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The influence of cell type on the production of filaments by herpes simplex virus type 2
Comp. lmmun. Microbiol. infect. Dis., Vol. 2, pp. 191-199. '~ Pergamon Press Ltd., 1979. Printed in Great Britain
0147-9571/79/0901-0191 $02.00/0
THE I N F L U E N C E OF CELL TYPE ON THE P R O D U C T I O N OF F I L A M E N T S BY H E R P E S S I M P L E X V I R U S TYPE 2 ISABEL W. SMITH, J. F. PEUTHERER a n d OONAGH G. GRAY Department of Bacteriology, Medical School, University of Edinburgh, Teviot Place, Edinburgh, U.K. Abstract The production of filaments by herpes simplex virus type 2 was studied in BHK, Vero and HEp-2 cell lines and human embryonic and adult cell strains. It was found that filaments were only produced in the cell lines. Possible explanations are discussed and the literature covering the production of filaments, tubules and stacked lamellae by members of the herpes group is reviewed. Key words: Filaments, H SV-2, cell strains, cell lines, electron microscopy, ultrastructure of nucleus INFLUENCE DU TYPE DE CELLULE SUR LA PRODUCTION DES FILAMENTS PAR LE HERPESVIRUS SIMPLEX TYPE 2 R~sam~-La production des filaments par le herpesvirus simplex type 2 a ere etudiee sur les lignees cellulaires BHK, Vero et HEP-2 et sur des cellules diploides d'embryons et d'adultes humains. Nous avons remarque que les filaments n'&aient produits que dans les lign+escellulaires. Nous discutons ici les explications possibles et passons en revue la litterature couvrant la production des filaments, des tubules et des lamelles entass6es sous Faction des membres du groupe herpes. Mots-clef;s': Filaments, HVS-2, cellules diploides, lign6es cellulaires, microscopic 61ectronique, ultrastructure du noyau
INTRODUCTION E l e c t r o n m i c r o s c o p i c a l studies o f cells infected with various herpesviruses have revealed the presence o f a n u m b e r o f different structures in the cell nucleus in a d d i t i o n to the virus. These can be g r o u p e d into three, o r p o s s i b l y four, categories. T h e b r a i n s o f mice infected with herpes simplex virus (HSV) were f o u n d to c o n t a i n tubules o f 100-nm d i a m e t e r a s s o c i a t e d with viral capsids in the nucleus [1]. Similar structures have been n o t e d in B H K cells infected with Class III ts m u t a n t s o f HSV-2 i n c u b a t e d at the n o n - p e r m i s s i v e t e m p e r a t u r e [2], with M a r e k ' s disease virus [3], infectious l a r y n g o t r a c h e i t i s virus [4], a n d Luck6's virus [5]. T h e d i a g r a m (Fig. l a ) represents t h e structures f o u n d in cells infected with Luck6's virus. T h e t o t a l d i a m e t e r o f the t u b u l e is 65 nm. The central electron-dense filament has a d i a m e t e r o f 35 n m ; c o r r e s p o n d i n g to the core o f the virion. B o t h the inner core a n d the 65-nm t u b u l e have been isolated f r o m cells a n d e x a m i n e d after negative staining. The inner 35-nm filament a n d the inner core o f the virion b o t h h a d a g r a n u l a r a p p e a r a n c e . The 65-nm t u b u l e showed a helical structure with c a p s o m e r e s identical to those o f the virion. This structure w o u l d a p p e a r to be c o m p o s e d o f c a p s o m e r e s t h a t have a s s e m b l e d into a long tubule r a t h e r t h a n into capsids. Similar a b e r r a n t forms have been recognised with p o l y o m a virus [6]. T h e d i m e n s i o n s o f the tubules a n d inner filament have v a r i e d in different reports, d e p e n d i n g on the infecting virus. A similar p o l y h e a d has been f o u n d in cells infected with Herpesvirus saimiri [7-9]. The a p p e a r a n c e differed f r o m t h a t described a b o v e in t h a t the 12-nm lamella o f the t u b u l e h a d 191
192
ISABEL W. SMITH, J. F. PEUTHERERand OONAGH G. GRAY
IIIU a
b
c
d
e
Fig. 1. Diagrammatic representation of structures found in the nuclei of cells infected with members of the herpesvirus group: (a) tubules, (b) polyhead, (c) stacked lamellae, (d) longitudinal view of two filaments or microtubules and (e) cross-section of (d).
subunits on its inner surface and rounded ends could be seen occasionally (Fig. lb). The subunits correspond in size to the viral capsomeres and in some micrographs capsomeres could be seen in the lumen of the tubule. These structures would appear to be aberrant viral capsids. Class III ts mutants of HSV-2 produced similar structures when grown in BHK cells at the non-permissive temperature [2]. The feature illustrated in Fig. lc has been called stacked lamellae; they have been reported in cells infected with Herpesvirus saimiri [9, 10, 11]. The lamellae are each about 12-nm wide, show a periodicity of 6.5 nm and are separated by 20-25-nm electron dense particles. Comparable structures were produced when HSV was grown in human amnion cells in the presence of fluorophenylalanine (FPA) [12]. Filaments resembling stacked lamellae were detected only after 46-hr incubation. Although the published micrographs are of low magnification, the structures are of about the same magnitude as those described for Herpesvirus saimiri. The third type of filament or microtubule is that associated with HSV-2. These filaments are shown in longitudinal section in Fig. ld and in cross-section in Fig. le. Published reports agree that the filaments have a diameter of 16 nm and that the centre to centre spacing in cross-section is 28 nm. These structures have been shown by optical diffraction analysis to have a helical structure [13]. Examination of BHK cells infected with ts mutants of HSV-2 showed that at the non-permissive temperature, filaments were produced irrespective of D N A synthesis [2]. Longitudinal sections of similar filaments have been reported in Jijoye cells containing the Epstein-Barr virus [14], and cells infected with guinea-pig cytomegalovirus [15] and guinea-pig herpes-like virus [16]. The present report examines the production of these filaments or microtubules by HSV-2 in different host cells.
MATERIALS AND METHODS Virus
Herpes simplex virus 8300 was isolated from a skin biopsy from the neck of a patient with a generalised herpetic infection [17]. This isolate, and a number of others including one from the genital area, were characterised as type 2 viruses by the methods described previously [18]. Four of the virus strains described in this latter paper were used in the present study for comparative purposes. Isolates 0027, 0083 and 0330 were classified as
Filament production by HSV-2
193
HSV-2 and isolate 1190 as HSV-1. All had been isolated from male patients with herpetic infections of the genital tract.
Cells The continuous cell lines BHK, Vero and HEp-2 were all available in this department. The human embryo liver, skin and fibroblasts were set up as diploid cultures in this department. The synovial cell culture was obtained from Dr. M. Norval of this department. All the cells were grown on Eagle's medium [19] supplemented with 5~ newborn calf serum for the cell lines and 10~ foetal calf serum for the cell strains. All the cells used were mycoplasma-free.
Growth of virus The stock viruses were passed twice in the cells before inoculating a monolayer culture in a 100-ml bottle. In all but the synovial cells, the medium was removed, inoculum added and allowed to adsorb for 4 hr at 35°C. Ten millilitres of medium were added and incubation continued for 72 hr to ensure that all the cells were infected with virus. The synovial cells had to be treated with hyaluronidase to remove the hyaluronic acid before inoculation with HSV; 80 ~/ml of bovine hyaluronidase (Sigma, Type 1) in complete medium was added to these cells [20].
Preparation of cells for electronmicroscopy The infected and control monolayers were removed with glass beads, centrifuged at 500 g for 5 min, resuspended in Dulbecco's salt solution A, centrifuged at 500 g for 5 min, resuspended in 2~o glutaraldehyde, post-fixed in osmium tetroxide and embedded in araldite [21]. Ultrathin sections were cut on an LKB1 microtome, stained with uranyl acetate and lead citrate and viewed at 30,000 in an Hitachi 12A electron microscope. The neck biopsy was fixed in 2~o glutaraldehyde and processed as above.
RESULTS Figure 2a is an ultrathin section of the skin biopsy. The host cell chromatin is clumped at the nuclear membrane and the centre of the nucleus is packed with herpes virions. No filaments could be seen in this nucleus when viewed at higher magnification nor in any of the other cells examined, even those containing very few virus particles. The virus isolated from the patient was grown in human embryo liver cells (Fig. 2b). Again no filaments were observed but the cellular chromatin was marginated; many virions were found in the nucleus and the nuclear membrane showed infolding. It was noted that the virions varied, some appeared to have a dense core, others were empty and a few had a complete double membrane derived by budding through the nuclear membrane which is preserved. A similar appearance was found when this virus was propagated in human embryo skin cells, fibroblasts and adult synovial cells. In a continuous human cell line (HEp-2) isolate 8300 produced virus and filaments in the
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ISABEL W. SMITH, J. F. PEUTHERER and OONAGH G. GRAY
Fig. 2. Ultrathin section of (a) skin biopsy × 30,600 and (b) h u m a n embryo liver cell infected with isolate 8300 x 15,300; empty (e), full (f) and enveloped (g) particles can be seen within the nucleus.
Filament production by HSV-2
195
Fig. 3. Ultrathin section of (a) HEp-2 cells infected with isolate 8300 x 45,000 showing a virion and filaments : (b) BHK cells infected with isolate 8300× 30,000 showing a cluster of full and empty virions and longitudinal filaments and (c) BHK cells infected with isolate 8300× 37,500 showing filaments cut in cross-section. nucleus. F i g u r e 3a shows l o n g i t u d i n a l filaments a n d a g r o u p in cross-section. T h e nuclear m e m b r a n e has not been preserved but one virion a n d m a r g i n a t e d c h r o m a t i n can be seen indicating the p o s i t i o n o f the m e m b r a n e . In B H K cells (Fig. 3b) a g a i n l o n g i t u d i n a l filaments, virions a n d m a r g i n a t e d c h r o m a t i n can be seen but a g a i n the nuclear m e m b r a n e is not preserved. F i g u r e 3c is also f r o m an infected B H K cell showing g o o d examples o f filaments in cross-section. These filaments are 16-nm wide a n d have a c e n t r e - t o - c e n t r e spacing o f 25 n m in cross-section. V e r o cells infected with 8300 p r o d u c e d virus a n d nuclear filaments. T h e o t h e r viruses were all e x a m i n e d in the s a m e series o f cell cultures. T a b l e 1 shows that filaments were always p r o d u c e d by H S V - 2 in B H K , Vero a n d H E p - 2 cells but never in the h u m a n e m b r y o o r a d u l t synovial cells. The HSV- 1 strain p r o d u c e d virus but no filaments in a n y o f the cells. U n i n o c u l a t e d cells were e x a m i n e d for all the cell lines a n d strains used b u t no filaments were observed.
196
ISABELW. SMITH,J. F. PEUTHERERand OONAGHG. GRAY Table 1. The results of electronmicroscopical examination of cells infected with HSV-1 and HSV-2 Filament production with: HSV-2
HSV-I
Cells
0027
BHK
+
+
+
+
-
Vero
+ + . . -
+ +
+ + . .
+ +
-
HEp-2 Human embryo fibroblasts Human embryo l i v e r Human embryo skin Adult synovial
0083
. .
0330
. . -
.
8300
1190
. -
+ = Filaments observed in nucleus.
DISCUSSION Despite the fact that HSV-2 can produce stacked lamellae [12] and tubules or polyheads [2], only filaments or microtubules were found in this study. This could be due to the fact that the cultures were not treated with F P A [12] and were incubated at 35°C. The stacked lamellae and tubules or polyhead only seem to be produced when the g r o w t h cycle is blocked so they m a y represent aberrant assemblies o f viral components. The detection o f filaments in the nuclei o f cells infected with herpes simplex virus has been used to distinguish H S V 1 and 2 [18]. It has been suggested [22] that routine electronmicroscopy o f B H K infected cells could be used to type HSV in a diagnostic laboratory. F r o m the results presented here it would appear that the type o f cell used to grow the virus plays an important part in the p r o d u c t i o n o f the filaments and that direct electronmicroscopy o f biopsy material cannot be used to determine the type o f the virus. It has been shown that B H K cells [2, 18, 22], Vero cells [23], HEp-2 cells [24], the chorioallantoic m e m b r a n e [25] and mouse brain [1] will allow filament production whereas h u m a n e m b r y o skin, liver and fibroblasts and adult synovial cells do not. These observations on h u m a n cell strains are in agreement with published work on the infection o f h u m a n e m b r y o lung cells with HSV-2 [26]. As it has been shown that only 7-10~o o f infected nuclei contain filaments [22], it was necessary to cut at least two blocks and examine m a n y grids from each preparation before recording a negative result. This procedure was followed in the present study. In addition, the observations were confirmed with fresh preparations o f the h u m a n embryonic fibroblasts and liver cells, but this was not possible with the skin or synovial cell cultures due to a shortage o f cells. Filaments were not seen in the uninfected control cells nor in the preparations o f cells infected with HSV-1. This is in agreement with previous observations [18, 22]. It has been shown that the n u m b e r o f infected Vero cells with filaments was maximal at 12 hr post-infection [23] but that the p r o p o r t i o n decreased thereafter. This finding could be relevant to the failure to detect filaments in the h u m a n cell strains after 72 hr incubation. In this laboratory it was sometimes necessary to search for quite a while to find filaments in Vero cells at 72 hr post-infection. However, before this work was begun it was established
Filament production by HSV-2
197
that in B H K cells, filaments were first noted at 14 hr post-infection--the time at which the culture had produced one infectious virus per infected cell and that filaments continued to be present until 72 hr post-infection. The production of filaments by HSV does not therefore depend solely on the type of virus but also on the host cell. Filaments are produced in BHK, Vero and HEp-2 cells and chick and mouse tissue. They were not found in the neck skin biopsy, foetal fibroblasts or adult synovial cells, although virus was produced in all these cells. The concentration of calf serum in the growth medium for the cell strains was double that of the cell lines. Previous observations [18] were made on BHK cells grown on Eagle's medium supplemented with 10~ calf serum so that the concentration of serum would not explain the lack of filaments in the cell strains. However, foetal calf serum was used for cell strains and newborn calf serum for cell lines. HEp-2 cells are a cell line in contrast to the other human cell strains used in this study. The growth and metabolism of HEp-2 cells differs from that of diploid cells and could affect viral replication. Similarly B H K and Vero cells are cell lines and they also support the production of filaments. The previously reported [1, 25] detection of filaments in mouse and chicken tissue might be related to the fact that most mice and chickens are known to carry endogenous retroviruses. Filaments have been reported in guinea pig cells (which are known to carry a retrovirus) when they are infected with guinea pig herpes-like virus [16]. These filaments are 18 nm in diameter, slightly larger than the 10-16 nm quoted for HSV-2 but the authors consider they resemble the filaments found in chicken tissue [25]. Other reports of similar filaments have been published. Herpes sylvilagus produces 15-nm filaments in a rabbit kidney cell line [27]; the Jijoye cells show fibrils [14] and guinea-pig cytomegalovirus shows 10-13-nm dense strands separated by 5-8-nm spaces [15]. In cross-section there is not a clear lattice structure but a beaded appearance, the beads being separated by 5-8-nm. In all these cases the filaments are produced in cells which are continuous or carry a retrovirus. It would be interesting to try to propagate the guinea-pig viruses in primary cells free from retroviruses to see if filaments are still produced. It has been shown that viruses of the herpes group do not always produce the same effect in different cells. Equine abortion virus when grown on primary horse kidney (HOK) cells does not produce syncytia, whereas it does on the equine dermis (ED) cell line. At the ultrastructural level there are also differences. Fibrillar material which looks to have the diameter of viral cores is found free in the H O K cells but membrane bound in ED cells. The viral yield, measured after the disruption of the cells is very similar. However, from the electron micrographs it appears that spontaneous release of the virus is less in ED cells [28]. Herpes simplex virus produces a more extensive cytopathic effect in primary rabbit kidney cells than in KB cells [29] and the authors consider that the rabbit kidney cells were more sensitive to HSV. Human embryo fibroblasts were shown to be more sensitive to, and to produce more HSV, than KB cells (a cell line) [30]. It is possible that diploid cells allow a greater assembly and spontaneous release of virus than occurs in other cell cultures. Filament production could occur in cells in which the synthesis of viral components occurs but the products are not assembled into complete virions. Further studies will be concerned with the yields of virus from the different types of cell discussed in this paper.
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ISABEL W. SMITH, J. F. PEUTHERER and OONAGH G. GRAY
In this l a b o r a t o r y f i l a m e n t s h a v e b e e n d e t e c t e d at the s a m e t i m e as t h e virus p a r t i c l e s a n d this is in a g r e e m e n t w i t h t h e p u b l i s h e d w o r k in V e r o cells [23]. W i t h g u i n e a - p i g h e r p e s virus, h o w e v e r , f i l a m e n t s a r e d e t e c t e d e a r l i e r t h a n t h e v i r i o n s [16]. T h e a u t h o r s s p e c u l a t e o n the n a t u r e a n d f u n c t i o n o f the f i l a m e n t s s u g g e s t i n g t h a t t h e y a r e e i t h e r a n early s t a g e in virus g r o w t h o r a n a c c u m u l a t i o n o f viral p r o t e i n . R e c e n t l y [2] it has b e e n s h o w n t h a t ts m u t a n t s p r o d u c e f i l a m e n t s in B H K cells at t h e n o n - p e r m i s s i v e t e m p e r a t u r e i r r e s p e c t i v e o f the ability o f t h e m u t a n t to s y n t h e s i s e D N A at t h a t t e m p e r a t u r e . T h i s w o u l d a p p e a r to s u p p o r t the i d e a t h a t the f i l a m e n t s a r e p r o t e i n in n a t u r e . Acknowledgements--We thank Dr. Mary Norval for the synovial ceils and D. O. Brown and E. J. Gowans for the human embryo cells.
REFERENCES 1. Murphy, F. A., Harrison, A. K. and Whitefield, S. G., Intranuclear formation of filaments in herpesvirus hominis infection of mice, Archiv ges. Viru~'lbrsch. 21, 463-468 (1967). 2. Aitkinson, M. A., Barr, S. and Timbury, M. C., The fine structure of cells infected with temperature-sensitive mutants of herpes simplex virus type 2, J. gen. Virology, 40, 103-119 (1978). 3. Campbell, J. G. and Woode, G. N., Demonstration of a herpes-type virus in short-term cultured blood lymphocytes associated with Marek's disease, J. reed. Microbiol., 3, 463-473 (1970). 4. Watrach, A. M., Intranuclear filaments associated with infectious laryngotracheitis virus, Virology, 18, 324-327 (1962). 5. Stackpole, C. W. and Mizell, M., Electron microscopic observations of herpes-type virus-related structures in frog renal adenocarcinomas, Virology 36, 63-72 (1968). 6. Howatson, A. F. and Almeida, J. D., Observations on the fine structure of polyoma virus. J. biophys, biochem. Cytol., 8, 823-834 (1960). 7. Morgan, D. G., Achong, B. C. and Epstein, M. A., Unusual intranuclear tubular structures associated with maturation of Herpesvirus saimiri in monkey kidney cell cultures, Br. J. Cancer, 27, 434-440 (1973). 8. Friedman, A., Coward, J. E. and Morgan, C., Electron microscopic study of the development of Herpesvirus saimiri, Virology, 69, 810-815 (1976). 9. Tralka, T. S., Costa, J. and Rabson, A., Electron microscopic study of Herpesvirus saimiri, Virology, 80, 158-165 (1977). 10. Morgan, D. G., Achong, B. G. and Epstein, M. A., Morphological observations on the replication of Herpesvirus saimiri in monkey kidney cell cultures, J. gen. Virology, 32, 461 470 (1976). 11. Banfield, W. G., Lee, C. W., Tralka, T. S. and Rabson, A. S., Lamella-particle complexes in nuclei of owl monkey kidney cultures infected with Herpesvirus saimiri, J. natn. Cancer Inst. 58, 1421-1425 (1977). 12. Chitwood, L. A. and Bracken, E. C., Replication of herpes simplex virus in a metabolically imbalanced system, Virology, 24, 116-120 (1964). 13. Kitamura, M., Miyazono, J., Mori, R. and Oda, H., Optical diffraction analysis of electron microscope image of tubular structures found in cells infected with herpes simplex virus type 2, J. gen. Virology, 41, 167-170 (1978). 14. Toshima, S., Takagi, N., Minowada, J., Moore, G. E. and Sandberg, A. A., Electron microscopic and cytogenetic studies of cells derived from Burkitt's Lymphoma, Cancer Res., 27, 753 771 (1967). 15. Middlekamp, J. N., Patrizi, G. and Reed, C. A., Light and electron-microscopical studies of the guinea pig cytomegalovirus, J. Ultrastruct. Res., 18, 85-101 (1967). 16. Fong, C. K. and Hsiung, G. D., Morphogenic studies of herpesvirus and oncornavirus from leukaemic and normal guinea pigs, Fedn. Proc. Fedn. Am. Socs exp. Biol., 36, 232(~2327 (1977). 17. Peutherer, J. F., Smith, I. W. and Robertson, D. H. H. Virological studies in a case of necrotising balanitis due to a generalised primary infection with herpes simplex virus, type 2, Br. J. vener. Dis. 55, 48-51 (1979). 18. Smith, I. W., Peutherer, J. F. and Robertson, D. H. H., Characterization of genital strains of Herpesvirus hominis, Br. J. vener. Dis., 49, 385-390 (1973). 19. Smith, I. W., Cell, tissue and organ culture. In Medical Microbiolog~ Cruickshank, R., Duguid, J. P., Marmion, B. P. and Swain, R. H. A. (Eds), 12th edition, Vol. 2, p. 209. Churchill Livingstone, Edinburgh (1975).
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20. Clarris, B. J., Fraser, J. R. E. and Rodda, S. J., Effect of cell-bound hyaluronic acid on the infectivity of Newcastle disease virus for human synovial cells in vitro, Ann. rheum. Dis., 33, 240-242 (1974). 21. Glauert, A. M., The fixation and embedding of biological specimens. In Techniques for Electron Microscopy Kay, D. (Ed.), 2nd edition, p. 166. Blackwell Scientific Publications, London (1965). 22. Young, S. K., Rowe, N. H. and Sanderlin, K. C., Herpes simplex virus types 1 and 2 in clinical infections: differences observed by electronmicroscopy, J. infect. Dis., 135, 486-489 (1977). 23. Oda, H. and Mori, R., Electron microscope observations on tubular structures in cells infected with herpes simplex virus type 2, Archs Virology, 511, 159-168 (1976). 24. Schwartz, J. and Roizman, B., Similarities and differences in the development of laboratory strains and freshly isolated strains of herpes simplex virus in HEp-2 cells: electron microscopy, J. Virology, 4, 879-889 (1969). 25. Couch, E. F. and Nahmias, A. J., Filamentous structures of type 2 Herpesvirus hominis infection of the chorioallantoic membrane, J. Virology, 3, 228-232 (1969). 26. Cabral, G. A. and Schaffer, P. A., Electron microscope studies of temperature-sensitive mutants of herpes simplex virus type 2, J. Virology, 18, 727-737 (1976). 27. Heine, U. and Hinze, H. C., Morphological studies on Herpes sylvilagus in rabbit kidney cell cultures, Cancer Res., 32, 1340-1350 (1972). 28. Fong, C. K. Y. and Hsiung, G. D., Development of an equine herpes virus in two cell culture systems. Light and electronmicroscopy, Infect. Immun., 6~ 865-876 (1972). 29. Waddell, G. H. and Sigel, M. M., Factors association with the response of the cell to herpes simplex virus infections, Archiv ges. Viru~[brsch., 19, 130-142 (1966). 30. Albanese, M., Bynoe, M. L. and Tyrrell, D. A. J., Studies of a strain of herpes virus isolated from a case of upper respiratory disease, Archiv ges. Viru~forsch., 18, 356-362 (1966).
0147-9571/79/0901-0191 $02.00/0
THE I N F L U E N C E OF CELL TYPE ON THE P R O D U C T I O N OF F I L A M E N T S BY H E R P E S S I M P L E X V I R U S TYPE 2 ISABEL W. SMITH, J. F. PEUTHERER a n d OONAGH G. GRAY Department of Bacteriology, Medical School, University of Edinburgh, Teviot Place, Edinburgh, U.K. Abstract The production of filaments by herpes simplex virus type 2 was studied in BHK, Vero and HEp-2 cell lines and human embryonic and adult cell strains. It was found that filaments were only produced in the cell lines. Possible explanations are discussed and the literature covering the production of filaments, tubules and stacked lamellae by members of the herpes group is reviewed. Key words: Filaments, H SV-2, cell strains, cell lines, electron microscopy, ultrastructure of nucleus INFLUENCE DU TYPE DE CELLULE SUR LA PRODUCTION DES FILAMENTS PAR LE HERPESVIRUS SIMPLEX TYPE 2 R~sam~-La production des filaments par le herpesvirus simplex type 2 a ere etudiee sur les lignees cellulaires BHK, Vero et HEP-2 et sur des cellules diploides d'embryons et d'adultes humains. Nous avons remarque que les filaments n'&aient produits que dans les lign+escellulaires. Nous discutons ici les explications possibles et passons en revue la litterature couvrant la production des filaments, des tubules et des lamelles entass6es sous Faction des membres du groupe herpes. Mots-clef;s': Filaments, HVS-2, cellules diploides, lign6es cellulaires, microscopic 61ectronique, ultrastructure du noyau
INTRODUCTION E l e c t r o n m i c r o s c o p i c a l studies o f cells infected with various herpesviruses have revealed the presence o f a n u m b e r o f different structures in the cell nucleus in a d d i t i o n to the virus. These can be g r o u p e d into three, o r p o s s i b l y four, categories. T h e b r a i n s o f mice infected with herpes simplex virus (HSV) were f o u n d to c o n t a i n tubules o f 100-nm d i a m e t e r a s s o c i a t e d with viral capsids in the nucleus [1]. Similar structures have been n o t e d in B H K cells infected with Class III ts m u t a n t s o f HSV-2 i n c u b a t e d at the n o n - p e r m i s s i v e t e m p e r a t u r e [2], with M a r e k ' s disease virus [3], infectious l a r y n g o t r a c h e i t i s virus [4], a n d Luck6's virus [5]. T h e d i a g r a m (Fig. l a ) represents t h e structures f o u n d in cells infected with Luck6's virus. T h e t o t a l d i a m e t e r o f the t u b u l e is 65 nm. The central electron-dense filament has a d i a m e t e r o f 35 n m ; c o r r e s p o n d i n g to the core o f the virion. B o t h the inner core a n d the 65-nm t u b u l e have been isolated f r o m cells a n d e x a m i n e d after negative staining. The inner 35-nm filament a n d the inner core o f the virion b o t h h a d a g r a n u l a r a p p e a r a n c e . The 65-nm t u b u l e showed a helical structure with c a p s o m e r e s identical to those o f the virion. This structure w o u l d a p p e a r to be c o m p o s e d o f c a p s o m e r e s t h a t have a s s e m b l e d into a long tubule r a t h e r t h a n into capsids. Similar a b e r r a n t forms have been recognised with p o l y o m a virus [6]. T h e d i m e n s i o n s o f the tubules a n d inner filament have v a r i e d in different reports, d e p e n d i n g on the infecting virus. A similar p o l y h e a d has been f o u n d in cells infected with Herpesvirus saimiri [7-9]. The a p p e a r a n c e differed f r o m t h a t described a b o v e in t h a t the 12-nm lamella o f the t u b u l e h a d 191
192
ISABEL W. SMITH, J. F. PEUTHERERand OONAGH G. GRAY
IIIU a
b
c
d
e
Fig. 1. Diagrammatic representation of structures found in the nuclei of cells infected with members of the herpesvirus group: (a) tubules, (b) polyhead, (c) stacked lamellae, (d) longitudinal view of two filaments or microtubules and (e) cross-section of (d).
subunits on its inner surface and rounded ends could be seen occasionally (Fig. lb). The subunits correspond in size to the viral capsomeres and in some micrographs capsomeres could be seen in the lumen of the tubule. These structures would appear to be aberrant viral capsids. Class III ts mutants of HSV-2 produced similar structures when grown in BHK cells at the non-permissive temperature [2]. The feature illustrated in Fig. lc has been called stacked lamellae; they have been reported in cells infected with Herpesvirus saimiri [9, 10, 11]. The lamellae are each about 12-nm wide, show a periodicity of 6.5 nm and are separated by 20-25-nm electron dense particles. Comparable structures were produced when HSV was grown in human amnion cells in the presence of fluorophenylalanine (FPA) [12]. Filaments resembling stacked lamellae were detected only after 46-hr incubation. Although the published micrographs are of low magnification, the structures are of about the same magnitude as those described for Herpesvirus saimiri. The third type of filament or microtubule is that associated with HSV-2. These filaments are shown in longitudinal section in Fig. ld and in cross-section in Fig. le. Published reports agree that the filaments have a diameter of 16 nm and that the centre to centre spacing in cross-section is 28 nm. These structures have been shown by optical diffraction analysis to have a helical structure [13]. Examination of BHK cells infected with ts mutants of HSV-2 showed that at the non-permissive temperature, filaments were produced irrespective of D N A synthesis [2]. Longitudinal sections of similar filaments have been reported in Jijoye cells containing the Epstein-Barr virus [14], and cells infected with guinea-pig cytomegalovirus [15] and guinea-pig herpes-like virus [16]. The present report examines the production of these filaments or microtubules by HSV-2 in different host cells.
MATERIALS AND METHODS Virus
Herpes simplex virus 8300 was isolated from a skin biopsy from the neck of a patient with a generalised herpetic infection [17]. This isolate, and a number of others including one from the genital area, were characterised as type 2 viruses by the methods described previously [18]. Four of the virus strains described in this latter paper were used in the present study for comparative purposes. Isolates 0027, 0083 and 0330 were classified as
Filament production by HSV-2
193
HSV-2 and isolate 1190 as HSV-1. All had been isolated from male patients with herpetic infections of the genital tract.
Cells The continuous cell lines BHK, Vero and HEp-2 were all available in this department. The human embryo liver, skin and fibroblasts were set up as diploid cultures in this department. The synovial cell culture was obtained from Dr. M. Norval of this department. All the cells were grown on Eagle's medium [19] supplemented with 5~ newborn calf serum for the cell lines and 10~ foetal calf serum for the cell strains. All the cells used were mycoplasma-free.
Growth of virus The stock viruses were passed twice in the cells before inoculating a monolayer culture in a 100-ml bottle. In all but the synovial cells, the medium was removed, inoculum added and allowed to adsorb for 4 hr at 35°C. Ten millilitres of medium were added and incubation continued for 72 hr to ensure that all the cells were infected with virus. The synovial cells had to be treated with hyaluronidase to remove the hyaluronic acid before inoculation with HSV; 80 ~/ml of bovine hyaluronidase (Sigma, Type 1) in complete medium was added to these cells [20].
Preparation of cells for electronmicroscopy The infected and control monolayers were removed with glass beads, centrifuged at 500 g for 5 min, resuspended in Dulbecco's salt solution A, centrifuged at 500 g for 5 min, resuspended in 2~o glutaraldehyde, post-fixed in osmium tetroxide and embedded in araldite [21]. Ultrathin sections were cut on an LKB1 microtome, stained with uranyl acetate and lead citrate and viewed at 30,000 in an Hitachi 12A electron microscope. The neck biopsy was fixed in 2~o glutaraldehyde and processed as above.
RESULTS Figure 2a is an ultrathin section of the skin biopsy. The host cell chromatin is clumped at the nuclear membrane and the centre of the nucleus is packed with herpes virions. No filaments could be seen in this nucleus when viewed at higher magnification nor in any of the other cells examined, even those containing very few virus particles. The virus isolated from the patient was grown in human embryo liver cells (Fig. 2b). Again no filaments were observed but the cellular chromatin was marginated; many virions were found in the nucleus and the nuclear membrane showed infolding. It was noted that the virions varied, some appeared to have a dense core, others were empty and a few had a complete double membrane derived by budding through the nuclear membrane which is preserved. A similar appearance was found when this virus was propagated in human embryo skin cells, fibroblasts and adult synovial cells. In a continuous human cell line (HEp-2) isolate 8300 produced virus and filaments in the
194
ISABEL W. SMITH, J. F. PEUTHERER and OONAGH G. GRAY
Fig. 2. Ultrathin section of (a) skin biopsy × 30,600 and (b) h u m a n embryo liver cell infected with isolate 8300 x 15,300; empty (e), full (f) and enveloped (g) particles can be seen within the nucleus.
Filament production by HSV-2
195
Fig. 3. Ultrathin section of (a) HEp-2 cells infected with isolate 8300 x 45,000 showing a virion and filaments : (b) BHK cells infected with isolate 8300× 30,000 showing a cluster of full and empty virions and longitudinal filaments and (c) BHK cells infected with isolate 8300× 37,500 showing filaments cut in cross-section. nucleus. F i g u r e 3a shows l o n g i t u d i n a l filaments a n d a g r o u p in cross-section. T h e nuclear m e m b r a n e has not been preserved but one virion a n d m a r g i n a t e d c h r o m a t i n can be seen indicating the p o s i t i o n o f the m e m b r a n e . In B H K cells (Fig. 3b) a g a i n l o n g i t u d i n a l filaments, virions a n d m a r g i n a t e d c h r o m a t i n can be seen but a g a i n the nuclear m e m b r a n e is not preserved. F i g u r e 3c is also f r o m an infected B H K cell showing g o o d examples o f filaments in cross-section. These filaments are 16-nm wide a n d have a c e n t r e - t o - c e n t r e spacing o f 25 n m in cross-section. V e r o cells infected with 8300 p r o d u c e d virus a n d nuclear filaments. T h e o t h e r viruses were all e x a m i n e d in the s a m e series o f cell cultures. T a b l e 1 shows that filaments were always p r o d u c e d by H S V - 2 in B H K , Vero a n d H E p - 2 cells but never in the h u m a n e m b r y o o r a d u l t synovial cells. The HSV- 1 strain p r o d u c e d virus but no filaments in a n y o f the cells. U n i n o c u l a t e d cells were e x a m i n e d for all the cell lines a n d strains used b u t no filaments were observed.
196
ISABELW. SMITH,J. F. PEUTHERERand OONAGHG. GRAY Table 1. The results of electronmicroscopical examination of cells infected with HSV-1 and HSV-2 Filament production with: HSV-2
HSV-I
Cells
0027
BHK
+
+
+
+
-
Vero
+ + . . -
+ +
+ + . .
+ +
-
HEp-2 Human embryo fibroblasts Human embryo l i v e r Human embryo skin Adult synovial
0083
. .
0330
. . -
.
8300
1190
. -
+ = Filaments observed in nucleus.
DISCUSSION Despite the fact that HSV-2 can produce stacked lamellae [12] and tubules or polyheads [2], only filaments or microtubules were found in this study. This could be due to the fact that the cultures were not treated with F P A [12] and were incubated at 35°C. The stacked lamellae and tubules or polyhead only seem to be produced when the g r o w t h cycle is blocked so they m a y represent aberrant assemblies o f viral components. The detection o f filaments in the nuclei o f cells infected with herpes simplex virus has been used to distinguish H S V 1 and 2 [18]. It has been suggested [22] that routine electronmicroscopy o f B H K infected cells could be used to type HSV in a diagnostic laboratory. F r o m the results presented here it would appear that the type o f cell used to grow the virus plays an important part in the p r o d u c t i o n o f the filaments and that direct electronmicroscopy o f biopsy material cannot be used to determine the type o f the virus. It has been shown that B H K cells [2, 18, 22], Vero cells [23], HEp-2 cells [24], the chorioallantoic m e m b r a n e [25] and mouse brain [1] will allow filament production whereas h u m a n e m b r y o skin, liver and fibroblasts and adult synovial cells do not. These observations on h u m a n cell strains are in agreement with published work on the infection o f h u m a n e m b r y o lung cells with HSV-2 [26]. As it has been shown that only 7-10~o o f infected nuclei contain filaments [22], it was necessary to cut at least two blocks and examine m a n y grids from each preparation before recording a negative result. This procedure was followed in the present study. In addition, the observations were confirmed with fresh preparations o f the h u m a n embryonic fibroblasts and liver cells, but this was not possible with the skin or synovial cell cultures due to a shortage o f cells. Filaments were not seen in the uninfected control cells nor in the preparations o f cells infected with HSV-1. This is in agreement with previous observations [18, 22]. It has been shown that the n u m b e r o f infected Vero cells with filaments was maximal at 12 hr post-infection [23] but that the p r o p o r t i o n decreased thereafter. This finding could be relevant to the failure to detect filaments in the h u m a n cell strains after 72 hr incubation. In this laboratory it was sometimes necessary to search for quite a while to find filaments in Vero cells at 72 hr post-infection. However, before this work was begun it was established
Filament production by HSV-2
197
that in B H K cells, filaments were first noted at 14 hr post-infection--the time at which the culture had produced one infectious virus per infected cell and that filaments continued to be present until 72 hr post-infection. The production of filaments by HSV does not therefore depend solely on the type of virus but also on the host cell. Filaments are produced in BHK, Vero and HEp-2 cells and chick and mouse tissue. They were not found in the neck skin biopsy, foetal fibroblasts or adult synovial cells, although virus was produced in all these cells. The concentration of calf serum in the growth medium for the cell strains was double that of the cell lines. Previous observations [18] were made on BHK cells grown on Eagle's medium supplemented with 10~ calf serum so that the concentration of serum would not explain the lack of filaments in the cell strains. However, foetal calf serum was used for cell strains and newborn calf serum for cell lines. HEp-2 cells are a cell line in contrast to the other human cell strains used in this study. The growth and metabolism of HEp-2 cells differs from that of diploid cells and could affect viral replication. Similarly B H K and Vero cells are cell lines and they also support the production of filaments. The previously reported [1, 25] detection of filaments in mouse and chicken tissue might be related to the fact that most mice and chickens are known to carry endogenous retroviruses. Filaments have been reported in guinea pig cells (which are known to carry a retrovirus) when they are infected with guinea pig herpes-like virus [16]. These filaments are 18 nm in diameter, slightly larger than the 10-16 nm quoted for HSV-2 but the authors consider they resemble the filaments found in chicken tissue [25]. Other reports of similar filaments have been published. Herpes sylvilagus produces 15-nm filaments in a rabbit kidney cell line [27]; the Jijoye cells show fibrils [14] and guinea-pig cytomegalovirus shows 10-13-nm dense strands separated by 5-8-nm spaces [15]. In cross-section there is not a clear lattice structure but a beaded appearance, the beads being separated by 5-8-nm. In all these cases the filaments are produced in cells which are continuous or carry a retrovirus. It would be interesting to try to propagate the guinea-pig viruses in primary cells free from retroviruses to see if filaments are still produced. It has been shown that viruses of the herpes group do not always produce the same effect in different cells. Equine abortion virus when grown on primary horse kidney (HOK) cells does not produce syncytia, whereas it does on the equine dermis (ED) cell line. At the ultrastructural level there are also differences. Fibrillar material which looks to have the diameter of viral cores is found free in the H O K cells but membrane bound in ED cells. The viral yield, measured after the disruption of the cells is very similar. However, from the electron micrographs it appears that spontaneous release of the virus is less in ED cells [28]. Herpes simplex virus produces a more extensive cytopathic effect in primary rabbit kidney cells than in KB cells [29] and the authors consider that the rabbit kidney cells were more sensitive to HSV. Human embryo fibroblasts were shown to be more sensitive to, and to produce more HSV, than KB cells (a cell line) [30]. It is possible that diploid cells allow a greater assembly and spontaneous release of virus than occurs in other cell cultures. Filament production could occur in cells in which the synthesis of viral components occurs but the products are not assembled into complete virions. Further studies will be concerned with the yields of virus from the different types of cell discussed in this paper.
198
ISABEL W. SMITH, J. F. PEUTHERER and OONAGH G. GRAY
In this l a b o r a t o r y f i l a m e n t s h a v e b e e n d e t e c t e d at the s a m e t i m e as t h e virus p a r t i c l e s a n d this is in a g r e e m e n t w i t h t h e p u b l i s h e d w o r k in V e r o cells [23]. W i t h g u i n e a - p i g h e r p e s virus, h o w e v e r , f i l a m e n t s a r e d e t e c t e d e a r l i e r t h a n t h e v i r i o n s [16]. T h e a u t h o r s s p e c u l a t e o n the n a t u r e a n d f u n c t i o n o f the f i l a m e n t s s u g g e s t i n g t h a t t h e y a r e e i t h e r a n early s t a g e in virus g r o w t h o r a n a c c u m u l a t i o n o f viral p r o t e i n . R e c e n t l y [2] it has b e e n s h o w n t h a t ts m u t a n t s p r o d u c e f i l a m e n t s in B H K cells at t h e n o n - p e r m i s s i v e t e m p e r a t u r e i r r e s p e c t i v e o f the ability o f t h e m u t a n t to s y n t h e s i s e D N A at t h a t t e m p e r a t u r e . T h i s w o u l d a p p e a r to s u p p o r t the i d e a t h a t the f i l a m e n t s a r e p r o t e i n in n a t u r e . Acknowledgements--We thank Dr. Mary Norval for the synovial ceils and D. O. Brown and E. J. Gowans for the human embryo cells.
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20. Clarris, B. J., Fraser, J. R. E. and Rodda, S. J., Effect of cell-bound hyaluronic acid on the infectivity of Newcastle disease virus for human synovial cells in vitro, Ann. rheum. Dis., 33, 240-242 (1974). 21. Glauert, A. M., The fixation and embedding of biological specimens. In Techniques for Electron Microscopy Kay, D. (Ed.), 2nd edition, p. 166. Blackwell Scientific Publications, London (1965). 22. Young, S. K., Rowe, N. H. and Sanderlin, K. C., Herpes simplex virus types 1 and 2 in clinical infections: differences observed by electronmicroscopy, J. infect. Dis., 135, 486-489 (1977). 23. Oda, H. and Mori, R., Electron microscope observations on tubular structures in cells infected with herpes simplex virus type 2, Archs Virology, 511, 159-168 (1976). 24. Schwartz, J. and Roizman, B., Similarities and differences in the development of laboratory strains and freshly isolated strains of herpes simplex virus in HEp-2 cells: electron microscopy, J. Virology, 4, 879-889 (1969). 25. Couch, E. F. and Nahmias, A. J., Filamentous structures of type 2 Herpesvirus hominis infection of the chorioallantoic membrane, J. Virology, 3, 228-232 (1969). 26. Cabral, G. A. and Schaffer, P. A., Electron microscope studies of temperature-sensitive mutants of herpes simplex virus type 2, J. Virology, 18, 727-737 (1976). 27. Heine, U. and Hinze, H. C., Morphological studies on Herpes sylvilagus in rabbit kidney cell cultures, Cancer Res., 32, 1340-1350 (1972). 28. Fong, C. K. Y. and Hsiung, G. D., Development of an equine herpes virus in two cell culture systems. Light and electronmicroscopy, Infect. Immun., 6~ 865-876 (1972). 29. Waddell, G. H. and Sigel, M. M., Factors association with the response of the cell to herpes simplex virus infections, Archiv ges. Viru~[brsch., 19, 130-142 (1966). 30. Albanese, M., Bynoe, M. L. and Tyrrell, D. A. J., Studies of a strain of herpes virus isolated from a case of upper respiratory disease, Archiv ges. Viru~forsch., 18, 356-362 (1966).