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DNA and histone synthesis in butyrate-inhibited BSC-1 cells infected with SV40
VIROLOGY
116,196-206
(1982)
DNA and Histone Synthesis in Butyrate-Inhibited Cells Infected with SV40 ELLEN
DANIELL,l
Department
J. LAWRENCE
of Molecular Received
Biology, June
BURG,
University 17, 1981;
AND
of California, accepted
August
BSC-1
MARTHA Berkeley,
J. .FEDOR
California
9.4720
20, 1981
The effect of butyrate on DNA synthesis, histone synthesis, and virus production in SV40-infected monkey cells has been studied. When cells in which DNA and histone synthesis have been blocked by treatment with butyrate are infected with SV40, DNA synthesis is induced to a level at least equal to that in untreated infected cells. Both viral and cellular DNA sequences are replicated. Viral DNA synthesized is predominantly SV40 Form I. The amount of viral DNA which accumulates is reduced by lo-fold in the presence of butyrate, and the yield of infectious virus is lowered loo-fold. Histone synthesis in these cells is stimulated concomitantly with DNA synthesis. The hyperacetylation of histones characteristic of butyrate-treated cells is unchanged by SV40 infection; the newly synthesized histones are hyperacetylated. Thus the mechanism by which butyrate blocks DNA and histone synthesis is not directly related to the hyperacetylation of histones.
minichromosomes which are the templates for viral transcription and replication (Griffith, 1975; Pett et al., 1975). SV40 infection stimulates both cellular DNA synthesis and histone synthesis, and the histones made are deposited on viral as well as cellular DNA (Kit et al., 1967; Ritzi and Levine, 1969; Winocour and Robbins, 1970). SV40 and cellular DNA replication share a number of features, including a tight dependence on protein synthesis, bidirectional replication from each origin, and use of host DNA polymerase a (Fareed and Davoli, 1977). In the present work we investigated the effect of butyrate on SV40 infection of BSC-1 cells to ascertain whether the block to cellular DNA replication and histone synthesis would inhibit SV40 replication. This study was begun as a complement to the demonstration that adenoviruses replicate in butyrate-treated cells with full efficiency, accompanied by no detectable cellular DNA or histone synthesis (Daniell, 1980). The differences between these two types of viruses in mode of replication and hostvirus interaction are highlighted by the results reported here.
INTRODUCTION
Addition of millimolar amounts of butyrate to the medium of tissue culture cells rapidly results in increased levels of histone acetylation (Vidali et al., 1978), due to the inhibition of histone deacetylases (Candid0 et al., 1978; Cousens et al., 1979; Sealy and Chalkley, 1978; Boffa et al., 1978). After longer periods of treatment, cell DNA synthesis and cell division cease (Riggs et al., 1977; Hagopian et al., 1977), as does histone synthesis (Daniell, 1980). These long-term effects do not appear to result directly from the hyperacetylation of histones (Rubenstein et al., 1979) and kinetics of shutoff of DNA synthesis are consistent with a block at a particular stage in the cell cycle (Daniel& 1980). Both immediate and long-term effects are readily reversible; cells remain viable and return to normal when fed with butyratefree medium. The DNA of simian virus 40 (SV40) is associated with histones both in mature virus particles and in the intracellular 1 To whom
reprint
requests
should
0042-6822/82/010196-11$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
be addressed. 196
SV40 IN BUTYRATE-TREATED
MATERIALS
AND
METHODS
Cells and viruses. BSC-1 (monkey kidney) cells were cultured as monolayers in Dulbecco’s medium (DME) supplemented with 5% calf serum. Plaque-purified SV40 (small plaque strain) was propagated and titrated on BSC-1 cells. Infections were performed at a multiplicity of 10 PFU/cell unless otherwise stated. For infection, medium was removed from cell monolayers, and dilutions of virus in phosphatebuffered saline (PBS) were added to the cells. As SV40 stocks were prepared and stored in DME, mock-infected cells were treated with appropriate dilutions of DME into PBS. Conditioned medium was added back to the cells after 2 hr of adsorption. Sodium butyrate (Baker) was added to tissue culture medium as a dilution of an 0.5 M stock in sterile PBS. A final concentration of 6.5 mM butyrate was used to inhibit DNA synthesis and cell division in BSC-1 cells. Butyrate was added 24 hr before infection unless otherwise stated. Virus production was measured by scraping cells into the medium, freezing, and thawing four times, and sonicating before plaque titration on BSC-1 cells. DNA synthesis in infected- and uninfected cells. Cells were labeled with [3H]thymidine (1 &i/ml, 58 Ci/mmol) for 1 hr at times indicated; incorporated label was measured as previously described (Daniell, 1980), and counts were normalized to counts per minute per lo5 cells. Shutoff of DNA synthesis was operationally defined as a level of thymidine incorporation 5-10% that in untreated, rapidly growing (i.e., nonconfluent) cells. Histone synthesis and acetylation. To determine whether newly synthesized histone was accumulating in nuclei under various conditions, cells were labeled for 5 hr with 1 &i/ml L-[14C(U)]arginine (298 mCi/mmol) in DME plus 5% dialyzed fetal calf serum. Isolation and extraction of nuclei with sulfuric acid followed by acetone precipitation of acid-soluble proteins and electrophoresis on the, acid-urea gel system of Panyim and Chalkley (1969) have been described previously (Kit and Daniell, 1978).
CELLS
197
Histone acetylation was determined by ad!dition of 10 &i/ml [14C]acetate (58.7 mCi/mmol) to the medium for 1 hr, followed by electrophoresis of acid extracted histones on Triton X-loo-acid-urea gels (Fedor and Daniell, 1980). Different schedules of butyrate treatment, infection, and labeling are described in the text and figure legends. DNA synthesis in cell lysates. Lysates were prepared and DNA synthesis measured by the method of Fraser and Huberman (1977). Butyrate-treated cells were treated with 6.5 mM butyrate for 3-24 hr before infection with SV40 (5 PFU/cell). Cell lysates were prepared 30 hr postinfection, and DNA synthesis measured as incorporation of [a-32P]dTTP (7.5 &i/ml) into TCA-precipitable counts. The final concentration of KC1 in the reaction mix was 18 mM; all other components are as described in Fraser and Huberman (1977). Isolation of DNA. Preparation of total cellular and viral DNA from isolated nuclei has been described (Daniel1 and Mullenbach, 1978). Probes for hybridization experiments were prepared by this method from cells labeled with [3H]thymidine 2836 hr postinfection, followed by sonication to reduce the size of the DNA to an average of 300 nucleotides. Alternatively, low-molecular-weight, predominantly viral DNA was separated from high-molecular-weight cellular DNA by the method of Hirt (1967). In uninfected cells, 94-98% of cellular DNA remains in the 1 M NaCl-SDS insoluble pellet. Ninety to ninety-five percent of SV40 DNA in infected cells is in the soluble “supernatant” fraction (Kay and Singer, 1977; Hirt, 1967). Gel electrophoresis and jluorography. Electrophoresis was performed on 1% agarose gels, as described (Daniel1 and Mullenbach, 1978). To detect [3H]thymidine counts by autoradiography, gels were dehydrated in methanol (double the gel volume for three times 45 min), then soaked in 2 vol of a 5% (w/v) solution of PPO (2,5diphenyloxozole; Packard) in methanol for l--l’/2 hr (Laskey and Mills, 1975). The gel was rinsed in water, and soaked in water with 6% glycerol (v/v), dried under vac-
198
DANIELL,
BURG,
uum at room temperature, and autoradiographed with Kodak XR-5 film. After autoradiography, gels impregnated with PPO could be sliced and counted by Cerenkov counting. The efficiency of counting by this method is about 40%, and is more reproducible than dissolving and counting agarose gel slices.
RESULTS
Effect of butyrate on monkey cells. The minimal concentration of butyrate needed to shut off cellular DNA synthesis was determined to be 6.5 mM for BSC-1 cells. As observed for other cell lines, DNA synthesis declined to minimal levels (~5% normal) in one cell-doubling time. Cells maintained for 72 hr in this concentration of butyrate resumed DNA synthesis, cell division and normal morphology within 24 hr after replacing the medium with fresh or conditioned butyrate-free medium. Some increased sensitivity to long treatment with butyrate was noted if cells had been passaged for several months after thawing. When cell death or failure to resume growth when cultured without butyrate was observed in uninfected cultures maintained throughout an experiment, the results were discarded. Overall rates of protein and RNA synthesis did not change significantly in butyrate-treated cells (less than 10%; data not shown). Histone synthesis (measured as acid-extractable counts in nuclei, and analyzed by gel electrophoresis and fluorography) is shut off by more than 90%. The experiments reported here were performed with BSC-1 cells, in which induction of cellular DNA synthesis is less than in CV-1 and other monkey cell types (Ritzi and Levine, 1969). Yield of infectious virus is reduced in butyrate-treated cells. The effect of butyrate on virus yield was investigated by infecting BSC-1 cells which had been pretreated with butyrate, and by infection of normal cells, adding butyrate only after virus adsorption. Results of representative experiments are shown in Table 1. The
AND
FEDOR
TABLE
1
YIELDOFINFECTIOUSSV~O FROMBUTYRATETREATEDCELLS Virus Multiplicity of infection 10 0.1 50 10
+Butyrate 32” 1.2” 75” 206
yield
(PFU/cell)
-Butyrate 1800 190 2000 1500
-/+ 56 160 26 75
‘Cells which were not pretreated with butyrate were infected. 6.5 mM sodium butyrate was added when medium was replaced after virus adsorption. b Cells were pretreated with 6.5 mM butyrate for 24 hr and the conditioned medium with butyrate was replaced after virus adsorption.
number of plaque-forming units produced in butyrate-treated cells under either schedule of butyrate treatment was significantly lower than the yield in untreated cells. At low multiplicities of infection, the difference is greater than lOOfold. Experiments in which butyrate was added only after infection are most instructive, as efficiency of virus adsorption or confluency of cells are not variable. Clearly virus multiplication is inhibited by the presence of butyrate in the medium. DNA synthesis in butyrate-treated cells following SViO infection. Incorporation of [3H]thymidine in a 1-hr pulse was measured at various times after infection of BSC-1 cells with SV40. In the experiment shown in Fig. 1, untreated cultures nearing confluency were used so that thymidine incorporation was low before infection, allowing significant stimulation of total DNA synthesis in those cultures as well as in butyrate-inhibited cells. Figure 1 illustrates that thymidine incorporation is induced by SV40 in butyrate-treated cells with similar kinetics to those observed in normal confluent cells. Peak levels of incorporation were consistently higher in butyrate-treated cells. The use of rH]thymidine incorporation in vivo to compare levels of DNA synthesis in infected and uninfected cells is subject
SV40
0
5
10
IN BUTYRATE-TREATED
CELLS
15 20 Z!j 30 Hours after infection
35
199
40
45
50
FIG. 1. DNA synthesis in BSC-1 cells infected with !W40 in the presence of butyrate. Cells were labeled for 1 hr with rH]thymidine and harvested at times indicated, incorporation was measured as described and normalized to cpm per lo5 cells. The four curves represent: (0) butyrate-treated cells;, (A) butyrate-treated cells, SV40 infected, (0) untreated cells; (a) untreated cells, SV40 infected.
to complication from changing pool sizes, kinase levels and cell permeability changes. To demonstrate that SV40 infection really restored the ability of cells to replicate DNA, we employed a cell-free system. Hagopian et al. (1977) demonstrated that a crude cell lysate of HeLa cells which have been exposed to butyrate for 24 hr is completely inactive in incorporation of deoxytriphosphates or thymidine (6% the activity of normal lysates). Figure 2 shows that lysates from uninfected BSC-1 cells treated with butyrate have little DNA replication activity, while lysates from SV40infected butyrate treated cells have activity equal to normal cells. Characterization of DNA synthesized. The DNA synthesized in butyrate-treated infected cells was characterized by hybridization, Hirt extraction and by gel electrophoresis. DNA from cells labeled with [3H]thymidine from 28 to 34 hr postinfection was hybridized to SV40 and to BSC-1 DNA immobilized on filters. The
results, shown in Table 2, indicate that in butyrate-treated cells, a large fraction of the newly synthesized DNA consists of cellular sequences. EDNA was extracted from cells labeled for 1 hr at various times after infection by the method of Hirt (1967). This method separates viral (low molecular weight) from host cell DNA, and has been employed in previous studies of cellular and viral DNA synthesis in SV40-infected cells (Kay and Singer, 1977; Ritzi and Levine, 1969). However, this method proved not to be useful for comparing incorporation into cellular and viral sequences in the presence of butyrate. Electrophoretic analysis of the DNA in Hirt supernatants revealed that in butyrate-treated cells, a large fraction of the counts in these preparations is in a band of higher molecular weight than viral DNA. Figure 3 shows Hirt supernatants prepared from cells labeled for 1 hr at various times after infection. Gel bands were excised and counted to deter-
DANIELL,
200
BURG,
AND
FEDOR
35
abcdefgh
Cdl
sv40 (II)
sv40 (I)
Cdl
sv40
0
0
5
10
15 Time,
20
25
30
sv40
min
FIG. 2. Incorporation of [&‘P]TTP into DNA in a cell lysate. Lysates were prepared 36 hr after infection (48 hr after butyrate addition) as described under Materials and Methods, and incorporation of TTP was measured as a function of time: (0) untreated BSC-1 cells; (0) BSC-1 cells treated with butyrate; (A) SV40-infected cells without butyrate; (0) SV40 infected cells, treated with butyrate.
mine the fraction of DNA in viral bands (Table 3). At 22 hr after infection no viral DNA is detectable in butyrate-treated cells (lane a). At 28 hr only 40% of the TABLE
FIG. 3. Analysis of Hirt supernatant DNA from BSC-1 cells infected with SV40 in the presence or absence of butyrate. Cells were labeled with [qlthymidine for 1 hr prior to harvest by Hirt fractionation. DNA from supernatant fractions was purified by extraction and precipitation and subjected to gel electrophoresis through lo-cm agarose gels (1%) for 16 hr at 20 V. At the top is the photograph of the ethidium stained gel, below is the autoradiograph of the same gel. DNA from an equal number of cells was loaded onto each slot. (a, c, e, and g) + butyrate, and (b, d, f, and h) - butyrate. (a, b) 22 hr after infection, (c, d) 28 hr after infection, (e, f) 38 hr after infection, and (g, h) 46 hr after infection.
2
HYBRIDIZATIONOFLABELEDDNAFROMINFECTEDBUTYRATE-TREATEDANDUNTREATEDCELLS TO SV40 AND TO BSC DNA”
Specific activity (X10* cpm/pg)
Probe BSCSVIO (+B) BSCSV40 (-B) BSC (Mock) “Hybridization incubated with blank.
9 3 4
cpm hybridized filter
to
BSC
sv40
4650 1400 3596
1070 3360 122
was carried out by the method of Denhardt three filters: one carrying 50 pg of BSC-1 DNA,
SVIO/BSC .23 2.4 .03
(1966). 5 X lo4 counts of each probe was one carrying 10 pg of SV40 DNA, and one
SV40 IN BUTYRATE-TF1EATED TABLE
CELLS
201
3
DISTRIBUTIONOFNEWLYSYNTHESIZED DNA II‘ITOHIRTSUPERNATANTS ANDPELLETS ATDIFFERENTTIMESAFTF.RSV~O INFECTION Labeling period” (hr after infection)
Thymidine incorporationb (X10’ cpm)
Counts in Hirt supernatant (% total incorporation)
Percentage SV40 in supernatant’
Butyrate
sv40
Mock (22)
+ -
-
0.06 0.73
>15d 2
22
+ -
+ +
0.48 1.6
30 57
12 82
28
+ -
+ +
1.25 1.5
49 70
40 92
37
+ -
+ +
2.9 2.1
65 78
60 95
46
+ -
+ +
1.8 2.9
68 80
80 95
-
a All labeling periods were 1 hr long, ending at the time specified. * Normalized to counts per lo5 cells. ’ Gels were sliced and counted after fluorography and the fraction of the total which was present in SV40 DNA (Forms I, II, and II) was calculated. ’ The distribution of label during Hirt fractionation of butyrate-treated uninfected cells was highly variable ranging from 15 to 50% in different experiments.
counts in the Hirt supernatant of butyrate-treated cells (lane c) are in viral bands, whereas in untreated cells, 92% of the label is viral (lane d). At later times, the proportion of viral DNA in supernatants from butyrate-treated cells increases. The “upper band” DNA has been further characterized and its cellular origin confirmed. Hybridization of SV40 probe to Southern blots of these gels show that viral DNA does not hybridize to this band. Figure 4 illustrates that this band is present in Hirt supernatants from uninfected cells (lane c and d), and has a molecular weight greater than 21 kb. Electrophoresis on lower-percentage gels shows that the material is diffuse but all of it runs more slowly than adenovirus type 5 DNA used as a marker of 35 kb (data not shown). When this material is eluted from gels and treated with a variety of restriction endonucleases, no discrete fragments are generated, though the DNA is cleaved into small pieces. This not only confirms that
the material is not viral, but suggests that it. represents a nonspecific assortment of cellular sequences. In the samples in Fig. 4, cells were lab’eled for 15 hr (from 23 to 38 hr after infection or mock infection). The fluorograph (lanes c’-g’) shows that label alecumulates in the cellular material as a result of SV40 infection, and to a far greater extent in butyrate-treated than in untreated cells. The amount of SV40 (Forms I and II) per cell is less in butyrate-treated cells at all times, although the amount of label in a pulse surpasses that in untreated cells after 32 hr. It is also clear from Figs. 3 and 4 that the SV40 synthesized in butyrate-treated cells is predominantly Form I (supercoiled). To ascertain whether viral DNA turns over at an increased rate in butyratetreated cells, we compared DNA from cells labeled for different lengths of time. Two identical cultures were labeled for 1 and for 6 hr, respectively, and harvested by the Hirt procedure or by extraction of whole
DANIELL,
202
a
35 kb
21 kb 14kb
BURG,
AND
FEDOR
c'
bcdefg
d'
f'
g'
‘\ -/-
FIG. 4. Electrophoresis of Hirt supernatants from infected and mock-infected cells. Samples were prepared, electrophoresed, and fluorographed as in Fig. 3 except that the labeling was from 23 to 38 hr after infection. (a) Ad5 DNA marker; (b, e) Ad5 DNA cleaved with BarnHI; (c, c’) mockinfected cells, butyrate added 35 hr before start of labeling; (d, d’) mock-infected cells, no butyrate; (f, f’) SV40-infected cells, butyrate added 12 hr prior to infection; and (g, g’) SV40-infected cells, no butyrate.
cells. The physical amount of DNA in each sample is the same, and the ratios between long and short label represent the rate of TABLE ACCUMULATION
Butyrate
DNA Hirt Hirt
OF [3H]T~~~~~~~~
fraction
supernatantb pellet!
SV40"
Celld Hirt supernatant* Hirt pelletb
accumulation of counts in any fraction. Results of a typical experiment are shown in Table 4. Three- or fourfold more counts 4 IN DNA
FROM INFECTED
CELLS~
6 hr (x10-3)
1 hr (x10-3)
140
34 18 3.9
73 8.9 22.0 35
11
SV40"
8.6
Celld
1.1
3.6 11.2 1.8 3.0 0.29
6/l 4.1 4.1 2.3 6.1 3.1 6.0 2.9 3.8
“DNA was isolated by Hirt fractionation from cells labeled for 1 hr or for 6 hr prior to harvesting (37 hr after infection). After phenol extraction and precipitation, aliquots of supernatant material were electrophoresed through 1% agarose, stained, photographed, treated with PPO, and sliced and counted as described under Materials and Methods. b TCA-precipitable counts in 100 pl of Hirt supernatant or Hirt pellet resuspended in a volume equal to the supernatant. ‘Sum of Form I and Form II. d The “high-molecular-weight” band from Hirt supernatants.
SV40
IN BUTYRATE-TREATED
(a) 1
2
3
4
5
203
CELLS
(b’
1
2
3
4
5
Hl
FIG. 5. Histone synthesis in butyrate-treated SV40-infected cells. Acid-urea gel of aeid-extractable nuclear proteins labeled with [%]arginine from 218 to 36 hr after infection (or mock infection). (a) Amido black-stained gel, photographed. (b) Autoradiograph: 1, SV40-infected cells, 5 X lo3 cpm loaded; 2, uninfected cells, butyrate added 40 hr before labeling, 600 cpm loaded; 3, uninfected cells, no butyrate (1.5 X lo3 epm); 4, SV40-infected cells, butyrate added 12 hr before infection (40 hr before labeling) (1.5 X lo3 cpm); and 5, SV40-infected. cells (1 X lo3 cpm).
accumulated in Hirt supernatants during the long-labeling period than during the short-labeling period, the treated cells showing a somewhat higher ratio than untreated cells. There is no significant difference between butyrate-treated cells and untreated cells in the relative amount of labeling of SV40 DNA during longand short-labeling intervals. In butyratetreated cells the ratio of long to short labeling intervals. In butyrate-treated cells the ratio of long to short labeling of the cellular band of the Hirt supernatant is higher than in untreated cells, and the Hirt pellet does not show the six-fold accumulation of counts detected in untreated cell chromosomal DNA. These anomalies may reflect fragmentation of newly synthesized high-molecular-weight DNA (Hirt pellet material) contributing to the Hirt supernatant. Histone synthesis and acetylation in infected cells. Histone synthesis, or at least accumulation of newly made histones in the nucleus (see Groppi and Coffino, 1980) is coupled to cellular DNA synthesis (Borun et al., 1967). SV40 infection stimulates
histone synthesis as well as cellular DNA synthesis, although there does not appear to be strict temporal coupling between histone and DNA synthesis in infected cells (Kay and Singer, 1977). Since SV40 infection removes the butyrate-induced block to DNA synthesis, it was of interest w:hether histone synthesis would also ensue. Incorporation of [‘“Cl arginine into acidsoluble protein in nuclei was measured in butyrate-treated cells at various times after infection. In a labeling period 30-36 hr after infection, SV40-infected butyratetr#eated cells showed an amount of incorporation into this material equal to that in untreated infected cells. Earlier in infe’ction of butyrate-treated cells, as in uninfected cells, no histone synthesis was detectable (data not shown). Gel electrophoresis confirmed the identity of the newly synthesized material as histone (Fig. 5). The newly made histones in butyrate-treated infected cells appeared to be hyperacetylated, as judged by the absence of a discrete band of histone H4 on an acid-urea gel. This is character-
204
DANIELL,
BURG,
istic of highly acetylated histone populations analyzed in this gel system, caused by the distribution of H4 into several species with different mobilities. Histone acetylation in butyrate-treated infected cells was further studied by labeling cells with [14C]acetate. Two different protocols were employed. In the first, cells were treated with butyrate for 15 hr prior to infection, which proceeded in the presence of butyrate, and [14C]acetate was added to the medium 30 hr after infection, for 1 hr before harvesting. Acid soluble nuclear proteins were electrophoresed on Triton X-loo-acid-urea gels to discriminate histone species with high levels of acetylation. Figure 6 shows the stained gel (a-d) and autoradiogram (al-d’) of SV40infected and uninfected cells in the presence and absence of butyrate. The most distinct differences between hyperacetylated and normal histones appear in H4 and H2b in this gel system (Franklin and Zweidler, 1977). Clearly, SV40 infection has no effect on either the bulk level of acetylation (compare a and c) or the incorporation of [14C]acetate into highly acetylated species (a’ and c’) in butyratetreated cells. Comparison of lanes b’ and d’ shows that a pulsed acetate label enters more highly acetylated species of histones in SV40-infected than uninfected cells. An enhancement of acetylation in the histones associated with SV40 minichromosomes has been described previously (La Bella et al., 1979). In a complementary analysis, cells were infected with SV40 in the absence of butyrate and at 36 hr after infection, pulse labeled with [14C]acetate in the presence and absence of butyrate. These data show that SV40 infection has no effect on the immediate block to deacetylation imposed by butyrate. This data is not presented here, as the autoradiograph is virtually identical to that in Figs. 6a’-d’. DISCUSSION
We have shown that monkey cells inhibited by butyrate can be infected by SV40, and that cellular DNA and histone
AND
FEDOR a
b
c
d
a’
b’
c’
d’
H2b H4
FIG. 6. Acetylation of histones in butyrate-treated infected cells. Triton X-loo-acid-urea gel of acid-extractable nuclear proteins from butyrate treated (a, c) or untreated (b, d) cells labeled with [%]acetate for 1 hr before harvesting. (a, b); SV40 infected, (c, d) uninfected. (a-d) Amido black-stained gel; (a’-d’) autoradiograph of same gel.
synthesis are induced by infection, although we cannot say that they are stimulated to the same degree. In vitro experiments confirm that the ability of the blocked cells to incorporate deoxytriphosphates into DNA has been restored. However, the effects of butyrate have not been completely reversed. The butyrate-induced hyperacetylation of histones which is due to inhibition of histone deacetylase is unchanged by SV40 infection. In fact, the newly synthesized histones are hyperacetylated. The DNA replication which ensues during SV40 infection in the presence of butyrate is not entirely normal. The amount of viral DNA per cell at any time is reduced 4- to lo-fold, and the onset of viral DNA synthesis is later in butyrate-treated cells (Fig. 3; Table 3). Much of the cellular DNA synthesized is of low enough molecular weight to accumulate in a Hirt supernatant fraction. We have observed that 20% or more of the low residual level of rH]thymidine incorporated into cellular DNA in butyrate-inhibited uninfected cells segregates into the supernatant in a Hirt fractionation (Table 3). This may indicate
SV40 IN BUTYRATE-TREATED
that any cellular DNA synthesized in the presence of butyrate is fragmented and that SV40 stimulates synthesis of fragmented DNA. There is no evidence that newly synthesized viral DNA is broken down at a high rate. The nature of the block to DNA synthesis in butyrate-treated cells is not well understood. Rubenstein et al. (1979) have presented evidence that the inhibition is not directly related to histone hyperacetylation. Our studies show that the time it takes for DNA synthesis and cell division to cease in an unsynchronized population of cells is proportional to cell doubling time, suggesting a cell-cyle block (Daniell, 1980). The proposition that the block to DNA synthesis is a secondary effect is supported by the finding that SV40 infection can overcome this block without affecting histone acetylation. Further study of the newly synthesized cellular DNA, for example by determining its molecular weight under denaturing conditions, is warranted. Possibly the abnormal nature of the histones in these cells prevents proper chromatin segregation and results in loss of short newly replicated pieces of DNA from the chromosome. It is unclear why there is such a low yield of SV40 from these cells. The 50- to loo-fold decrease in infectious virus cannot be accounted for by the decrease in viral DNA. The SV40 DNA which is made has normal or near-normal superhelical density indicating a normal stoichiometry of association with histones (Germond et ah, 1975). Possibly the minichromosomes containing such highly acetylated histones cannot be packaged into infectious virions. This is particularly interesting because SV40 DNA in virions and in minichromosomes is associated with histones with a high degree of acetylation (Schaffhausen and Benjamin, 19’76; La Bella et ab, 1979). There may be limits to the degree of acetylation allowable for packaging and infectivity which the butyrate-induced hyperacetylation surpasses. Alternatively, hyperacetylated minichromosomes may not be properly transcribed. Analysis of late viral mRNAs and
CELLS
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capsid proteins in butyrate-treated cells, and physical quantitation of virus particles (as opposed to infectivity assays) will shed light on these questions. ACKNOWLEDGMENTS This material is based in part upon work supported by the National Science Foundation under Grant PCM’7821098, by BRSG Grant RR-7006 from the Biomedical Research Support Program, Division of Research Resources, National Institutes of Health, and by NIH National Research Service Award GM07232 from the National Institutes of General Medical Sciences. The authors thank Tom Wilkie for technical assistance and Barbara Kellogg for her pa,tient attention to revisions. REFERENCES BOFFA, L. C., VIDALI, G., MANN, R. S., and ALLFREY, V. G. (19’78). Suppression of, histone deacetylation in viva and in vitro by sodium butyrate. J. Biol. Chem. 253,3364-3366. BORUN, T. W., SCHARFF, M. D., and ROBBINS, E. (1967). Rapidly labeled, polysome-associated RNA having the properties of histone messenger. Proc. Nat. Ad Sci USA 68, 19’77-1983. OINDIDO, E. P. M., REEVES, R., and DAVIE, J. R. (1978). Sodium butyrate inhibits histone deaeetylation in cultured cells. Cell 14, 105-113. COUSENS, L. S., GALLWITZ, G., and ALBERTS, B. M. (1979). Different accessibilities in chromatin to histone acetylase. J. Biol. Chem. 254,1716-1723. DANIELL, E., and MULLENBACH, T. (1978). Synthesis of defective viral DNA in HeLa cells infected with adenovirus type 3. J. viral. 26, 61-70. DANIELL, E. (1980). Cells inhibited with n-butyrate support adenovirus replication. Virology 107, 514519. DENHARDT, D. (1966). A membrane filter technique for the detection of complementary DNA. B&hem Biophys.
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23, 641-646.
F.AREED, G. C., and DAVOLI, D. (1977). Molecular biology of papova viruses. Annu. Rev. B&hem. 46, 471-552. FEDOR, M., and DANIELL, E. (1980). Acetylation of histone-like proteins of Adenovirus type 5. J. viral. 35,637-643. FRANKLIN, S. G., and ZWEIDLER, A. (1977). Non-allelic variants of histone 2a, 2b and 3 in mammals. Nature
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FRASER, J. M. K., and HUBERMAN, J. A. (1977). In vitro HeLa cell DNA synthesis similarity to in tivo replication. J. Mol. Biol. 117, 249-272. GERMOND,
J. E., HIRT, B., OUDET,
P., GROSS-BELLARD,
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BURG, AND FEDOR
M., and CHAMBON, P. (1975). Folding of the DNA double helix in chromatin-like structures from simian virus 40. Proc. Nat. Acad Sci. USA 72,18431847. GRIFFITH, J. D. (1975). Chromatin structure deduced from a minichromosome. Science 187,1202-1203. GROPPI, V. E., and COFFINO,P. (1980). Gl and S-phase mammalian cells synthesize histones at equivalent rates. CeZZ21, 195-204. HAGOPIAN, H. K., RIGGS, M. G., SCHWARTZ,L. A., and INGRAM, V. M. (1977). Effect of n-butyrate on DNA synthesis in chick fibroblasts and HeLa cells. CeU 12, 855-860. HIRT, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26, 365-369.
KAY, A. C., and SINGER, M. F. (1977). Histone synthesis during infection of monkey kidney cells with simian virus 40. Nucleic Acids Res. 4, 3371-3386. KIT, G., and DANIELL, E. (1978). Adenovirus core protein synthesis in the absence of viral DNA synthesis late in infection. J. viral. 27, 74-80. KIT, S., DE TORRES, R. A., DUBBS, D. R., and SALUI, M. L. (1967). Induction of cellular deoxyribonucleic acid synthesis by simian virus 40. J. firol. 1, 738746. LA BELLA, F., VIDALI, G., and VESCO, C. (1979). Histone acetylation in CV-1 cells infected with simian virus 40. virology 96, 564-575. LASKEY, R. A., and MILLS, A. D. (1975). Quantitative film detection of 3H and 14Cpolyacrylamide gels by fluorography. Eur. 3. B&hem. 56,335-341. PANYIM, S., and CHALKLEY, R. (1969). High resolution acrylamide gel electrophoresis of histones. Arch. B&hem. Biqphys. 130,337-346.
PETT, D. M., ESTES, M. K., and PAGANO, J. S. (1975). Structural proteins of simian virus 40. I. Histone characteristics of low-molecular-weight polypeptides. J. I%-ol. 15, 379-385. RIGGS, M. G., WHITTAKER, R. G., NEUMANN, J. R., and INGRAM, V. M. (1977). n-Butyrate causes histone modification in HeLa and Friend erythroleukemia cells. Nature (London) 268,462-464. RITZI, E., and LEVINE, A. J. (1969). Deoxyribonucleic acid replication in simian virus IO-infected cells. III. Comparison of simian virus 40 lytic infection in three different monkey kidney cell lines. J. viro2 5, 686-692. RUBENSTEIN, P., SEALY, L., MARSHALL, S., and CHALKLEY, R. (1979). Cellular protein synthesis and inhibition of cell division are independent of butyrate-induced histone hyperacetylation. Nature
(London)
280, 692-693.
SEALY, L., and CHALKLEY, R. (1978). Effect of sodium butyrate on histone modification. Cell 14,115-121. SCHAFFHAUSEN, B. S., and BENJAMIN, T. L. (1976). Deficiency in histone acetylation in nontransforming host range mutants of polyoma virus. Proc. Nat. Acad. Sci. USA 73, 1092-1096. VIDALI, G., BOFFA, L. C., BRADBURY, E. M., and ALLFREY, V. G. (1978). Butyrate suppression of histone deacetylation leads to accumulation of multiacetylated forms of histones H3 and H4 and increased DNase I sensitivity of the associated DNA sequences. Proc. Nat. Acad Sci USA 75,2239-2243. WINOCOUR, E., and ROBBINS, E. (1970). Histone synthesis in polyoma- and SV40-infected cells. viralogy 40,307-315.
116,196-206
(1982)
DNA and Histone Synthesis in Butyrate-Inhibited Cells Infected with SV40 ELLEN
DANIELL,l
Department
J. LAWRENCE
of Molecular Received
Biology, June
BURG,
University 17, 1981;
AND
of California, accepted
August
BSC-1
MARTHA Berkeley,
J. .FEDOR
California
9.4720
20, 1981
The effect of butyrate on DNA synthesis, histone synthesis, and virus production in SV40-infected monkey cells has been studied. When cells in which DNA and histone synthesis have been blocked by treatment with butyrate are infected with SV40, DNA synthesis is induced to a level at least equal to that in untreated infected cells. Both viral and cellular DNA sequences are replicated. Viral DNA synthesized is predominantly SV40 Form I. The amount of viral DNA which accumulates is reduced by lo-fold in the presence of butyrate, and the yield of infectious virus is lowered loo-fold. Histone synthesis in these cells is stimulated concomitantly with DNA synthesis. The hyperacetylation of histones characteristic of butyrate-treated cells is unchanged by SV40 infection; the newly synthesized histones are hyperacetylated. Thus the mechanism by which butyrate blocks DNA and histone synthesis is not directly related to the hyperacetylation of histones.
minichromosomes which are the templates for viral transcription and replication (Griffith, 1975; Pett et al., 1975). SV40 infection stimulates both cellular DNA synthesis and histone synthesis, and the histones made are deposited on viral as well as cellular DNA (Kit et al., 1967; Ritzi and Levine, 1969; Winocour and Robbins, 1970). SV40 and cellular DNA replication share a number of features, including a tight dependence on protein synthesis, bidirectional replication from each origin, and use of host DNA polymerase a (Fareed and Davoli, 1977). In the present work we investigated the effect of butyrate on SV40 infection of BSC-1 cells to ascertain whether the block to cellular DNA replication and histone synthesis would inhibit SV40 replication. This study was begun as a complement to the demonstration that adenoviruses replicate in butyrate-treated cells with full efficiency, accompanied by no detectable cellular DNA or histone synthesis (Daniell, 1980). The differences between these two types of viruses in mode of replication and hostvirus interaction are highlighted by the results reported here.
INTRODUCTION
Addition of millimolar amounts of butyrate to the medium of tissue culture cells rapidly results in increased levels of histone acetylation (Vidali et al., 1978), due to the inhibition of histone deacetylases (Candid0 et al., 1978; Cousens et al., 1979; Sealy and Chalkley, 1978; Boffa et al., 1978). After longer periods of treatment, cell DNA synthesis and cell division cease (Riggs et al., 1977; Hagopian et al., 1977), as does histone synthesis (Daniell, 1980). These long-term effects do not appear to result directly from the hyperacetylation of histones (Rubenstein et al., 1979) and kinetics of shutoff of DNA synthesis are consistent with a block at a particular stage in the cell cycle (Daniel& 1980). Both immediate and long-term effects are readily reversible; cells remain viable and return to normal when fed with butyratefree medium. The DNA of simian virus 40 (SV40) is associated with histones both in mature virus particles and in the intracellular 1 To whom
reprint
requests
should
0042-6822/82/010196-11$02.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
be addressed. 196
SV40 IN BUTYRATE-TREATED
MATERIALS
AND
METHODS
Cells and viruses. BSC-1 (monkey kidney) cells were cultured as monolayers in Dulbecco’s medium (DME) supplemented with 5% calf serum. Plaque-purified SV40 (small plaque strain) was propagated and titrated on BSC-1 cells. Infections were performed at a multiplicity of 10 PFU/cell unless otherwise stated. For infection, medium was removed from cell monolayers, and dilutions of virus in phosphatebuffered saline (PBS) were added to the cells. As SV40 stocks were prepared and stored in DME, mock-infected cells were treated with appropriate dilutions of DME into PBS. Conditioned medium was added back to the cells after 2 hr of adsorption. Sodium butyrate (Baker) was added to tissue culture medium as a dilution of an 0.5 M stock in sterile PBS. A final concentration of 6.5 mM butyrate was used to inhibit DNA synthesis and cell division in BSC-1 cells. Butyrate was added 24 hr before infection unless otherwise stated. Virus production was measured by scraping cells into the medium, freezing, and thawing four times, and sonicating before plaque titration on BSC-1 cells. DNA synthesis in infected- and uninfected cells. Cells were labeled with [3H]thymidine (1 &i/ml, 58 Ci/mmol) for 1 hr at times indicated; incorporated label was measured as previously described (Daniell, 1980), and counts were normalized to counts per minute per lo5 cells. Shutoff of DNA synthesis was operationally defined as a level of thymidine incorporation 5-10% that in untreated, rapidly growing (i.e., nonconfluent) cells. Histone synthesis and acetylation. To determine whether newly synthesized histone was accumulating in nuclei under various conditions, cells were labeled for 5 hr with 1 &i/ml L-[14C(U)]arginine (298 mCi/mmol) in DME plus 5% dialyzed fetal calf serum. Isolation and extraction of nuclei with sulfuric acid followed by acetone precipitation of acid-soluble proteins and electrophoresis on the, acid-urea gel system of Panyim and Chalkley (1969) have been described previously (Kit and Daniell, 1978).
CELLS
197
Histone acetylation was determined by ad!dition of 10 &i/ml [14C]acetate (58.7 mCi/mmol) to the medium for 1 hr, followed by electrophoresis of acid extracted histones on Triton X-loo-acid-urea gels (Fedor and Daniell, 1980). Different schedules of butyrate treatment, infection, and labeling are described in the text and figure legends. DNA synthesis in cell lysates. Lysates were prepared and DNA synthesis measured by the method of Fraser and Huberman (1977). Butyrate-treated cells were treated with 6.5 mM butyrate for 3-24 hr before infection with SV40 (5 PFU/cell). Cell lysates were prepared 30 hr postinfection, and DNA synthesis measured as incorporation of [a-32P]dTTP (7.5 &i/ml) into TCA-precipitable counts. The final concentration of KC1 in the reaction mix was 18 mM; all other components are as described in Fraser and Huberman (1977). Isolation of DNA. Preparation of total cellular and viral DNA from isolated nuclei has been described (Daniel1 and Mullenbach, 1978). Probes for hybridization experiments were prepared by this method from cells labeled with [3H]thymidine 2836 hr postinfection, followed by sonication to reduce the size of the DNA to an average of 300 nucleotides. Alternatively, low-molecular-weight, predominantly viral DNA was separated from high-molecular-weight cellular DNA by the method of Hirt (1967). In uninfected cells, 94-98% of cellular DNA remains in the 1 M NaCl-SDS insoluble pellet. Ninety to ninety-five percent of SV40 DNA in infected cells is in the soluble “supernatant” fraction (Kay and Singer, 1977; Hirt, 1967). Gel electrophoresis and jluorography. Electrophoresis was performed on 1% agarose gels, as described (Daniel1 and Mullenbach, 1978). To detect [3H]thymidine counts by autoradiography, gels were dehydrated in methanol (double the gel volume for three times 45 min), then soaked in 2 vol of a 5% (w/v) solution of PPO (2,5diphenyloxozole; Packard) in methanol for l--l’/2 hr (Laskey and Mills, 1975). The gel was rinsed in water, and soaked in water with 6% glycerol (v/v), dried under vac-
198
DANIELL,
BURG,
uum at room temperature, and autoradiographed with Kodak XR-5 film. After autoradiography, gels impregnated with PPO could be sliced and counted by Cerenkov counting. The efficiency of counting by this method is about 40%, and is more reproducible than dissolving and counting agarose gel slices.
RESULTS
Effect of butyrate on monkey cells. The minimal concentration of butyrate needed to shut off cellular DNA synthesis was determined to be 6.5 mM for BSC-1 cells. As observed for other cell lines, DNA synthesis declined to minimal levels (~5% normal) in one cell-doubling time. Cells maintained for 72 hr in this concentration of butyrate resumed DNA synthesis, cell division and normal morphology within 24 hr after replacing the medium with fresh or conditioned butyrate-free medium. Some increased sensitivity to long treatment with butyrate was noted if cells had been passaged for several months after thawing. When cell death or failure to resume growth when cultured without butyrate was observed in uninfected cultures maintained throughout an experiment, the results were discarded. Overall rates of protein and RNA synthesis did not change significantly in butyrate-treated cells (less than 10%; data not shown). Histone synthesis (measured as acid-extractable counts in nuclei, and analyzed by gel electrophoresis and fluorography) is shut off by more than 90%. The experiments reported here were performed with BSC-1 cells, in which induction of cellular DNA synthesis is less than in CV-1 and other monkey cell types (Ritzi and Levine, 1969). Yield of infectious virus is reduced in butyrate-treated cells. The effect of butyrate on virus yield was investigated by infecting BSC-1 cells which had been pretreated with butyrate, and by infection of normal cells, adding butyrate only after virus adsorption. Results of representative experiments are shown in Table 1. The
AND
FEDOR
TABLE
1
YIELDOFINFECTIOUSSV~O FROMBUTYRATETREATEDCELLS Virus Multiplicity of infection 10 0.1 50 10
+Butyrate 32” 1.2” 75” 206
yield
(PFU/cell)
-Butyrate 1800 190 2000 1500
-/+ 56 160 26 75
‘Cells which were not pretreated with butyrate were infected. 6.5 mM sodium butyrate was added when medium was replaced after virus adsorption. b Cells were pretreated with 6.5 mM butyrate for 24 hr and the conditioned medium with butyrate was replaced after virus adsorption.
number of plaque-forming units produced in butyrate-treated cells under either schedule of butyrate treatment was significantly lower than the yield in untreated cells. At low multiplicities of infection, the difference is greater than lOOfold. Experiments in which butyrate was added only after infection are most instructive, as efficiency of virus adsorption or confluency of cells are not variable. Clearly virus multiplication is inhibited by the presence of butyrate in the medium. DNA synthesis in butyrate-treated cells following SViO infection. Incorporation of [3H]thymidine in a 1-hr pulse was measured at various times after infection of BSC-1 cells with SV40. In the experiment shown in Fig. 1, untreated cultures nearing confluency were used so that thymidine incorporation was low before infection, allowing significant stimulation of total DNA synthesis in those cultures as well as in butyrate-inhibited cells. Figure 1 illustrates that thymidine incorporation is induced by SV40 in butyrate-treated cells with similar kinetics to those observed in normal confluent cells. Peak levels of incorporation were consistently higher in butyrate-treated cells. The use of rH]thymidine incorporation in vivo to compare levels of DNA synthesis in infected and uninfected cells is subject
SV40
0
5
10
IN BUTYRATE-TREATED
CELLS
15 20 Z!j 30 Hours after infection
35
199
40
45
50
FIG. 1. DNA synthesis in BSC-1 cells infected with !W40 in the presence of butyrate. Cells were labeled for 1 hr with rH]thymidine and harvested at times indicated, incorporation was measured as described and normalized to cpm per lo5 cells. The four curves represent: (0) butyrate-treated cells;, (A) butyrate-treated cells, SV40 infected, (0) untreated cells; (a) untreated cells, SV40 infected.
to complication from changing pool sizes, kinase levels and cell permeability changes. To demonstrate that SV40 infection really restored the ability of cells to replicate DNA, we employed a cell-free system. Hagopian et al. (1977) demonstrated that a crude cell lysate of HeLa cells which have been exposed to butyrate for 24 hr is completely inactive in incorporation of deoxytriphosphates or thymidine (6% the activity of normal lysates). Figure 2 shows that lysates from uninfected BSC-1 cells treated with butyrate have little DNA replication activity, while lysates from SV40infected butyrate treated cells have activity equal to normal cells. Characterization of DNA synthesized. The DNA synthesized in butyrate-treated infected cells was characterized by hybridization, Hirt extraction and by gel electrophoresis. DNA from cells labeled with [3H]thymidine from 28 to 34 hr postinfection was hybridized to SV40 and to BSC-1 DNA immobilized on filters. The
results, shown in Table 2, indicate that in butyrate-treated cells, a large fraction of the newly synthesized DNA consists of cellular sequences. EDNA was extracted from cells labeled for 1 hr at various times after infection by the method of Hirt (1967). This method separates viral (low molecular weight) from host cell DNA, and has been employed in previous studies of cellular and viral DNA synthesis in SV40-infected cells (Kay and Singer, 1977; Ritzi and Levine, 1969). However, this method proved not to be useful for comparing incorporation into cellular and viral sequences in the presence of butyrate. Electrophoretic analysis of the DNA in Hirt supernatants revealed that in butyrate-treated cells, a large fraction of the counts in these preparations is in a band of higher molecular weight than viral DNA. Figure 3 shows Hirt supernatants prepared from cells labeled for 1 hr at various times after infection. Gel bands were excised and counted to deter-
DANIELL,
200
BURG,
AND
FEDOR
35
abcdefgh
Cdl
sv40 (II)
sv40 (I)
Cdl
sv40
0
0
5
10
15 Time,
20
25
30
sv40
min
FIG. 2. Incorporation of [&‘P]TTP into DNA in a cell lysate. Lysates were prepared 36 hr after infection (48 hr after butyrate addition) as described under Materials and Methods, and incorporation of TTP was measured as a function of time: (0) untreated BSC-1 cells; (0) BSC-1 cells treated with butyrate; (A) SV40-infected cells without butyrate; (0) SV40 infected cells, treated with butyrate.
mine the fraction of DNA in viral bands (Table 3). At 22 hr after infection no viral DNA is detectable in butyrate-treated cells (lane a). At 28 hr only 40% of the TABLE
FIG. 3. Analysis of Hirt supernatant DNA from BSC-1 cells infected with SV40 in the presence or absence of butyrate. Cells were labeled with [qlthymidine for 1 hr prior to harvest by Hirt fractionation. DNA from supernatant fractions was purified by extraction and precipitation and subjected to gel electrophoresis through lo-cm agarose gels (1%) for 16 hr at 20 V. At the top is the photograph of the ethidium stained gel, below is the autoradiograph of the same gel. DNA from an equal number of cells was loaded onto each slot. (a, c, e, and g) + butyrate, and (b, d, f, and h) - butyrate. (a, b) 22 hr after infection, (c, d) 28 hr after infection, (e, f) 38 hr after infection, and (g, h) 46 hr after infection.
2
HYBRIDIZATIONOFLABELEDDNAFROMINFECTEDBUTYRATE-TREATEDANDUNTREATEDCELLS TO SV40 AND TO BSC DNA”
Specific activity (X10* cpm/pg)
Probe BSCSVIO (+B) BSCSV40 (-B) BSC (Mock) “Hybridization incubated with blank.
9 3 4
cpm hybridized filter
to
BSC
sv40
4650 1400 3596
1070 3360 122
was carried out by the method of Denhardt three filters: one carrying 50 pg of BSC-1 DNA,
SVIO/BSC .23 2.4 .03
(1966). 5 X lo4 counts of each probe was one carrying 10 pg of SV40 DNA, and one
SV40 IN BUTYRATE-TF1EATED TABLE
CELLS
201
3
DISTRIBUTIONOFNEWLYSYNTHESIZED DNA II‘ITOHIRTSUPERNATANTS ANDPELLETS ATDIFFERENTTIMESAFTF.RSV~O INFECTION Labeling period” (hr after infection)
Thymidine incorporationb (X10’ cpm)
Counts in Hirt supernatant (% total incorporation)
Percentage SV40 in supernatant’
Butyrate
sv40
Mock (22)
+ -
-
0.06 0.73
>15d 2
22
+ -
+ +
0.48 1.6
30 57
12 82
28
+ -
+ +
1.25 1.5
49 70
40 92
37
+ -
+ +
2.9 2.1
65 78
60 95
46
+ -
+ +
1.8 2.9
68 80
80 95
-
a All labeling periods were 1 hr long, ending at the time specified. * Normalized to counts per lo5 cells. ’ Gels were sliced and counted after fluorography and the fraction of the total which was present in SV40 DNA (Forms I, II, and II) was calculated. ’ The distribution of label during Hirt fractionation of butyrate-treated uninfected cells was highly variable ranging from 15 to 50% in different experiments.
counts in the Hirt supernatant of butyrate-treated cells (lane c) are in viral bands, whereas in untreated cells, 92% of the label is viral (lane d). At later times, the proportion of viral DNA in supernatants from butyrate-treated cells increases. The “upper band” DNA has been further characterized and its cellular origin confirmed. Hybridization of SV40 probe to Southern blots of these gels show that viral DNA does not hybridize to this band. Figure 4 illustrates that this band is present in Hirt supernatants from uninfected cells (lane c and d), and has a molecular weight greater than 21 kb. Electrophoresis on lower-percentage gels shows that the material is diffuse but all of it runs more slowly than adenovirus type 5 DNA used as a marker of 35 kb (data not shown). When this material is eluted from gels and treated with a variety of restriction endonucleases, no discrete fragments are generated, though the DNA is cleaved into small pieces. This not only confirms that
the material is not viral, but suggests that it. represents a nonspecific assortment of cellular sequences. In the samples in Fig. 4, cells were lab’eled for 15 hr (from 23 to 38 hr after infection or mock infection). The fluorograph (lanes c’-g’) shows that label alecumulates in the cellular material as a result of SV40 infection, and to a far greater extent in butyrate-treated than in untreated cells. The amount of SV40 (Forms I and II) per cell is less in butyrate-treated cells at all times, although the amount of label in a pulse surpasses that in untreated cells after 32 hr. It is also clear from Figs. 3 and 4 that the SV40 synthesized in butyrate-treated cells is predominantly Form I (supercoiled). To ascertain whether viral DNA turns over at an increased rate in butyratetreated cells, we compared DNA from cells labeled for different lengths of time. Two identical cultures were labeled for 1 and for 6 hr, respectively, and harvested by the Hirt procedure or by extraction of whole
DANIELL,
202
a
35 kb
21 kb 14kb
BURG,
AND
FEDOR
c'
bcdefg
d'
f'
g'
‘\ -/-
FIG. 4. Electrophoresis of Hirt supernatants from infected and mock-infected cells. Samples were prepared, electrophoresed, and fluorographed as in Fig. 3 except that the labeling was from 23 to 38 hr after infection. (a) Ad5 DNA marker; (b, e) Ad5 DNA cleaved with BarnHI; (c, c’) mockinfected cells, butyrate added 35 hr before start of labeling; (d, d’) mock-infected cells, no butyrate; (f, f’) SV40-infected cells, butyrate added 12 hr prior to infection; and (g, g’) SV40-infected cells, no butyrate.
cells. The physical amount of DNA in each sample is the same, and the ratios between long and short label represent the rate of TABLE ACCUMULATION
Butyrate
DNA Hirt Hirt
OF [3H]T~~~~~~~~
fraction
supernatantb pellet!
SV40"
Celld Hirt supernatant* Hirt pelletb
accumulation of counts in any fraction. Results of a typical experiment are shown in Table 4. Three- or fourfold more counts 4 IN DNA
FROM INFECTED
CELLS~
6 hr (x10-3)
1 hr (x10-3)
140
34 18 3.9
73 8.9 22.0 35
11
SV40"
8.6
Celld
1.1
3.6 11.2 1.8 3.0 0.29
6/l 4.1 4.1 2.3 6.1 3.1 6.0 2.9 3.8
“DNA was isolated by Hirt fractionation from cells labeled for 1 hr or for 6 hr prior to harvesting (37 hr after infection). After phenol extraction and precipitation, aliquots of supernatant material were electrophoresed through 1% agarose, stained, photographed, treated with PPO, and sliced and counted as described under Materials and Methods. b TCA-precipitable counts in 100 pl of Hirt supernatant or Hirt pellet resuspended in a volume equal to the supernatant. ‘Sum of Form I and Form II. d The “high-molecular-weight” band from Hirt supernatants.
SV40
IN BUTYRATE-TREATED
(a) 1
2
3
4
5
203
CELLS
(b’
1
2
3
4
5
Hl
FIG. 5. Histone synthesis in butyrate-treated SV40-infected cells. Acid-urea gel of aeid-extractable nuclear proteins labeled with [%]arginine from 218 to 36 hr after infection (or mock infection). (a) Amido black-stained gel, photographed. (b) Autoradiograph: 1, SV40-infected cells, 5 X lo3 cpm loaded; 2, uninfected cells, butyrate added 40 hr before labeling, 600 cpm loaded; 3, uninfected cells, no butyrate (1.5 X lo3 epm); 4, SV40-infected cells, butyrate added 12 hr before infection (40 hr before labeling) (1.5 X lo3 cpm); and 5, SV40-infected. cells (1 X lo3 cpm).
accumulated in Hirt supernatants during the long-labeling period than during the short-labeling period, the treated cells showing a somewhat higher ratio than untreated cells. There is no significant difference between butyrate-treated cells and untreated cells in the relative amount of labeling of SV40 DNA during longand short-labeling intervals. In butyratetreated cells the ratio of long to short labeling intervals. In butyrate-treated cells the ratio of long to short labeling of the cellular band of the Hirt supernatant is higher than in untreated cells, and the Hirt pellet does not show the six-fold accumulation of counts detected in untreated cell chromosomal DNA. These anomalies may reflect fragmentation of newly synthesized high-molecular-weight DNA (Hirt pellet material) contributing to the Hirt supernatant. Histone synthesis and acetylation in infected cells. Histone synthesis, or at least accumulation of newly made histones in the nucleus (see Groppi and Coffino, 1980) is coupled to cellular DNA synthesis (Borun et al., 1967). SV40 infection stimulates
histone synthesis as well as cellular DNA synthesis, although there does not appear to be strict temporal coupling between histone and DNA synthesis in infected cells (Kay and Singer, 1977). Since SV40 infection removes the butyrate-induced block to DNA synthesis, it was of interest w:hether histone synthesis would also ensue. Incorporation of [‘“Cl arginine into acidsoluble protein in nuclei was measured in butyrate-treated cells at various times after infection. In a labeling period 30-36 hr after infection, SV40-infected butyratetr#eated cells showed an amount of incorporation into this material equal to that in untreated infected cells. Earlier in infe’ction of butyrate-treated cells, as in uninfected cells, no histone synthesis was detectable (data not shown). Gel electrophoresis confirmed the identity of the newly synthesized material as histone (Fig. 5). The newly made histones in butyrate-treated infected cells appeared to be hyperacetylated, as judged by the absence of a discrete band of histone H4 on an acid-urea gel. This is character-
204
DANIELL,
BURG,
istic of highly acetylated histone populations analyzed in this gel system, caused by the distribution of H4 into several species with different mobilities. Histone acetylation in butyrate-treated infected cells was further studied by labeling cells with [14C]acetate. Two different protocols were employed. In the first, cells were treated with butyrate for 15 hr prior to infection, which proceeded in the presence of butyrate, and [14C]acetate was added to the medium 30 hr after infection, for 1 hr before harvesting. Acid soluble nuclear proteins were electrophoresed on Triton X-loo-acid-urea gels to discriminate histone species with high levels of acetylation. Figure 6 shows the stained gel (a-d) and autoradiogram (al-d’) of SV40infected and uninfected cells in the presence and absence of butyrate. The most distinct differences between hyperacetylated and normal histones appear in H4 and H2b in this gel system (Franklin and Zweidler, 1977). Clearly, SV40 infection has no effect on either the bulk level of acetylation (compare a and c) or the incorporation of [14C]acetate into highly acetylated species (a’ and c’) in butyratetreated cells. Comparison of lanes b’ and d’ shows that a pulsed acetate label enters more highly acetylated species of histones in SV40-infected than uninfected cells. An enhancement of acetylation in the histones associated with SV40 minichromosomes has been described previously (La Bella et al., 1979). In a complementary analysis, cells were infected with SV40 in the absence of butyrate and at 36 hr after infection, pulse labeled with [14C]acetate in the presence and absence of butyrate. These data show that SV40 infection has no effect on the immediate block to deacetylation imposed by butyrate. This data is not presented here, as the autoradiograph is virtually identical to that in Figs. 6a’-d’. DISCUSSION
We have shown that monkey cells inhibited by butyrate can be infected by SV40, and that cellular DNA and histone
AND
FEDOR a
b
c
d
a’
b’
c’
d’
H2b H4
FIG. 6. Acetylation of histones in butyrate-treated infected cells. Triton X-loo-acid-urea gel of acid-extractable nuclear proteins from butyrate treated (a, c) or untreated (b, d) cells labeled with [%]acetate for 1 hr before harvesting. (a, b); SV40 infected, (c, d) uninfected. (a-d) Amido black-stained gel; (a’-d’) autoradiograph of same gel.
synthesis are induced by infection, although we cannot say that they are stimulated to the same degree. In vitro experiments confirm that the ability of the blocked cells to incorporate deoxytriphosphates into DNA has been restored. However, the effects of butyrate have not been completely reversed. The butyrate-induced hyperacetylation of histones which is due to inhibition of histone deacetylase is unchanged by SV40 infection. In fact, the newly synthesized histones are hyperacetylated. The DNA replication which ensues during SV40 infection in the presence of butyrate is not entirely normal. The amount of viral DNA per cell at any time is reduced 4- to lo-fold, and the onset of viral DNA synthesis is later in butyrate-treated cells (Fig. 3; Table 3). Much of the cellular DNA synthesized is of low enough molecular weight to accumulate in a Hirt supernatant fraction. We have observed that 20% or more of the low residual level of rH]thymidine incorporated into cellular DNA in butyrate-inhibited uninfected cells segregates into the supernatant in a Hirt fractionation (Table 3). This may indicate
SV40 IN BUTYRATE-TREATED
that any cellular DNA synthesized in the presence of butyrate is fragmented and that SV40 stimulates synthesis of fragmented DNA. There is no evidence that newly synthesized viral DNA is broken down at a high rate. The nature of the block to DNA synthesis in butyrate-treated cells is not well understood. Rubenstein et al. (1979) have presented evidence that the inhibition is not directly related to histone hyperacetylation. Our studies show that the time it takes for DNA synthesis and cell division to cease in an unsynchronized population of cells is proportional to cell doubling time, suggesting a cell-cyle block (Daniell, 1980). The proposition that the block to DNA synthesis is a secondary effect is supported by the finding that SV40 infection can overcome this block without affecting histone acetylation. Further study of the newly synthesized cellular DNA, for example by determining its molecular weight under denaturing conditions, is warranted. Possibly the abnormal nature of the histones in these cells prevents proper chromatin segregation and results in loss of short newly replicated pieces of DNA from the chromosome. It is unclear why there is such a low yield of SV40 from these cells. The 50- to loo-fold decrease in infectious virus cannot be accounted for by the decrease in viral DNA. The SV40 DNA which is made has normal or near-normal superhelical density indicating a normal stoichiometry of association with histones (Germond et ah, 1975). Possibly the minichromosomes containing such highly acetylated histones cannot be packaged into infectious virions. This is particularly interesting because SV40 DNA in virions and in minichromosomes is associated with histones with a high degree of acetylation (Schaffhausen and Benjamin, 19’76; La Bella et ab, 1979). There may be limits to the degree of acetylation allowable for packaging and infectivity which the butyrate-induced hyperacetylation surpasses. Alternatively, hyperacetylated minichromosomes may not be properly transcribed. Analysis of late viral mRNAs and
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capsid proteins in butyrate-treated cells, and physical quantitation of virus particles (as opposed to infectivity assays) will shed light on these questions. ACKNOWLEDGMENTS This material is based in part upon work supported by the National Science Foundation under Grant PCM’7821098, by BRSG Grant RR-7006 from the Biomedical Research Support Program, Division of Research Resources, National Institutes of Health, and by NIH National Research Service Award GM07232 from the National Institutes of General Medical Sciences. The authors thank Tom Wilkie for technical assistance and Barbara Kellogg for her pa,tient attention to revisions. REFERENCES BOFFA, L. C., VIDALI, G., MANN, R. S., and ALLFREY, V. G. (19’78). Suppression of, histone deacetylation in viva and in vitro by sodium butyrate. J. Biol. Chem. 253,3364-3366. BORUN, T. W., SCHARFF, M. D., and ROBBINS, E. (1967). Rapidly labeled, polysome-associated RNA having the properties of histone messenger. Proc. Nat. Ad Sci USA 68, 19’77-1983. OINDIDO, E. P. M., REEVES, R., and DAVIE, J. R. (1978). Sodium butyrate inhibits histone deaeetylation in cultured cells. Cell 14, 105-113. COUSENS, L. S., GALLWITZ, G., and ALBERTS, B. M. (1979). Different accessibilities in chromatin to histone acetylase. J. Biol. Chem. 254,1716-1723. DANIELL, E., and MULLENBACH, T. (1978). Synthesis of defective viral DNA in HeLa cells infected with adenovirus type 3. J. viral. 26, 61-70. DANIELL, E. (1980). Cells inhibited with n-butyrate support adenovirus replication. Virology 107, 514519. DENHARDT, D. (1966). A membrane filter technique for the detection of complementary DNA. B&hem Biophys.
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