Efficient Transcription of a Compact Nucleoprotein Complex Isolated ...

JOURNAL OF VIROLOGY, Aug. 1980, p. 371-381 0022-538X/80/08-0371/11$02.00/0

Vol. 35, No. 2

Efficient Transcription of a Compact Nucleoprotein Complex Isolated from Purified Simian Virus 40 Virions JOHN N. BRADY,* CHRISTIAN LAVIALLE, AND NORMAN P. SALZMAN Laboratory of Biology of Viruses, National Institute ofAllergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20205

Simian virus 40 (SV40) virions were dissociated in vitro by treatment with ethylene glycol-bis-N-N'-tetraacetic acid and dithiothreitol. The compact nucleoprotein core released as a result of the dissociation had a sedimentation value of 110 to 115S compared with a value of 240S for intact virions. The viral cores contained a fraction of the viral proteins VP, and VP2 in addition to the proteins found associated with the viral minichromosome, i.e., VP3 and histones H2A, H2B H3, and H4. Our results suggest that the association of VP,, VP2, or both with the viral minichromosome, in addition to maintaining a highly compact structure, modifies the transcriptional properties of the nucleoprotein complex. In the presence of saturating amounts of Escherichia coli RNA polymerase, 95 to 100% of the SV40 nucleoprotein cores were able to form transcriptional complexes. Sedimentation analysis of the core transcriptional complex indicated that the initiation and elongation of nascent RNA chains occurred on the compact SV40 core. Cesium chloride density gradient analysis of the SV40 virion core before and after transcription indicated that no substantial loss of protein occurred during the process of transcription. RNA synthesized from SV40 cores was a fairly homogeneous 16 to 18S species with an average chain length of approximately 2,300 nucleotides. Hybridization analysis of this RNA indicated that specific recognition of RNA polymerase promoter sites was preserved, since transcription was asymmetric, occurring preferentially on the "early" SV40 DNA strand. The rate of incorporation of ribonucleoside triphosphates into acid-insoluble RNA with SV40 cores as the template was 70 to 95% of that obtained with supercoiled SV40 form I DNA. SV40 minichromosomes, under identical transcription assay conditions, had an incorporation rate which was 20% of that obtained with SV40 form I DNA. These results show that association of protein VP, or VP2 or both enhances the transcriptional activity and suggest that these "late" viral proteins may play a role in the regulation of expression of the SV40 genome. Present evidence indicates that most, if not all, eucaryotic genes which are actively involved in RNA transcription contain nucleosomes (11, 14, 21, 40, 47), the basic units of chromatin. Since nucleosomes serve as efficient inhibitors of initiation of RNA synthesis and elongation of RNA chains (3, 7, 48), experiments have been carried out to determine how DNA-histone interactions are modified in active regions of chromatin. DNase I nuclease digestion studies indicate that the chromatin structure in actively transcribing genes is more accessible to cleavage than is that of the inactive regions of chromatin (14, 47). Association of nonhistone proteins (8, 19, 24, 43), loss of histone HI (4, 9, 23), and acetylation of histones (37) in transcriptionally active genes have also been demonstrated. However, the means by which the RNA polymerase enzyme transcribes through the intra-nucleosomal DNA sequences is not clear. The study of this basic

aspect of transcription has been complicated by the absence of a simple controlled system in which interactions between DNA, polymerase, histone, and nonhistone proteins in a transcriptionally active complex could be analyzed. Due to the relative simplicity of simian virus 40 (SV40) nucleoprotein complexes and the ability to isolate SV40 DNA, DNA-histone (minichromosome), and DNA-histone viral protein (core) complexes from purified SV40 virions, we feel that the transcriptional system described in this paper offers a model system for the study of eucaryotic transcription. With purified SV40 virions and Escherichia coli RNA polymerase, only a very limited number of proteins and a well-characterized SV40 DNA molecule are present. Comparative studies of the transcriptional properties of papovavirus DNA and minichromosomes have been reported by a number of investigators (16-18, 33, 34). However, the 371

372

BRADY, LAVIALLE, AND SALZMAN

SV40 core transcriptional complex that we describe is unique and is dependent upon a mild, in vitro dissociation of SV40 virions which leaves a select group of virion proteins associated with the DNA-histone complex (1, 2). The results presented in this paper suggest that the association of these virion proteins with the minichromosome converts the complex to a more efficient template for RNA synthesis. This is the first suggestive evidence that "late" viral proteins of SV40 may play a role in the regulation of expression of the SV40 genome. MATERIALS AND METHODS Viruses and cells. Wild-type SV40 (strain 776) was originally obtained from K. Takemoto. Virus was grown in BSC-1 cells. Infection and purification of SV40 virions. Confluent monolayers of BSC-1 cells, maintained in Eagle minimal essential medium supplemented with 0.03% glutamine, 10% fetal calf serum, and streptomycin, penicillin, ampicillin, and mycostatin were infected with wild-type SV40 at a multiplicity of infection of 10 PFU/cell. After a 2-h adsorption at 37°C, Eagle minimal essential medium supplemented with 1 to 2% fetal calf serum was added. After 7 to 8 days, infected cells and supernatant were collected, and the virus was purified through the equilibrium CsCl gradient step as described previously (22). Viral bands were collected after CsCl isopycnic banding, diluted to a density of 1.2 g/cm3 with sterile phosphate-buffered saline, and layered onto a four-step CsCl gradient consisting of 0.9 ml of 1.35, 1.32, 1.29, and 1.26 g/cm3 layered in an SW50.1 nitrocellulose centrifuge tube. Gradients were centrifuged at 35,000 rpm for 3 h. Viral bands were collected and dialyzed against sterile 0.01 M Tris buffer (pH 7.4) at 4°C. Virus was stored at 4°C until used. All solutions used in the in vitro experiments described below were sterile. Dissociation of SV40 virions: preparation of SV40 cores and minichromosomes. Preparation of SV40 nucleoprotein cores was carried out by ethylene glycol-bis-N-N'-tetraacetic acid (EGTA)-dithiothreitol (DTT) dissociation of SV40 virions as recently described for polyoma (2), except that the disruption was performed at 320C for 30 min. The preparation of minichromosomes from SV40 virions was carried out by the method of Christiansen et al. (5). Velocity sedimentation of nucleoprotein complexes. Dissociated SV40 virions were layered onto isokinetic 10 to 30% sucrose gradients containing 5 mM EGTA, 3 mM DTT, and 150 mM NaCl in 10 mM Tris-hydrochloride (pH 7.8) and 0.05% Triton X-100. Centrifugation was in an SW41 rotor at 39,000 rpm (4°C) for the time indicated in the figure legends. Isokinetic gradients were prepared by the method of McCarty et al. (30). In vitro transcription of SV40 DNA and nucleoprotein complexes. Each reaction contained, in a total volume of 100 Id, 50 mM Tris (pH 7.8), 1 mM DTT, 6 mM MgC92, 150 mM KCl, 37.5 mM NaCl, 0.005% Triton X-100, 0.1 mM EDTA, 1.25 mM EGTA, 1 mM each ATP, GTP, and CTP, 0.1 mM UTP, and

J. VIROL. 50 /LCi of [3H]UTP (35 Ci/mmol; New England Nuclear Corp.). The concentration of DNA, as SV40 form I DNA or in the form of nucleoprotein complexes, ranged from 0.5 to 50 ,g/ml. The concentration of DNA used in a given experiment is designated in the

figure legends. E. coli RNA polymerase (Holoenzyme; Worthington Biochemicals Corp.) was added at a molar enzyme/DNA ratio of 10 to 60, depending upon the experimental conditions. Enzyme and DNA were preincubated for 5 min at 37°C in the above buffer minus the ribonucleoside triphosphates; 50 d1 of buffer with ribonucleoside triphosphates was then added to initiate the assay. For kinetic assays, at the appropriate time 10 to 20 ul of transcription assay mix was removed and added to 50 ,l of polymerase-stopping buffer containing 1% sodium dodecyl sulfate (SDS)0.15 M NaCl-5 mM EDTA in 10 mM Tris buffer (pH 7.4); 50 pl of each sample was then spotted on a 3 MM filter disk (Whatman), dried, and trichloroacetic acid precipitated, and counts per minute were determined. RNA purification. Polymerase-stopping buffer was added to stop the transcription reaction and remove protein from the DNA. An equal volume of phenol-chloroform-isoamyl alcohol (50:50:1) was added, mixed for 5 min at room temperature, and centrifuged at 2,500 rpm in a Sorvall swinging-bucket centrifuge for 10 min. The upper phase was removed, carrier RNA was added to 50,ug/ml, and 2 volumes of 95% ethanol (-20°C) was added. Precipitation was carried out overnight at -20°C. The RNA precipitate was pelleted, washed with ethanol, and vacuum dried. The RNA pellet was suspended and precipitated once more and then treated with DNase (Worthington Biochemicals Corp.; iodoacetic acid treated to remove RNase activity), purified by G-50 fine Sephadex chromatography, and stored in polymerase-stopping buffer at -20°C. Size analysis of purified [3H]RNA. Purified RNA samples were boiled for 2 min and immersed in an ice bath to minimize the extent of reannealing. Samples were layered onto isokinetic 10 to 30% sucrose g'radients containing 0.1% SDS-0.15 M NaCl in 10 mM Tris buffer (pH 7.4). Centrifugation was in an SW41 rotor at 23,000 rpm (200C) for 16 to 20 h. RNA (28, 18 and 4S) was used as marker. RNA samples were also analyzed by agarose gel electrophoresis after denaturation with glyoxal as described by McMaster and Carmichael (31). Hybridization ofRNA to the separated strands of SV40 DNA fragments. The separated strands of the two SV40 DNA fragments generated by cleavage with BamHI and HpaII restriction endonucleases (Boehringer Mannheim Corp.) were electrophoresed, transferred to nitrocellulose paper (Schleicher and Schuell Co.), and hybridized as previously described (41). After autoradiography and hybridization, nitrocellulose strips were cut in 1-mm slices and counted in nonaqueous scintillation fluid. The position of DNA strand fragments was determined by ethidium bromide staining of the agarose gel before transfer. Agarose gel electrophoresis of SV40 DNA. DNA samples were electrophoresed on a 1.5% agarose slab gel at 35 V for 15 h with a 40 mM Tris acetate (pH 7.9)-5 mM sodium acetate-i mM sodium EDTA

VOL. 35, 1980

TRANSCRIPTION OF SV40 NUCLEOPROTEIN COMPLEX

buffer. The gel was stained with ethidium bromide and photographed under a UV light source. SV40 DNA, treated with EcoRI under partial digest conditions, was used as a marker for supercoiled, relaxed, circular, and linear SV40 DNAs (forms I, II, and III, respectively). SDS-polyacrylamide gel electrophoresis. SDSpolyacrylamide gel electrophoresis was performed essentially as described previously (2). RESULTS

Sedimentation analysis, electrophoresis, and electron microscopy of SV40 cores and minichromosomes. After dissociation of SV40 virions by EGTA-DTT (pH 8.7) or glycine-DTT (pH 9.85), the nucleoprotein complexes were compared on parallel sucrose gradients (Fig. 1). SV40 minichromosomes, prepared by the method of Christiansen et al. (5), sedimented as a 50 to 55S complex. SV40 nucleoprotein cores, however, sedimented as a larger complex of 110 to 115S. SV40 virions (240S) were pelleted under identical conditions. To further analyze the nucleoprotein complexes, peak fractions of each were pooled and analyzed by SDS-polyacrylamide gel electrophoresis (Fig. 2). Figure 2A shows the electrophoretic pattern of the 110S SV40 core isolated from the sucrose gradient shown in Fig. 1. The appearance of viral proteins (VPI, VP2, and VP3) and histone proteins (VP4, VP5, VP6, and VP7) was evident, although the amount of viral protein per unit of DNA was reduced when compared with that of intact SV40 virions (see below). The two protein bands migrating between VP1 and VP2 were most likely minor cellular protein contaminants or proteolytic degradation products of VP,. The amount of these proteins varied from one virion preparation to another, although the high transcriptional activity of the SV40 core was constantly observed. Figure 2B shows the electrophoretic profile of SV40 minichromosomes as isolated in Fig. 1. In contrast to the nucleoprotein core, the minichromosomes contained only VP3 and histone proteins VP4, VP5, VP6, and VP7; no VP1 or VP2 was present. The electrophoretic profile of the minichromosome is in agreement with the results of Christiansen et al. (5), and the presence of viral proteins with the EGTA-DTT core is in agreement with a previous report on polyoma (2). Based on density gradient analysis of fornaldehyde-fixed SV40 cores and minichromosomes, we estimate that the SV40 core contains approximately 34% ofthe total viral protein (p = 1.40 g/cm3), whereas the SV40 minichromosome contains about 13% of the total viral protein (p = 1.495 g/cm3). It is important to note that no naked DNA (p = 1.70 g/cm3) was detected in either core or minichromosome preparations.

373

12 0L

084

10 20 30 FRACTION NUMBER 20

l

B

16

40

55S

21S

1

120-

08 4-

40 10 20 30 FRACTION NUMBER FIG. 1. Velocity sedimentation of in vitro-dissociated SV40 virions. [3HJTdR-labeled SV40 virions were dissociated by treatment with: (A) 5mMEGTA3 mM DTT-0.15 M NaCi in 50mM This (pH 8.7) for 30 min at 320C or (B) 3 mM DTT-0.075 M NaCl in 100 mM glycine (pH 9.85) for 5 min at 370C as described by Christiansen et al. (5). After dissociation, 2.5% Triton X-100 was added to a final concentration of0.05%, and reactions were transferred to an ice bucket. Samples were layered onto isokinetic 10 to 30% sucrose gradients and centrifuged in an SW41 rotor at 40,000 rpm for 2 h (40C). The sedimentation position of the 21S marker SV40 DNA is indicated.

Electron microscopy of the nucleoprotein core and minichromosome with the dissociation procedures described has been reported previously (2, 5). The 50 to 55S minichromosome appears as the typical "beads on a string" chromatin structure with an average of 21 nucleosomes per DNA molecule. In contrast, the nucleoprotein core was found to be a compact chromatin structure with an average diameter of 30 to 50 nm. SV40 DNA, cores, and minichromosomes

374

J. VIROL.

BRADY, LAVIALLE, AND SALZMAN

66% is a conservative estimate since in most cases the core was 80 to 95% as efficient as naked SV40 DNA. Additionally, we checked the transcriptional activity of eight different virus preparations. In all cases, the SV40 core was more efficient than the minichromosomes as a template for transcription, and the result shown in Fig. 3 is the least difference seen between the two templates. Similar kinetic assays on preinitiated template-polymerase complexes in the presence of 2 ug of rifampin per ml demonstrated a 7% reduction in activity for the DNA template, whereas the core incorporation of [3H]UMP was reduced by 33% (data not shown). These results most likely indicate that at least a portion of the increased activity of the core as a template for transcription is due to the increased number of initiations occurring on the core during the transcription.

Determination of size of [3H]RNA synthesized on SV40 DNA and core. For a further comparison of the SV40 DNA and core as templates for RNA synthesis, RNA synthesized analysis of SV40 core and minichromosome nucleoprotein complexes. Samples were isolated as described in the legend to Fig. 1 and prepared for electrophoresis as previously described (2). (A) 11OS SV40 core; (B) 55S SV40 minichromosome.

150-

C., 0

templates for transcription. Based on SDS-polyacrylamide gel analysis and electron microscopy, it is clear that there are sharp differences in protein composition and degree of compaction between the nucleoprotein core and minichromosome. We were interested in determining the effect that the presence of VP1 and VP2 and compactness of the core would have on the transcription capabilities of the complex. [14C]thymidine (TdR)-labeled SV40 cores and minichromosomes were isolated from gradients as shown in Fig. 1. ['4C]TdR-labeled SV40 form I DNA (ethidium bromide-CsCl purified from the same virion preparation) was diluted so that an equal number of ['4C]TdR counts per minute was added to each transcription assay for the SV40 DNA, core, and minichromosome. The results of this experiment are shown in Fig. 3. As expected, the rate of incorporation of [3H]UMP with the SV40 minichromosome was approximately 20% of that of naked SV40 DNA. This is in agreement with the results of previous investigations (33). Quite surprisingly, however, we found that the SV40 core was a more efficient template for transcription. The rate of incorporation of [3H]UMP with the SV40 core was equal to 66% of that of SV40 form I DNA (Fig. 3). The as

a

100

_

0 u

z

CL50

TIME OF INCUBATION (min.)

FIG. 3. Transcription kinetics of SV40 DNA, core, and minichromosome. Viral DNA or nucleoprotein complex (5-pg DNA equivalent of each) was added to a polymerase preincubation mix and incubated for 5 min at 37°C; ribonucleoside triphosphate mix was then added to initiate transcription, and samples were taken at various intervals and processed as described in the text. [14CJTdR-labeled SV40 cores and minichromosomes were isolated from gradients as described in the legend to Fig. 1. [14C]TdR-labeled SV40 form I DNA was purified by ethidium bromideCsCI centrifugation from the same virion preparation as that used for core and minichromosome isolation. Symbols: 0, SV40 form I DNA; 0, SV40 core; E, SV40

minichromosome.

VOL. 35, 1980

TRANSCRIPTION OF SV40 NUCLEOPROTEIN COMPLEX

from the two templates was purified and analyzed by velocity sedimentation. The size of RNA synthesized on the SV40 core was smaller and more homogeneous than that synthesized on SV40 form I DNA (Fig. 4). RNA synthesized from SV40 DNA ranged from lOS to greater than 35S, with an average size of approxiinately 21 to 22S. RNA synthesized from SV40 cores

x

C;-

0q

FRACTION NUMBER FIG. 4. Sedimentation analysis for RNA synthesized by SV40 form I DNA and SV40 core. RNA synthesized from either SV40 form I DNA or SV40 cores was purified as described in the text, and the final ethanol pellet was suspended in 0.15 M NaCl-5 mM EDTA-1% SDS in 10 mM Tris buffer (pH 7.8). The RNA sample was incubated in a boiling-water bath for 2 min to denature the RNA and then transferred immediately to an ice bath to prevent reannealing. Samples were layered onto isokinetic 10 to 30% sucrose gradients containing 0.15 M NaCI-5 mM EDTA-0.2% SDS in 10 mM Tris buffer (pH 7.8) and centrifuged in an SW41 rotor at 23,000 rpm for 16 h (200C). Arrows indicate sedimentation position of marker 28S, 18S, and 4S RNA run in a parallel gradient. (A) RNA synthesized from SV40 form I DNA; (B) RNA synthesized from SV40 core.

375

had a sedimentation range of 10 to 28S, with a more defined peak size of 16 to 18S. The average length of the RNA chains synthesized from the SV40 core was 2,300 nucleotides, approximately 44% of the total length of the DNA molecule. Glyoxal-denatured RNA samples were also electrophoresed on a 1.5% agarose gel. The results of the electrophoresis were similar to those described above; the core RNA consistently showed a more homogeneous population of RNA molecules than that synthesized from the SV40 form I DNA template (data not shown). Transcription assays across core sucrose gradient. To demonstrate that the template involved in core transcription was indeed in the form of a core, SV40 virions were dissociated and centrifuged through a sucrose gradient as described above. After centrifugation, fractions were collected and assayed for ['4C]TdR DNA counts per minute (Fig. 5A) and transcriptional capabilities (Fig. 5B); all of the transcriptional activity in the dissociated SV40 preparation was found to be located at the position of the SV40 core DNA. No transcriptional activity was detected in the region of the SV40 minichromosomes or free SV40 DNA. We did notice, however, that the heavy side of the SV40 core peak was slightly more efficient in RNA synthesis than was the lighter side. This may reflect a heterogeneity in the core particles, in which the cores containing a full complement of proteins were more efficient in transcription than were core particles which had lost a portion of the protein. Initiation of RNA synthesis on SV40 cores. Since the above experiment indicates that the DNA involved in SV40 transcription was contained in the core particle at the beginning of the transcription, we were interested in determining whether we could detect the growth of nascent RNA chains on the compact core particle. SV40 cores were incubated for various lengths of time in the transcription assay mix and then analyzed by sucrose velocity sedimentation. If the intact core is actually the template, then one would expect to see the [3H]UMP RNA label and ['4C]TdR core DNA label cosediment as transcription is initiated. After longer incubation, the presence of nascent RNA chains should increase the sedimentation value of the complex. When RNA transcription was allowed to proceed for 1 min, both the [3H]UMP and ['4C]TdR cosedimented in the sucrose gradient (Fig. 6A). As synthesis was allowed to proceed for 2 and 5 min, the [3H]UMP label was found to sediment at a faster rate. The increased sedimentation rate was presumably due to the growth of RNA chains on the SV40 core complex.

BRADY, LAVIALLE, AND SALZMAN As a separate part of the experiment, [14C]TdR-labeled cores were incubated in a transcription assay mix containing cold triphosphates, and the RNA chains were allowed to elongate for 30 min (Fig. 6B). Control SV40 cores which were incubated in the transcription assay mix376

J. VIROL.

50 A 40-

30

10

x

20 30 40 FRACTION NUMBER

50

20

o) 20

B 16-

10

-6 10 20 30 FRACTION NUMBER B

40

12

FRACTION NUMBER

FIG. 6. Intato oftasrpionaddtria 4

40 10

030-

20 30 FRACTION NUMBER

40

FIG. 6. Initiation of transcription and determination ofpercentage ofSV40 cores involved in transcrip0. tion. [14C]TdR-labeled SV40 virions were dissociated o20as described in the legend to Fig. 1. (A) Virion preparation (25 Al, 5 ug of DNA) was added to preincubation mix with E. coli polymerase and incubated at 10 37°C for 5 min. Ribonucleoside triphosphate mix (50 id) was added to each reaction and incubated at 37°C. At 1, 2, and 5 min, EDTA was added to 5 mM to stop the reaction, and then the mixture was trans20 30 10o 40 ferred to an ice bath. (B) SV40 cores were treated as FRACTION NUMBER in (A), except that ribonucleoside triphosphate mix FIG. 5. Transcri]ption assay of gradient fr-actions contained cold triphosphates, and the enzymel used to Purify SV40 core. SV40 virions were disso- DNA ratioonly was increased to 50:1. Reactions were ciated and sedimented through a sucrose gradient as layered onto isokinetic 10 to 30% sucrose gradients described in the legend to Fig. 1, except the centrifu- and centrifuged as described in the legend to Fig. 5. gation time was decreased to 90 min. Samples of 25 [4C]TdR per minute were determined directly pul were taken from each fraction to determine counts on samplescounts gradient fractions. [3HIUMP counts per minute and transcriptional potential. (A) counts- per minute from were determined by trichloroacetic acid per-minute profile of ["4CJTdR-labeled core complex; precipitation. Symbols: (A) , I min; *, 2 min; 0, 5 (B) trichloroacetic acid-precipitable [3HJUMP incor- min; arrow indicates the sedimentation position of porated in gradient fractions. A 25-gl sample of each 11OS SV40 core; (B) , 110S SV40 virion core; 0, gradient fraction was added to an equal volume of sedimentation of SV40 virion core after a 30preincubation mix containing E. coli RNA polymer- min incubationprofile ase and incubated for 5 min at 37°C; 50,ul of ribon- triphosphates. with polymerase and ribonucleoside ucleoside triphosphate mix was then added, and reactions were returned to 37°Cfor30min. Polymerasestopping buffer (equal volume) was then added to ture without ribonucleoside triphosphate sedistop the reaction, and a sample was removed to mented at 110 to 115S. However, after incubadetermine trichloroacetic acid-precipitable counts tion in the complete transcription assay mixture per minute as described in the text. for 30 min, the [14C]TdR profile was quite dif-

VOL. 35, 1980

TRANSCRIPTION OF SV40 NUCLEOPROTEIN COMPLEX

ferent. No ['4C]TdR radioactivity was detected in the position of the SV40 core. Instead, the [I4C]TdR label sedimented as a heavier complex, presumably due to the growth of RNA chains on the SV40 core. Since no radioactivity was found in the position of SV40 core after transcription, this experiment further indicates that the core must be a very efficient template since all of the particles were apparently involved in the process of transcription. These experiments reveal two important aspects of the SV40 core transcription process. First, the cosedimentation of the [3H]UMP and ['4C]TdR labels indicates that transcription is initiated on the intact core particles; no loss of protein is indicated. Furthermore, separate CsCl density determinations on core transcription complexes elongated for 5 to 30 min and then digested with RNase showed no shift in density, indicating that no loss of protein from the cores occurs during elongation of the RNA chains (data not shown). Our experiments indicate that the SV40 core is an efficient template for transcription since under an appropriate, but not excessive, enzyme/DNA ratio, all of the core particles are apparently involved in transcription. Agarose gel electrophoresis of SV40 core DNA. Another important aspect of understanding the transcriptional activity of the SV40 core was to determine whether any cleavage of the DNA was occurring during transcription. Although form II or III SV40 DNA is a less efficient template for transcription (16, 27), similar studies in the presence of viral and histone proteins have not been conducted. In addition, virus-associated endonucleases have been reported with the papovaviruses (10, 32), so we thought that it was important to determine the nature of the DNA before and after transcription. SV40 core DNA and form I DNA before and after transcription were therefore analyzed by agarose gel electrophoresis. No cleavage of DNA was detected as a result of the transcription process (Fig. 7). Both SV40 form I DNA and SV40 core DNA contained the same amount of form I DNA before and after transcription. To estimate the amount of form I DNA after transcription, we found it necessary to treat with RNase (Fig. 7E and F) since the presence of RNA (Fig. 7C and D) caused the DNA pattern to be somewhat diffuse. Hybridization of SV40 core RNA to early and late SV40 DNA fragments. After cleavage of SV40 DNA with BamHI and HpaII restriction enzymes, the DNA was found in two fragments, A and B. The A fragment contains the early coding sequences of SV40, and the B

A

B

C

D

E

377

F

II

III

I

FIG. 7. Agarose gel electrophoresis of DNA templates before and after transcription. SV40 form I DNA or SV40 cores (5 pg of each) were added to the transcription assay as described in the legend to Fig. 8. Equal samples of the untranscribed DNA templates and transcribed templates were then treated with 1% SDS, phenol-chloroform-isoamyl alcohol extracted, and ethanol precipitated. The DNA from the transcription reactions was divided into two portions, one of which was treated with RNase. A 500-ng amount of each sample was loaded onto a 1.5% agarose gel slab and electrophoresed at 35 Vfor 15 h with 40 mM Tris-acetate (pH 7.9)-5 mM sodium acetate-1 mM sodium EDTA buffer. (A) SV40 form I DNA control; (B) SV40 core DNA control; (C) transcribed SV40 form I DNA; (D) transcribed SV40 core DNA; (E) transcribed, RNase-treated SV40 F1 DNA; (F) transcribed, RNase-treated SV40 core DNA; (I) form I supercoiled SV40 DNA; (II) form II relaxed, circular SV40 DNA; (III) form III linear SV40 DNA. The gel was stained with ethidium bromide and photographed under a UV light source.

fragment contains the late coding sequences of the genome. After agarose gel electrophoresis, the two DNA strands of the A and B fragments were separated and transferred to nitrocellulose for hybridization as described above. To determine whether a particular segment or strand of the viral genome was preferentially transcribed, purified core [3H]RNA was hybridized to the separated BamHI and HpaII fragments. [3H]RNA synthesized on SV40 form I DNA served as a control. No significant difference in the hybridization pattern of DNA [3H]RNA and core [3H]RNA was detected (Fig. 8). Hybridization of both RNA preparations was asymmetric with a pronounced template preference for the early SV40 DNA strand. No hybridization was detected to the late SV40 DNA strand. However, the presence of a large excess of early RNA as compared with late RNA transcripts during the hybridization reaction could generate RNA duplex molecules and effectively block the reaction of late RNA transcripts with DNA bound to nitrocellulose. We are currently conducting

BRADY, LAVIALLE, AND SALZMAN

378

A

40 e

EA LA I

LB

EB

I

I

_

30 a-.20 20

10, 10 20 FRACTION NUMBER

3

0.

FRACTION NUMBER

FIG. 8. Hybridization of RNA to BamHI and HpaII restriction fragments of SV40. Hybridization, separation of DNA strands, and autoradiography were performed as described previously (40). Purified [3H]RNA synthesized from SV40 form I DNA (A) or from SV40 cores (B) was used in the analysis. After hybridization and autoradiography, nitrocellulose strips were cut in 1-mm slices and counted in nonaqueous scintillation fluid. The position of DNA fragments was determined by comparison with a control DNA nitrocellulose strip. EA, Early strand, A fragment; LA, late strand, A fragment; LB, late strand, B fragment; EB, early strand, B fragment.

RNA self-hybridization experiments to clarify this point.

DISCUSSION The association of SV40 DNA with cellular histones in the basic chromatin minichromosome structure has been demonstrated by a number of investigators (5, 15, 35, 38, 39). The structure of the nucleosome is the basic repeating unit of chromatin in which eight histone molecules are associated with approximately 145 to 200 base pairs of DNA. Since earlier studies revealed that virtually all of the viral DNA, both in infected cells and isolated from mature virions, had this same basic structure, it seemed likely that this was the native structure of the

J. VIROL.

viral chromatin. It is now clear that the structure of both virion-derived and infected cell-derived SV40 nucleoprotein complexes is much more complex than the simple minichromosome structure. Varshavsky et al. (44-46) and Nedospasov et al. (36) have isolated nucleoprotein complexes from SV40-infected cells and demonstrated that the bulk of viral chromatin found late in the infection cycle is in a nuclease-resistant condensed 30-nm complex due to the association of Hi with the viral minichromosome. More recent studies by Garber et al. (13) under conditions in which no virion breakdown occurs during extraction have provided the first evidence that similar condensed chromatin structures are intermediates in virion maturation since they can be chased efficiently into mature virions (42). Fernandez-Munoz et al. (12), in a similas analysis, have shown by gel electrophoresis that, as the condensed nucleoprotein complex is packaged into mature virions, a loss of HI occurs. Similar studies by La Bella and Vesco (20) support the loss of HI from the nucleoprotein complex as the process of virion formation occurs. Recent studies with polyoma (1, 2) and the results presented in this paper show that, under the mild EGTA-DTT dissociation conditions, a compact core is released from the virion. In agreement with other investigators (25), we found no Hi associated with the compact nucleoprotein core. Instead, the compact nucleoprotein complex contained a select species of viral proteins in place of the Hi. Thus, it appears that, as the viral chromatin is packaged into the mature virion, an exchange of Hi and viral protein occurs. The exchange apparently does not affect the compact structure of the chromatin which is required for encapsidation (28) but does alter the transcription potential of the complex. The results presented in this paper demonstrate that the SV40 nucleoprotein core released from purified virions by in vitro EGTA-DTT dissociation are efficient templates for RNA transcription. Kinetic incorporation of [3H]UMP into acid-insoluble RNA shows that the core approaches the efficiency of SV40 forn I DNA as a template for RNA synthesis. SV40 minichromosomes, which lack VP, and VP2, have an incorporation rate which is 20% of that obtained with SV40 DNA. These results suggest that the association of VP, or VP2 or both with the viral minichromosome, in addition to maintaining a highly compact structure, enhances the transcriptional activity of the nucleoprotein complex. However, since minichromosomes and cores are isolated by different techniques, we cannot rule out the possibility that they differ in the amount or arrangement of VP3 or histones

VOL. 35, 1980

TRANSCRIPTION OF SV40 NUCLEOPROTEIN COMPLEX

or both and that this accounts for the observed difference in physical and transcriptional properties. Transcription studies of temperature-sensitive viral protein mutants of SV40 should allow us to distinguish among these possibilities. Initiation of transcription and chain elongation occur on the intact SV40 core; no detectable loss of protein is observed during either process. The RNA synthesized from SV40 cores has an average ch*i length of 2,300 nucleotides. Since a mean value of 21 nucleosomes has been found associated with the 5,200-base pair SV40 form I DNA (5), this implies that the RNA polymerase has transcribed through 9 to 10 nucleosomes, although we cannot rule out the possibility that the nucleosomes are sliding along the DNA as transcription occurs. The possibffity that we are synthesizing the RNA from SV40 form I DNA present in the sample has been eliminated since we show that all of the transcriptional activity in the dissociated virion preparation cosediments with the nucleoprotein core. Another finding of potential significance is that this RNA is more homogeneous than the RNA synthesized on SV40 form I DNA. Whether this represents a greater selectivity in promoter recognition or more precise termination is presently under study. Hybridization analysis of core RNA demonstrates that transcription is asymmetric, occurring preferentially on the early SV40 form I DNA strand. Since transcription of SV40 form I DNA is also asymmetric, these results suggest that specific recognition of at least some RNA polymerase initiation sites is preserved in the compact SV40 nucleoprotein core. It is interesting to compare the properties of the SV40 nucleoprotein core with those of the transcriptionally active regions of eucaryotic chromatin. Experimental evidence indicates that actively transcribing regions of eucaryotic genes contain nucleosomes (14,21,40,47). These same sequences, however, are apparently different from the bulk chromatin nucleosomes since they are much more accessible to DNase I digestion (14, 47). Still other experiments indicate that the transcriptionally active regions contain less Hi histone (4, 9, 23), show a distinct increase in histone acetylation (37), and contain certain nonhistone proteins, some of which are metabolically unstable and are different from nonhistone proteins found associated with the bulk of chromatin (8, 19). The SV40 core complex contains many of these same characteristics. As indicated above, the basic structure of the viral chromatin is the nucleosome-DNA complex referred to as the minichromosome. Previous investigators have shown that the histones found inside the virion are acetylated (25). In addition,

379

the SV40 core complex is extremely sensitive to nuclease digestion (M. Seidman, personal communication). Finally, the apparent exchange of H1 for the nonhistone viral proteins during the process of encapsidation would represent an additional property of transcriptionally active chromatin. It is important to note that the addition of viral protein to the acetylated histoneDNA complex is apparently a prerequisite for the enhanced transcriptional activity since the DNA-histone complex, even though it contains acetylated histones, has only 20% of the transcriptional capability of SV40 form I DNA. Mathis et al. (29) have shown that the in vivo acetylation of histones does not enhance the transcriptional activity of the viral minichromosome. Our studies suggest that the late viral structural proteins may have a regulatory function in addition to their structural involvement in the outer capsid protein shell of the virion. Since the addition of VP1 and VP2 to the histone-DNA complex apparently occurs during the encapsidation of the virion, the transition to a transcriptionally active core template would not be expressed late in the infection cycle, but rather during the early phase of the next infection cycle. In studies with polyoma, after absorption, penetration, and transport of the virion to the cell nuclei, the virion is uncoated very shortly after passing through the nuclear membrane (26). One theory for polyoma or SV40 uncoating is that, as the virion passes through the nuclear membrane, the virion-associated Ca2+ is sequestered by the membrane (6). Upon nuclear entry, uncoating occurs by the action of biological reducing agents such as cysteine or glutathione releasing the capsid proteins and nucleoprotein core to initiate transcriptional and replicative events. The in vitro EGTA-DTT dissociation procedure could be considered to duplicate the in vivo uncoating event. Evidence for the appearance of a nucleoprotein core during the early infection cycle has been obtained with polyoma (V. D. Winston and R. A. Consigli, personal communication). To successfully initiate the infection cycle, the nucleoprotein complex must compete with cellular chromatin for available RNA polymerase molecules. A template such as the SV40 core described in this paper should increase the efficiency of early viral transcription. Reconstitution experiments, in which the viral proteins are added back to the basic minichromosome structure, are in progress. Our preliminary results indicate that the viral proteins have a very high affinity for the minichromosome. Transcriptional studies of temperature-sensitive

380

BRADY, LAVIALLE, AND SALZMAN

viral protein mutants of SV40 are also important for a more complete understanding of the function of viral proteins in the processes of transcription of the SV40 core complex. These types of studies should ultimately allow us to determine the nature of the interaction between the DNA-histone complex and the nonhistone viral proteins. LITERATURE CITED 1. Brady, J. N., V. D. Winston, and R. A. Consigli. 1977.

2.

3. 4. 5.

6. 7.

8.

9.

10. 11.

12.

13. 14.

15.

16.

17. 18.

Dissociation of polyoma virus by the chelation of calcium ions found associated with purified virions. J. Virol. 23:717-724. Brady, J. N., V. D. Winston, and R. A. Consigli. 1978. Characterization of a DNA-protein complex and capsomere subunits derived from polyoma by treatment with ethyleneglycol-bis-N,N'-tetraacetic acid and dithiothreitol. J. Virol. 27:193-204. Bustin, M. 1978. Binding of E. coli RNA polymerase to chromatin subunits. Nucleic Acids Res. 5:925-932. Chiu, N., R. Baserga, and J. J. Furth. 1977. Composition and template activity of chromatin fractionated by isoelectric focusing. Biochemistry 16:4796-4802. Christiansen, G., T. Landers, J. Griffith, and P. Berg. 1977. Characterization of components released by alkali disruption of simian virus 40. J. Virol. 21:1079-1084. Consigli, R. A., and M. S. Center. 1978. Recent advances in polyoma virus research. Crit. Rev. Microbiol. 6:263-299. Cremisi, C., A. Chestier, C. Dauguet, and M. Yaniv. 1977. Transcription of SV40 nucleoprotein complexes in vitro. Biochem. Biophys. Res. Commun. 78:74-82. Defer, N., M. Crepin, C. Terrioux, J. Kruh, and F. Gros. 1979. Comparison of non-histone proteins selectively associated with nucleosomes with proteins released during limited DNase digestions. Nucleic Acids Res. 6:953-966. Djondjurov, L., E. Ivanova, and R. Tsanev. 1979. Two chromatin fractions with different metabolic properties of non-histone proteins and of newly synthesized RNA. Eur. J. Biochem. 97:133-139. Dooley, D. C., M. T. Ryzlak, and H. L. Ozer. 1976. Endonucleases and simian virus 40 virions: origin of a virion-associated endonuclease. J. Virol. 17:352-362. Felsenfeld, G. 1978. Chromatin. Nature (London) 271: 115-122. Fernandez-Munoz, R., M. Coca-Prados, and M.-T. Hsu. 1979. Intracellular forms of simian virus 40 nucleoprotein complexes. I. Methods of isolation and chara terization in CV-1 cells. J. Virol. 29:612-623. Garber, E., M. Seidman, and A. J. Levine. 1978. The detection and characterization of multiple forms of SV40 nucleoprotein complexes. Virology 90:305-316. Garel, A., and R. Axel. 1976. Selective digestion of transcriptionally active ovalbumin genes from oviduct nuclei. Proc. Natl. Acad. Sci. U.S.A. 73:3966-3970. Germond, J. E., B. Hirt, P. Oudet, M. Gross-Bellard, and P. Chambon. 1975. Folding of the DNA double helix in chromatin-like structures from simian virus 40. Proc. Natl. Acad. Sci. U.S.A. 72:1843-1847. Hale, P., and J. Lebowitz. 1978. Binding and transcription of simian virus 40 DNA by DNA-dependent RNA polymerase from Escherichia coli. J. Virol. 25:298-304. Hall, M. R. 1977. Transcription and reisolation of the simian virus 40 nucleoprotein complex. Biochem. Biophys. Res. Commun. 76:698-704. Huang, E.-S., M. Nonoyama, and J. S. Pagano. 1972. Structure and function of the polypeptides in simian virus 40. II. Transcription of subviral deoxynucleoprotein complexes in vitro. J. Virol. 9:930-937.

J. VIROL. 19. Jackson, J. B., J. M. Pollock, Jr., and R. L. Rill. 1979. Chromatin fraction procedure that yields nucleosomes containing near-stoichiometric amounts of high mobility group nonhistone chromosomal proteins. Biochemistry 18:3739-3748. 20. La Bella, F., and C. Vesco. 1980. Late modifications of simian virus 40 chromatin during the lytic cycle occur in an immature form of virion. J. Virol. 33:1138-1150. 21. Lacy, E., and R. Axel. 1975. Analysis of DNA of isolated chromatin subunits. Proc. Natl. Acad. Sci. U.S.A. 72: 3978-3982. 22. Lebowitz, J., C. F. Garon, M. C. Y. and N. P. Salzman. 1976. Chemical modification of simian virus 40 DNA by reaction with a water-soluble carbodiimide. J. Virol. 18:205-210. 23. Letansky, K. 1978. Nuclear proteins in genetically active and inactive parts of chromatin. FEBS Lett. 89:93-97. 24. Levy, B., W. Connor, and G. Dixon. 1979. A subset of trout testis nucleosomes enriched in transcribed DNA sequences contains high mobility group proteins as major structural components. J. Biol. Chem. 254:608620. 25. MacGregor, J. P., Y. H. Chen, D. A. Goldstein, and M. R. Hall. 1978. Properties of the histones associated with simian virus 40 replicative form nucleoprotein complex. Biochem. Biophys. Res. Commun. 83:814821. 26. MacKay, R. L., and R. A. Consigli. 1976. Early events in polyoma virus infection: attachment, penetration, and nuclear entry. J. Virol. 19:620-636. 27. Mandel, J. L., and P. Chambon. 1974. Animal DNAdependent RNA polymerases. Eur. J. Biochem. 41:367378. 28. Martin, R. G. 1977. On the nucleoprotein core of simian virus 40. Virology 83:433-437. 29. Mathis, D. J., P. Oudet, B. Wasylyk, and P. Chambon. 1978. Effect of histone acetylation on structure and in vitro transcription of chromatin. Nucleic Acids Res. 5: 3523-3547. 30. McCarty, K. S., Jr., R. T. Vollmer, and K. S. McCarty. 1974. Improved computer program data for the resolution and fractionation of macromolecules by isokinetic sucrose density gradient sedimentation. Anal. Biochem. 61:165-183. 31. McMaster, G. K., and G. G. Carmichael. 1977. Analysis of single- and double-stranded nucleic acids on polyacrylamide and agarose gels by using glyoxal and acridine orange. Proc. Natl. Acad. Sci. U.S.A. 74:4835-4838. 32. McMillen, J., M. S. Center, and R. A. Consigli. 1976. Origin of the polyoma virus-associated endonuclease. J. Virol. 17:127-131. 33. Meneguzzi, G., N. Chenciner, and G. Milanesi. 1979. Transcription of nucleosomal DNA in SV40 minichromosomes by eukaryotic and prokaryotic RNA polymerases. Nucleic Acids Res. 6:2947-2960. 34. Meneguzzi, G., P. F. Pignatti, G. B. Brodano, and G. Milanesi. 1978. Minichromosome from BK virus as a template for transcription in vitro. Proc. Natl. Acad. Sci. U.S.A. 75:1126-1130. 35. Muller, U., H. Zentgraf, I. Eicken, and W. Keller. 1978. Higher order structure of simian virus 40 chromatin. Science 201:406-415. 36. Nedospasov, S. A., V. V. Bakayev, and G. P. Georgiev. 1978. Chromosome of the mature virion of simian virus 40 contains HI histone. Nucleic Acids Res. 5: 2847-2860. 37. Nelson, D., M. E. Perry, and R. Chalkley. 1979. A correlation between nucleosome spacer region susceptibility to DNase I and histone acetylation. Nucleic Acids Res. 6:561-574. 38. Polisky, B., and B. McCarthy. 1975. Location of histones on simian virus 40 DNA. Proc. Natl. Acad. Sci. U.S.A. 72:2895-2899. 39. Ponder, B. A. J., and L. V. Crawford. 1977. The ar-

C#en,

VOL. 35, 1980

40. 41.

42.

43. 44.

TRANSCRIPTION OF SV40 NUCLEOPROTEIN COMPLEX

rangement of nucleosomes in nucleoprotein complexes from polyoma virus and SV40. Cell 11:35-49. Reeves, R., and A. Jones. 1976. Genomic transcriptional activity and structure of chromatin. Nature (London) 260:495-500. Salzman, N. P., E. H. Birkenmeier, N. Chiu, E. May, M. F. Radonovich, and M. Shani. 1977. Regulation of transciption during the SV40 lytic cycle. INSERM (Paris) 69:243-252. Seidman, M., E. Garber, and A. J. Levine. 1979. Parameters affecting the stability of SV40 virions during the extraction of nucleoprotein complexes. Virology 95: 256-259. Stein, G. S., T. C. Spelsberg, and L J. Kleinsmith. 1974. Nonhistone chromosomal proteins and gene regulation. Science 183:817-824. Varshavsky, A. J., S. A. Nedospasov, V. V. Schmatchenko, V. V. Bakayev, P. M. Chumackov,

and G. P. Georgiev. 1977. Compact form of SV40 viral resistant to nuclease: possible implications for chromatin structure. Nucleic Acids Res. 4:3303-3325. Varshavsky, A. J., 0. H. Sundin, and M. J. Bohn. 1978. SV40 viral minichromosomes: preferential exposure of the origin of replication as probed by restriction endonucleases. Nucleic Acids Res. 5:3469-3477. Var8havsky, A. J., 0. Sundin, and M. Bohn. 1979. A stretch of "late" SV40 viral DNA about 400 bp long which includes the origin of replication is specifically exposed in SV40 minichromosomes. Cell 16:453-466. Weintraub, IL, and KL Groudine. 1976. Chromosomal subunits in active genes have an altered conformation. Science 193:848-856. WIlliamson, P., and G. Felsenfeld. 1978. Transcription of histone-covered T7 DNA by Escherichia coli RNA polymerase. Biochemistry 17:5695-5704. minichromosome is

45.

46.

47. 48.

381