Vol. 17, No. 1
JOURNAL OF VIROLOGY, Jan. 1976, p. 183-190 Copyright 01976 American Society for Microbiology
Printed in U.S.A.
Kinetics of Cell Fusion Induced by a Syncytia-Producing Mutant of Herpes Simplex Virus Type I STANLEY PERSON,* ROBERT W. KNOWLES, G. SULLIVAN READ, SUSAN C. WARNER, AND VINCENT C. BOND Biophysics Department, The Pennsylvania State University, University Park, Pennsylvania 16802 Received for publication 22 July 1975
We have isolated a number of plaque-morphology mutants from a strain of herpes simplex virus type I which, unlike the wild type, cause extensive cell fusion during a productive viral infection. After the onset of fusion, there is an exponential decrease in the number of single cells as a function of time after infection. At a multiplicity of infection (MOI) of 3.8 plaque-forming units per cell, fusion begins 5.3 h after infection with the number of single cells decreasing to 10% of the original number 10.2 h after infection. As the MOI is gradually increased from 0.4 to 8, the onset of fusion occurs earlier during infection. However, when the MOI is increased from 8 to 86, the onset of fusion does not occur any earlier. The rate of fusion is independent of the MOI for an MOI greater than 1. The rate of fusion varies linearly with initial cell density up to 3.5 x 104 cells/cm' and is independent of initial cell density at higher cell concentrations. To assay cell fusion we have developed a simple quantitative assay using a Coulter counter to measure the number of single cells as a function of time after infection. Data obtained using a Coulter counter are similar to those obtained with a microscope assay.
Membrane fusion is thought to be a widespread biological phenomenon. It may be important in a number of endo- and exocytotic processes, muscle and bone cell development, fertilization, neural transmitter release, and cell fusion induced by a number of enveloped animal viruses (9, 10). Anticipating that similar mechanisms may underlie these fusion events, we have chosen to study virus-induced cell fusion. The limited size of a viral genome and the ease of obtaining mutants are valuable for a genetic and biochemical study. Virus-induced cell fusion has been divided operationally into fusion from within and fusion from without (1). Fusion from within occurs during a productive viral infection at a low multiplicity of infection (MOI), whereas fusion from without occurs in the absence of viral gene expression when a markedly larger MOI is used. We have chosen to study fusion from within using mutants of herpes simplex virus (HSV). Early reports indicated that, although wildtype HSV does not cause significant cell fusion, variants, possibly mutants, do cause fusion (3, 4, 6, 7, 13, 15). More recently, a spontaneously occurring fusion mutant of HSV was isolated, reverted to wild type, and mapped with reference to a number of conditional lethal mutations (2). The fusion mutation is not condition-
ally lethal and should be thought of as a plaque-morphology mutation. We have isolated a number of these mutants from a wild-type strain of HSV type I, KOS, and have begun to characterize their infections of secondary cultures of human embryonic lung (HEL) cells. We use the terminology of Brown et al. (2) in designating the mutants as syn because of their ability to form syncytia, cells each containing many nuclei, during infection. The most common assay for virus-induced cell fusion has been to fix and stain cells and record the average number of nuclei per cell as a function of time after adding virus. This number minus one has been called the fusion index (8), or alternatively the average number of fusion events per cell (1). It is a parameter which reflects the net result of all possible types of fusion events-fusion between two mononucleated cells, fusion between mononucleated and multinucleated cells, and fusion between two multinucleated cells. We began our studies using this type of assay but soon found that it was unsuitable for our purposes. Besides being tedious, it includes a possible subjective bias since personal judgment is required to distinguish adjacent unfused cells from small syncytia. In addition, multinucleated cells containing a large number
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of nuclei cannot be scored accurately. Therefore, we were unable to follow fusion long enough to determine the shape of the fusion kinetics curve. As an alternative assay, we harvested cells with trypsin-EDTA and counted the number of multinucleated cells in the suspension with a Coulter counter. Although this approach alleviated some of the problems inherent in the visual assay, it was also unsatisfactory because of the complex shape of the fusion kinetics curve. We found in preliminary experiments that the number of multinucleated cells increases when fusion begins, but reaches a maximum value 2 to 3 h later and thereafter decreases. The decrease is probably due to fusion between multinucleated cells. These difficulties were overcome by using the Coulter counter to measure the disappearance of single cells as a function of time after infection. Single cell (mononucleated cell; used throughout this paper in this sense) disappearance is presumed to be due to their fusion into multinucleated cells. By focusing on the initial fusion event of single cells with their neighbors, this assay measures a conceptually simpler parameter of cell fusion than the fusion index. Kohn (5) noted an inverse relationship between the fusion index and the percentage of single unfused cells and recognized that the disappearance of single cells could be used as a measure of fusion. Using this assay we find that after an initial lag period the number of mononucleated cells decreases exponentially with time after infection. The slope of this curve on a semi-logarithmic plot is the average probability per unit time that a mononucleated cell will fuse with a neighbor. We have used this assay to compare wild-type (KOS) and syn mutant infections and to investigate the dependence of cell fusion upon the MOI of the mutant virus and the initial cell density. MATERIALS AND METHODS Cells and virus. HEL cells obtained from the Hershey Medical Center (The Pennsylvania State University, Hershey, courtesy of Fred Rapp) were supplied by John Docherty (Department of Microbiology, The Pennsylvania State University, University Park). Type I HSV, strain KOS, was obtained from Priscilla Schaffer (Baylor College of Medicine, Houston, Tex.). We have isolated several mutants of KOS which cause extensive syncytia formation in an otherwise normal infection. One of these mutants, syn 20, was used in the experiments reported here. The procedure for isolation of the mutants will be fully described in a later paper. Briefly, KOS was mutagenized by growth for 24 h on HEL monolayers in the presence of 10 ,g of
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N-methyl-N'-nitro-N-nitrosoguanidine per ml. Mutagenized virus were then diluted into a suspension of HEL cells in growth medium, which was then plated (approximately 10 infected cells and 3 x 104 uninfected cells per well) in 96-well tissue culture plates (Falcon Plastics, Oxnard, Calif.). After incubation at 34 C for 2.5 days, the wells were scanned with a microscope, and the contents of wells containing syncytia were harvested. Mutants were isolated by serial dilution and plaque-purified twice before preparing stocks. Media and solutions. The medium, used for all experiments and for growth of cell and virus stock cultures, contained F12 powdered medium (GIBCO, Long Island, N.Y.); 10% (vol/vol) fetal bovine serum (Flow Laboratories, Rockville, Md.); penicillin G sodium and streptomycin sulfate (Nutritional Biochemicals, Cleveland, Ohio), 0.1 g/liter; CaCl2.2H20, 0.1 g/liter; and NaHCO,, 2 g/liter. It was supplemented with the 20 amino acids and the nucleosides uridine, adenosine, and guanosine at 15 mg/liter. The pH was adjusted to 7.3. Growth medium containing 0.5% methyl cellulose was used for routine plaque assays. A tricine-buffered saline solution (TBS) was used to wash cells and dilute virus suspensions. It contained NaCl (8 g/liter), KCl (0.38 g/liter), MgCl .6H,O (0.1 g/liter), CaCl2 .2H20 (0.1 g/liter), and tricine (4.5 g/liter) in double-distilled water. The pH was adjusted to 7.3. For routine harvesting of cells we used a trypsinEDTA solution containing 0.005% (wt/vol) trypsin (Nutritional Biochemicals, Cleveland, catalog no. 1308A) and 10-3 M EDTA in TBS. To harvest infected cells for the Coulter counter fusion assay we used a trypsin-EDTA solution containing 0.025% trypsin and 10-2 M EDTA. A saline solution (NaCl, 9 g/liter, in double-distilled water) was used to dilute cell suspensions for Coulter counting. Growth and maintenance of cell cultures. Primary HEL cells were subcultured 11 times (P11) and stored at -75 C in growth medium containing 10% (vol/vol) glycerol. At monthly intervals cells were thawed, put into 16-ounce prescription bottles containing 20 ml of growth medium, and incubated at 37 C. After a few hours the medium was changed to remove the glycerol, and incubation continued until the cells reached confluency. Cells were then subcultured at 3.5-day intervals until P20. At P20 all cells were discarded and a new vial of P11 cells was put into use. Cells in 16-ounce bottles were routinely harvested by decanting the medium, washing twice with 10 ml of TBS, and adding 5 ml of trypsin-EDTA solution. After 5 min, 5 ml of growth medium was added, the solution was pipetted vigorously to remove cells from the glass, and the cell concentration was determined by Coulter counter. In a typical cell passage about 107 cells were harvested per 16-ounce bottle and used to seed new bottles with 2 x 101 cells. By the next day the cell number typically decreased to 1.4 x 10" cells per bottle and then increased with a doubling time of 22 h. After 3.5 days the cultures were just entering
VOL. 17, 1976 stationary phase. Growth curves were determined using a Coulter counter. Growth and maintenance of virus stocks. KOS and syn 20 viruses were plaque-purified twice and grown into single primary stocks. Primary stocks containing about 109 plaque-forming units (PFU) were stored in growth medium containing 10% (vol/ vol) glycerol at - 75 C. Stocks for experiments were prepared from the primary stock by infecting cells grown to 80% confluency in roller bottles (about 7 x 107 cells and 805 cm2 of surface area per bottle). Cell layers were washed with 50 ml of TBS for 10 min and exposed for 1 h to 10 ml of a virus suspension containing 106 PFU. The virus suspension was then removed and replaced by 100 ml of growth medium, and incubation was continued for 2 days at 34 C. Infected cells were then scraped into the medium and subjected to three cycles of freezing and thawing to facilitate viral release. Cell debris was pelleted by centrifugation at 7,500 x g for 5 min, and the virus was pelleted by centrifugation at 54,000 x g for 1 h in a type 30 fixed angle Beckman rotor. Viral pellets were resuspended in a small volume of growth medium containing 10% glycerol and stored at - 75 C until used. Coulter counter fusion assay. Cells in late logarithmic phase were harvested, seeded into 2-ounce prescription bottles at a density of 106 cells/bottle, and incubated at 37 C. After 40 h the bottles contained on the average 1.2 x 106 cells (5.7 x 104 cells/ cm2), corresponding to about 90% confluency. At this time the medium was removed and the cell layer was washed twice with 3 ml of TBS. Cells were then infected by adding 0.2 ml of a virus suspension for 1 h at 30 C. Unless otherwise specified we added 0.2 ml of a virus suspension containing 3 x 107 PFU/ml which resulted in an average MOI of 3.8. We measured the disappearance of free virus from virus suspensions during attachment for 1 h. The average adsorption was 75% and varied no more than 15%. After adsorption unattached virus was removed, 5 ml of growth medium was added to each bottle, and the infected cells were incubated at 34 C. At various times after infection individual bottles were harvested. The cell layer was washed twice with 3 ml of TBS, and 0.9 ml of trypsin-EDTA solution was added for 5 min. This was followed by the addition of 0.9 ml of growth medium. The cells were suspended by vigorous pipetting and fixed by adding 0.2 ml of formalin. Cells were then counted, using a Coulter counter (model A, Coulter Electronics, Hialeah, Fla.). The extent of fusion was determined by measuring the number of cells with pulse heights terminating in the threshold interval 10-15. This number is a measure of unfused single cells. Microscope fusion assay. Cells with radioactively labeled DNA were prepared by growing them in medium containing 1 UCi of [3H thymidine (New England Nuclear Corp., Boston) per ml for 3 days at 37 C. Labeled and unlabeled cells were harvested in late logarithmic phase, mixed, and seeded into 35-mm petri dishes containing cover slips (22 by 22 mm). Each dish received 2.5 x 10' cells of which 8% were labeled. After 8 h of incubation at 37 C, the cells were
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washed twice with 1.5 ml of TBS and infected by adding onto each cover slip 0.05 ml of a virus suspension containing 5 x 107 PFU/ml. After 1 h of adsorption at 30 C unadsorbed virus was removed, 2 ml of growth medium was added to each dish, and the cells were incubated at 34 C. Under these conditions the MOI was 4. At various times after infection, cover slips were removed from the incubation chamber, the cells were fixed with a solution of TBS:formalin (9:1, vol/vol), and autoradiographs were prepared. After development the cells were stained with Harris hematoxylin, destained with acid alcohol, and counter-stained with eosin Y. Cells were then scanned to determine the extent of fusion by measuring the fraction of labeled cells remaining unfused.
RESULTS Cell size distribution of uninfected HEL cells. A Coulter counter can be used to measure the total number of cells in a population or, alternatively, to measure only those cells of particular sizes. We decided to focus our attention on the initial fusion events of single cells. Therefore, we measured the disappearance of single cells from a narrow size (threshold) interval as a function of time after infection. We wanted to use a size interval corresponding to small single cells so that fusion between any two mononucleated cells would produce a binucleated cell too large to be counted in this interval. Therefore it was necessary to determine the size distribution of uninfected HEL cells, grown and harvested under conditions used in the fusion assay experiments. Such a distribution is shown in Fig. 1. Microscopic
0.30D 0.25-J U
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FIG. 1. The size distribution of uninfected HEL cells. Uninfected HEL cells were harvested with trypsin-EDTA and counted with a Coulter counter. Cell counts were made at several threshold settings and the fraction of cells in each threshold interval was calculated. Threshold intervals correspond to different cell sizes and increase from left to right.
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examination confirmed that these uninfected cells existed totally as single cells after the harvesting procedures. We note that the threshold interval 10-15 corresponds to small single cells and includes 32% of the total cell population in this experiment. Decrease in number of single cells as a measure of fusion. We examined the decrease in the number of single cells after infection as a possible fusion assay. We harvested infected cells with trypsin-EDTA and used the Coulter counter to measure the number of cells in the threshold interval 10-15 (Fig. 2). In this experiment this number remained constant until 5.5 h after infection and then decreased exponentially, reaching 10% of the initial number at 11 h after infection. As a check on the Coulter counter assay we used the microscope assay to measure the fraction of cells remaining unfused as a function of time after infection. Prior to I
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TIME AFTER INFECTION (hours)
FIG. 2. The decrease in number of single cells as a measure of fusion. HEL cells in 2-ounce bottles or on cover slips were infected with syn 20 at an MOI of approximately 4. Similar cell densities (3 x 104
cells/cm2) and times of growth of cells prior to infection (8 h) were used in the two experiments. For the Coulter counter, cells were harvested at various times after infection and counted (0). We measured the number of cells, N, in the threshold 10-15, which corresponds to small single cells (Fig. 1). Counts taken during the first 5 h after infection were averaged and the average number was taken as the initial cell number, designated N.. The fraction, N/N., is plotted in Fig. 2-6. Cover slips were processed and examined under the microscope (x). At various times after infection cells were examined to determine the fraction remaining unfused. Zero time corresponds to the time when cells began incubation at 34 C after adsorption.
infection [3H thymidine-labeled and unlabeled cells were harvested, mixed, and seeded into Petri dishes containing cover slips. Eight percent of the mixed cells were labeled, and these cells served as a random sample of the cell population to be scored. Autoradiographs were prepared and cover slips were scanned using a microscope. Cells with labeled nuclei were scored either as remaining unfused or as being part of a multinucleated cell. The results, also shown in Fig. 2, are the same as those obtained with the Coulter counter. In these experiments similar cell densities (3 x 104 cells/cm2) were used. This lower cell density was used for the Coulter counter because we could not distinguish adjacent unfused cells from small syncytia above this cell density using the microscope assay. In addition, cells were incubated only 8 h at 37 C before the addition of virus. The Coulter counter assay would be inaccurate if cells were selectively lost from monolayers before harvesting with trypsin-EDTA or if the harvesting procedure did not remove all the cells from the glass. To test these possibilities, cells were labeled with ["4C Ileucine prior to infection, and the radioactivity released by harvesting was compared to the number of unfused single cells at various times after infection. The radioactivity remained essentially constant throughout the experiment, whereas the number of single cells decreased to less than 10% of the initial number (Fig. 3). Kinetics of cell fusion during syn 20 infection. The data obtained with the Coulter counter fusion assay were quite reproducible. Using the procedure described above we obtained initial cell counts in the threshold interval 10-15 of about 6,000 decreasing to about 400 at 11.5 h after infection. In 10 experiments whose average values for cell density and MOI were 5.7 x 104 cells/cm 2 and 3.8 PFU/cell, respectively, the onset of fusion varied from 4.6 to 5.8 h (average value 5.3 h) and the time required for cell counts to decrease to 10% of their initial value varied from 4.4 to 5.3 h (average value 4.9 h). The maximum variation in cell density and MOI was ±20% among the replicate experiments. In several experiments we used other threshold intervals (15-20 and 20-25) and obtained similar results. This assay provides a measurement of the kinetics of the initial fusion events which single cells undergo during syncytia formation. The decrease in the number of single cells obeys the relation N/N. = e At, where N is the number of single cells in the threshold interval 10-15 at any time, t, after the onset of fusion and No is the initial number of single cells in the thresh-
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A
from 0.4 to 8, cell fusion started at earlier times after infection. Increasing the MOI to 86 did not, however, cause fusion to occur any earlier than it did at an MOI of 8, indicating that there is a minimum time needed before fusion can begin. The rate of fusion, shown by the slope of each curve, is independent of MOI until an MOI is reached that results in a significant fraction of cells being uninfected. At an MOI of 0.4, the
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FIG. 3. Control experiments on the use of the Coulter counter in the fusion assay. Cells in 2-ounce bottles were prelabeled with ["'C]leucine and then infected with syn 20 at an MOI of 3.7. At various times after infection cells were harvested and the fraction remaining unfused was determined with the Coulter counter as in Fig. 2 (0). A portion of the cell suspension was also analyzed for radioactivity (0).
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old. The differential form of this equation, dN/dt AN, gives the rate of fusion of single cells at any time, t. We note that the average value of the slope of the fusion kinetics curve, A, was 0.47 per h for the 10 replicate experiments. Comparison of wild-type (KOS) and syn 20 infections. Parallel experiments in which HEL cells were infected with either wild type or syn 20 were performed to compare the extent of fusion caused by the two viruses. The change in number of single cells after infection is shown in Fig. 4. Although there is a small but reproducible decrease in the number of single cells after the wild-type infection, which has also been observed with the microscope, there is a striking difference between wild-type and mutant infections. The number of cells for the wild-type infection decreases to about 80% of the initial value and remains at that level, whereas cells infected with syn 20 decrease to less than 10% of their initial number. This experiment also indicates that the decrease in number of single cells ceases about 12 h after infection with
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FIG. 4. A comparison of wild-type KOS and syn 20 infections using the Coulter counter single cell fusion assay. HEL cells were infected at an MOI of 4.1 with KOS (x), or 3.3 for syn 20 (0). At various times after infection the cells were harvested and the fraction remaining unfused was determined with the Coulter counter as in Fig. 2.
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20. Effect of MOI on kinetics of cell fusion. One TIME AFTER INFECTION (hours) variable we found that affected the kinetics of FIG. 5. The effect of the MOI on the kinetics of cell cell fusion was the MOI. Figure 5 shows the fusion with syn 20. HEL cells were infected with results from experiments in which cells were syn 20 at the indicated MOL At various times after infected with syn 20 at an MOI of 0.4 to 86. The infection the cells were harvested and the fraction number of remaining single cells was measured remaining unfused was determined with the Coulter with a Coulter counter. As the MOI increased counter as in Fig. 2. syn
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slope is about half the value found at higher MOI. Effect of the initial cell density on the kinetics of cell fusion. Most experiments done with a Coulter counter employed an average initial cell density of 5.7 x 10' cells/cm2. The rate of cell fusion (A) was also determined for a number of initial cell densities, and the results are shown in Fig. 6. At low cell densities the microscope assay was primarily used because the number of cells was too small to obtain accurate data with a Coulter counter. At high densities the Coulter counter assay was used because of the difficulty of distinguishing adjacent unfused cells from fused cells, in a nearly confluent monolayer, using the microscope. The decrease in the number of single cells appeared to be exponential with time for all of the cell densities used. The data in Fig. 6 indicate that the rate of fusion is approximately linearly dependent on cell density up to 3.5 x 104 cells/cm2 and is independent of cell density at higher cell concentrations. The onset of fusion is also influenced by cell density. At an average initial cell density of 5.7 x 104 cells/cm2 and an MOI of 3.8, fusion began 5.3 h after infection. At cell densities less than 2.5 x 104 cells/cm2 and a similar MOI fusion began 4 h after infection.
DISCUSSION We have developed a simple quantitative assay for cell fusion to study syncytia formation caused by mutants of HSV. In this assay we measure with a Coulter counter the decrease in number of small single cells during viral infection with a syn mutant. We harvest infected cells with trypsin-EDTA to separate unfused cells and thereby provide an operational definition of cell fusion; cells are considered to be fused if they are not separated into single cells by the harvesting procedure. In 10 replicate experiments, at an average MOI of 3.8, fusion began at 5.3 h after infection. The number of single cells then decreased exponentially, reaching 10% of the initial value 4.9 h later. The exponential decrease stopped 12 h after infection, perhaps due to the breakdown of large syncytia into fragments during harvesting. At similar cell densities the Coulter counter and microscope assays for the fraction of cells remaining unfused at various times after infection gave essentially identical results. When cells were labeled with [14C leucinee prior to infection, we found that the radioactivity remained constant in contrast to the marked decrease in single cell number. This shows that
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FIG. 6. The dependence of the rate of cell fusion on the initial cell density. HEL cells were infected at an MOI of approximately 4 and assayed for cell fusion at various times after infection using a Coulter counter (0) or microscope (0). In each experiment the fraction of single cells remaining unfused was determined and plotted as a function of time after infection. The slopes of these curves (A) were calculated, after the onset of fusion, and are plotted as a function of the initial cell density.
cells were not lost from the monolayer prior to harvesting, and that cells were reproducibly removed from the glass by harvesting. We conclude that using a Coulter counter to measure the disappearance of single cells is a valid assay for cell fusion. The fusion assay presented here measures the kinetics of initial fusion events which single cells undergo during syncytia formation. Because most fusion events involve a mononucleated cell inside the threshold interval and a mononucleated or multinucleated cell outside the threshold interval, the disappearance of one mononucleated cell from the threshold interval reflects the occurrence of a single fusion event. Although we cannot rigorously derive an analytical expression for fusion kinetics, empirically we find that the decrease in the number of single cells is exponential with time, after the onset of fusion. A similar relationship was noted when a Coulter counter was used to measure the disappearance of single cells in an assay for cell adhesion (12). The exponential nature of the fusion kinetics curve indicates that, at any time after the onset of fusion, the probability that a single cell will fuse during the next instant is independent of whether or not the other cells have already fused. Although the slope of the fusion curve allows us to determine the rate of cell fusion, we do not know what the rate-limiting step is. It may be related to the rate of
VOL. 17, 1976
synthesis and subsequent incorporation of fusion-causing molecules into the plasma membrane, to the rate at which particular portions of plasma membranes of two neighboring cells come into close contact, or to a number of other factors. The slope of the fusion curve (X) varies with experimental conditions such as initial cell density and MOI. The average cell density for experiments shown in Fig. 3-5 and for replicate experiments of that type was 5.7 x 104 cells/ cm2, which represents a 90% confluent monolayer. For this cell density A = 0.47 per h. A is independent of cell density for values greater than 3.5 x 104 cells/cm2. At lower cell densities, where an appreciable fraction of cells are not touching, X is proportional to cell density. Kohn (5) has also noted that the fusion index was dependent on cell density at densities less than about 3.0 x 104 cells/cm2, but no effect was observed at higher densities. The slope of the fusion kinetics curve is about 0.5 per h for an MOI >1 (at a cell concentration of 5.7 x 104 cells/cm2) and about one-half this value at an MOI of 0.4. Using the Poisson relationship one can calculate that only about 33% of the cells are infected at an average MOI of 0.4. Perhaps the slower rate of fusion is due to a smaller probability of fusion of infected and uninfected cells relative to that observed when all cells are infected. The onset of fusion is influenced by the MOI of the mutant virus. The minimum time between virus adsorption and the onset of fusion that we observed was 4 h when an MOI of 8 or 86 was used. Since a minimum time of this duration is required, the results reported here are characteristic of fusion from within. Although we have not observed fusion from without with HEL cells infected with syn 20 at an MOI as high as 86, Nii and Kamahora (7) have reported fusion from without caused by another variant of HSV with FL cells. It is difficult for us to determine accurately the MOI used in their experiments, and it is possible that we would observe fusion from without by using a larger MOI. At a lower MOI the time before the onset of fusion was dependent on MOI, increasing progressively from a minimum of 4 h to a maximum of 7.5 h as the MOI decreased from 8 to 0.4. Using a low MOI, but one which results in nearly all of the cells being infected (MOI of 1.8, for example), the onset of fusion was 6 to 6.5 h. A possible interpretation of the dependence of the onset of fusion on MOI is that a cell has to accumulate a certain number of mutant mole-
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cules in its plasma membrane for fusion to occur. This number could be obtained at earlier times at a higher MOI if there is increased transcription and translation due to an increased number of viral genomes per cell. The onset of fusion is also influenced by cell density. At lower cell densities the onset of fusion occurs earlier than at higher cell densities. At the lower cell densities all of the cells are in the exponential growth phase, which may explain the earlier onset of fusion. In an earlier report, Roizman (11) described another strain of HSV, called MP, which produced syncytia when plated on FL or HEp-2 cells at less than 0.01 PFU/cell, but produced little cell fusion when almost all cells were infected. He suggested that infected cells could fuse only with uninfected cells. This is clearly not the case with syn 20 on HEL cells. Preliminary experiments in our laboratory indicate that MP will fuse a monolayer of HEL, HEp-2, or BHK-21 cells infected at an MOI greater than 1. The Coulter counter assay provides a simple quantitative measure of cell fusion. It has been useful in following the effects of several inhibitors of cell fusion, including 2-deoxyglucose and glucosamine (Knowles and Person, manuscript in preparation), as well as for monitoring fusion of cells mixedly infected with two syn mutants in genetic studies which are currently in progress in our laboratory. In addition, the assay should be easily modified for use with other cell and virus systems. ACKNOWLEDGMENTS This work was supported by a grant from AEC, AT(11-1)3419, and by Public Health Service grant Al 11513 from the National Institute of Allergy and Infectious Diseases. We acknowledge discussions of data and manuscript with colleagues Tom Holland, Paul Keller, and William D. Taylor. Preliminary experiments by Paul Keller were most helpful in the development of the fusion assay. LITERATURE CITED 1. Bratt, M. A., and W. R. Gallaher. 1969. Preliminary analysis of the requirements for fusion from within and fusion from without by Newcastle Disease Virus. Proc. Natl. Acad. Sci. U. S. A. 64:536-543. 2. Brown, S. M., D. H. Ritchie, and J. H. Subak-Sharpe. 1973. Genetic studies with herpes simplex virus type I. The isolation of temperature-sensitive mutants, their arrangement into complementation groups and recombination analysis leading to a linkage map. J. Gen. Virol. 18:329-346. 3. Gray, A., T. Tokumaru, and T. F. McN. Scott. 1958. Different cytopathic effects observed in HeLa cells infected with HSV. Arch. Gesamte Virusforsch. 8:59-76. 4. Hoggan, M. D., and B. Roizman. 1959. The isolation and properties of a variant of herpes simplex producing multinucleated giant cells in monolayer cultures in the
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presence of antibody. Am. J. Hyg. 70:208-219. 5. Kohn, A. 1965. Polykaryocytosis induced by NDV in monolayers of animal cells. Virology 26:228-245. 6. Munk, K., and D. Donner. 1963. Cytopathic effect and plaque morphology of several herpes virus strains. Arch. Gesamte Virusforsch. 13:529-540. 7. Nii, S., and J. Kamahora. 1961. Cytopathic changes induced by herpes simplex virus. Biken J. 4:255-270. 8. Okada, Y., and J. Takodoro. 1962. Analysis of giant polynuclear cell formation caused by HVJ virus from Ehrlich's ascites tumor cells. II. Quantitative analysis of giant polynuclear cell formation. Exp. Cell Res.
26:108-118. 9. Poste, G. 1972. Mechanisms of virus induced cell fusion. Int. Rev. Cytol. 33:157-252. 10. Poste, G., and A. C. Allison. 1973. Membrane fusion. Biochim. Biophys. Acta 300:421-465.
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Roizman, B. 1962. Polykaryocytosis. Cold Spring Harbor Symp. Quant. Biol. 27:327-341. 12. Roseman, S., W. Rothman, B. Walther, R. Ohman, and 11.
J. Umbreit. 1974. Measurement of cell-free interactions, p. 597-611. In S. Fleisher and L. Packer (ed.), Methods in enzymology, vol. 32, part B. Academic Press Inc., New York. 13. Schneweis, K.-E. 1962. Cytopathic effects of HSV. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 186:467-477. 14. Tokumaru, F. 1968. The nature of toxins of HSV. I. Syncytial giant cell producing components in tissue culture. Arch. Gesamte Virusforsch. 24:104-122. 15. Wheeler, C. E., Jr. 1964. Biologic comparison of a syncytial and a small giant cell-forming strain of Herpes Simplex. J. Immunol. 93:749-756.
JOURNAL OF VIROLOGY, Jan. 1976, p. 183-190 Copyright 01976 American Society for Microbiology
Printed in U.S.A.
Kinetics of Cell Fusion Induced by a Syncytia-Producing Mutant of Herpes Simplex Virus Type I STANLEY PERSON,* ROBERT W. KNOWLES, G. SULLIVAN READ, SUSAN C. WARNER, AND VINCENT C. BOND Biophysics Department, The Pennsylvania State University, University Park, Pennsylvania 16802 Received for publication 22 July 1975
We have isolated a number of plaque-morphology mutants from a strain of herpes simplex virus type I which, unlike the wild type, cause extensive cell fusion during a productive viral infection. After the onset of fusion, there is an exponential decrease in the number of single cells as a function of time after infection. At a multiplicity of infection (MOI) of 3.8 plaque-forming units per cell, fusion begins 5.3 h after infection with the number of single cells decreasing to 10% of the original number 10.2 h after infection. As the MOI is gradually increased from 0.4 to 8, the onset of fusion occurs earlier during infection. However, when the MOI is increased from 8 to 86, the onset of fusion does not occur any earlier. The rate of fusion is independent of the MOI for an MOI greater than 1. The rate of fusion varies linearly with initial cell density up to 3.5 x 104 cells/cm' and is independent of initial cell density at higher cell concentrations. To assay cell fusion we have developed a simple quantitative assay using a Coulter counter to measure the number of single cells as a function of time after infection. Data obtained using a Coulter counter are similar to those obtained with a microscope assay.
Membrane fusion is thought to be a widespread biological phenomenon. It may be important in a number of endo- and exocytotic processes, muscle and bone cell development, fertilization, neural transmitter release, and cell fusion induced by a number of enveloped animal viruses (9, 10). Anticipating that similar mechanisms may underlie these fusion events, we have chosen to study virus-induced cell fusion. The limited size of a viral genome and the ease of obtaining mutants are valuable for a genetic and biochemical study. Virus-induced cell fusion has been divided operationally into fusion from within and fusion from without (1). Fusion from within occurs during a productive viral infection at a low multiplicity of infection (MOI), whereas fusion from without occurs in the absence of viral gene expression when a markedly larger MOI is used. We have chosen to study fusion from within using mutants of herpes simplex virus (HSV). Early reports indicated that, although wildtype HSV does not cause significant cell fusion, variants, possibly mutants, do cause fusion (3, 4, 6, 7, 13, 15). More recently, a spontaneously occurring fusion mutant of HSV was isolated, reverted to wild type, and mapped with reference to a number of conditional lethal mutations (2). The fusion mutation is not condition-
ally lethal and should be thought of as a plaque-morphology mutation. We have isolated a number of these mutants from a wild-type strain of HSV type I, KOS, and have begun to characterize their infections of secondary cultures of human embryonic lung (HEL) cells. We use the terminology of Brown et al. (2) in designating the mutants as syn because of their ability to form syncytia, cells each containing many nuclei, during infection. The most common assay for virus-induced cell fusion has been to fix and stain cells and record the average number of nuclei per cell as a function of time after adding virus. This number minus one has been called the fusion index (8), or alternatively the average number of fusion events per cell (1). It is a parameter which reflects the net result of all possible types of fusion events-fusion between two mononucleated cells, fusion between mononucleated and multinucleated cells, and fusion between two multinucleated cells. We began our studies using this type of assay but soon found that it was unsuitable for our purposes. Besides being tedious, it includes a possible subjective bias since personal judgment is required to distinguish adjacent unfused cells from small syncytia. In addition, multinucleated cells containing a large number
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of nuclei cannot be scored accurately. Therefore, we were unable to follow fusion long enough to determine the shape of the fusion kinetics curve. As an alternative assay, we harvested cells with trypsin-EDTA and counted the number of multinucleated cells in the suspension with a Coulter counter. Although this approach alleviated some of the problems inherent in the visual assay, it was also unsatisfactory because of the complex shape of the fusion kinetics curve. We found in preliminary experiments that the number of multinucleated cells increases when fusion begins, but reaches a maximum value 2 to 3 h later and thereafter decreases. The decrease is probably due to fusion between multinucleated cells. These difficulties were overcome by using the Coulter counter to measure the disappearance of single cells as a function of time after infection. Single cell (mononucleated cell; used throughout this paper in this sense) disappearance is presumed to be due to their fusion into multinucleated cells. By focusing on the initial fusion event of single cells with their neighbors, this assay measures a conceptually simpler parameter of cell fusion than the fusion index. Kohn (5) noted an inverse relationship between the fusion index and the percentage of single unfused cells and recognized that the disappearance of single cells could be used as a measure of fusion. Using this assay we find that after an initial lag period the number of mononucleated cells decreases exponentially with time after infection. The slope of this curve on a semi-logarithmic plot is the average probability per unit time that a mononucleated cell will fuse with a neighbor. We have used this assay to compare wild-type (KOS) and syn mutant infections and to investigate the dependence of cell fusion upon the MOI of the mutant virus and the initial cell density. MATERIALS AND METHODS Cells and virus. HEL cells obtained from the Hershey Medical Center (The Pennsylvania State University, Hershey, courtesy of Fred Rapp) were supplied by John Docherty (Department of Microbiology, The Pennsylvania State University, University Park). Type I HSV, strain KOS, was obtained from Priscilla Schaffer (Baylor College of Medicine, Houston, Tex.). We have isolated several mutants of KOS which cause extensive syncytia formation in an otherwise normal infection. One of these mutants, syn 20, was used in the experiments reported here. The procedure for isolation of the mutants will be fully described in a later paper. Briefly, KOS was mutagenized by growth for 24 h on HEL monolayers in the presence of 10 ,g of
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N-methyl-N'-nitro-N-nitrosoguanidine per ml. Mutagenized virus were then diluted into a suspension of HEL cells in growth medium, which was then plated (approximately 10 infected cells and 3 x 104 uninfected cells per well) in 96-well tissue culture plates (Falcon Plastics, Oxnard, Calif.). After incubation at 34 C for 2.5 days, the wells were scanned with a microscope, and the contents of wells containing syncytia were harvested. Mutants were isolated by serial dilution and plaque-purified twice before preparing stocks. Media and solutions. The medium, used for all experiments and for growth of cell and virus stock cultures, contained F12 powdered medium (GIBCO, Long Island, N.Y.); 10% (vol/vol) fetal bovine serum (Flow Laboratories, Rockville, Md.); penicillin G sodium and streptomycin sulfate (Nutritional Biochemicals, Cleveland, Ohio), 0.1 g/liter; CaCl2.2H20, 0.1 g/liter; and NaHCO,, 2 g/liter. It was supplemented with the 20 amino acids and the nucleosides uridine, adenosine, and guanosine at 15 mg/liter. The pH was adjusted to 7.3. Growth medium containing 0.5% methyl cellulose was used for routine plaque assays. A tricine-buffered saline solution (TBS) was used to wash cells and dilute virus suspensions. It contained NaCl (8 g/liter), KCl (0.38 g/liter), MgCl .6H,O (0.1 g/liter), CaCl2 .2H20 (0.1 g/liter), and tricine (4.5 g/liter) in double-distilled water. The pH was adjusted to 7.3. For routine harvesting of cells we used a trypsinEDTA solution containing 0.005% (wt/vol) trypsin (Nutritional Biochemicals, Cleveland, catalog no. 1308A) and 10-3 M EDTA in TBS. To harvest infected cells for the Coulter counter fusion assay we used a trypsin-EDTA solution containing 0.025% trypsin and 10-2 M EDTA. A saline solution (NaCl, 9 g/liter, in double-distilled water) was used to dilute cell suspensions for Coulter counting. Growth and maintenance of cell cultures. Primary HEL cells were subcultured 11 times (P11) and stored at -75 C in growth medium containing 10% (vol/vol) glycerol. At monthly intervals cells were thawed, put into 16-ounce prescription bottles containing 20 ml of growth medium, and incubated at 37 C. After a few hours the medium was changed to remove the glycerol, and incubation continued until the cells reached confluency. Cells were then subcultured at 3.5-day intervals until P20. At P20 all cells were discarded and a new vial of P11 cells was put into use. Cells in 16-ounce bottles were routinely harvested by decanting the medium, washing twice with 10 ml of TBS, and adding 5 ml of trypsin-EDTA solution. After 5 min, 5 ml of growth medium was added, the solution was pipetted vigorously to remove cells from the glass, and the cell concentration was determined by Coulter counter. In a typical cell passage about 107 cells were harvested per 16-ounce bottle and used to seed new bottles with 2 x 101 cells. By the next day the cell number typically decreased to 1.4 x 10" cells per bottle and then increased with a doubling time of 22 h. After 3.5 days the cultures were just entering
VOL. 17, 1976 stationary phase. Growth curves were determined using a Coulter counter. Growth and maintenance of virus stocks. KOS and syn 20 viruses were plaque-purified twice and grown into single primary stocks. Primary stocks containing about 109 plaque-forming units (PFU) were stored in growth medium containing 10% (vol/ vol) glycerol at - 75 C. Stocks for experiments were prepared from the primary stock by infecting cells grown to 80% confluency in roller bottles (about 7 x 107 cells and 805 cm2 of surface area per bottle). Cell layers were washed with 50 ml of TBS for 10 min and exposed for 1 h to 10 ml of a virus suspension containing 106 PFU. The virus suspension was then removed and replaced by 100 ml of growth medium, and incubation was continued for 2 days at 34 C. Infected cells were then scraped into the medium and subjected to three cycles of freezing and thawing to facilitate viral release. Cell debris was pelleted by centrifugation at 7,500 x g for 5 min, and the virus was pelleted by centrifugation at 54,000 x g for 1 h in a type 30 fixed angle Beckman rotor. Viral pellets were resuspended in a small volume of growth medium containing 10% glycerol and stored at - 75 C until used. Coulter counter fusion assay. Cells in late logarithmic phase were harvested, seeded into 2-ounce prescription bottles at a density of 106 cells/bottle, and incubated at 37 C. After 40 h the bottles contained on the average 1.2 x 106 cells (5.7 x 104 cells/ cm2), corresponding to about 90% confluency. At this time the medium was removed and the cell layer was washed twice with 3 ml of TBS. Cells were then infected by adding 0.2 ml of a virus suspension for 1 h at 30 C. Unless otherwise specified we added 0.2 ml of a virus suspension containing 3 x 107 PFU/ml which resulted in an average MOI of 3.8. We measured the disappearance of free virus from virus suspensions during attachment for 1 h. The average adsorption was 75% and varied no more than 15%. After adsorption unattached virus was removed, 5 ml of growth medium was added to each bottle, and the infected cells were incubated at 34 C. At various times after infection individual bottles were harvested. The cell layer was washed twice with 3 ml of TBS, and 0.9 ml of trypsin-EDTA solution was added for 5 min. This was followed by the addition of 0.9 ml of growth medium. The cells were suspended by vigorous pipetting and fixed by adding 0.2 ml of formalin. Cells were then counted, using a Coulter counter (model A, Coulter Electronics, Hialeah, Fla.). The extent of fusion was determined by measuring the number of cells with pulse heights terminating in the threshold interval 10-15. This number is a measure of unfused single cells. Microscope fusion assay. Cells with radioactively labeled DNA were prepared by growing them in medium containing 1 UCi of [3H thymidine (New England Nuclear Corp., Boston) per ml for 3 days at 37 C. Labeled and unlabeled cells were harvested in late logarithmic phase, mixed, and seeded into 35-mm petri dishes containing cover slips (22 by 22 mm). Each dish received 2.5 x 10' cells of which 8% were labeled. After 8 h of incubation at 37 C, the cells were
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washed twice with 1.5 ml of TBS and infected by adding onto each cover slip 0.05 ml of a virus suspension containing 5 x 107 PFU/ml. After 1 h of adsorption at 30 C unadsorbed virus was removed, 2 ml of growth medium was added to each dish, and the cells were incubated at 34 C. Under these conditions the MOI was 4. At various times after infection, cover slips were removed from the incubation chamber, the cells were fixed with a solution of TBS:formalin (9:1, vol/vol), and autoradiographs were prepared. After development the cells were stained with Harris hematoxylin, destained with acid alcohol, and counter-stained with eosin Y. Cells were then scanned to determine the extent of fusion by measuring the fraction of labeled cells remaining unfused.
RESULTS Cell size distribution of uninfected HEL cells. A Coulter counter can be used to measure the total number of cells in a population or, alternatively, to measure only those cells of particular sizes. We decided to focus our attention on the initial fusion events of single cells. Therefore, we measured the disappearance of single cells from a narrow size (threshold) interval as a function of time after infection. We wanted to use a size interval corresponding to small single cells so that fusion between any two mononucleated cells would produce a binucleated cell too large to be counted in this interval. Therefore it was necessary to determine the size distribution of uninfected HEL cells, grown and harvested under conditions used in the fusion assay experiments. Such a distribution is shown in Fig. 1. Microscopic
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FIG. 1. The size distribution of uninfected HEL cells. Uninfected HEL cells were harvested with trypsin-EDTA and counted with a Coulter counter. Cell counts were made at several threshold settings and the fraction of cells in each threshold interval was calculated. Threshold intervals correspond to different cell sizes and increase from left to right.
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examination confirmed that these uninfected cells existed totally as single cells after the harvesting procedures. We note that the threshold interval 10-15 corresponds to small single cells and includes 32% of the total cell population in this experiment. Decrease in number of single cells as a measure of fusion. We examined the decrease in the number of single cells after infection as a possible fusion assay. We harvested infected cells with trypsin-EDTA and used the Coulter counter to measure the number of cells in the threshold interval 10-15 (Fig. 2). In this experiment this number remained constant until 5.5 h after infection and then decreased exponentially, reaching 10% of the initial number at 11 h after infection. As a check on the Coulter counter assay we used the microscope assay to measure the fraction of cells remaining unfused as a function of time after infection. Prior to I
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FIG. 2. The decrease in number of single cells as a measure of fusion. HEL cells in 2-ounce bottles or on cover slips were infected with syn 20 at an MOI of approximately 4. Similar cell densities (3 x 104
cells/cm2) and times of growth of cells prior to infection (8 h) were used in the two experiments. For the Coulter counter, cells were harvested at various times after infection and counted (0). We measured the number of cells, N, in the threshold 10-15, which corresponds to small single cells (Fig. 1). Counts taken during the first 5 h after infection were averaged and the average number was taken as the initial cell number, designated N.. The fraction, N/N., is plotted in Fig. 2-6. Cover slips were processed and examined under the microscope (x). At various times after infection cells were examined to determine the fraction remaining unfused. Zero time corresponds to the time when cells began incubation at 34 C after adsorption.
infection [3H thymidine-labeled and unlabeled cells were harvested, mixed, and seeded into Petri dishes containing cover slips. Eight percent of the mixed cells were labeled, and these cells served as a random sample of the cell population to be scored. Autoradiographs were prepared and cover slips were scanned using a microscope. Cells with labeled nuclei were scored either as remaining unfused or as being part of a multinucleated cell. The results, also shown in Fig. 2, are the same as those obtained with the Coulter counter. In these experiments similar cell densities (3 x 104 cells/cm2) were used. This lower cell density was used for the Coulter counter because we could not distinguish adjacent unfused cells from small syncytia above this cell density using the microscope assay. In addition, cells were incubated only 8 h at 37 C before the addition of virus. The Coulter counter assay would be inaccurate if cells were selectively lost from monolayers before harvesting with trypsin-EDTA or if the harvesting procedure did not remove all the cells from the glass. To test these possibilities, cells were labeled with ["4C Ileucine prior to infection, and the radioactivity released by harvesting was compared to the number of unfused single cells at various times after infection. The radioactivity remained essentially constant throughout the experiment, whereas the number of single cells decreased to less than 10% of the initial number (Fig. 3). Kinetics of cell fusion during syn 20 infection. The data obtained with the Coulter counter fusion assay were quite reproducible. Using the procedure described above we obtained initial cell counts in the threshold interval 10-15 of about 6,000 decreasing to about 400 at 11.5 h after infection. In 10 experiments whose average values for cell density and MOI were 5.7 x 104 cells/cm 2 and 3.8 PFU/cell, respectively, the onset of fusion varied from 4.6 to 5.8 h (average value 5.3 h) and the time required for cell counts to decrease to 10% of their initial value varied from 4.4 to 5.3 h (average value 4.9 h). The maximum variation in cell density and MOI was ±20% among the replicate experiments. In several experiments we used other threshold intervals (15-20 and 20-25) and obtained similar results. This assay provides a measurement of the kinetics of the initial fusion events which single cells undergo during syncytia formation. The decrease in the number of single cells obeys the relation N/N. = e At, where N is the number of single cells in the threshold interval 10-15 at any time, t, after the onset of fusion and No is the initial number of single cells in the thresh-
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A
from 0.4 to 8, cell fusion started at earlier times after infection. Increasing the MOI to 86 did not, however, cause fusion to occur any earlier than it did at an MOI of 8, indicating that there is a minimum time needed before fusion can begin. The rate of fusion, shown by the slope of each curve, is independent of MOI until an MOI is reached that results in a significant fraction of cells being uninfected. At an MOI of 0.4, the
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FIG. 3. Control experiments on the use of the Coulter counter in the fusion assay. Cells in 2-ounce bottles were prelabeled with ["'C]leucine and then infected with syn 20 at an MOI of 3.7. At various times after infection cells were harvested and the fraction remaining unfused was determined with the Coulter counter as in Fig. 2 (0). A portion of the cell suspension was also analyzed for radioactivity (0).
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old. The differential form of this equation, dN/dt AN, gives the rate of fusion of single cells at any time, t. We note that the average value of the slope of the fusion kinetics curve, A, was 0.47 per h for the 10 replicate experiments. Comparison of wild-type (KOS) and syn 20 infections. Parallel experiments in which HEL cells were infected with either wild type or syn 20 were performed to compare the extent of fusion caused by the two viruses. The change in number of single cells after infection is shown in Fig. 4. Although there is a small but reproducible decrease in the number of single cells after the wild-type infection, which has also been observed with the microscope, there is a striking difference between wild-type and mutant infections. The number of cells for the wild-type infection decreases to about 80% of the initial value and remains at that level, whereas cells infected with syn 20 decrease to less than 10% of their initial number. This experiment also indicates that the decrease in number of single cells ceases about 12 h after infection with
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FIG. 4. A comparison of wild-type KOS and syn 20 infections using the Coulter counter single cell fusion assay. HEL cells were infected at an MOI of 4.1 with KOS (x), or 3.3 for syn 20 (0). At various times after infection the cells were harvested and the fraction remaining unfused was determined with the Coulter counter as in Fig. 2.
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20. Effect of MOI on kinetics of cell fusion. One TIME AFTER INFECTION (hours) variable we found that affected the kinetics of FIG. 5. The effect of the MOI on the kinetics of cell cell fusion was the MOI. Figure 5 shows the fusion with syn 20. HEL cells were infected with results from experiments in which cells were syn 20 at the indicated MOL At various times after infected with syn 20 at an MOI of 0.4 to 86. The infection the cells were harvested and the fraction number of remaining single cells was measured remaining unfused was determined with the Coulter with a Coulter counter. As the MOI increased counter as in Fig. 2. syn
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slope is about half the value found at higher MOI. Effect of the initial cell density on the kinetics of cell fusion. Most experiments done with a Coulter counter employed an average initial cell density of 5.7 x 10' cells/cm2. The rate of cell fusion (A) was also determined for a number of initial cell densities, and the results are shown in Fig. 6. At low cell densities the microscope assay was primarily used because the number of cells was too small to obtain accurate data with a Coulter counter. At high densities the Coulter counter assay was used because of the difficulty of distinguishing adjacent unfused cells from fused cells, in a nearly confluent monolayer, using the microscope. The decrease in the number of single cells appeared to be exponential with time for all of the cell densities used. The data in Fig. 6 indicate that the rate of fusion is approximately linearly dependent on cell density up to 3.5 x 104 cells/cm2 and is independent of cell density at higher cell concentrations. The onset of fusion is also influenced by cell density. At an average initial cell density of 5.7 x 104 cells/cm2 and an MOI of 3.8, fusion began 5.3 h after infection. At cell densities less than 2.5 x 104 cells/cm2 and a similar MOI fusion began 4 h after infection.
DISCUSSION We have developed a simple quantitative assay for cell fusion to study syncytia formation caused by mutants of HSV. In this assay we measure with a Coulter counter the decrease in number of small single cells during viral infection with a syn mutant. We harvest infected cells with trypsin-EDTA to separate unfused cells and thereby provide an operational definition of cell fusion; cells are considered to be fused if they are not separated into single cells by the harvesting procedure. In 10 replicate experiments, at an average MOI of 3.8, fusion began at 5.3 h after infection. The number of single cells then decreased exponentially, reaching 10% of the initial value 4.9 h later. The exponential decrease stopped 12 h after infection, perhaps due to the breakdown of large syncytia into fragments during harvesting. At similar cell densities the Coulter counter and microscope assays for the fraction of cells remaining unfused at various times after infection gave essentially identical results. When cells were labeled with [14C leucinee prior to infection, we found that the radioactivity remained constant in contrast to the marked decrease in single cell number. This shows that
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FIG. 6. The dependence of the rate of cell fusion on the initial cell density. HEL cells were infected at an MOI of approximately 4 and assayed for cell fusion at various times after infection using a Coulter counter (0) or microscope (0). In each experiment the fraction of single cells remaining unfused was determined and plotted as a function of time after infection. The slopes of these curves (A) were calculated, after the onset of fusion, and are plotted as a function of the initial cell density.
cells were not lost from the monolayer prior to harvesting, and that cells were reproducibly removed from the glass by harvesting. We conclude that using a Coulter counter to measure the disappearance of single cells is a valid assay for cell fusion. The fusion assay presented here measures the kinetics of initial fusion events which single cells undergo during syncytia formation. Because most fusion events involve a mononucleated cell inside the threshold interval and a mononucleated or multinucleated cell outside the threshold interval, the disappearance of one mononucleated cell from the threshold interval reflects the occurrence of a single fusion event. Although we cannot rigorously derive an analytical expression for fusion kinetics, empirically we find that the decrease in the number of single cells is exponential with time, after the onset of fusion. A similar relationship was noted when a Coulter counter was used to measure the disappearance of single cells in an assay for cell adhesion (12). The exponential nature of the fusion kinetics curve indicates that, at any time after the onset of fusion, the probability that a single cell will fuse during the next instant is independent of whether or not the other cells have already fused. Although the slope of the fusion curve allows us to determine the rate of cell fusion, we do not know what the rate-limiting step is. It may be related to the rate of
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synthesis and subsequent incorporation of fusion-causing molecules into the plasma membrane, to the rate at which particular portions of plasma membranes of two neighboring cells come into close contact, or to a number of other factors. The slope of the fusion curve (X) varies with experimental conditions such as initial cell density and MOI. The average cell density for experiments shown in Fig. 3-5 and for replicate experiments of that type was 5.7 x 104 cells/ cm2, which represents a 90% confluent monolayer. For this cell density A = 0.47 per h. A is independent of cell density for values greater than 3.5 x 104 cells/cm2. At lower cell densities, where an appreciable fraction of cells are not touching, X is proportional to cell density. Kohn (5) has also noted that the fusion index was dependent on cell density at densities less than about 3.0 x 104 cells/cm2, but no effect was observed at higher densities. The slope of the fusion kinetics curve is about 0.5 per h for an MOI >1 (at a cell concentration of 5.7 x 104 cells/cm2) and about one-half this value at an MOI of 0.4. Using the Poisson relationship one can calculate that only about 33% of the cells are infected at an average MOI of 0.4. Perhaps the slower rate of fusion is due to a smaller probability of fusion of infected and uninfected cells relative to that observed when all cells are infected. The onset of fusion is influenced by the MOI of the mutant virus. The minimum time between virus adsorption and the onset of fusion that we observed was 4 h when an MOI of 8 or 86 was used. Since a minimum time of this duration is required, the results reported here are characteristic of fusion from within. Although we have not observed fusion from without with HEL cells infected with syn 20 at an MOI as high as 86, Nii and Kamahora (7) have reported fusion from without caused by another variant of HSV with FL cells. It is difficult for us to determine accurately the MOI used in their experiments, and it is possible that we would observe fusion from without by using a larger MOI. At a lower MOI the time before the onset of fusion was dependent on MOI, increasing progressively from a minimum of 4 h to a maximum of 7.5 h as the MOI decreased from 8 to 0.4. Using a low MOI, but one which results in nearly all of the cells being infected (MOI of 1.8, for example), the onset of fusion was 6 to 6.5 h. A possible interpretation of the dependence of the onset of fusion on MOI is that a cell has to accumulate a certain number of mutant mole-
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cules in its plasma membrane for fusion to occur. This number could be obtained at earlier times at a higher MOI if there is increased transcription and translation due to an increased number of viral genomes per cell. The onset of fusion is also influenced by cell density. At lower cell densities the onset of fusion occurs earlier than at higher cell densities. At the lower cell densities all of the cells are in the exponential growth phase, which may explain the earlier onset of fusion. In an earlier report, Roizman (11) described another strain of HSV, called MP, which produced syncytia when plated on FL or HEp-2 cells at less than 0.01 PFU/cell, but produced little cell fusion when almost all cells were infected. He suggested that infected cells could fuse only with uninfected cells. This is clearly not the case with syn 20 on HEL cells. Preliminary experiments in our laboratory indicate that MP will fuse a monolayer of HEL, HEp-2, or BHK-21 cells infected at an MOI greater than 1. The Coulter counter assay provides a simple quantitative measure of cell fusion. It has been useful in following the effects of several inhibitors of cell fusion, including 2-deoxyglucose and glucosamine (Knowles and Person, manuscript in preparation), as well as for monitoring fusion of cells mixedly infected with two syn mutants in genetic studies which are currently in progress in our laboratory. In addition, the assay should be easily modified for use with other cell and virus systems. ACKNOWLEDGMENTS This work was supported by a grant from AEC, AT(11-1)3419, and by Public Health Service grant Al 11513 from the National Institute of Allergy and Infectious Diseases. We acknowledge discussions of data and manuscript with colleagues Tom Holland, Paul Keller, and William D. Taylor. Preliminary experiments by Paul Keller were most helpful in the development of the fusion assay. LITERATURE CITED 1. Bratt, M. A., and W. R. Gallaher. 1969. Preliminary analysis of the requirements for fusion from within and fusion from without by Newcastle Disease Virus. Proc. Natl. Acad. Sci. U. S. A. 64:536-543. 2. Brown, S. M., D. H. Ritchie, and J. H. Subak-Sharpe. 1973. Genetic studies with herpes simplex virus type I. The isolation of temperature-sensitive mutants, their arrangement into complementation groups and recombination analysis leading to a linkage map. J. Gen. Virol. 18:329-346. 3. Gray, A., T. Tokumaru, and T. F. McN. Scott. 1958. Different cytopathic effects observed in HeLa cells infected with HSV. Arch. Gesamte Virusforsch. 8:59-76. 4. Hoggan, M. D., and B. Roizman. 1959. The isolation and properties of a variant of herpes simplex producing multinucleated giant cells in monolayer cultures in the
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presence of antibody. Am. J. Hyg. 70:208-219. 5. Kohn, A. 1965. Polykaryocytosis induced by NDV in monolayers of animal cells. Virology 26:228-245. 6. Munk, K., and D. Donner. 1963. Cytopathic effect and plaque morphology of several herpes virus strains. Arch. Gesamte Virusforsch. 13:529-540. 7. Nii, S., and J. Kamahora. 1961. Cytopathic changes induced by herpes simplex virus. Biken J. 4:255-270. 8. Okada, Y., and J. Takodoro. 1962. Analysis of giant polynuclear cell formation caused by HVJ virus from Ehrlich's ascites tumor cells. II. Quantitative analysis of giant polynuclear cell formation. Exp. Cell Res.
26:108-118. 9. Poste, G. 1972. Mechanisms of virus induced cell fusion. Int. Rev. Cytol. 33:157-252. 10. Poste, G., and A. C. Allison. 1973. Membrane fusion. Biochim. Biophys. Acta 300:421-465.
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Roizman, B. 1962. Polykaryocytosis. Cold Spring Harbor Symp. Quant. Biol. 27:327-341. 12. Roseman, S., W. Rothman, B. Walther, R. Ohman, and 11.
J. Umbreit. 1974. Measurement of cell-free interactions, p. 597-611. In S. Fleisher and L. Packer (ed.), Methods in enzymology, vol. 32, part B. Academic Press Inc., New York. 13. Schneweis, K.-E. 1962. Cytopathic effects of HSV. Zentralbl. Bakteriol. Parasitenkd. Infektionskr. Hyg. Abt. 1 Orig. 186:467-477. 14. Tokumaru, F. 1968. The nature of toxins of HSV. I. Syncytial giant cell producing components in tissue culture. Arch. Gesamte Virusforsch. 24:104-122. 15. Wheeler, C. E., Jr. 1964. Biologic comparison of a syncytial and a small giant cell-forming strain of Herpes Simplex. J. Immunol. 93:749-756.
