Japan. J. Microbiol. Vol. 20 (6), 499-505, 1976
Purification
of Host DNA Synthesis-Suppressing
(DSF) Produced Tomoko
by Infection
K. YAMAMOTO', Tomonori
with Measles
MINAGAWA, and
Hiroo
Factor Virus IIDA
Departmentof Microbiology,Hokkaido UniversitySchoolof Medicine, Sapporo (Received for publication, May 6, 1976)
ABSTRACT
Host DNA synthesis-suppressing factor (DSF) produced into culture fluid of cloned HeLa cells (HeLa C-9) infected with a small plaque variant of Toyoshima strain of measles virus was purified by precipitation with ammonium sulfate, chromatography on CM-cellulose and DEAE-cellulose, and gel-filtration on Sephadex G-100 and G-200. The specific activity of the finally purified DSF was 302 units/mg of protein representing approximately 300-fold purification. The molecular weight of DSF was estimated to be about 55 000. By isoelectric focusing, two kinds of DSF having isoelectric points of 4.24 and 5.24 were detectable. The purified DSF was able to suppress host DNA synthesis of HeLa cells, continuous human lymphoid cells (NC-37), mouse L cells and Meth-A cells derived from an ascitic tumor of the mouse. The activity of the purified DSF was inactivated by heating at 56 C for 30 min or by treatment with trypsin.
It has been recognized that cellular DNA synthesis is inhibited by infection of several RNA viruses before the onset of morphological cellular damage. In many cases, the inhibition of cellular DNA synthesis follows inhibition of protein synthesis [2, 5, 7, 14]. On the other hand, reovirus type 3 [4, 6], measles virus [12] and pancreatic necrosis virus [9] inhibit cellular DNA synthesis without detectable effects upon RNA and protein syntheses. In previous reports [11, 12] , we demonstrated that the inhibition of host DNA synthesis induced by infection of measles virus was mediated by a DNA. synthesis-suppressing factor (DSF), a nonvirion component, produced into culture fluid after infection. The biological activity of DSF appeared to be associated with protein since the activity was destroyed by treatment with trypsin and pronase but not with RNase, DNase, phospholipase C or D, and the production of DSF was blocked by Requests for reprints should be addressed to Dr. Tomonori Minagawa, Department of Microbiology, Hokkaido University School of Medicine, Nishi 7chome, Kita 15-jo, Kita-ku, Sapporo, 060 Japan. 'Present address: Faculty of Pharmaceutical Science, Chiba University, Chiba. 499
cycloheximide. The production of DSF was closely correlated with the replication of measles virus in HeLa cells. Ultravioletinactivated and antiserum-neutralized measles virus failed to induce DSF production. In addition, DSF was not produced by poliovirus type 1 or vesicular stomatitis virus which showed severe cytopathic effects. On the other hand, certain cellular factors were involved in the production of DSF since some cell lines such as Vero cells and CCRF-CEM cells and some clones obtained from HeLa cells produced a markedly lower amount of DSF despite the fact that growth of measles virus was similar (in preparation). The role of cellular function in the production of DSF remained to be elucidated. The results so far obtained would imply that the production of DSF may be dependent on both replication of measles virus and cellular functions but not on any fortuitous agents such as mycoplasma or other viruses. The present report describes the purification of DSF produced in HeLa C-9 cells infected with a small plaque variant of Toyoshima strain and some properties of the purified DSF.
T.K.
500
MATERIALS
AND
YAMAMOTO,
T. MINAGAWA
METHODS
Cell cultures. Cloned HeLa cells (HeLa C-9) and mouse L cells (L-MS) were cultivated in Eagle's minimum essential medium (MEM, Nissui Seiyaku Co., Tokyo) supplemented with 5% calf serum. Suspension cultures of continuous human lymphoid cell lines (NC-37, CCRF-CEM and MOLT-4) and mouse Meth-A cells were cultivated -in RPMI 1640 medium (Nissui Seiyaku Co.) supplemented with 10% fetal calf serum. Contamination by mycoplasma was checked by inoculating culture fluids of these cells into Bacto-PPLO agar (Difco, U.S.A.). Virus. A small plaque variant of Toyoshima strain of measles virus (TSP) which had been purified by plaque selection on Vero cells was grown in HeLa cell cultures. After the infected cultures were frozen and thawed and centrifuged at 5000 rpm for 20 min, the supernatant was stored at 80 C. Preparationof crudeDSF. HeLa C-9 monolayer cultures in Roux bottles (10 x 15 cm)
Fig.1.
Production
cuhurefluidof small
of DSF HeLa
plaque
Toyoshima
cultu.res of HeLa
Thereafter, vested. after
infection
iRoculated
at an input multiplicity
activities with
(○ − 〇)
(● 一 ●)and the infectivities of progeny TSP (○ … ○)and TLP(● … ●)were according [11].
to the mcthods
with of O.1.
fluid was har-
in the harvested TSP
a
strain. Monolayer
every day the culture
DSF
with
a large plaque
C-9 cells were
or TLP
were inoculated with TSP at a multiplicity of about 0.1 and maintained with MEM containing 2°/ calf serum. One day after infection, the culture fluid was replaced with serum-free MEM, and the cultures were harvested after further incubation for 2 days. These cultures were stored at 20 C prior to processing for purification of DSF. The growth of measles virus and production of DSF in HeLa C-9 cells are shown in Figure 1. Measurement of DSF activity. Routinely, the DSF activity was assayed in continuous human lymphoid cells (NC-37). One milliliter of NC-37 cells (2 x 106 cells) in a polypropylene tube (1.2 x 7.5 cm, Falcon 2026) was inoculated with 1 ml of a DSF sample serially diluted with maintenance medium (RPMI 1640 medium containing 5% fetal calf serum) and incubated at 37 C for 16 hr. After that, the culture was labeled with 3H-thymidine (2 15 Ci per mmole) at 37 C for 30 min. At the end of the labeling period, the culture was cooled in ice, centrifuged at 3000 rpm for 10 min, and then the cell pellet was resuspended in 0.01 M phosphate-buffered saline (PBS). The resuspended sample was precipitated with 5% trichloroacetic acid (TCA) and then incorporation of 3H-thymidine into the TCA-insoluble materials was determined as previously described [12]. The ratio of suppression of host DNA synthesis was calculated as follows: [counts per minute
virus into
C-9 cclls infected
variant(TSP)and
variant(TLP)of
either TSP
and progeny
AND H. IIDA
described
samples and
TLP
virus of assayed prcviously
Fig. 2. Dose response of the purified DSF in terms of suppression of host DNA synthesis of NC-37 cells. One milliliter of an NC-37 cell suspension (2 x 106 cells) was mixed with an equal volume of serially diluted DSF and incubated for 16 hr, and then labeled with 3H-thymidine (2 pCi/ml).
PURIFICATION
Table
These
values
were
calculated
1.
from
Summary
two
of the
experiments
(cpm) of control minus cpm of experiment] / [cpm of control] x 100 (%). The dilution of DSF sample that caused 50% suppression of the DNA synthesis was defined as the DSF titer (unit/ml). An example of the relationship between the suppression of host DNA synthesis and log DSF dose is shown in Figure 2. Protein determination. Protein concentration was determined either by the method of Lowry et al [10] or absorbance measurement at 280 nm with bovine serum albumin as the standard. Isoelectricfocusing. An LKB 8101, 110 ml ampholine electrofocusing column was filled with 20 fractions of 5 ml of 1.9% carrier ampholites covering a pH range from 3 to 10 in sucrose solution of densities varying from 40% to 0%. Five-tenths milliliters of a DSF sample was incorporated into one fraction in the middle portion of the column. A current of 500 V was maintained at 4 C for 64 hr. On termination of the run, fractionation was carried out and each fraction was dialyzed twice against 0.01 M phosphate buffer (pH 7.4), and then DSF activity in each fraction was determined. Reagents. Bovine serum albumin, cytochrome c, RNase, DNase, soybean trypsin inhibitor (Sigma Chemical Co.), crystalline trypsin (Difco Laboratories) and phospholipase C (Worthington Biochemical Co.) were employed in these experiments. RESULTS Purificationof DSF Purification of DSF was carried out by ammonium sulfate precipitation, CMcellulose chromatography, DEAE-cellulose
OF
501
DSF
purification
which
of DSF
used
different
starting
materials.
chromatography and Sephadex G-100 and G-200 gel-filtration. The total quantity, the specific activity per mg of protein, and the percentage of recovery of DSF at each stage of the purification procedures are summarized in Table 1. All the procedures were performed at 4 C. Stage 1: Concentrationwith ammoniumsulfate. About 9 liters of culture fluid were centrifuged at 10 000 rpm for 20 min. The supernatant, the starting material, containing 1.03 mg/ml of protein was fractionated at 4 C with solid ammonium sulfate. The precipitate formed at 0 to 33.3% saturation of ammonium sulfate was collected by centrifugation at 10 000 rpm for 60 min, dissolved in 143 ml of 0.01 Mphosphate buffer (pH 5.5), and dialyzed at 4 C against the same buffer. The precipitate formed during dialysis was removed by centrifugation and the supernatant was again dialyzed against the same buffer, and the dialyzed sample was further centrifuged at 25 000 rpm for 3 hr in order to remove virus particles. The supernatant that contained DSF units of 44.4% and protein representing 4.5% of the starting material was used for the following stage of purification. Stage 2: Chromatographyon CM-cellulose. The DSF solution concentrated by precipitation with ammonium sulfate was divided into two parts and run slowly through two sets of CM-cellulose column (1.0 x 13.8 cm) equilibrated with 0.01 M phosphate buffer (pH 5.5). The columns were then washed with 30 to 50 ml of the same buffer. The activity of DSF was not detected in these fractions. After that, 0.01 M phosphate buffer (pH 8.0) was added for elution of
502
T. K. YAMAMOTO,
T. MINAGAWA
DSF. DSF activity was eluted in fractions of 3 to 5 at pH 6.26 to 7.95 as shown in Table 2. Fractions 3 to 5 were pooled and used for the next stage of purification. Stage 3: Chromatographyon DEAE-cellulose. The pooled fractions from CM-cellulose columns were dialyzed three times against 0.01 M Tris buffer (pH 7.5) and applied to a DEAE-cellulose column (1.5 x 20 cm) equilibrated with the same buffer. DSF Table 2. Elution the CM-cellulose
of DSF from column
A DSF sample obtained after precipitation with ammonium sulfate was divided into two parts (73 ml and 70 ml) and applied to two sets of CM-cellulose columns (a and b). Tenmilliliter fractions were collected and DSF activity in the fractions were assayed. Parentheses indicate the pH value.
Fig.3.
Chromatography
cellulose sample
obtained
was
of
column.
applied
Thirty from
to
a
chromatographed following phosphate buffer
(pH
0.01M
phosphate
0.5 and of
M
at
the
a
a rate
buffer
Five-milliliter at 280
fractions
were
fractions
assayed.
and
with
the
ml/hr.(A):0.01
(pH
nm(●)and
DSF
column
buffer(pH 6.0).(B):0.01M 6.0)containing O. I M
NaCL
a
column
elution
of 33
was chromatographed by stepwise elution. When 0.01 M phosphate buffer (pH 6.0) containing 0.1 M NaCl was added to the column, more than 90% of the total DSF activity bound to the column was eluted in fractions 20 to 24, and the peak of DSF activity corresponded to the major protein peak, as shown in Figure 3. Fractions 20 to 24 were pooled and used for the next stage of purification. Stage 4: Gel-filtration on Sephadex G-100. The pooled fractions from the DEAE-cellulose column were concentrated 10-fold by dialysis against 25% polyethylene glycol G-6000 and loaded onto the top of a Sephadex G-100 column (2.6 x 37.5 cm) equilibrated with 0.01 M phosphate buffer (pH 7.4), and the same buffer was added for elution of DSF. Five-milliliter fractions were collected at a flow rate of 6.6 ml/hr (Fig. 4). The main DSF peak from fractions 20 to 23 was pooled and concentrated for the next stage of purification. Stage 5: Gel-filtration on Sephadex G-200. The pooled and concentrated fractions from stage 4 were loaded onto a Sephadex G-200 column (2.6 x 38.5 cm) equilibrated with 0.01 M phosphate buffer (pH 7.4). Figure 5 shows the elution pattern obtained when DSF was eluted with the same buffer at a
DEAE-
of
CM.cellulose
stepwis
absorbance the
on
DEAE-cellulose by
buffers
DSF
millilitcrs
AND H. IIDA
phosphate NaCl.(C):
6.0) were DSF
containing collected titers(○)
M
Fig.4.
Ge1-filtration
column.
A
cellulose
column.
column
DSF
and
of
sample was
at
fractions
280 were
on
a Sephadex
obtained applied
from to
nm(●)and assayed.
7.4)at were
the
Sephadex
chromatographed
phosphate buffer(pH Five-milliliter fractions ance
DSF
with a rate collected
DSF
G-100 DEAEG-100 0.01
M
of 6.6 ml/hr. and absorb-
titers(○)of
the
PURIFICATION
Fig.5.
Gel-filtratiлn
column. dex to
A
G-100 a
DSF
of DSF
sample
column
Sephadex
O.Ol
at a rate
of 6.o
ml/hr.
collected DSF
and
Table
3.
of the
at
fractions
of enzymes purified
the
Sephaapplied
chrлmato-
buH℃r(pH
Five-milliliter
the
Effects
and
phosphate
G-200
and
cл1u.mn,
M
absorbance
titers(○)in
from
concentrated
G-200
with
a Sephadex
obtained
was
graphed
on
280 were
on
fractions
7.4) wcre
nm(●)and assayed。
the activity
OF
DSF
503
Fig. 6. Isoelectric focusing of DSF. A DSF sample containing 400 units obtained from the final step of purification was electrofocused. Detailed methods are described in the text. After the isoelectric focusing of DSF, 2-ml fractions were collected and eachfraction was dialyzed against 0.01Mphosphate buffer (pH 7.4), and then DSF activity (0) was determined. Table 4. Effects of the purified DNA synthesis of various cell
DSF on cultures
DSF
The finally purified DSF (302 units/mg of protein) was incubated with various enzymes at 37 C for 1 hr, and then assayed the remaining activity of DSF. Soybean trypsin inhibitor (10 mg/ml) was added after the treatment with trypsin.
flow rate of 6.0 ml/hr. The DSF peak from fractions 26 to 29 was collected. The final specific activity of the purified DSF obtained in this stage was 302 units/mg of protein, the value of the original material being increased 330-fold. Determinationof Molecular Weightof DSF The molecular weight of the purified DSF was determined by gel-filtration of Sephadex G-100 and G-200 using marker proteins of bovine serum albumin and cytochrome c according to the method of Andrews [1]. The results indicated that the
One milliliter of 1.0 unit or 10 units of the purified DSF (302 units/mg of protein), crude DSF and control medium was inoculated into various cell cultures (1 x 106 cells). After 16-hr treatment, these cells were labeled, fractionated and counted by the methods described in the text.
purified DSF had a molecular weight of approximately 55 000. Determination of Isoelectric Points of DSF Isoelectric focusing was performed on the DSF obtained from gel-filtration on Sephadex G-100 and approximately 40% of the input DSF activity was recovered. DSF activity was detectable in two peaks containing different percentage of the total activity with isoelectric points of 4.24 and 5.24 (Fig. 6).
504
T.K.
YAMAMOTO,
T. MINAGAWA
SomePropertiesof the PurifiedDSF The finally purified DSF was incubated with various enzymes at 37 C. Table 3 shows that DSF activity was destroyed by trypsin but not by DNase, RNase, or phospholipase C. These results agree with our previous report concerning the effect of these enzymes on a crude DSF preparation from infected Vero cells [12]. Furthermore, as is clear in Table 3, the purified DSF was as heatlabile as was the crude DSF (unpublished data) . The ultraviolet extinction spectrum of the finally purified DSF was typical for protein, having a maximum at 280 nm and a minimum at 252 nm. The ultraviolet absorption ratio of 280 nm to 260 nm was 1.65, hence the possibility of presence of nucleic acid in this material was very low. Furthermore, effects of the finally purified DSF on DNA. synthesis of cell cultures derived from different animal species were investigated. As indicated in Table 4, the purified DSF remarkably suppressed the cellular DNA synthesis of HeLa cells and NC-37 cells, but not that of CCRF-CEM cells. Similarly, the cellular DNA synthesis of mouse L cells and Meth-A cells derived from an ascitic tumor of a BALB/c mouse induced by 20methylcholanthrene was suppressed depending upon the dose of DSF added. These host range in terms of sensitivity to the effect of purified DSF is identical with that of crude DSF. Thus it is concluded that DSF purified under these conditions has the same properties as crude DSF. DISCUSSION Purification of DSF produced from HeLa C-9 cells infected with a small plaque variant of Toyoshima strain of measles virus has been carried out. The finally obtained DSF has a specific activity of 302 units/mg of protein and is practically purified from impurity protein. Under these conditions, DSF behaves as a single molecular species with a molecular weight of 55 000. However, the result obtained from the isoelectric focusing experiments shows that the purified DSF is heterogeneous in isoelectric points (Fig. 6). At present, it is not clear whether or not the heterogeneity of the purified DSF is due to different molecular species. In a previous paper [11], it was reported
AND H.
IIDA
that a crude DSF sample obtained from Vero cells infected with measles virus had molecular weights of 45 000, 20 000 and 3 000 daltons and the nature of a heatstable material. These results conflict with the present report. These discrepancies might be due to the difference in the source of DSF and/or contaminating materials in the crude DSF. The phenomenon of suppression of host DNA synthesis by measles virus has also been reported by some investigators. Zweiman and Miller [15] reported that autoclaved measles virus could inhibit the cellular DNA synthesis of phytohemagglutinin (PHA)stimulated lymphocytes and that the suppressive activity was found in both dialyzable and nondialyzable fractions of autoclaved measles virus. DSF also inhibited the cellular DNA synthesis of PHA-stimulated lymphocytes of mice (unpublished data) and continuous human lymphoid cells (Table 4). However, our DSF is different from the inhibitor reported by Zweiman and Miller since DSF is a heat-labile molecule whose suppressive activity is associated with protein (Table 3). Sullivan et al [13] also demonstrated that partially purified measles virus inhibited 3H-thymidine incorporation by PHA-stimulated human lymphocytes and discussed that a DSF-like substance was not included in the supernatant fluid of infected Vero cells. We assume that their system may have been insufficient for assay of the DSF activity since the titer of DSF produced in culture fluid of infected Vero cells is very low, 1 to 0.5 units/ml of culture fluid. Therefore, it will not be negated that the inhibition of cellular DNA synthesis by infection with measles virus might be due to DSF. Lindahl-Magnusson et al [8] reported. that a mouse interferon preparation inhibited the growth of mouse embryo and mouse kidney cells, revealing thereby speciesspecificity of the action. However, the purified DSF prepared from infected HeLa cells was able to suppress not only the cellular DNA synthesis of HeLa and human lymphoid cells but also that of cells derived from mice (Table 4). Thus, it can be concluded that the suppression caused by DSF is distinguishable from the effect of interferon.
PURIFICATION
Furthermore, DSF is different from a factor produced by mycoplasma reported by Callewaert et al [3], because DSF is inactivated by heating at 56 C for 30 min while the mycoplasma factor is stable after heating at 60 C for 30 min. However, the possibility that the production of DSF may result from an interaction between undetectable agents or repressed cellular gene and replication of measles virus can not be excluded. DSF is also produced in human lymphoid cells (NC-37) after infection with measles virus. We are now carrying out purification of DSF derived from NC-37 cells infected with measles virus to compare with the purified DSF derived from infected HeLa cells. ACKNOWLEDGMENT This investigation was supported by a Grant-inAide for Scientific Research from the Ministry of Education, Science and Culture, Japan. REFERENCES [1]
Andrews P. 1964. Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem. J. 91: 222-233.
[2 ]
Bablanian, R. 1975. Structural and functional alterations in cultured cells infected with cytocidal viruses. Progr. Med. Virol. 19: 40-83.
[ 3 ] Callewaert, D.M., Kaplan, J., Peterson, W.D., Jr., and Lightbody, J.J. 1975. Suppression of lymphocyte activation by a factor produced by Mycoplasmaarginini. J. Immunol. 115: 1662-1664. [ 4 ] Cox, D.C., and Shaw, J.E. 1974. Inhibition of the initiation of cellular DNA synthesis after reovirus infection. J. Virol. 13: 760-761. [5]
Ensminger, W.D., and Tamm, I. 1970. The step in cellular DNA synthesis blocked by New-
OF
DSF
505
castle disease or Mengovirus infection. Virology 40: 152-165. [ 6 ] Hand, R., Ensminger, W.D., and Tamm, I. 1971. Cellular DNA replication in infection with cytocidal RNA virus. Virology 44: 527-536. [ 7 ] Lai, M.T., Werenne, J.J., and Joklik, W. 1973. The preparation of virus top component and its effect on host DNA and protein synthesis. Virology 54: 237-244. [ 8 ] Lindahl-Magnusson, P., Leary, P., and Gresser, I. 1971. Interferon and cell division. VI. Inhibitory effect of interferon on the multiplication of mouse embryo and mouse kidney cells in primary cultures. Proc. Soc. Exp. Biol. Med. 138: 1044-1050. [9]
Lothrop, D., and Nicholson, B.L. 1974. Inhibition of cellular DNA synthesis in cells infected with infectious pancreatic necrosis virus. J. Virol. 14: 485-492. [10] Lowry, 0.H., Rosebrough, N. J., Farr, A.L., and Randall, R. J. 1951. Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193: 267-275. [11] Minagawa, T., Nakaya, C., and Iida, H. 1974. Host DNA synthesis-suppression factor in culture fluid of tissue cultures infected with measles virus. J. Virol. 13: 1118-1125. [12] Minagawa, T., Nakaya, C., Oguma, K., and Iida, H. 1973. Inhibition of host deoxyribonucleic acid synthesis after infection of ultraviolet-irradiated measles virus. Japan. J. Microbiol. 17: 237-241. [13] Sullivan, J.L., Barry, D.W., Albrecht, P., and Lucus, S.J. 1975. Inhibition of lymphocytes stimulation by measles virus. J. Immunol. 114: 1458 1461. [14] Yaoi, Y., Mitsui, H., and Amano, M. 1970. Effect of UV-irradiated vesicular stomatitis virus on nucleic acid synthesis in chick embryo cells. J. Gen. Virol. 8: 165-172. [15]
Zweiman, B., and Miller, M.F. 1974. Effect of non-viable measles virus on proliferating human lymphocytes. Int. Arch. Allergy Appl. Immunol. 46: 822-833.
Purification
of Host DNA Synthesis-Suppressing
(DSF) Produced Tomoko
by Infection
K. YAMAMOTO', Tomonori
with Measles
MINAGAWA, and
Hiroo
Factor Virus IIDA
Departmentof Microbiology,Hokkaido UniversitySchoolof Medicine, Sapporo (Received for publication, May 6, 1976)
ABSTRACT
Host DNA synthesis-suppressing factor (DSF) produced into culture fluid of cloned HeLa cells (HeLa C-9) infected with a small plaque variant of Toyoshima strain of measles virus was purified by precipitation with ammonium sulfate, chromatography on CM-cellulose and DEAE-cellulose, and gel-filtration on Sephadex G-100 and G-200. The specific activity of the finally purified DSF was 302 units/mg of protein representing approximately 300-fold purification. The molecular weight of DSF was estimated to be about 55 000. By isoelectric focusing, two kinds of DSF having isoelectric points of 4.24 and 5.24 were detectable. The purified DSF was able to suppress host DNA synthesis of HeLa cells, continuous human lymphoid cells (NC-37), mouse L cells and Meth-A cells derived from an ascitic tumor of the mouse. The activity of the purified DSF was inactivated by heating at 56 C for 30 min or by treatment with trypsin.
It has been recognized that cellular DNA synthesis is inhibited by infection of several RNA viruses before the onset of morphological cellular damage. In many cases, the inhibition of cellular DNA synthesis follows inhibition of protein synthesis [2, 5, 7, 14]. On the other hand, reovirus type 3 [4, 6], measles virus [12] and pancreatic necrosis virus [9] inhibit cellular DNA synthesis without detectable effects upon RNA and protein syntheses. In previous reports [11, 12] , we demonstrated that the inhibition of host DNA synthesis induced by infection of measles virus was mediated by a DNA. synthesis-suppressing factor (DSF), a nonvirion component, produced into culture fluid after infection. The biological activity of DSF appeared to be associated with protein since the activity was destroyed by treatment with trypsin and pronase but not with RNase, DNase, phospholipase C or D, and the production of DSF was blocked by Requests for reprints should be addressed to Dr. Tomonori Minagawa, Department of Microbiology, Hokkaido University School of Medicine, Nishi 7chome, Kita 15-jo, Kita-ku, Sapporo, 060 Japan. 'Present address: Faculty of Pharmaceutical Science, Chiba University, Chiba. 499
cycloheximide. The production of DSF was closely correlated with the replication of measles virus in HeLa cells. Ultravioletinactivated and antiserum-neutralized measles virus failed to induce DSF production. In addition, DSF was not produced by poliovirus type 1 or vesicular stomatitis virus which showed severe cytopathic effects. On the other hand, certain cellular factors were involved in the production of DSF since some cell lines such as Vero cells and CCRF-CEM cells and some clones obtained from HeLa cells produced a markedly lower amount of DSF despite the fact that growth of measles virus was similar (in preparation). The role of cellular function in the production of DSF remained to be elucidated. The results so far obtained would imply that the production of DSF may be dependent on both replication of measles virus and cellular functions but not on any fortuitous agents such as mycoplasma or other viruses. The present report describes the purification of DSF produced in HeLa C-9 cells infected with a small plaque variant of Toyoshima strain and some properties of the purified DSF.
T.K.
500
MATERIALS
AND
YAMAMOTO,
T. MINAGAWA
METHODS
Cell cultures. Cloned HeLa cells (HeLa C-9) and mouse L cells (L-MS) were cultivated in Eagle's minimum essential medium (MEM, Nissui Seiyaku Co., Tokyo) supplemented with 5% calf serum. Suspension cultures of continuous human lymphoid cell lines (NC-37, CCRF-CEM and MOLT-4) and mouse Meth-A cells were cultivated -in RPMI 1640 medium (Nissui Seiyaku Co.) supplemented with 10% fetal calf serum. Contamination by mycoplasma was checked by inoculating culture fluids of these cells into Bacto-PPLO agar (Difco, U.S.A.). Virus. A small plaque variant of Toyoshima strain of measles virus (TSP) which had been purified by plaque selection on Vero cells was grown in HeLa cell cultures. After the infected cultures were frozen and thawed and centrifuged at 5000 rpm for 20 min, the supernatant was stored at 80 C. Preparationof crudeDSF. HeLa C-9 monolayer cultures in Roux bottles (10 x 15 cm)
Fig.1.
Production
cuhurefluidof small
of DSF HeLa
plaque
Toyoshima
cultu.res of HeLa
Thereafter, vested. after
infection
iRoculated
at an input multiplicity
activities with
(○ − 〇)
(● 一 ●)and the infectivities of progeny TSP (○ … ○)and TLP(● … ●)were according [11].
to the mcthods
with of O.1.
fluid was har-
in the harvested TSP
a
strain. Monolayer
every day the culture
DSF
with
a large plaque
C-9 cells were
or TLP
were inoculated with TSP at a multiplicity of about 0.1 and maintained with MEM containing 2°/ calf serum. One day after infection, the culture fluid was replaced with serum-free MEM, and the cultures were harvested after further incubation for 2 days. These cultures were stored at 20 C prior to processing for purification of DSF. The growth of measles virus and production of DSF in HeLa C-9 cells are shown in Figure 1. Measurement of DSF activity. Routinely, the DSF activity was assayed in continuous human lymphoid cells (NC-37). One milliliter of NC-37 cells (2 x 106 cells) in a polypropylene tube (1.2 x 7.5 cm, Falcon 2026) was inoculated with 1 ml of a DSF sample serially diluted with maintenance medium (RPMI 1640 medium containing 5% fetal calf serum) and incubated at 37 C for 16 hr. After that, the culture was labeled with 3H-thymidine (2 15 Ci per mmole) at 37 C for 30 min. At the end of the labeling period, the culture was cooled in ice, centrifuged at 3000 rpm for 10 min, and then the cell pellet was resuspended in 0.01 M phosphate-buffered saline (PBS). The resuspended sample was precipitated with 5% trichloroacetic acid (TCA) and then incorporation of 3H-thymidine into the TCA-insoluble materials was determined as previously described [12]. The ratio of suppression of host DNA synthesis was calculated as follows: [counts per minute
virus into
C-9 cclls infected
variant(TSP)and
variant(TLP)of
either TSP
and progeny
AND H. IIDA
described
samples and
TLP
virus of assayed prcviously
Fig. 2. Dose response of the purified DSF in terms of suppression of host DNA synthesis of NC-37 cells. One milliliter of an NC-37 cell suspension (2 x 106 cells) was mixed with an equal volume of serially diluted DSF and incubated for 16 hr, and then labeled with 3H-thymidine (2 pCi/ml).
PURIFICATION
Table
These
values
were
calculated
1.
from
Summary
two
of the
experiments
(cpm) of control minus cpm of experiment] / [cpm of control] x 100 (%). The dilution of DSF sample that caused 50% suppression of the DNA synthesis was defined as the DSF titer (unit/ml). An example of the relationship between the suppression of host DNA synthesis and log DSF dose is shown in Figure 2. Protein determination. Protein concentration was determined either by the method of Lowry et al [10] or absorbance measurement at 280 nm with bovine serum albumin as the standard. Isoelectricfocusing. An LKB 8101, 110 ml ampholine electrofocusing column was filled with 20 fractions of 5 ml of 1.9% carrier ampholites covering a pH range from 3 to 10 in sucrose solution of densities varying from 40% to 0%. Five-tenths milliliters of a DSF sample was incorporated into one fraction in the middle portion of the column. A current of 500 V was maintained at 4 C for 64 hr. On termination of the run, fractionation was carried out and each fraction was dialyzed twice against 0.01 M phosphate buffer (pH 7.4), and then DSF activity in each fraction was determined. Reagents. Bovine serum albumin, cytochrome c, RNase, DNase, soybean trypsin inhibitor (Sigma Chemical Co.), crystalline trypsin (Difco Laboratories) and phospholipase C (Worthington Biochemical Co.) were employed in these experiments. RESULTS Purificationof DSF Purification of DSF was carried out by ammonium sulfate precipitation, CMcellulose chromatography, DEAE-cellulose
OF
501
DSF
purification
which
of DSF
used
different
starting
materials.
chromatography and Sephadex G-100 and G-200 gel-filtration. The total quantity, the specific activity per mg of protein, and the percentage of recovery of DSF at each stage of the purification procedures are summarized in Table 1. All the procedures were performed at 4 C. Stage 1: Concentrationwith ammoniumsulfate. About 9 liters of culture fluid were centrifuged at 10 000 rpm for 20 min. The supernatant, the starting material, containing 1.03 mg/ml of protein was fractionated at 4 C with solid ammonium sulfate. The precipitate formed at 0 to 33.3% saturation of ammonium sulfate was collected by centrifugation at 10 000 rpm for 60 min, dissolved in 143 ml of 0.01 Mphosphate buffer (pH 5.5), and dialyzed at 4 C against the same buffer. The precipitate formed during dialysis was removed by centrifugation and the supernatant was again dialyzed against the same buffer, and the dialyzed sample was further centrifuged at 25 000 rpm for 3 hr in order to remove virus particles. The supernatant that contained DSF units of 44.4% and protein representing 4.5% of the starting material was used for the following stage of purification. Stage 2: Chromatographyon CM-cellulose. The DSF solution concentrated by precipitation with ammonium sulfate was divided into two parts and run slowly through two sets of CM-cellulose column (1.0 x 13.8 cm) equilibrated with 0.01 M phosphate buffer (pH 5.5). The columns were then washed with 30 to 50 ml of the same buffer. The activity of DSF was not detected in these fractions. After that, 0.01 M phosphate buffer (pH 8.0) was added for elution of
502
T. K. YAMAMOTO,
T. MINAGAWA
DSF. DSF activity was eluted in fractions of 3 to 5 at pH 6.26 to 7.95 as shown in Table 2. Fractions 3 to 5 were pooled and used for the next stage of purification. Stage 3: Chromatographyon DEAE-cellulose. The pooled fractions from CM-cellulose columns were dialyzed three times against 0.01 M Tris buffer (pH 7.5) and applied to a DEAE-cellulose column (1.5 x 20 cm) equilibrated with the same buffer. DSF Table 2. Elution the CM-cellulose
of DSF from column
A DSF sample obtained after precipitation with ammonium sulfate was divided into two parts (73 ml and 70 ml) and applied to two sets of CM-cellulose columns (a and b). Tenmilliliter fractions were collected and DSF activity in the fractions were assayed. Parentheses indicate the pH value.
Fig.3.
Chromatography
cellulose sample
obtained
was
of
column.
applied
Thirty from
to
a
chromatographed following phosphate buffer
(pH
0.01M
phosphate
0.5 and of
M
at
the
a
a rate
buffer
Five-milliliter at 280
fractions
were
fractions
assayed.
and
with
the
ml/hr.(A):0.01
(pH
nm(●)and
DSF
column
buffer(pH 6.0).(B):0.01M 6.0)containing O. I M
NaCL
a
column
elution
of 33
was chromatographed by stepwise elution. When 0.01 M phosphate buffer (pH 6.0) containing 0.1 M NaCl was added to the column, more than 90% of the total DSF activity bound to the column was eluted in fractions 20 to 24, and the peak of DSF activity corresponded to the major protein peak, as shown in Figure 3. Fractions 20 to 24 were pooled and used for the next stage of purification. Stage 4: Gel-filtration on Sephadex G-100. The pooled fractions from the DEAE-cellulose column were concentrated 10-fold by dialysis against 25% polyethylene glycol G-6000 and loaded onto the top of a Sephadex G-100 column (2.6 x 37.5 cm) equilibrated with 0.01 M phosphate buffer (pH 7.4), and the same buffer was added for elution of DSF. Five-milliliter fractions were collected at a flow rate of 6.6 ml/hr (Fig. 4). The main DSF peak from fractions 20 to 23 was pooled and concentrated for the next stage of purification. Stage 5: Gel-filtration on Sephadex G-200. The pooled and concentrated fractions from stage 4 were loaded onto a Sephadex G-200 column (2.6 x 38.5 cm) equilibrated with 0.01 M phosphate buffer (pH 7.4). Figure 5 shows the elution pattern obtained when DSF was eluted with the same buffer at a
DEAE-
of
CM.cellulose
stepwis
absorbance the
on
DEAE-cellulose by
buffers
DSF
millilitcrs
AND H. IIDA
phosphate NaCl.(C):
6.0) were DSF
containing collected titers(○)
M
Fig.4.
Ge1-filtration
column.
A
cellulose
column.
column
DSF
and
of
sample was
at
fractions
280 were
on
a Sephadex
obtained applied
from to
nm(●)and assayed.
7.4)at were
the
Sephadex
chromatographed
phosphate buffer(pH Five-milliliter fractions ance
DSF
with a rate collected
DSF
G-100 DEAEG-100 0.01
M
of 6.6 ml/hr. and absorb-
titers(○)of
the
PURIFICATION
Fig.5.
Gel-filtratiлn
column. dex to
A
G-100 a
DSF
of DSF
sample
column
Sephadex
O.Ol
at a rate
of 6.o
ml/hr.
collected DSF
and
Table
3.
of the
at
fractions
of enzymes purified
the
Sephaapplied
chrлmato-
buH℃r(pH
Five-milliliter
the
Effects
and
phosphate
G-200
and
cл1u.mn,
M
absorbance
titers(○)in
from
concentrated
G-200
with
a Sephadex
obtained
was
graphed
on
280 were
on
fractions
7.4) wcre
nm(●)and assayed。
the activity
OF
DSF
503
Fig. 6. Isoelectric focusing of DSF. A DSF sample containing 400 units obtained from the final step of purification was electrofocused. Detailed methods are described in the text. After the isoelectric focusing of DSF, 2-ml fractions were collected and eachfraction was dialyzed against 0.01Mphosphate buffer (pH 7.4), and then DSF activity (0) was determined. Table 4. Effects of the purified DNA synthesis of various cell
DSF on cultures
DSF
The finally purified DSF (302 units/mg of protein) was incubated with various enzymes at 37 C for 1 hr, and then assayed the remaining activity of DSF. Soybean trypsin inhibitor (10 mg/ml) was added after the treatment with trypsin.
flow rate of 6.0 ml/hr. The DSF peak from fractions 26 to 29 was collected. The final specific activity of the purified DSF obtained in this stage was 302 units/mg of protein, the value of the original material being increased 330-fold. Determinationof Molecular Weightof DSF The molecular weight of the purified DSF was determined by gel-filtration of Sephadex G-100 and G-200 using marker proteins of bovine serum albumin and cytochrome c according to the method of Andrews [1]. The results indicated that the
One milliliter of 1.0 unit or 10 units of the purified DSF (302 units/mg of protein), crude DSF and control medium was inoculated into various cell cultures (1 x 106 cells). After 16-hr treatment, these cells were labeled, fractionated and counted by the methods described in the text.
purified DSF had a molecular weight of approximately 55 000. Determination of Isoelectric Points of DSF Isoelectric focusing was performed on the DSF obtained from gel-filtration on Sephadex G-100 and approximately 40% of the input DSF activity was recovered. DSF activity was detectable in two peaks containing different percentage of the total activity with isoelectric points of 4.24 and 5.24 (Fig. 6).
504
T.K.
YAMAMOTO,
T. MINAGAWA
SomePropertiesof the PurifiedDSF The finally purified DSF was incubated with various enzymes at 37 C. Table 3 shows that DSF activity was destroyed by trypsin but not by DNase, RNase, or phospholipase C. These results agree with our previous report concerning the effect of these enzymes on a crude DSF preparation from infected Vero cells [12]. Furthermore, as is clear in Table 3, the purified DSF was as heatlabile as was the crude DSF (unpublished data) . The ultraviolet extinction spectrum of the finally purified DSF was typical for protein, having a maximum at 280 nm and a minimum at 252 nm. The ultraviolet absorption ratio of 280 nm to 260 nm was 1.65, hence the possibility of presence of nucleic acid in this material was very low. Furthermore, effects of the finally purified DSF on DNA. synthesis of cell cultures derived from different animal species were investigated. As indicated in Table 4, the purified DSF remarkably suppressed the cellular DNA synthesis of HeLa cells and NC-37 cells, but not that of CCRF-CEM cells. Similarly, the cellular DNA synthesis of mouse L cells and Meth-A cells derived from an ascitic tumor of a BALB/c mouse induced by 20methylcholanthrene was suppressed depending upon the dose of DSF added. These host range in terms of sensitivity to the effect of purified DSF is identical with that of crude DSF. Thus it is concluded that DSF purified under these conditions has the same properties as crude DSF. DISCUSSION Purification of DSF produced from HeLa C-9 cells infected with a small plaque variant of Toyoshima strain of measles virus has been carried out. The finally obtained DSF has a specific activity of 302 units/mg of protein and is practically purified from impurity protein. Under these conditions, DSF behaves as a single molecular species with a molecular weight of 55 000. However, the result obtained from the isoelectric focusing experiments shows that the purified DSF is heterogeneous in isoelectric points (Fig. 6). At present, it is not clear whether or not the heterogeneity of the purified DSF is due to different molecular species. In a previous paper [11], it was reported
AND H.
IIDA
that a crude DSF sample obtained from Vero cells infected with measles virus had molecular weights of 45 000, 20 000 and 3 000 daltons and the nature of a heatstable material. These results conflict with the present report. These discrepancies might be due to the difference in the source of DSF and/or contaminating materials in the crude DSF. The phenomenon of suppression of host DNA synthesis by measles virus has also been reported by some investigators. Zweiman and Miller [15] reported that autoclaved measles virus could inhibit the cellular DNA synthesis of phytohemagglutinin (PHA)stimulated lymphocytes and that the suppressive activity was found in both dialyzable and nondialyzable fractions of autoclaved measles virus. DSF also inhibited the cellular DNA synthesis of PHA-stimulated lymphocytes of mice (unpublished data) and continuous human lymphoid cells (Table 4). However, our DSF is different from the inhibitor reported by Zweiman and Miller since DSF is a heat-labile molecule whose suppressive activity is associated with protein (Table 3). Sullivan et al [13] also demonstrated that partially purified measles virus inhibited 3H-thymidine incorporation by PHA-stimulated human lymphocytes and discussed that a DSF-like substance was not included in the supernatant fluid of infected Vero cells. We assume that their system may have been insufficient for assay of the DSF activity since the titer of DSF produced in culture fluid of infected Vero cells is very low, 1 to 0.5 units/ml of culture fluid. Therefore, it will not be negated that the inhibition of cellular DNA synthesis by infection with measles virus might be due to DSF. Lindahl-Magnusson et al [8] reported. that a mouse interferon preparation inhibited the growth of mouse embryo and mouse kidney cells, revealing thereby speciesspecificity of the action. However, the purified DSF prepared from infected HeLa cells was able to suppress not only the cellular DNA synthesis of HeLa and human lymphoid cells but also that of cells derived from mice (Table 4). Thus, it can be concluded that the suppression caused by DSF is distinguishable from the effect of interferon.
PURIFICATION
Furthermore, DSF is different from a factor produced by mycoplasma reported by Callewaert et al [3], because DSF is inactivated by heating at 56 C for 30 min while the mycoplasma factor is stable after heating at 60 C for 30 min. However, the possibility that the production of DSF may result from an interaction between undetectable agents or repressed cellular gene and replication of measles virus can not be excluded. DSF is also produced in human lymphoid cells (NC-37) after infection with measles virus. We are now carrying out purification of DSF derived from NC-37 cells infected with measles virus to compare with the purified DSF derived from infected HeLa cells. ACKNOWLEDGMENT This investigation was supported by a Grant-inAide for Scientific Research from the Ministry of Education, Science and Culture, Japan. REFERENCES [1]
Andrews P. 1964. Estimation of the molecular weights of proteins by Sephadex gel-filtration. Biochem. J. 91: 222-233.
[2 ]
Bablanian, R. 1975. Structural and functional alterations in cultured cells infected with cytocidal viruses. Progr. Med. Virol. 19: 40-83.
[ 3 ] Callewaert, D.M., Kaplan, J., Peterson, W.D., Jr., and Lightbody, J.J. 1975. Suppression of lymphocyte activation by a factor produced by Mycoplasmaarginini. J. Immunol. 115: 1662-1664. [ 4 ] Cox, D.C., and Shaw, J.E. 1974. Inhibition of the initiation of cellular DNA synthesis after reovirus infection. J. Virol. 13: 760-761. [5]
Ensminger, W.D., and Tamm, I. 1970. The step in cellular DNA synthesis blocked by New-
OF
DSF
505
castle disease or Mengovirus infection. Virology 40: 152-165. [ 6 ] Hand, R., Ensminger, W.D., and Tamm, I. 1971. Cellular DNA replication in infection with cytocidal RNA virus. Virology 44: 527-536. [ 7 ] Lai, M.T., Werenne, J.J., and Joklik, W. 1973. The preparation of virus top component and its effect on host DNA and protein synthesis. Virology 54: 237-244. [ 8 ] Lindahl-Magnusson, P., Leary, P., and Gresser, I. 1971. Interferon and cell division. VI. Inhibitory effect of interferon on the multiplication of mouse embryo and mouse kidney cells in primary cultures. Proc. Soc. Exp. Biol. Med. 138: 1044-1050. [9]
Lothrop, D., and Nicholson, B.L. 1974. Inhibition of cellular DNA synthesis in cells infected with infectious pancreatic necrosis virus. J. Virol. 14: 485-492. [10] Lowry, 0.H., Rosebrough, N. J., Farr, A.L., and Randall, R. J. 1951. Protein measurement with the Folin-phenol reagent. J. Biol. Chem. 193: 267-275. [11] Minagawa, T., Nakaya, C., and Iida, H. 1974. Host DNA synthesis-suppression factor in culture fluid of tissue cultures infected with measles virus. J. Virol. 13: 1118-1125. [12] Minagawa, T., Nakaya, C., Oguma, K., and Iida, H. 1973. Inhibition of host deoxyribonucleic acid synthesis after infection of ultraviolet-irradiated measles virus. Japan. J. Microbiol. 17: 237-241. [13] Sullivan, J.L., Barry, D.W., Albrecht, P., and Lucus, S.J. 1975. Inhibition of lymphocytes stimulation by measles virus. J. Immunol. 114: 1458 1461. [14] Yaoi, Y., Mitsui, H., and Amano, M. 1970. Effect of UV-irradiated vesicular stomatitis virus on nucleic acid synthesis in chick embryo cells. J. Gen. Virol. 8: 165-172. [15]
Zweiman, B., and Miller, M.F. 1974. Effect of non-viable measles virus on proliferating human lymphocytes. Int. Arch. Allergy Appl. Immunol. 46: 822-833.
