Vol. 23, No. 1 Printed in U.S.A.
JOURNAL OF VIROLOGY, July 1977, p. 213-215 Copyright © 1977 American Society for Microbiology
Ammonium Chloride Inhibits Cell Fusion Induced by syn Mutants of Herpes Simplex Virus Type 1 THOMAS C. HOLLAND AND STANLEY PERSON* Biophysics Laboratory, Department of Biochemistry and Biophysics, The Pennsylvania State University, University Park, Pennsylvania 16802
Received for publication 5 January 1977
Cell fusion induced by syn mutants of herpes simplex virus type 1 is inhibited by NH4Cl. The inhibition of fusion is both rapid and rapidly reversible and appears to be due to the NH4+ ion. Virus production was not significantly altered by NH4Cl, although cell growth was greatly diminished.
NH4Cl caused a noticeable decrease in the rate of fusion of cells infected with syn 20, a mutant of the KOS strain of HSV-1 (6); 20 mM NH4Cl caused an almost complete inhibition of fusion. When 20 mM NH4Cl was added to the cultures 6 h after infection (1 h after fusion had begun), the cells were inhibited from further fusion (Fig. lb). Cell fusion was measured by assaying the fraction of single cells remaining unfused as a function of time after infection, as described previously (6). Similar experiments were also performed with either (NH4)2SO4, NaCl, or RbCl. Only (NH4)2S04 was able to inhibit fusion, suggesting that the inhibition is associated with the NH4+ ion and not with the Cl- ion. Related experiments have shown that here. In Fig. la NH4Cl was added to the cultures 1 Li+, K+, and Cs+ are unable to inhibit fusion h after infection. As shown in the figure, 5 mM (T. C. Holland, M.S. thesis, The Pennsylvania
Although the hallmark of infections with most wild-type strains of herpes simplex virus type 1 (HSV-1) is the presence of individual rounded cells, some strains that cause extensive cell fusion (3, 4, 7, 8) have been isolated. Mutants that cause extensive cell fusion (syn mutants) have been isolated from wild-type strains that cause little fusion (2, 6). Fusion caused by HSV-1 can occur at a low multiplicity of infection and requires viral protein synthesis. This has been termed fusion from within (1). In the course of characterizing syn mutants isolated in our laboratory, it was found that fusion could be rapidly and reversibly inhibited by NH4Cl. These experiments are reported
I
b
0
LA. 0.j
2
0
wOa6
I
I
I
1
0~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Un 1.0
D 1,(
I
z
J 0 2
w
U
0 .
t 0.2
z
4
0
* no NH4CI 20 mM NH4CI added at 6 hr
i
a
U 49 I&.
8 10 4 6 TIME AFTER INFECTION (hr)
2
10 4 6 8 TIMlE AFTER INFECTION (hr)
12
FIG. 1. Effect of NH4Cl on cell fusion. Log-phase HEL cells in 2-ounce (ca. 56.7-ml) prescription bottles (1.2 x 106 cells per bottle) were infected with the syncytia-producing mutant syn 20 at an attached multiplicity of infection of 4 PFU per cell, as described previously (6). After removal of unattached virus, growth medium was added, and the cultures were incubated at 340C. Zero hours after infection is defined as the time of addition of growth medium. (a) At 1 h after infection, 1.0 M NH4Cl was added to a final concentration of 0, 5, 10, or 20 mM. (b) At 6 h after infection, 1.0 M NH4Cl was added to one set of cultures to a final concentration of 20 mM. Cell fusion was assayed by harvesting cells with a trypsin-EDTA solution and using a Coulter counter to determine the fraction of single cells remaining as a function of time after infection. Cells were considered to be fused if they were not separated into single cells by the harvesting procedure (6). 213
214 0
J. VIROL.
NOTES
010 XX xX
0
z hrefe w- 0.4 010m
-J 10o/
-J
EControl (no NHrCe O Mtee xe added at I hr a 0.2 xc20 mMwNH4CI 2 oF20 mMNH4Cl fromw Ihr to7 hr 0 oL.
0
-
0mM
0
thereafterfromw Ihr to 9 hr,
rpteN20mMoNH4CI 0 mM thereafter Is.t
NH~~~~~ C2m
6 4 8 10 TIME AFTER INFECTION (hr)
2
40
12
8
120
60 200
NC 4
FIG. 2. Effect of removal of NH4Cl at 7 and 9 h after infection. HEL cells were infected with syn 20 and assayed for cell fusion' as described in the legend T I ME ( hours) to Fig. 1. NH4Cl was added to a final concentration of 20 mM to two sets of cultures.- It was removed fr-om 4. Effect ofNH4Cl on cell growth. Two-ounce these sets of cultures at 7 and 9 h after infection, (ca.FIG. 56.7-ml) prescription bottles were seeded with respectively. Nothing was added to the third (control) 1.0 x 105 cells in 5.0 ml of medium and incubated set of cultures. at 37°C. At the time indicated by the arrow (30.5 h after incubation), 1.0 M NH4Cl was added to one10 l half of the bottles to a final concentration of 20 mM. Periodically the cell number was determined by harvesting the cells and counting them with a Coulter counter (see legend to Fig. 1). /x I-
z
107
O,x
i
Id o control x 20mM NH4CI
106
0
_x
5
U
10
15
U
20
25
3U
30
31
35
TIME AFTER INFECTION (hr)
FIG. 3. Effect of NH4Cl on virus growth. Cells infected, and NH4Cl was added to the indicated cultures (final concentration, 20 mM) as described in the legend to Fig. 1. At the times indicated, cultures were removed from the incubator and frozen. After three cycles of freezing and thawing, virus was titered by plaque assay on HEL cells, as described previously (6). were
State University, University Park, 1975). NH4C1 has been found to inhibit fusion produced by all of the syn mutants isolated in this laboratory. Fusion of HEp-2 or BHK-21 cells infected with syn 20 was also inhibited by NH4Cl (data not shown). Figure 2 shows that when NH4Cl was added to the medium to a final concentration of 20 mM 1 h after infection and removed by changing the medium 7 h after infection (well after untreated control cultures have begun to fuse), fusion began almost immediately and proceeded at the normal rate. A similar result was seen when
the inhibitor was present from 1 to 9 h after infection. Thus, the effect of on syn 20NH4Co induced cell fusion is both rapid and rapidly reversible. Virus growth curves for syn 20 were determined in the presence of 20 mM NH4C(, the used in the fusion inhibihighest cubatiio)1.an tion experiments. As shown in Fig. 3, there was very little effect on virus growth; virus production was reduced by 0 to 50% in replicate experiments. The effect of the same concentration of NH4clon cell growth was noticeably greater (Fig. 4). The doubling time of the control culture was 22 h; that of the treated culture was nearly 200 h. The morphology of human embryonic lung (HEL) cell sbot not affected by
NH4Cl.
The plating efficiencies of both the syn mutant, syn 20, and the wild-type strain KOS were unchanged when plaqued on HEL cells at 34°C in the presence of 20 mM NH4Cl. However, in presence of NH4Cl plaques produced the were smaller than control plaques and contained no syncytia (Holland, M.S. thesis). The synthesis of virus-specified proteins and glycoproteins was determined in the presence and absence of 20 mMNH4Cle syn 20-infected cells were labeled from 4 to 24 h after infection with either l4C-amino acids [14tcglUCoSor amine, and the proteins and glycoproteins were
VOL. 23, 1977
examined by sodium dodecyl sulfate-gel electrophoresis, as described earlier (5). No significant differences were observed in the protein or glycoprotein profiles labeled in the presence or absence of NH4Cl (Holland, M.S. thesis).
The lack of effect of NH4Cl on virus growth and virus-specific protein synthesis suggests that it does not act by inhibition of synthesis of a virus-specific macromolecule essential for fusion. This, together with the rapidity of the effect and its reversal, suggests that NH4C1 interferes directly with some component of the fusion mechanism. This component is most likely to be a macromolecule or supramolecular structure associated with the plasma membrane. We thank Susan C. Warner for excellent technical assistance and Robert W. Knowles, Paul M. Keller, and G. Sullivan Read for helpful discussions during the course of this
work. These studies were supported by U.S. Energy Research and Development Administration grant E(11-1)-3419 and by Public Health Service grant no. AI-11513 from the National Institute of Allergy and Infectious Diseases. T.C.H. is supported by Public Health Service predoctoral training grant GM-01015 from the National Institute of General Medical Sciences.
NOTES
215
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. 8:5976. 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 presence of antibody. Am. J. Hyg. 70:208-219. 5. Knowles, R. W., and S. Person. 1976. Effects of 2deoxyglucose, glucosamine, and mannose on cell fusion and the glycoproteins of herpes simplex virus. J. Virol. 18:644-651. 6. Person, S., R. W. Knowles, G. S. Read, S. C. Warner, and V. C. Bond. 1976. Kinetics of cell fusion induced by a syncytia-producing mutant of herpes simplex virus type I. J. Virol. 17:183-190. 7. Schneweis, K.-E. 1962. Cytopathic effects of HSV. Zentralbl. Bakteriol. Parasitenkd. Infectionskr. Hyg. Abt. 1 Orig. 186:467-477. 8. 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, July 1977, p. 213-215 Copyright © 1977 American Society for Microbiology
Ammonium Chloride Inhibits Cell Fusion Induced by syn Mutants of Herpes Simplex Virus Type 1 THOMAS C. HOLLAND AND STANLEY PERSON* Biophysics Laboratory, Department of Biochemistry and Biophysics, The Pennsylvania State University, University Park, Pennsylvania 16802
Received for publication 5 January 1977
Cell fusion induced by syn mutants of herpes simplex virus type 1 is inhibited by NH4Cl. The inhibition of fusion is both rapid and rapidly reversible and appears to be due to the NH4+ ion. Virus production was not significantly altered by NH4Cl, although cell growth was greatly diminished.
NH4Cl caused a noticeable decrease in the rate of fusion of cells infected with syn 20, a mutant of the KOS strain of HSV-1 (6); 20 mM NH4Cl caused an almost complete inhibition of fusion. When 20 mM NH4Cl was added to the cultures 6 h after infection (1 h after fusion had begun), the cells were inhibited from further fusion (Fig. lb). Cell fusion was measured by assaying the fraction of single cells remaining unfused as a function of time after infection, as described previously (6). Similar experiments were also performed with either (NH4)2SO4, NaCl, or RbCl. Only (NH4)2S04 was able to inhibit fusion, suggesting that the inhibition is associated with the NH4+ ion and not with the Cl- ion. Related experiments have shown that here. In Fig. la NH4Cl was added to the cultures 1 Li+, K+, and Cs+ are unable to inhibit fusion h after infection. As shown in the figure, 5 mM (T. C. Holland, M.S. thesis, The Pennsylvania
Although the hallmark of infections with most wild-type strains of herpes simplex virus type 1 (HSV-1) is the presence of individual rounded cells, some strains that cause extensive cell fusion (3, 4, 7, 8) have been isolated. Mutants that cause extensive cell fusion (syn mutants) have been isolated from wild-type strains that cause little fusion (2, 6). Fusion caused by HSV-1 can occur at a low multiplicity of infection and requires viral protein synthesis. This has been termed fusion from within (1). In the course of characterizing syn mutants isolated in our laboratory, it was found that fusion could be rapidly and reversibly inhibited by NH4Cl. These experiments are reported
I
b
0
LA. 0.j
2
0
wOa6
I
I
I
1
0~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
Un 1.0
D 1,(
I
z
J 0 2
w
U
0 .
t 0.2
z
4
0
* no NH4CI 20 mM NH4CI added at 6 hr
i
a
U 49 I&.
8 10 4 6 TIME AFTER INFECTION (hr)
2
10 4 6 8 TIMlE AFTER INFECTION (hr)
12
FIG. 1. Effect of NH4Cl on cell fusion. Log-phase HEL cells in 2-ounce (ca. 56.7-ml) prescription bottles (1.2 x 106 cells per bottle) were infected with the syncytia-producing mutant syn 20 at an attached multiplicity of infection of 4 PFU per cell, as described previously (6). After removal of unattached virus, growth medium was added, and the cultures were incubated at 340C. Zero hours after infection is defined as the time of addition of growth medium. (a) At 1 h after infection, 1.0 M NH4Cl was added to a final concentration of 0, 5, 10, or 20 mM. (b) At 6 h after infection, 1.0 M NH4Cl was added to one set of cultures to a final concentration of 20 mM. Cell fusion was assayed by harvesting cells with a trypsin-EDTA solution and using a Coulter counter to determine the fraction of single cells remaining as a function of time after infection. Cells were considered to be fused if they were not separated into single cells by the harvesting procedure (6). 213
214 0
J. VIROL.
NOTES
010 XX xX
0
z hrefe w- 0.4 010m
-J 10o/
-J
EControl (no NHrCe O Mtee xe added at I hr a 0.2 xc20 mMwNH4CI 2 oF20 mMNH4Cl fromw Ihr to7 hr 0 oL.
0
-
0mM
0
thereafterfromw Ihr to 9 hr,
rpteN20mMoNH4CI 0 mM thereafter Is.t
NH~~~~~ C2m
6 4 8 10 TIME AFTER INFECTION (hr)
2
40
12
8
120
60 200
NC 4
FIG. 2. Effect of removal of NH4Cl at 7 and 9 h after infection. HEL cells were infected with syn 20 and assayed for cell fusion' as described in the legend T I ME ( hours) to Fig. 1. NH4Cl was added to a final concentration of 20 mM to two sets of cultures.- It was removed fr-om 4. Effect ofNH4Cl on cell growth. Two-ounce these sets of cultures at 7 and 9 h after infection, (ca.FIG. 56.7-ml) prescription bottles were seeded with respectively. Nothing was added to the third (control) 1.0 x 105 cells in 5.0 ml of medium and incubated set of cultures. at 37°C. At the time indicated by the arrow (30.5 h after incubation), 1.0 M NH4Cl was added to one10 l half of the bottles to a final concentration of 20 mM. Periodically the cell number was determined by harvesting the cells and counting them with a Coulter counter (see legend to Fig. 1). /x I-
z
107
O,x
i
Id o control x 20mM NH4CI
106
0
_x
5
U
10
15
U
20
25
3U
30
31
35
TIME AFTER INFECTION (hr)
FIG. 3. Effect of NH4Cl on virus growth. Cells infected, and NH4Cl was added to the indicated cultures (final concentration, 20 mM) as described in the legend to Fig. 1. At the times indicated, cultures were removed from the incubator and frozen. After three cycles of freezing and thawing, virus was titered by plaque assay on HEL cells, as described previously (6). were
State University, University Park, 1975). NH4C1 has been found to inhibit fusion produced by all of the syn mutants isolated in this laboratory. Fusion of HEp-2 or BHK-21 cells infected with syn 20 was also inhibited by NH4Cl (data not shown). Figure 2 shows that when NH4Cl was added to the medium to a final concentration of 20 mM 1 h after infection and removed by changing the medium 7 h after infection (well after untreated control cultures have begun to fuse), fusion began almost immediately and proceeded at the normal rate. A similar result was seen when
the inhibitor was present from 1 to 9 h after infection. Thus, the effect of on syn 20NH4Co induced cell fusion is both rapid and rapidly reversible. Virus growth curves for syn 20 were determined in the presence of 20 mM NH4C(, the used in the fusion inhibihighest cubatiio)1.an tion experiments. As shown in Fig. 3, there was very little effect on virus growth; virus production was reduced by 0 to 50% in replicate experiments. The effect of the same concentration of NH4clon cell growth was noticeably greater (Fig. 4). The doubling time of the control culture was 22 h; that of the treated culture was nearly 200 h. The morphology of human embryonic lung (HEL) cell sbot not affected by
NH4Cl.
The plating efficiencies of both the syn mutant, syn 20, and the wild-type strain KOS were unchanged when plaqued on HEL cells at 34°C in the presence of 20 mM NH4Cl. However, in presence of NH4Cl plaques produced the were smaller than control plaques and contained no syncytia (Holland, M.S. thesis). The synthesis of virus-specified proteins and glycoproteins was determined in the presence and absence of 20 mMNH4Cle syn 20-infected cells were labeled from 4 to 24 h after infection with either l4C-amino acids [14tcglUCoSor amine, and the proteins and glycoproteins were
VOL. 23, 1977
examined by sodium dodecyl sulfate-gel electrophoresis, as described earlier (5). No significant differences were observed in the protein or glycoprotein profiles labeled in the presence or absence of NH4Cl (Holland, M.S. thesis).
The lack of effect of NH4Cl on virus growth and virus-specific protein synthesis suggests that it does not act by inhibition of synthesis of a virus-specific macromolecule essential for fusion. This, together with the rapidity of the effect and its reversal, suggests that NH4C1 interferes directly with some component of the fusion mechanism. This component is most likely to be a macromolecule or supramolecular structure associated with the plasma membrane. We thank Susan C. Warner for excellent technical assistance and Robert W. Knowles, Paul M. Keller, and G. Sullivan Read for helpful discussions during the course of this
work. These studies were supported by U.S. Energy Research and Development Administration grant E(11-1)-3419 and by Public Health Service grant no. AI-11513 from the National Institute of Allergy and Infectious Diseases. T.C.H. is supported by Public Health Service predoctoral training grant GM-01015 from the National Institute of General Medical Sciences.
NOTES
215
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. 8:5976. 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 presence of antibody. Am. J. Hyg. 70:208-219. 5. Knowles, R. W., and S. Person. 1976. Effects of 2deoxyglucose, glucosamine, and mannose on cell fusion and the glycoproteins of herpes simplex virus. J. Virol. 18:644-651. 6. Person, S., R. W. Knowles, G. S. Read, S. C. Warner, and V. C. Bond. 1976. Kinetics of cell fusion induced by a syncytia-producing mutant of herpes simplex virus type I. J. Virol. 17:183-190. 7. Schneweis, K.-E. 1962. Cytopathic effects of HSV. Zentralbl. Bakteriol. Parasitenkd. Infectionskr. Hyg. Abt. 1 Orig. 186:467-477. 8. 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.
