT
COMMUNICATIONS
FIGURES 1 AND 2
SHORT
COMMUNICATIONS
FIGURES
3 AND 4
813
814
SHORT
COMMUNICATIONS
FIGURES 5-8
SHORT
COMMUNICATIONS
Mate that they may be the counterpart of polyheads, polysheaths, or polytaiis which accumulate in bacteria infected with amber and t.s mutants of T-even bacteriophages (6). The appearance of nonenveloped capsids in the cytoplasm of cells devoid of nuclear changes suggests that HVS capsids may be actually assembled in the cytoplasm. Should this be confirmed, it will be the first instance of a herpes-type virus differentiating in the cytoplasm. ACKNOWLEDGMENTS This work was supported by United States Public Health Grants AI-06814 from the National Institute of Allergy and Infectious Diseases and International Research Fellowship Award Number FO, TW0188701, National Institutes of Health (A.F.).
3. 4. 5. 6.
7.
8.
9. 10.
REFERENCES 1. COUCH, E. F., and NAHMIAS, J. Viral. 3,228-232 (1969). 2. EPSTEIN, M. A., ACHONG, B. G., CHURCHILL, A.
11. 12.
815
E., and BIGGS, P. M., J. Nat. Cancer Inst. 41, 805811 (1968). FAWCE~, D. W., J. Biophys. Biochem. Cytol. 2, 725-742 (1956). HEINE, U., ABLASHI, D. V., and ARMSTRONG, G. R., Cancer Res. 31, 10191029 (1971). HEINE, LJ., and HINZE, H. C., Cancer Res. 32, 1340-1350 (1972). KELLENBERGER, E., EISERLING, F. A., and BOY DE LA TOUR, E., J. Ultrastruct. Res. 21, 335 360 (1968). MELENDEZ, L. V., HUNT, R. D., DANIEL, M. D., GARCIA, F. G., and FRASER, C. E. O., Lab. Anim. Care 19, 378-386 (1969). MORGAN, D. G., EPSTEIN, M. A., ACHONG, B. G., and MELENDEZ, L. V., Nature (London) 228, 170-172 (1970). MORGAN, D. G., ACHONG, B. G., and EPSTEIN, M. A., Brit. J. Cancer 27, 434-444 (1973). MURPHY, F. A., HARRISON, A. K., and WHITFIELD, S. G., Arch. Ges. Virusforsch. 21, 463 468 (1967). NII, S., MORGAN, C., and ROSE, H. M., J. Viral. 2, 517-536 (1968). WATRACH, A. M., Virology 18, 324-327 (1962).