Journal of Medical Virology 3:271-274 (1979)
Fusion of Cytomegalovirus Infected Fibroblasts to Form Multinucleate Giant Helen M. Garnett Department of Botany and Microbiology, University of the Witwatersrand, Johannesburg, South Africa
Multinucleate giant cells have been observed to arise by cell fusion in cultures of human embryonic fibroblasts infected with human cytomegalovirus (CMV), strain AD 1 69. The infected cells developed intranuclear and intracytoplasmic inclusions and began t o fuse three to four days postinfection when the majority of the cells were showing signs of a cytopathic effect. Key words: cytomegalovirus, multinucelate cell, fusion
I NTR OD UCTlO N
During the process of CMV replication in human embryonic fibroblasts the membrane of infected cells is altered in topography [Garnett, in press], in activity [Garnett, 19741. and in composition [Boldogh et al, 1977; Michelson-Fiske, 19771. Infection characteristically leads to a cytopathic effect characterised by cells rounding and the cells becoming less adhesive both to the neighbouring normal cells and to the substrate [Wright et al, 19681. Multinucleate giant cells are not generally observed in such cultures although it has been reported that infected cells can obtain a phagocytic property and engulf other infected cells, leading to the formation of large niultinucleate cells, which retained their phagocytic activity [Diosi et al, 19721. This paper reports on the formation of similar multinucleate giant cells by the process of fusion rather than phagocytosis. Manuscript received July 17, 1978. Address reprint requests to Helen M. Garnett, Department of Botany and Microbiology, University of Witwatersrand, Jan Smuts Avenue, Johannesburg, South Africa.
0146-6615/79/0304-0271,$01.10 01979 Alan R. Liss, Inc.
272
Garnett
MATERIALS AND METHODS Cell Culture
Human embryonic fibroblasts were routinely obtained from the lungs of 12-25week-old fetuses by the method of Hayfick and Moorhead [1961]. These were generally seeded in 75 cm2 Falcon flasks, although tube cultures were also prepared, using Eagle's minimal essential medium (MEM) supplemented with 10%fetal calf serum. Three to four days later the medium was changed to maintenance medium, this being MEM supplemented with 2% fetal calf serum. The cultures were routinely checked for the presence of mycoplasma by the method of Hayflick [1965] . Tube cultures of HeLa cells, Vero cells, and primary African green monkey kidney cells were grown on MEM containing 10% calf serum. Infection
When confluent (3-4 days after seeding), fibroblasts were washed with Hanks' balanced salt solution and exposed to lo7 plaque-forming units of CMV, strain AD 169, in 3 ml of MEM. After adsorption for 90 minutes at 37"C, maintenance medium was added. After seven days the medium was removed and the remaining cells scraped into 10 nil of fresh MEM. This was freeze-thawed three times and clarified by centrifugation at 3,OOOg for 10 minutes. Aliquots, 0.5 ml, of this extract and of the original supernatant were used to inoculate tube cultures of HeLa, Vero, African green monkey kidney cells, and human fibroblasts in a manner similar to that already described. Fibroblasts were also inoculated with herpes simplex virus. Light Microscopy
Observations of cell morphology were made with a Nikon inverted phase contrast microscope. For photography, cultures were fixed in methanol for 5 minutes, stained in May-Grunwald solution for 10 minutes and Giemsa for 20 minutes. A Zeiss photomicroscope was used for photography of stained cultures. Electron Microscopy
Five ml aliquots of the supernatant and the clarified extract from infected cells were spun at 100,OOOg for 1 hour at 4 O C . The supernatants were removed and the pellets resuspended in 0.3 1n1of 2.5% phosphotungstic acid. This material was then applied to Formvar-coated grids and viewed under an AEI electron microscope. RESULTS
The effect of CMV on human embryonic fibroblasts was generally as previously described [Wright et al, 19681. However, cells obtained from one particular 16-week-old fetus were noted to give a different sequence of events. The cells rounded up as normal after 18 hours, and intranuclear and intracytoplasmic inclusions developed, However three to four days postinfection, when 90% of the cells were showing signs of infection, evidence of fusion was noted. The process was followed under the phase-contrast microscope. The cells were not engulfed by other cells as no evidence of pseudopodia was seen. However, the membranes of adjoining cells appeared to break down to form a binucleate cell. Progressively more cells fused to give a multinucleate structure (Figs. 1 and 2). No destruction of the nucleus or the inclusion bodies was observed. This effect was reproducible in these cells, providing the inoculum contained a high titer of virus.
Fusion of CMV Infected Cells
273
Fig. 1. Multinucleate fibroblasts fusing t o form an even larger cell (X 500).
Fig. 2. Large multinucleate giant cell, resulting from fusion, observed seven days after challenge of a fibroblast culture with CMV ( X 500).
Herpes simplex inoculation of cultures from the same fetus gave the typical margination of chromatin. No fusion was observed. To ensure that no other viruses were contaminating the culture, the maintenance medium and free-thawed cell extracts from the infected cultures were used to inoculate Vero cells, primary monkey kidney cells, and HeLa cells. No evidence of viral infection was seen in any of these cells. Electron microscopic observations of the high-speed pellet obtained from the medium and the clarified cell extract revealed only particles with the typical marphology of the herpesviridae. DISCUSSION
The presence of large “owls-eye’’ cells is characteristic of human CMV infections in vivo [von Glahn and Pappenheimer, 19251. In vitro the virus is generally grown in fibroblasts which round up on infection and pull away from their neighbouring cells [Wright et al, 19681. However, another phenomenon has been noted in a culture where 90% of the cells were showing inclusions three to four days after infection. Binucleate cells arose from cell fusion. The fusion process proceeded, and large multinucleate giant cells resulted (Figs. 1 and 2). Particles with the morphology of CMV were isolated from the cells but no evidence of other viruses was obtained. Obviously the possibility exists that there was somethng unusual about the particular line of fibroblasts. However, they showed the normal cytopathogenic effect when inoculated with herpes simplex, and uninoculated cultures were successfully passaged some 35 times, during which time they retained their typical fibroblast morphology. The alternative assumption is that under certain conditions of infection the surface of infected cells is altered to favour cell fusion. New antigens have been detected in the membranes
274
Garnett
of CMV infected cells [Boldogh et al, 1977; Michelson-Fiske, 19771, and it may be that a particular level of these antigens triggers the fusion process. Alternatively, the altered distribution of normal membrane components could trigger the process. Recently a similar phenomenon has been noted during the isolation of CMV from several clinical specimens in other lines o f fibroblasts [Le ROUX,personal communication] and has been noted when CMV infected cells are exposed to aflatoxin B1 [Garnett, in press] . Thus it appears that multinucleate cells can arise by fusion in CMV infected cultures in vitro and it is possible that giant cells may arise in vivo by a similar process. ADDENDUM
Since the preparation o f this manuscript, a paper has been published reporting the formation o f syncytia some 40 days after the infection of MRC-5 fibroblasts by low-titer human CMV [Booth et al, 19781 . These authors suggested that prolonged abortive infection of cells by CMV may he the cause of the syncytia. In contrast, the formation of the multinucleate giant cells reported above is a rapid process. Although Booth et a1 [1978] mention possible “fusion factors,” no reference is made to observations of fusion, as was noted in the current studies. ACKNOWLEDGMENTS
This work was supported by a grant from the South African Medical Research Council. REFERENCES Boldogh I, Gonczol E, Vaczi L (1977): Cytomegalovirus induced membrane antigens in productively infected cells. Acta Microbiologica Academiae Scientarum Hungaricae 24: 21 -28. Booth JC, Beesley JE, Stern H (1978): Syncytium formation caused by human cytomegalovirus in human embryonic lung fibroblasts. Arch Virol 57: 143-152. Diosi P, Babusceac L, Gherman D (1972): Cytophagia in cell cultures infected with cytomegalovirus. Journal of Infectious Discases 125:669%671. Garnett HM (1974): A cytomegalovirus induced membrane alteration indicating a vital role for Ca2+ in virus replication. South African Journal of Science 70:93-94. von Glahn WC, I’appenheimer AM (1925): Intranuclear inclusions in visceral disease. American Journal of Pathology 1:445-466. Hayflick L (1965): Tissue cultures and mycoplasma. Texas Reports of Biology and Medicine. 23: 285-303. Hayflick L, Moorhead PS (1961): The serial cultivation of human diploid cell strains. Experiincntal Cell Research 25:585-621. Michelson-Fiske S (1977): Human cytomegalovirus. A review of developments between 1972 and 1976. Part 11: Experimental developments. Biomedicine 26:86-97. Wright HT, Kasten FH, McAllister RM ( 1 968): Human cytomcgalovirus. Observations of intracellular lesion development as revealed by phase contrast, time-lapse cinematography. Proceedings of the Society for Experimental Biology and Medicine 127:1032-1036.
Fusion of Cytomegalovirus Infected Fibroblasts to Form Multinucleate Giant Helen M. Garnett Department of Botany and Microbiology, University of the Witwatersrand, Johannesburg, South Africa
Multinucleate giant cells have been observed to arise by cell fusion in cultures of human embryonic fibroblasts infected with human cytomegalovirus (CMV), strain AD 1 69. The infected cells developed intranuclear and intracytoplasmic inclusions and began t o fuse three to four days postinfection when the majority of the cells were showing signs of a cytopathic effect. Key words: cytomegalovirus, multinucelate cell, fusion
I NTR OD UCTlO N
During the process of CMV replication in human embryonic fibroblasts the membrane of infected cells is altered in topography [Garnett, in press], in activity [Garnett, 19741. and in composition [Boldogh et al, 1977; Michelson-Fiske, 19771. Infection characteristically leads to a cytopathic effect characterised by cells rounding and the cells becoming less adhesive both to the neighbouring normal cells and to the substrate [Wright et al, 19681. Multinucleate giant cells are not generally observed in such cultures although it has been reported that infected cells can obtain a phagocytic property and engulf other infected cells, leading to the formation of large niultinucleate cells, which retained their phagocytic activity [Diosi et al, 19721. This paper reports on the formation of similar multinucleate giant cells by the process of fusion rather than phagocytosis. Manuscript received July 17, 1978. Address reprint requests to Helen M. Garnett, Department of Botany and Microbiology, University of Witwatersrand, Jan Smuts Avenue, Johannesburg, South Africa.
0146-6615/79/0304-0271,$01.10 01979 Alan R. Liss, Inc.
272
Garnett
MATERIALS AND METHODS Cell Culture
Human embryonic fibroblasts were routinely obtained from the lungs of 12-25week-old fetuses by the method of Hayfick and Moorhead [1961]. These were generally seeded in 75 cm2 Falcon flasks, although tube cultures were also prepared, using Eagle's minimal essential medium (MEM) supplemented with 10%fetal calf serum. Three to four days later the medium was changed to maintenance medium, this being MEM supplemented with 2% fetal calf serum. The cultures were routinely checked for the presence of mycoplasma by the method of Hayflick [1965] . Tube cultures of HeLa cells, Vero cells, and primary African green monkey kidney cells were grown on MEM containing 10% calf serum. Infection
When confluent (3-4 days after seeding), fibroblasts were washed with Hanks' balanced salt solution and exposed to lo7 plaque-forming units of CMV, strain AD 169, in 3 ml of MEM. After adsorption for 90 minutes at 37"C, maintenance medium was added. After seven days the medium was removed and the remaining cells scraped into 10 nil of fresh MEM. This was freeze-thawed three times and clarified by centrifugation at 3,OOOg for 10 minutes. Aliquots, 0.5 ml, of this extract and of the original supernatant were used to inoculate tube cultures of HeLa, Vero, African green monkey kidney cells, and human fibroblasts in a manner similar to that already described. Fibroblasts were also inoculated with herpes simplex virus. Light Microscopy
Observations of cell morphology were made with a Nikon inverted phase contrast microscope. For photography, cultures were fixed in methanol for 5 minutes, stained in May-Grunwald solution for 10 minutes and Giemsa for 20 minutes. A Zeiss photomicroscope was used for photography of stained cultures. Electron Microscopy
Five ml aliquots of the supernatant and the clarified extract from infected cells were spun at 100,OOOg for 1 hour at 4 O C . The supernatants were removed and the pellets resuspended in 0.3 1n1of 2.5% phosphotungstic acid. This material was then applied to Formvar-coated grids and viewed under an AEI electron microscope. RESULTS
The effect of CMV on human embryonic fibroblasts was generally as previously described [Wright et al, 19681. However, cells obtained from one particular 16-week-old fetus were noted to give a different sequence of events. The cells rounded up as normal after 18 hours, and intranuclear and intracytoplasmic inclusions developed, However three to four days postinfection, when 90% of the cells were showing signs of infection, evidence of fusion was noted. The process was followed under the phase-contrast microscope. The cells were not engulfed by other cells as no evidence of pseudopodia was seen. However, the membranes of adjoining cells appeared to break down to form a binucleate cell. Progressively more cells fused to give a multinucleate structure (Figs. 1 and 2). No destruction of the nucleus or the inclusion bodies was observed. This effect was reproducible in these cells, providing the inoculum contained a high titer of virus.
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Fig. 1. Multinucleate fibroblasts fusing t o form an even larger cell (X 500).
Fig. 2. Large multinucleate giant cell, resulting from fusion, observed seven days after challenge of a fibroblast culture with CMV ( X 500).
Herpes simplex inoculation of cultures from the same fetus gave the typical margination of chromatin. No fusion was observed. To ensure that no other viruses were contaminating the culture, the maintenance medium and free-thawed cell extracts from the infected cultures were used to inoculate Vero cells, primary monkey kidney cells, and HeLa cells. No evidence of viral infection was seen in any of these cells. Electron microscopic observations of the high-speed pellet obtained from the medium and the clarified cell extract revealed only particles with the typical marphology of the herpesviridae. DISCUSSION
The presence of large “owls-eye’’ cells is characteristic of human CMV infections in vivo [von Glahn and Pappenheimer, 19251. In vitro the virus is generally grown in fibroblasts which round up on infection and pull away from their neighbouring cells [Wright et al, 19681. However, another phenomenon has been noted in a culture where 90% of the cells were showing inclusions three to four days after infection. Binucleate cells arose from cell fusion. The fusion process proceeded, and large multinucleate giant cells resulted (Figs. 1 and 2). Particles with the morphology of CMV were isolated from the cells but no evidence of other viruses was obtained. Obviously the possibility exists that there was somethng unusual about the particular line of fibroblasts. However, they showed the normal cytopathogenic effect when inoculated with herpes simplex, and uninoculated cultures were successfully passaged some 35 times, during which time they retained their typical fibroblast morphology. The alternative assumption is that under certain conditions of infection the surface of infected cells is altered to favour cell fusion. New antigens have been detected in the membranes
274
Garnett
of CMV infected cells [Boldogh et al, 1977; Michelson-Fiske, 19771, and it may be that a particular level of these antigens triggers the fusion process. Alternatively, the altered distribution of normal membrane components could trigger the process. Recently a similar phenomenon has been noted during the isolation of CMV from several clinical specimens in other lines o f fibroblasts [Le ROUX,personal communication] and has been noted when CMV infected cells are exposed to aflatoxin B1 [Garnett, in press] . Thus it appears that multinucleate cells can arise by fusion in CMV infected cultures in vitro and it is possible that giant cells may arise in vivo by a similar process. ADDENDUM
Since the preparation o f this manuscript, a paper has been published reporting the formation o f syncytia some 40 days after the infection of MRC-5 fibroblasts by low-titer human CMV [Booth et al, 19781 . These authors suggested that prolonged abortive infection of cells by CMV may he the cause of the syncytia. In contrast, the formation of the multinucleate giant cells reported above is a rapid process. Although Booth et a1 [1978] mention possible “fusion factors,” no reference is made to observations of fusion, as was noted in the current studies. ACKNOWLEDGMENTS
This work was supported by a grant from the South African Medical Research Council. REFERENCES Boldogh I, Gonczol E, Vaczi L (1977): Cytomegalovirus induced membrane antigens in productively infected cells. Acta Microbiologica Academiae Scientarum Hungaricae 24: 21 -28. Booth JC, Beesley JE, Stern H (1978): Syncytium formation caused by human cytomegalovirus in human embryonic lung fibroblasts. Arch Virol 57: 143-152. Diosi P, Babusceac L, Gherman D (1972): Cytophagia in cell cultures infected with cytomegalovirus. Journal of Infectious Discases 125:669%671. Garnett HM (1974): A cytomegalovirus induced membrane alteration indicating a vital role for Ca2+ in virus replication. South African Journal of Science 70:93-94. von Glahn WC, I’appenheimer AM (1925): Intranuclear inclusions in visceral disease. American Journal of Pathology 1:445-466. Hayflick L (1965): Tissue cultures and mycoplasma. Texas Reports of Biology and Medicine. 23: 285-303. Hayflick L, Moorhead PS (1961): The serial cultivation of human diploid cell strains. Experiincntal Cell Research 25:585-621. Michelson-Fiske S (1977): Human cytomegalovirus. A review of developments between 1972 and 1976. Part 11: Experimental developments. Biomedicine 26:86-97. Wright HT, Kasten FH, McAllister RM ( 1 968): Human cytomcgalovirus. Observations of intracellular lesion development as revealed by phase contrast, time-lapse cinematography. Proceedings of the Society for Experimental Biology and Medicine 127:1032-1036.
