VIROLOGY
76, 146-151
(1977)
Cell Cycle-Dependent
Inhibition of Kirsten Murine Sarcoma-Leukemia Virus Release by Cytochalasin B SANDRA
Department
PANEM
Pathology, University of Chicago, Chicago, Illinois
of
60637
Accepted August 11,1976 Inhibition of Kirsten murine sarcoma-leukemia virus [KiMSV(KiMuLV)l release from chronically infected normal rat kidney cells (NRK-K) by cytochalasin B (CB) was dose dependent. Concentrations of CB between lo-25 pg/ml inhibited virus release without inhibiting mitosis and cell division. Maximal inhibition occurred within 4.5 hr of adding CB to logarithmically growing NRK-K cells, and inhibition was reversed within 3 hr of drug removal. CB inhibition of virus release from synchronously growing NRK-K cells was cell-cycle dependent and occurred during late G2 phase and mitosis. INTRODUCTION
cell line referred to as NRK-K cells (Panem and Schauf, 1974). A single cell clone, N, of NRK-K cells was used for these experiments between the sixth and seventeenth in vitro subculture generations. NRK-K cells were grown in 250 ml Falcon tissue culture flasks (Falcon, Oxnard, Calif.), 32-0~ glass pharmacy bottles, or glass roller bottles (Bellco Glass, Vineland, N.J.) at 37” in an atmosphere of 5% CO,. Growth medium consisted of Eagle’s minimal essential medium (MEM) supplemented with 10% fetal calf serum, 100 units per ml of penicillin and 100 pg/ml of streptomycin (Gibco, Grand Island, N.Y.). Cells were fed every 2 days, and at weekly intervals cells were subcultured with trypsin-EDTA and monitored for mycoplasma contamination (McClain and Kirsten, 1974). Only mycoplasma-free cells were used for these experiments. Cell synchrony. Logarithmically growing cells were synchronized by a doublethymidine block as described (Panem and K&ten, 1973). Cell division was determined by serial counting of cell number using trypan blue dye exclusion as a measure of cell viability. Briefly, cell division was inhibited by incubating cells in thymidine-rich medium (2 m&0 for 16 hr. Cell division then proceeded for 8 hr in medium containing 10 fl deoxyguanosine, deoxy-
The coordination of murine oncornavirus replication with the host cell cycle has been reported (Fischinger et al., 1975; Panem and Kirsten, 1973; Paskind et al., 1975). Although there are discrepancies concerning which events of viral replication are cell-cycle restricted, the release of virus concomitant with mitosis from murine cells synchronized by different methods has been observed under conditions of chronic and de nouo infection (Fischinger et al., 1975; Lerner et al., 1971; Panem and Kirsten, 1973; Paskind et al., 1975). As microtubule and microfilament action is prominent during the mitotic interval, the cell-cycle dependence of virus maturation was studied by examining the effects of two inhibitors of contractile element function on the release of KiMSV(KiMuLV) from chronically infected cells. The results show that inhibition of virus release can be separated from inhibition of mitosis and cell division by cytochalasin B. Cytochalasin B reversibly inhibits a cell-cycle dependent step of virus maturation. MATERIALS
AND
METHODS
Cells and virus. Infection of normal rat kidney cells with Kirsten murine sarcomaleukemia virus [KiMSV(KiMuLV)l resulted in a transformed, virus-producing 146 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
CB
INHIBITION
OF
adenosine, and deoxycytosine. Cell division was again blocked for 15 hr with thymidine-rich medium. The second time of thymidine removal was defined at 0-hr postrelease (p.r.). NRK-K cells show celfcycle times of S + G2/M/Gl = 8.5 hr/O.51.5 hr/2.5 hr = 12.5 hr, as previously determined (Panem and Schauf, 1974). Drugs,
isotopes,
and
reagents.
Cyto-
chalasin B (Calbiochem, San Diego, Calif.) (CB) was purchased as a lyophilized powder. CB was reconstituted in 100% ethanol at 5 mg/ml and stored at -20”. Vinblastine sulfate (Velban, Eli Lilly, Indianapolis, Ind.) (VB) was purchased in lyophilized form, reconstituted in growth medium, and used immediately. [3H]Thymidine 5’triphosphate (sp act 55 Ci/mmol) was purchased from New England Nuclear, Boston, Mass. TNE buffer consisted of 0.1 M NaCl, 10 mM Tris-hydrochloride (pH 7.3), and 1 mM EDTA. Phosphate-buffer saline (PBS) consisted of 0.137 M NaCl, 2.7 mM KCl, 8.1 mM Na,HPO,, 1.5 mM KH2P04, and 1% phenol red. RNA-dependent (RDDP). Spent
DNA-polymerase
145
KiMSV(KiMuLV)
action-mixture products were precipitated by the addition of 25 volumes of ice-cold 5% trichloroacetic acid-2% Na,P,O, (TCAPPi). The precipitates were collected by centrifugation at 2000 g for 5 min and washed 3 times by centrifugation with 5 ml of TCA-PPi. The final precipitate was washed once with 95% ethanol, drained, dissolved in NCS (Amersham-Searle, Arlington Heights, Ill.) and 10 ml of toluenebased liquid scintillation fluid. Radioactivity was determined in a Packard Tri-Carb liquid scintillation counter. Electron microscopy. Cells were prepared for electron microscopy by fixation for at least 1 hr with 25% glutaraldehydc in 0.1 M Na cacodylate, pH 7.3, at room temperature. Samples were then postfixed for 45 min with 1% osmium tetroxide in collidine buffer. Following dehydration with ethanol, and embedding in Epon, thin sections were stained with uranyl acetate and lead citrate and were viewed in an RCA-EMU 4 electron microscope as described (Schwartz et al., 1974).
assay
culture fluids were assayed for RDDP after clarification by centrifugation at 10,000 g for 20 min as described (Prochownik et al., 1975). Clarilied culture fluids were concentrated by ultracentrifugation through a 3.0-cm cushion of 5% (w/v) sucrose in TNE buffer at 100,000 g for 90 min. The pellet was resuspended in 50 ~1 of TMG buffer [lo mM Tris-hydrochloride (pH 7.81, 7 mM 2-mercaptoethanol, and 10% glycerol] and stored at -80”. Enzyme activity was measured with the synthetic template poly(rA) . oligo(dT),,,-,,, (Collaborative Research, Waltham, Mass.). Reaction mixtures (0.2 ml) consisted of 0.025% Nonidet-P40, 25 mM Tris-hydrochloride (pH 8.11, 7 mM 2-mercaptoethanol, 0.2 mM MnCl,, 2 pg of template, 60 n&f KCl, 0.0083 mM thymidine 5’-triphosphate (Sigma, St. Louis, MO.), 5 @!i of [“Hlthymidine 5’-triphosphate, and 10 ~1 of resuspended supernatant pellet in TMG buffer. Reactions were carried out for 40 min in a 37” water bath. The mixture were chilled quickly to O”, and 250 pg of bovine serum albumin was added as carrier. Re-
RESULTS
Inhibition cell diuision.
of virus
release,
mitosis
and
Cytochalasin B (CB) and vinblastine sulfate (VB) were tested for inhibitory activity. Neither inhibitor interfered with the incorporation of [3H]TMP by viral reverse transcriptase (data not shown). Inhibition of virus release was measured by the decrease of reverse transcriptase activity in spent culture fluids of drug-treated NRK-K cells (Kelloff et al., 1972). CB and VB inhibited KiMSV(KiMuLV) release from logarithmically growing NRK-K cells in a dose-dependent manner (Fig. 1). Inhibition by VI3 was always accompanied by inhibition of mitosis and cell division. In contrast, with 25 pg/ml of CB, mitosis was not inhibited whereas virus release was inhibited by at least 70%. As judged by the doubling in viable cell number using trypan blue exclusion to assess viability, 25 pg/ml of CB did not inhibit division of NRK-K cells during the time interval of these experiments. Although cell division was not impaired by CB, treated cells showed rounding. Rapid
and reversible
inhibition
by CB.
148
SANDRA
FIG. 1. Inhibition of virus release and cell division by cytochalasin B (CB) and vinblastine sulfate (VB). Replicate cultures of exponentially growing NRK-K cells were incubated in duplicate overnight with medium containing inhibitors. The next morning, the medium was collected, clarified, and assayed for RDDP. The cells were removed using trypsin-EDTA, and the number of viable cells was determined by the trypan blue exclusion test. Data are presented as the percentage of inhibition of RDDP and the percentage of inhibition of cell division relative to the untreated controls. At 100 pg/ml of CB and VB, cytotoxicity was detected.
CB (20 pglml) suppressed KiMSV(KiMuLV) release by 30% within 1.5 hr and by greater than 80% within 4.5 hr of its addition to logarithmically growing NRK-K cells (data not shown). Control cultures treated with growth medium containing 0.2% ethanol showed no inhibition of virus release. The reversibility of CB inhibition was tested. Exponentially growing NRK-K cells were incubated with growth medium for 1.5 hr. This medium was collected and cells were then incubated with CB (20 pg/ml) for 27 hr during which several 1.5hr medium collections were made. The 1.5~hr collections were processed in parallel for reverse transcriptase activity and the data were expressed relative to enzyme activity at the start of the experiment. Rapid CB inhibition of supernatant reverse transcriptase was observed within 3 hr. In addition, within 3 hr of the drug’s removal a peak of virus release was seen (Fig. 2). Although the population doubled in the course of the experiment, the burst of supernatant activity following CB removal was greater than could be explained by an increase in cell number and was consistent with re-
PANEM
versible inhibition of a late event of virus replication by CB. This suggested that budding viral forms accumulated in CBtreated cells which were completed on removal of the drug. In this, but not all experiments, partial recovery from CB inhibition was observed between 3-4.5 hr. When partial recovery occurred, the level of virus released in 1.5-hr intervals was constant for the duration of drug treatment (4.5-23 hr). As maximum inhibition attained with logarithmically growing cells did not exceed 85%, and as the cell number doubled during CB treatment, the low level of virus released between 4.5 and 23 hr probably reflects a small proportion of cells escaping CB inhibition, possibly due to differential drug uptake. To test whether CB inhibition occurred following formation of viral buds, NRK-K cells were examined by electron microscopy following CB removal. Replicate flasks of logarithmically growing NRK-K
0
3.0
22 25 TIME (hrs)
28
FIG. 2. Reversibility of CB inhibition. Logarithmically growing NRK-K cells were fed fresh growth medium, which was collected 1.5 hr later. Medium containing 20 pg/ml of CB was added, replaced at 3 and 4.5 hr, and cells were incubated overnight. Fresh drug-containing medium was added in the morning and collected 1.5 hr later. Cells were washed and refed fresh growth medium without drug at 1.5hr intervals for the next 6 hr. All 1.5-hr collections were processed in duplicate for RDDP activity. Data are presented as the percentage of RDDP relative to the activity in the collection made at the beginning of the experiment. The hatchmarks indicate the period during which CB was present.
CB INHIBITION
OF
cells were treated with 20 pg/ml of CB for 4 hr. Cells were washed twice and fed fresh growth medium. Samples were processed for microscopy at 0, 1.5, and 3 hr following drug removal, and the number of budding type C particles on 50-100 individual cells were determined for the three samples as well as an untreated control (Table 1). Although such experiments are limited by considerations of sample size, entrapment, and/or loss of extracellular particles during specimen preparation relative to control cultures, cells processed immediately after CB removal showed fewer budding and extracellular particles than the sample processed at 1.5 hr. After 3 hr, increased numbers of both viral forms were found. These data suggested that CB inhibition occurred prior to visually detectable condensation of maturing type C virions at the plasma membrane. Cell cycle-dependent inhibition of virus release. The release of KiMSV(KiMuLV)
from NRK-K cells synchronized by doublethymidine blockade occurs concomitant with mitosis after thymidine removal (Panem and Kirsten, 1973). The time of CB inhibition during virus replication was studied by incubating replicate cultures of NRK-K cells with CB (10 pg/ml) for decreasing times during synchronized
149
KiMSV(KiMuLV1
growth. In pilot experiments, this dose of CB resulted in 50-75% inhibition of virus release without cytotoxicity in synchronized cells. The growth medium with or without CB was changed in late G2 phase and collected 2 hr later at the end of mitosis. The 2-hr medium collections were analyzed for reverse transcriptase as a measure of virus release (Table 2). CB inhibited virus release by nearly 70% in cultures treated just prior to mitosis, regardless of the duration of drug treatment during S + G2 phases. However, cultures treated for the latter portion of the mitotic interval (lo-11 hr) showed less inhibition of virus release. The effectiveness of CB inhibition of virus release therefore coincided with its presence during the final events of virus maturation at the G2/M interphase. Decreased inhibition of virus release in cultures treated for a portion of the mitotic interval does not result from a shorter exposure of cells to the inhibitor, as judged from the level of inhibition achieved in TABLE CELL Time of CB treatment (hr p.r.1
2
CYCLE-DEPENDENT CYTOCHALASIN
INHIBITION B”
RDDP
cpm
T”H1TTP
BY
activity
% Control x
% Inhibition
iO-3
TABLE 1 APPEARANCE OF VIRAL-BUDDING CYTOCHALASIN B-TREATED Hours following CB removal”
Untreated
control
IN
Virionsb Budding
0.0 1.5 3.0
0
FORMS CELLS
24 88 36 60
Extracellular
43 108 110 140
Extracellular/ budding ratio 1.8 1.2 3.0 2.3
o Replicate cultures of logarithmically growing NRK-K cells were treated for 4 hr with 20 pg/ml of CB, washed, and incubated with fresh growth medium. After 0, 1.5 and 3.0 hr, cells were prepared for electron microscopy. * Fifty to one hundred cell sections were examined for budding and extracellular virions. Data are presented as the number of virions per 100 cell sections and as the ratio of extracellular to budding virions.
10-11 9-10 9-11 8-11 7-11 3-11
42.5 34.7 15.7 13.0 13.2 13.3 8.9
100.0 81.7 37.0 30.4 31.0 31.1 21.0
0.0 18.3 63.0 69.6 69.0 68.9 79.0
-
a Replicate cultures of logarithmically growing NRK-K cells were synchronized by double-thymidine blockade. At various times after release from thymidine blockade (postrelease: p.r.), cultures were treated with 10 pg/ml of CB. At 9 hr p.r., all cultures were fed fresh growth medium with or without CB as indicated. This medium was collected at 11 hr p.r. and assayed in duplicate for RDDP. Data are presented as the counts per minute of [3H]TTP incorporated x 10m3 in duplicate assays, as the percentage of RDDP relative to the untreated control, and as the percentage of inhibition of RDDP activity. Synchrony was monitored by examining samples from parallel cultures at hourly intervals for the percentage of mitotic cells. Between 9 and 11 hr p.r., 27.8% of the cells passed through mitosis.
150
SANDRA
cells receiving CB from 9-10 hr (Table 2). In the representative experiment presented in Table 2,27.8% of the cells passed through mitosis from 9-11 hr p.r. Similar results were obtained when replicate cultures were pulsed with CB for l- or 2-hr intervals during synchronous growth of NRK-K cells (Table 2; Panem and Kirsten, 1976). However, the current experimental design was judged superior to that of pulsing as the effect of CB was not instantaneously reversible as required in pulse experiments. DISCUSSION
Microtubules and microfilaments play prominent roles in nuclear and cellular division during the mitotic interval. VB poisons the mitotic spindle apparatus by binding to microtubular protein and disaggregating microtubules (Olmsted and Borisy, 1973). Inhibition of cell division by VB is therefore a consequence of its primary effect on microtubules. In contrast, the effects of CB differ from cell to cell, they may be pleiotropic within a given cell type, and the mode of CB action is still ill-defined (Carter, 1972; Burnside and Manasek, 1972; Croop and Holtzer, 1975; Pollard and Weihing, 1974; Holtzer and Sanger, 1972). However, CB has been effective in distinguishing events of nuclear and cytoplasmic division (Carter, 1972). In these experiments, VB and CB have been utilized in an attempt to correlate KiMSV(KiMuLV) maturation with other events during the mitotic interval. At concentrations of CB between lo-25 pg/ml, inhibition of KiMSV(KiMuLV) release occurred although nuclear division continued. CB can selectively dissociate the events of nuclear division and virus maturation at the plasma membrane. Although KiMSV(KiMuLV) release was separable from nuclear division, CB action was restricted to the G2/M interphase during synchronous growth of NRK-K cells. The cell-cycle dependency of CB inhibition therefore confirms the temporal programming of KiMSV(KiMuLV) replication. Electron microscopic examination of CBinhibited cells did not show an accumulation of budding forms although cultures
PANEM
released from CB inhibition produce elevated amounts of KiMSV(KiMuLV) shortly after drug removal. These data suggest that virion components accumulate in CB-treated cells prior to visually detectable condensation of KiMSV(KiMuLV) at the plasma membrane. Furthermore, although virus release ordinarily occurs at the G2/M interphase, the rapid appearance of virus in culture fluids following CB removal from logarithmically growing cells indicates that the final events of virus replication can occur in Gl phase. We conclude that the event or events which confer cell-cycle dependency on KiMSV(KiMuLV) replication occur prior to virus maturation. CB inhibits the uptake of glucosamine as well as glycoprotein and mucopolysaccharide synthesis in some cultured cells (Sanger and Holtzer, 1972). Although CB inhibition of glycoprotein and mucopolysaccharide synthesis may depend upon the cell type studied (Spooner and Conrad, 1975), it is possible that CB inhibits KiMSV(KiMuLV) release by interfering with glycosylation of viral proteins and/or their insertion into the plasma membrane. We have recently found that the major virion glycoprotein, gp71, which appears as a cell-surface component, is differentially expressed during synchronous NRKK cell growth with maximal expression occurring in late G2 phase (Schauf and Panem, 1976). Experiments are therefore planned to examine the appearance of viral glycoproteins in the cytoplasm and at the cell surface of synchronized NRK-K’ cells undergoing CB inhibition. Cell-cycle dependence of type C virus replication may result from restriction of one or several events. At present, induction of endogenous type C virus by bromodeoxyuridine (Schwartz et al., 1975; Besmer et al., 1974)) type C virus maturation (Panem and Kirsten, 1973; Paskind et al., 1975; Fischinger et al., 1971), immune lysis of infected cells (Cikes and Friberg, 1971; Lerner et al., 1971), and expression of p30 and gp71 (Schauf and Panem, 1976) have been reported as cell-cycle dependent in murine cells. Further experimentation is needed to determine whether other aspects
CB INHIBITION
of viral expression are cell-cycle restricted and the means by which temporal controls are exerted. ACKNOWLEDGMENTS We thank W. H. Kirsten for generous support and helpful criticism, and P. G. Spear for a critical reading of this manuscript. This work was supported by USPHS Grants No. CA 14898 and No. CA 14599 from the National Cancer Institute, and a special fellowship from the Leukemia Society of America. REFERENCES
Spring
Harbor
Symp.
Quant.
Biol.
39, 1103-
1107. BURNSIDE, B., and MANASEK, F. J. (1972). Cytochalasin B: Problems in interpreting its effect on cells. Develop.
Biol.
27, 443-444.
CARTER, S. (19721. Effects of cytochalasins on mammalian cells. Nature (London) 213, 261-264. CIKES, M., and FRIBERG, S. (1971). Expression of H-2 and Moloney leukemia virus-determined cell-surface antigens in synchronized cultures of a mouse cell line. Proc. Nut. Acad. Sci. USA 68,566-569. CROOP,J., and HOLTZER, H. (1975). Response of myogenic and tibrogenic cells to cytochalasin B and to colcemid. I. Light microscopic observations. J. Cell Biol.
65, 271-285. FISCHINGER, P. J., TUTTLE-FULLER,
N., HUPER, G., and BOLOGNESI, D. P. (1975). Mitosis is required for production of murine leukemia virus and structural proteins during de novo infection. J. Viral. 16, 267-274. HOLTZER, H., and SANGER, J. (1972). Cytochalasin B: Microfilaments, cell movement and what else? Develop.
Bill.
viral antigen, accessibility to antibody, and complement activation. Proc. Nat.Acad. Sci. USA 68, 2584-2588.
MCCLAIN, K., and KIRSTEN, W. H. (19741. Mouse leukemia virus growth in mouse cells contaminated with mycoplasma. Cancer Res. 34, 281-285. OLMSTED, J. B., and BORISY, G. G. (1973). Microtubules. Ann. Rev. Biochem. 42, 507-539. PANEM, S., and KIRSTEN, W. H. (19731. Release of mouse sarcoma-leukemia virus from synchronized cells. J. Nut. Cancer Inst. 50, 563-565. PANEM, S., and KIRSTJZN, W. H. (1976). Effects of metabolic inhibitors on oncornavirus production during synchronized cell growth. Bibl. Haemat. 43, 472-474.
BESMER, P., SMOTKIN, D., HAL~ELTINE, W., FAN, H., WILSON, A. T., PASKIND, M., WEINBERG, R., and BALTIMORE, D. (1974). Mechanism of induction of RNA tumor viruses by halogenated pyrimidines. Cold
151
OF KiMSV(KiMuLV)
27, 444-446.
KELLOFF, G. J., HATANAICE, M., and GILDEN, R. V. (1972). Assay of C-type infectivity by measurement of RNA-dependent DNA polymerase activity. Virology 48, 266-269. LERNER, R. A., OLDSTONE, M. B., and COOPER,N. R. (1971). Cell cycle dependent immune lysis of Moloney virus transformed lymphocytes: Presence of
PANEM, S., and SCHAUF, V. (1974). Cell-cycle dependent appearance of murine leukemia-sarcoma virus antigen. J. Viral. 13, 1169-1175. PASKIND, M. P., WEINBERG, R. A., and BALTIMORE, D. (1975). Dependence of Moloney murine leukemia virus production on cell growth. Virology 67. 242-248.
POLLARD, T., and WEIHING, R. (1974). Actin and myosin and cell movement. Crit. Rev. Biochem. 2, l-65. PROCHOWNIK, E. V., PANEM, S., ~~~KIRSTEN, W. H. (1975). Biological and physical modification of a murine oncornavirus by 2-deoxy-n-glucose. J. Virol. 15. 1323-1331. SANGER, J. W., and HOLTZER, H. (1972). Cytochalasin B: Effects on cell morphology, cell adhesion and mucopolysaccharide synthesis. Proc. Nat. Acad.
Sci.
USA
69, 253-257.
SCHAUF, V., and PANEM, S. (1976). Expression of murine oncornavirus proteins in synchronized cells. Virology 71, 619-623. SCHWARTZ, S. A., PANEM, S., STEFANSKI, E., and KIRSTEN, W. H. (1974). Endogenous type C particles from rat embryo cells treated with 5-bromodeoxyuridine. Cancer Res. 34, 2255-2259. SCHWARTZ, S. A., PANEM, S., and KIRSTEN, W. H. (1975). Distribution and virogenic effect of 5-bromodeoxyuridine in synchronized rat embryo cells. Proc. Nat. Sci. USA 72, 1829-1833. SPOONER,B. S., and CONRAD, G. W. (1975). The role of extracellular materials in cell movement. I. Inhibition of mucopolysaccharide synthesis does not stop ruffling membrane activity or cell movement J. Cell Biol. 65, 286-297.
76, 146-151
(1977)
Cell Cycle-Dependent
Inhibition of Kirsten Murine Sarcoma-Leukemia Virus Release by Cytochalasin B SANDRA
Department
PANEM
Pathology, University of Chicago, Chicago, Illinois
of
60637
Accepted August 11,1976 Inhibition of Kirsten murine sarcoma-leukemia virus [KiMSV(KiMuLV)l release from chronically infected normal rat kidney cells (NRK-K) by cytochalasin B (CB) was dose dependent. Concentrations of CB between lo-25 pg/ml inhibited virus release without inhibiting mitosis and cell division. Maximal inhibition occurred within 4.5 hr of adding CB to logarithmically growing NRK-K cells, and inhibition was reversed within 3 hr of drug removal. CB inhibition of virus release from synchronously growing NRK-K cells was cell-cycle dependent and occurred during late G2 phase and mitosis. INTRODUCTION
cell line referred to as NRK-K cells (Panem and Schauf, 1974). A single cell clone, N, of NRK-K cells was used for these experiments between the sixth and seventeenth in vitro subculture generations. NRK-K cells were grown in 250 ml Falcon tissue culture flasks (Falcon, Oxnard, Calif.), 32-0~ glass pharmacy bottles, or glass roller bottles (Bellco Glass, Vineland, N.J.) at 37” in an atmosphere of 5% CO,. Growth medium consisted of Eagle’s minimal essential medium (MEM) supplemented with 10% fetal calf serum, 100 units per ml of penicillin and 100 pg/ml of streptomycin (Gibco, Grand Island, N.Y.). Cells were fed every 2 days, and at weekly intervals cells were subcultured with trypsin-EDTA and monitored for mycoplasma contamination (McClain and Kirsten, 1974). Only mycoplasma-free cells were used for these experiments. Cell synchrony. Logarithmically growing cells were synchronized by a doublethymidine block as described (Panem and K&ten, 1973). Cell division was determined by serial counting of cell number using trypan blue dye exclusion as a measure of cell viability. Briefly, cell division was inhibited by incubating cells in thymidine-rich medium (2 m&0 for 16 hr. Cell division then proceeded for 8 hr in medium containing 10 fl deoxyguanosine, deoxy-
The coordination of murine oncornavirus replication with the host cell cycle has been reported (Fischinger et al., 1975; Panem and Kirsten, 1973; Paskind et al., 1975). Although there are discrepancies concerning which events of viral replication are cell-cycle restricted, the release of virus concomitant with mitosis from murine cells synchronized by different methods has been observed under conditions of chronic and de nouo infection (Fischinger et al., 1975; Lerner et al., 1971; Panem and Kirsten, 1973; Paskind et al., 1975). As microtubule and microfilament action is prominent during the mitotic interval, the cell-cycle dependence of virus maturation was studied by examining the effects of two inhibitors of contractile element function on the release of KiMSV(KiMuLV) from chronically infected cells. The results show that inhibition of virus release can be separated from inhibition of mitosis and cell division by cytochalasin B. Cytochalasin B reversibly inhibits a cell-cycle dependent step of virus maturation. MATERIALS
AND
METHODS
Cells and virus. Infection of normal rat kidney cells with Kirsten murine sarcomaleukemia virus [KiMSV(KiMuLV)l resulted in a transformed, virus-producing 146 Copyright All rights
0 1977 by Academic Press, Inc. of reproduction in any form reserved.
CB
INHIBITION
OF
adenosine, and deoxycytosine. Cell division was again blocked for 15 hr with thymidine-rich medium. The second time of thymidine removal was defined at 0-hr postrelease (p.r.). NRK-K cells show celfcycle times of S + G2/M/Gl = 8.5 hr/O.51.5 hr/2.5 hr = 12.5 hr, as previously determined (Panem and Schauf, 1974). Drugs,
isotopes,
and
reagents.
Cyto-
chalasin B (Calbiochem, San Diego, Calif.) (CB) was purchased as a lyophilized powder. CB was reconstituted in 100% ethanol at 5 mg/ml and stored at -20”. Vinblastine sulfate (Velban, Eli Lilly, Indianapolis, Ind.) (VB) was purchased in lyophilized form, reconstituted in growth medium, and used immediately. [3H]Thymidine 5’triphosphate (sp act 55 Ci/mmol) was purchased from New England Nuclear, Boston, Mass. TNE buffer consisted of 0.1 M NaCl, 10 mM Tris-hydrochloride (pH 7.3), and 1 mM EDTA. Phosphate-buffer saline (PBS) consisted of 0.137 M NaCl, 2.7 mM KCl, 8.1 mM Na,HPO,, 1.5 mM KH2P04, and 1% phenol red. RNA-dependent (RDDP). Spent
DNA-polymerase
145
KiMSV(KiMuLV)
action-mixture products were precipitated by the addition of 25 volumes of ice-cold 5% trichloroacetic acid-2% Na,P,O, (TCAPPi). The precipitates were collected by centrifugation at 2000 g for 5 min and washed 3 times by centrifugation with 5 ml of TCA-PPi. The final precipitate was washed once with 95% ethanol, drained, dissolved in NCS (Amersham-Searle, Arlington Heights, Ill.) and 10 ml of toluenebased liquid scintillation fluid. Radioactivity was determined in a Packard Tri-Carb liquid scintillation counter. Electron microscopy. Cells were prepared for electron microscopy by fixation for at least 1 hr with 25% glutaraldehydc in 0.1 M Na cacodylate, pH 7.3, at room temperature. Samples were then postfixed for 45 min with 1% osmium tetroxide in collidine buffer. Following dehydration with ethanol, and embedding in Epon, thin sections were stained with uranyl acetate and lead citrate and were viewed in an RCA-EMU 4 electron microscope as described (Schwartz et al., 1974).
assay
culture fluids were assayed for RDDP after clarification by centrifugation at 10,000 g for 20 min as described (Prochownik et al., 1975). Clarilied culture fluids were concentrated by ultracentrifugation through a 3.0-cm cushion of 5% (w/v) sucrose in TNE buffer at 100,000 g for 90 min. The pellet was resuspended in 50 ~1 of TMG buffer [lo mM Tris-hydrochloride (pH 7.81, 7 mM 2-mercaptoethanol, and 10% glycerol] and stored at -80”. Enzyme activity was measured with the synthetic template poly(rA) . oligo(dT),,,-,,, (Collaborative Research, Waltham, Mass.). Reaction mixtures (0.2 ml) consisted of 0.025% Nonidet-P40, 25 mM Tris-hydrochloride (pH 8.11, 7 mM 2-mercaptoethanol, 0.2 mM MnCl,, 2 pg of template, 60 n&f KCl, 0.0083 mM thymidine 5’-triphosphate (Sigma, St. Louis, MO.), 5 @!i of [“Hlthymidine 5’-triphosphate, and 10 ~1 of resuspended supernatant pellet in TMG buffer. Reactions were carried out for 40 min in a 37” water bath. The mixture were chilled quickly to O”, and 250 pg of bovine serum albumin was added as carrier. Re-
RESULTS
Inhibition cell diuision.
of virus
release,
mitosis
and
Cytochalasin B (CB) and vinblastine sulfate (VB) were tested for inhibitory activity. Neither inhibitor interfered with the incorporation of [3H]TMP by viral reverse transcriptase (data not shown). Inhibition of virus release was measured by the decrease of reverse transcriptase activity in spent culture fluids of drug-treated NRK-K cells (Kelloff et al., 1972). CB and VB inhibited KiMSV(KiMuLV) release from logarithmically growing NRK-K cells in a dose-dependent manner (Fig. 1). Inhibition by VI3 was always accompanied by inhibition of mitosis and cell division. In contrast, with 25 pg/ml of CB, mitosis was not inhibited whereas virus release was inhibited by at least 70%. As judged by the doubling in viable cell number using trypan blue exclusion to assess viability, 25 pg/ml of CB did not inhibit division of NRK-K cells during the time interval of these experiments. Although cell division was not impaired by CB, treated cells showed rounding. Rapid
and reversible
inhibition
by CB.
148
SANDRA
FIG. 1. Inhibition of virus release and cell division by cytochalasin B (CB) and vinblastine sulfate (VB). Replicate cultures of exponentially growing NRK-K cells were incubated in duplicate overnight with medium containing inhibitors. The next morning, the medium was collected, clarified, and assayed for RDDP. The cells were removed using trypsin-EDTA, and the number of viable cells was determined by the trypan blue exclusion test. Data are presented as the percentage of inhibition of RDDP and the percentage of inhibition of cell division relative to the untreated controls. At 100 pg/ml of CB and VB, cytotoxicity was detected.
CB (20 pglml) suppressed KiMSV(KiMuLV) release by 30% within 1.5 hr and by greater than 80% within 4.5 hr of its addition to logarithmically growing NRK-K cells (data not shown). Control cultures treated with growth medium containing 0.2% ethanol showed no inhibition of virus release. The reversibility of CB inhibition was tested. Exponentially growing NRK-K cells were incubated with growth medium for 1.5 hr. This medium was collected and cells were then incubated with CB (20 pg/ml) for 27 hr during which several 1.5hr medium collections were made. The 1.5~hr collections were processed in parallel for reverse transcriptase activity and the data were expressed relative to enzyme activity at the start of the experiment. Rapid CB inhibition of supernatant reverse transcriptase was observed within 3 hr. In addition, within 3 hr of the drug’s removal a peak of virus release was seen (Fig. 2). Although the population doubled in the course of the experiment, the burst of supernatant activity following CB removal was greater than could be explained by an increase in cell number and was consistent with re-
PANEM
versible inhibition of a late event of virus replication by CB. This suggested that budding viral forms accumulated in CBtreated cells which were completed on removal of the drug. In this, but not all experiments, partial recovery from CB inhibition was observed between 3-4.5 hr. When partial recovery occurred, the level of virus released in 1.5-hr intervals was constant for the duration of drug treatment (4.5-23 hr). As maximum inhibition attained with logarithmically growing cells did not exceed 85%, and as the cell number doubled during CB treatment, the low level of virus released between 4.5 and 23 hr probably reflects a small proportion of cells escaping CB inhibition, possibly due to differential drug uptake. To test whether CB inhibition occurred following formation of viral buds, NRK-K cells were examined by electron microscopy following CB removal. Replicate flasks of logarithmically growing NRK-K
0
3.0
22 25 TIME (hrs)
28
FIG. 2. Reversibility of CB inhibition. Logarithmically growing NRK-K cells were fed fresh growth medium, which was collected 1.5 hr later. Medium containing 20 pg/ml of CB was added, replaced at 3 and 4.5 hr, and cells were incubated overnight. Fresh drug-containing medium was added in the morning and collected 1.5 hr later. Cells were washed and refed fresh growth medium without drug at 1.5hr intervals for the next 6 hr. All 1.5-hr collections were processed in duplicate for RDDP activity. Data are presented as the percentage of RDDP relative to the activity in the collection made at the beginning of the experiment. The hatchmarks indicate the period during which CB was present.
CB INHIBITION
OF
cells were treated with 20 pg/ml of CB for 4 hr. Cells were washed twice and fed fresh growth medium. Samples were processed for microscopy at 0, 1.5, and 3 hr following drug removal, and the number of budding type C particles on 50-100 individual cells were determined for the three samples as well as an untreated control (Table 1). Although such experiments are limited by considerations of sample size, entrapment, and/or loss of extracellular particles during specimen preparation relative to control cultures, cells processed immediately after CB removal showed fewer budding and extracellular particles than the sample processed at 1.5 hr. After 3 hr, increased numbers of both viral forms were found. These data suggested that CB inhibition occurred prior to visually detectable condensation of maturing type C virions at the plasma membrane. Cell cycle-dependent inhibition of virus release. The release of KiMSV(KiMuLV)
from NRK-K cells synchronized by doublethymidine blockade occurs concomitant with mitosis after thymidine removal (Panem and Kirsten, 1973). The time of CB inhibition during virus replication was studied by incubating replicate cultures of NRK-K cells with CB (10 pg/ml) for decreasing times during synchronized
149
KiMSV(KiMuLV1
growth. In pilot experiments, this dose of CB resulted in 50-75% inhibition of virus release without cytotoxicity in synchronized cells. The growth medium with or without CB was changed in late G2 phase and collected 2 hr later at the end of mitosis. The 2-hr medium collections were analyzed for reverse transcriptase as a measure of virus release (Table 2). CB inhibited virus release by nearly 70% in cultures treated just prior to mitosis, regardless of the duration of drug treatment during S + G2 phases. However, cultures treated for the latter portion of the mitotic interval (lo-11 hr) showed less inhibition of virus release. The effectiveness of CB inhibition of virus release therefore coincided with its presence during the final events of virus maturation at the G2/M interphase. Decreased inhibition of virus release in cultures treated for a portion of the mitotic interval does not result from a shorter exposure of cells to the inhibitor, as judged from the level of inhibition achieved in TABLE CELL Time of CB treatment (hr p.r.1
2
CYCLE-DEPENDENT CYTOCHALASIN
INHIBITION B”
RDDP
cpm
T”H1TTP
BY
activity
% Control x
% Inhibition
iO-3
TABLE 1 APPEARANCE OF VIRAL-BUDDING CYTOCHALASIN B-TREATED Hours following CB removal”
Untreated
control
IN
Virionsb Budding
0.0 1.5 3.0
0
FORMS CELLS
24 88 36 60
Extracellular
43 108 110 140
Extracellular/ budding ratio 1.8 1.2 3.0 2.3
o Replicate cultures of logarithmically growing NRK-K cells were treated for 4 hr with 20 pg/ml of CB, washed, and incubated with fresh growth medium. After 0, 1.5 and 3.0 hr, cells were prepared for electron microscopy. * Fifty to one hundred cell sections were examined for budding and extracellular virions. Data are presented as the number of virions per 100 cell sections and as the ratio of extracellular to budding virions.
10-11 9-10 9-11 8-11 7-11 3-11
42.5 34.7 15.7 13.0 13.2 13.3 8.9
100.0 81.7 37.0 30.4 31.0 31.1 21.0
0.0 18.3 63.0 69.6 69.0 68.9 79.0
-
a Replicate cultures of logarithmically growing NRK-K cells were synchronized by double-thymidine blockade. At various times after release from thymidine blockade (postrelease: p.r.), cultures were treated with 10 pg/ml of CB. At 9 hr p.r., all cultures were fed fresh growth medium with or without CB as indicated. This medium was collected at 11 hr p.r. and assayed in duplicate for RDDP. Data are presented as the counts per minute of [3H]TTP incorporated x 10m3 in duplicate assays, as the percentage of RDDP relative to the untreated control, and as the percentage of inhibition of RDDP activity. Synchrony was monitored by examining samples from parallel cultures at hourly intervals for the percentage of mitotic cells. Between 9 and 11 hr p.r., 27.8% of the cells passed through mitosis.
150
SANDRA
cells receiving CB from 9-10 hr (Table 2). In the representative experiment presented in Table 2,27.8% of the cells passed through mitosis from 9-11 hr p.r. Similar results were obtained when replicate cultures were pulsed with CB for l- or 2-hr intervals during synchronous growth of NRK-K cells (Table 2; Panem and Kirsten, 1976). However, the current experimental design was judged superior to that of pulsing as the effect of CB was not instantaneously reversible as required in pulse experiments. DISCUSSION
Microtubules and microfilaments play prominent roles in nuclear and cellular division during the mitotic interval. VB poisons the mitotic spindle apparatus by binding to microtubular protein and disaggregating microtubules (Olmsted and Borisy, 1973). Inhibition of cell division by VB is therefore a consequence of its primary effect on microtubules. In contrast, the effects of CB differ from cell to cell, they may be pleiotropic within a given cell type, and the mode of CB action is still ill-defined (Carter, 1972; Burnside and Manasek, 1972; Croop and Holtzer, 1975; Pollard and Weihing, 1974; Holtzer and Sanger, 1972). However, CB has been effective in distinguishing events of nuclear and cytoplasmic division (Carter, 1972). In these experiments, VB and CB have been utilized in an attempt to correlate KiMSV(KiMuLV) maturation with other events during the mitotic interval. At concentrations of CB between lo-25 pg/ml, inhibition of KiMSV(KiMuLV) release occurred although nuclear division continued. CB can selectively dissociate the events of nuclear division and virus maturation at the plasma membrane. Although KiMSV(KiMuLV) release was separable from nuclear division, CB action was restricted to the G2/M interphase during synchronous growth of NRK-K cells. The cell-cycle dependency of CB inhibition therefore confirms the temporal programming of KiMSV(KiMuLV) replication. Electron microscopic examination of CBinhibited cells did not show an accumulation of budding forms although cultures
PANEM
released from CB inhibition produce elevated amounts of KiMSV(KiMuLV) shortly after drug removal. These data suggest that virion components accumulate in CB-treated cells prior to visually detectable condensation of KiMSV(KiMuLV) at the plasma membrane. Furthermore, although virus release ordinarily occurs at the G2/M interphase, the rapid appearance of virus in culture fluids following CB removal from logarithmically growing cells indicates that the final events of virus replication can occur in Gl phase. We conclude that the event or events which confer cell-cycle dependency on KiMSV(KiMuLV) replication occur prior to virus maturation. CB inhibits the uptake of glucosamine as well as glycoprotein and mucopolysaccharide synthesis in some cultured cells (Sanger and Holtzer, 1972). Although CB inhibition of glycoprotein and mucopolysaccharide synthesis may depend upon the cell type studied (Spooner and Conrad, 1975), it is possible that CB inhibits KiMSV(KiMuLV) release by interfering with glycosylation of viral proteins and/or their insertion into the plasma membrane. We have recently found that the major virion glycoprotein, gp71, which appears as a cell-surface component, is differentially expressed during synchronous NRKK cell growth with maximal expression occurring in late G2 phase (Schauf and Panem, 1976). Experiments are therefore planned to examine the appearance of viral glycoproteins in the cytoplasm and at the cell surface of synchronized NRK-K’ cells undergoing CB inhibition. Cell-cycle dependence of type C virus replication may result from restriction of one or several events. At present, induction of endogenous type C virus by bromodeoxyuridine (Schwartz et al., 1975; Besmer et al., 1974)) type C virus maturation (Panem and Kirsten, 1973; Paskind et al., 1975; Fischinger et al., 1971), immune lysis of infected cells (Cikes and Friberg, 1971; Lerner et al., 1971), and expression of p30 and gp71 (Schauf and Panem, 1976) have been reported as cell-cycle dependent in murine cells. Further experimentation is needed to determine whether other aspects
CB INHIBITION
of viral expression are cell-cycle restricted and the means by which temporal controls are exerted. ACKNOWLEDGMENTS We thank W. H. Kirsten for generous support and helpful criticism, and P. G. Spear for a critical reading of this manuscript. This work was supported by USPHS Grants No. CA 14898 and No. CA 14599 from the National Cancer Institute, and a special fellowship from the Leukemia Society of America. REFERENCES
Spring
Harbor
Symp.
Quant.
Biol.
39, 1103-
1107. BURNSIDE, B., and MANASEK, F. J. (1972). Cytochalasin B: Problems in interpreting its effect on cells. Develop.
Biol.
27, 443-444.
CARTER, S. (19721. Effects of cytochalasins on mammalian cells. Nature (London) 213, 261-264. CIKES, M., and FRIBERG, S. (1971). Expression of H-2 and Moloney leukemia virus-determined cell-surface antigens in synchronized cultures of a mouse cell line. Proc. Nut. Acad. Sci. USA 68,566-569. CROOP,J., and HOLTZER, H. (1975). Response of myogenic and tibrogenic cells to cytochalasin B and to colcemid. I. Light microscopic observations. J. Cell Biol.
65, 271-285. FISCHINGER, P. J., TUTTLE-FULLER,
N., HUPER, G., and BOLOGNESI, D. P. (1975). Mitosis is required for production of murine leukemia virus and structural proteins during de novo infection. J. Viral. 16, 267-274. HOLTZER, H., and SANGER, J. (1972). Cytochalasin B: Microfilaments, cell movement and what else? Develop.
Bill.
viral antigen, accessibility to antibody, and complement activation. Proc. Nat.Acad. Sci. USA 68, 2584-2588.
MCCLAIN, K., and KIRSTEN, W. H. (19741. Mouse leukemia virus growth in mouse cells contaminated with mycoplasma. Cancer Res. 34, 281-285. OLMSTED, J. B., and BORISY, G. G. (1973). Microtubules. Ann. Rev. Biochem. 42, 507-539. PANEM, S., and KIRSTEN, W. H. (19731. Release of mouse sarcoma-leukemia virus from synchronized cells. J. Nut. Cancer Inst. 50, 563-565. PANEM, S., and KIRSTJZN, W. H. (1976). Effects of metabolic inhibitors on oncornavirus production during synchronized cell growth. Bibl. Haemat. 43, 472-474.
BESMER, P., SMOTKIN, D., HAL~ELTINE, W., FAN, H., WILSON, A. T., PASKIND, M., WEINBERG, R., and BALTIMORE, D. (1974). Mechanism of induction of RNA tumor viruses by halogenated pyrimidines. Cold
151
OF KiMSV(KiMuLV)
27, 444-446.
KELLOFF, G. J., HATANAICE, M., and GILDEN, R. V. (1972). Assay of C-type infectivity by measurement of RNA-dependent DNA polymerase activity. Virology 48, 266-269. LERNER, R. A., OLDSTONE, M. B., and COOPER,N. R. (1971). Cell cycle dependent immune lysis of Moloney virus transformed lymphocytes: Presence of
PANEM, S., and SCHAUF, V. (1974). Cell-cycle dependent appearance of murine leukemia-sarcoma virus antigen. J. Viral. 13, 1169-1175. PASKIND, M. P., WEINBERG, R. A., and BALTIMORE, D. (1975). Dependence of Moloney murine leukemia virus production on cell growth. Virology 67. 242-248.
POLLARD, T., and WEIHING, R. (1974). Actin and myosin and cell movement. Crit. Rev. Biochem. 2, l-65. PROCHOWNIK, E. V., PANEM, S., ~~~KIRSTEN, W. H. (1975). Biological and physical modification of a murine oncornavirus by 2-deoxy-n-glucose. J. Virol. 15. 1323-1331. SANGER, J. W., and HOLTZER, H. (1972). Cytochalasin B: Effects on cell morphology, cell adhesion and mucopolysaccharide synthesis. Proc. Nat. Acad.
Sci.
USA
69, 253-257.
SCHAUF, V., and PANEM, S. (1976). Expression of murine oncornavirus proteins in synchronized cells. Virology 71, 619-623. SCHWARTZ, S. A., PANEM, S., STEFANSKI, E., and KIRSTEN, W. H. (1974). Endogenous type C particles from rat embryo cells treated with 5-bromodeoxyuridine. Cancer Res. 34, 2255-2259. SCHWARTZ, S. A., PANEM, S., and KIRSTEN, W. H. (1975). Distribution and virogenic effect of 5-bromodeoxyuridine in synchronized rat embryo cells. Proc. Nat. Sci. USA 72, 1829-1833. SPOONER,B. S., and CONRAD, G. W. (1975). The role of extracellular materials in cell movement. I. Inhibition of mucopolysaccharide synthesis does not stop ruffling membrane activity or cell movement J. Cell Biol. 65, 286-297.
