Transcription of the cellular DNA sequences in a cloned host-substituted SV40 dna variant

VIROLOGY

94,

82-94

(1979)

Transcription

of the Cellular DNA Sequences in a Cloned Host-Substituted SV40 DNA Variant

J. R. HARTMAN,l Departments

of Virology

0. LAUB,* Y. ALONI,” and *Genetics,

Weizmann

Institute

AND

E. WINOCOUR

qf Science, Rehovot, Israel

Accepted October 3, 1978

The transcription of a cloned host-substituted SV40 genome of defined structure was studied in cells coinfected with wild-type virus and in Gz vitro reactions with Sarkosyl nuclear extracts (transcription complex preparations) of the coinfected cells. Evidence for the transcription of the monkey DNA sequences in substituted SV40 was obtained in both systems. Efforts to detect similar transcripts in uninfected cells, or in cells infected with wild type SV40 alone, were not successful. Both the highly reiterated and nonreiterated types of cellular DNA sequences (which are linked in the genome of the cloned substituted SV40 variant) were transcribed in the coinfected cells and the RNA transcripts were detected in the nuclear and in the cytoplasmic fractions. Relative to the amount of wild-type SV40 RNA, 40% of the RNA synthesized after in vitro incubation of transcription complex preparations, hybridized with substituted SV40 cellular DNA sequences. In contrast, only 15% of the nuclear RNA and 4% of the cytoplasmic RNA from intact cells hybridized with the cellular DNA derived from substituted SV40. The sucrose gradient sedimentation profile of the host-substituted SV40 RNA was uniquely different from and more heterogeneous than that of wild-type SV40 RNA. RNA homologous to the host DNA in the substituted SV40 variant was associated only with lighter (disomal and monosomal) ribosomal fractions. INTRODUCTION

Defective substituted SV40 variants, whose genomes consist of covalently linked viral and host DNA sequences, arise during the high-multiplicity serial passage of wildtype virus in monkey cells (reviewed in Winocour et al., 1974). The substituted SV40 DNAs that have been examined so far share a number of common features. The genome is composed of repeated segments, each of which contains the same or subsets of the same host and viral sequences; the viral sequences are derived from a small part of the wild-type genome (15% or less) which contains the origin of the replication; and the host DNA sequences may be either of the nonreiterated or highly reiterated or of both types (Rozenblatt et al., 1973; Frenkel et al., 1974; Lee et al., 1975; Davoli et al., 1977; Rao and Singer, 1977; Oren et al., 1978). Independently generated stocks of substituted SV40 may share common host 1 To whom request for reprints should be addressed. 0042~6822/79/050082-13$02.00/0

Copyright 8 1979by AcademicPress, Inc. All rights of reproductionin any form reserved.

82

sequences or may contain sequences that differ from population to population. A large proportion of independently isolated substituted SV40 stocks share common host sequences of the highly reiterated type (Oren et aZ., 1976; Rosenberg et al., 1977; Oren et al., 1978) which is derived from the (xcomponent segment of the monkey genome (M. Rosenberg et aE., 1977; H. Rosenberg et aZ., 1978). Although defective with respect to independent replication, substituted SV40 multiplies well in cells coinfected with wildtype virus and ultimately becomes the dominant component of the viral yield. Since all missing functions are supplied by the wild-type helper virus, and complementation between substituted variants and other types of defective viruses has not been reported, there is no evident reason why the substituted genome should be transcribed in the coinfected cell. Nevertheless, the possible transcription of such radically altered viral DNA molecules

SUBSTITUTED

SV40 TRANSCRIPTION

would be a matter of considerable biological interest that may help clarify the interplay between viral and cellular transcription promotors and aberrations of the RNA processing mechanisms. Moreover, the establishment of SV40 vectors, carrying inserted genes of another organism, as plasmid-like entities in mammalian cells will require knowledge of how alterations in DNA structure affects the mode of transcription. In this study, we have investigated the transcription of a cloned substituted SV40 genome of defined structure. We will show that the inserted host DNA sequences, which are both of the nonreiterated and reiterated type, are transcribed in cells coinfected with the variant and wild-type viruses. Evidence for the transcription of host-substituted SV40 DNA has also recently been reported by others (Kuff et az., 1978). MATERIALS

AND METHODS

Virus and cells. Confluent monolayers of monkey BSC-1 cells were infected with wild-type SV40 (a plaque-purified stock of strain ‘777)at a multiplicity of about 50 PFU/ cell, as described in Lavi and Winocour (1972). The substituted SV40 stocks T8/777, CVl-l-P5, and Rh 911 (Oren et al., 1976) were used undiluted; the cloned substituted F161 stock was diluted 1:5 prior to infection. The F161 stock contains a single species of a substituted variant (called F161F) and wild-type SV40 helper virus. The derivation and cloning of the F161 stock is described in Oren et al. (1978). Viral DNA preparations. Wild-type and substituted SV40 DNAs were extracted from infected cells by the Hirt procedure (Hirt, 1967) and the closed circular component I molecules were purified by cesium chloride-ethidium bromide equilibrium density gradients and by sedimentation in 5-20% neutral sucrose gradients as described elsewhere (Oren et al., 1976). The preparation and purification of the cloned substituted SV40 DNA called F161F is described in Oren et al. (1978). Briefly, F161F DNA was separated from wild type SV40 DNA by treatment with endo *R

83

EcoRl followed by equilibrium sedimentation in cesium chloride-ethidium bromide density gradients. The digestion of F161F DNA by the restriction endonucleases indicated in the text, the separation of the cleavage products by agarose and polyacrylamide gel electrophoresis, and the recovery of the fragments from the gel by electroelution were carried out as described in Oren et al. (1978). Cell fractionation and RNA extraction. Nuclei and cytoplasm were separated by treatment with NP-40 and sodium deoxycholate (Penman, 1966). For preparation of total cellular RNA, the monolayers were lysed with buffer containing 0.1 M NaCI, 0.01 M Tris.HCI, pH 7.4,l n-J4 EDTA, 1% SDS, and 300 pg/ml polyvinyl sulfate. RNA was extracted with phenol-chloroformisoamylalcohol(50:49: 1) and precipitated by 2 M LiCI. The pellet was resuspended in TKM (25 mJ4 KCI, 2.5 n-u?4MgC&, 50 m&f Tris .HCI, pH 6.7) and digested with 25 pg/ml DNase (Worthington, RNase-free electrophoretically purified) at 2” for 1 hr. The digest was extracted with phenol-chloroform and precipitated with 2 vol of ethanol. Where indicated, labeled RNA was sedimented through a linear 15-30% sucrose gradient in SDS buffer (0.1 M NaCl, 10 mM Tris*HCl, pH 7.4, 1 mikf EDTA, and 0.5% SDS) in the Spine0 SW 27.1 rotor at 25,000 rpm, 20”, for 18 hr. Preparation of Sarkosyl nuclear extracts from infected cells. At 48 hr postinfection, cells were scraped off the plates, washed, and resuspended in cold PBS. Nuclei were isolated by centrifugation after treatment of the cells with 0.5% NP-40 and resuspended in buffer containing 50 mJ4 Tris *HCl, pH 7.9, 50 mM KCl, 0.5 mM DTT, and 0.4 1M NaCl. Transcriptional complexes with endogenous RNA polymerase activity were prepared as described in Laub and Aloni (1976). Briefly, the nuclei were disrupted by adding 1 vol of a solution containing 0.5% Sarkosyl (sodium lauryl sarcosinate) and 0.4 NaCl (final volume, -2 ml/ lo7 nuclei). The mixture was centrifuged at 30,000 g for 30 min at 2”. The supernatant was carefully removed from the chromatin pellet and dialyzed for 2-3 hr against 0.15 M

HARTMAN ET AL.

84

(NH&SO,, 5 m&’ KCl, 30 m&f HEPESNaOH, pH 8.0. RNA synthesis by Sarkosyl nuclear extracts. The standard “reaction mixture” is detailed in Laub and Aloni (1976). After incubation at 26” for l-2 hr, the RNA was extracted with SDS-phenol-chloroform isoamylalcohol at room temperature and collected by ethanol precipitation. The precipitate was resuspended in TKM and digested with DNase as above. The digest was reextracted with SDS-phenol and the extract was passed through a Sephadex G-100 column (1.1 x 55 cm). The material excluded with the void volume was collected and stored under ethanol. Preparation of [3H]cRNA. [3H]cRNA complementary to wild-type SV40 DNA and to substituted F161F DNA was synthesized by Escherichia coli RNA polymerase, as described by Oren et al. (1976), except that the [3H]cRNA product was purified on a Sephadex G-100 column. The specific activity of the [3H]cRNA was calculated to be lo7 cpmlpg. After self-annealing at 68” for 24 hr in 4 x SSC (1 x SSC is 0.15 M NaCl, 0.015&f sodium citrate) 0.5% SDS, 64% of the [“H]cRNA made off F161F DNA was resistant to RNase treatment; [“H]cRNA made off wild-type SV40 DNA was lo- 15% resistant to RNase treatment after selfannealing under the above conditions (Aloni, 1972). Preparations of labeled RNA enriched for sequences complementary to the host DNA components in substituted SV40 DNAs. The procedure of Oren et al. (1976)

was followed. Briefly, the RNA was reduced to an average size of 4-6 S by controlled alkali cleavage. Viral sequences were removed by two to three successive cycles of exhaustive hybridization (in 4 x SSC, 0.5% SDS, at 68” for 20-24 hr) to 10 pg wild-type SV40 DNA immobilized in nitrocellulose filters. RNA-RNA

hybridization

in solution.

[3H]cRNA probes were mixed with an excess of cellular RNA and denatured by boiling for 7 min in 0.1% SDS and 5 m&f EDTA. Hybridization reactions were carried out at 68” for 20 hr in 2 x SSC. At the end of incubation, each sample was divided into

FIG. 1. A restriction endonuclease cleavage map of the cloned substituted SV40 genome designated F161F (from Oren al., 1978, and reprinted with permission). The inner circle shows the order of the end0.R H&(11 + III) cleavage products numbered l-7. The inner arrows denote the H&II (J) and Him111 (1) cleavage sites. The shaded segments denote the distribution of host DNA sequences; fragments I, 4, and 5 contain host sequences of the nonreiterated type whereas fragment 6 contains host sequences of the highly reiterated type. The viral sequences (unshaded areas) are derived solely from within a segment of the wild-type SV40 genome (0.59-0.73 map units) which contains the origin of replication at 0.67 map units. The cleavage site of end0.R BglI is also located at 0.67 map units on the wild-type genome.

et

two: one part was directly precipitated with 10% TCA while the second part was treated with 50 pg/ml RNase for 1 hr at room temperature and then precipitated with 10% TCA. Hybridization

to DNA fragments

in gels.

The technique of Southern (1975) was used as described in Oren et al. (1978). RESULTS

The Host Substituted

SV4.0 DNA

In most of the foregoing experiments, we utilized a cloned substituted SV40 variant (designated F161F) whose DNA structure has been described in detail elsewhere (Oren et al., 1978). The basic features of this DNA, which are illustrated by the restriction endonuclease cleavage map shown in Fig. 1, are as follows: The closed circular genome consists of four

SUBSTITUTED

SV40 TRANSCRIPTION

85

SV40 DNA by size (85% of unit-length SV40) and by the absence of the cleavage site for End0.R EcoRl. Consequently, the proportion of wild type to variant DNA can be determined by agarose gel electrophoresis before and after EcoRl treatment (Oren et al., 1978). By this test, the closed circular DNA progeny in monkey cells infected with the F161 stock was found to consist (at 48 hr postinfection) of F161F DNA and wild-type SV40 DNA in a ratio of at least 5:l. Since about 60% of the total DNA sequences in the F161F genome are of host origin, the proportion of host DNA I I I I sequences in the total viral yield of the 30 60 90 12 coinfected cells (wild-type SV40 DNA plus variant DNA) is estimated to be about 50% Time (min) (60% of 83%). The other defective SV40 FIG. 2. Kinetics of in vitro RNA synthesis. Sarkosyl populations used in the following transcripextracts, from the nuclei of BSC-1 monkey cells in- tion studies are uncloned stocks which fected with F161 virus were prepared and incubated contain a substantially lower proportion of in vitro as described in Laub and Aloni (1976). The substituted variants. reactions were carried out in the presence (open circles) and in the absence (closed circles) of 2 mgiml heparin. At the indicated times, 50-~1 aliquots of the reactions were analyzed for the incorporation of [3H]UTP into acid-precipitable material.

tandemly repeated segments, each of which contains covalently linked viral and monkey DNA sequences. The viral sequences in each subunit are derived from within a highly limited portion of the wild-type SV40 genome which contains the origin of replication, The monkey DNA sequences in the F161F genome are derived from both the highly reiterated and nonreiterated fractions of the cellular genome. It will be noted from Fig. 1 that cleavage of F161F DNA by the restriction endonuclease H&(11 + III) generates DNA fragments some of which contain solely host DNA sequences, either of the reiterated (fragment 6) or nonreiterated (fragment 4) type. Since the F161F variant, like other substituted SV40 variants, is defective with respect to independent replication, the monkey BSC-1 cells were infected with a virus stock (designated F161) which contains the variant F161F substituted virus and wild-type SV40 helper virus. F161F DNA can be distinguished from wild-type

Transcription in Vitro of Substituted SVhO Host Sequences by Sarkosyl Nuclear Extracts of Infected Cells

Sarkosyl and other detergent extracts of SV40-infected cell nuclei incorporate radiolabeled nucleotides into SV40-related RNA during in vitro incubation (Shmookler et al., 1974; Gariglio and Mousset, 1975; Green and Brooks, 19’75; Laub and Aloni, 1976; Ferdinand et al., 1977). This activity appears to be due to the presence of extractable viral transcription complexes which continue, upon in vitro incubation, to extend RNA chains initiatedin vivo (Laub and Aloni, 1976; Ferdinand et al., 1977). Since cellular mechanisms which process the primary SV40 transcripts in vivo (Aloni, 1974) are unlikely to function during in vitro incubation of the viral transcriptional complex, our initial experiments to detect the transcription of substituted SV40 host sequences were based upon the use of Sarkosyl nuclear extracts of infected cells. Figure 2 shows the kinetics of incorporation of [3H]UTP into acid-precipitable material during the incubation of Sarkosyl nuclear extracts prepared from FlGl-infected monkey cells at 48 hr postinfection. Hepa-

86

HARTMAN ET AL,.

rin, an inhibitor of the initiation of RNA synthesis (Cox, 1973) had no demonstrable effect on the activity of the extracts, similar to the results obtained with Sarkosyl extracts of cells infected with wild-type viruses (Laub and Aloni, 1976). In separate experiments, no evidence was obtained for the incorporation of [32P]ATP (labeled to high specific activity in the y-position) into RNA during in vitro incubation of extracts from either wild-type SV40-infected or FlGl-infected cells (data not shown). These results confirm the conclusion of Laub and Aloni (1976) and Ferdinand et ccl. (19’77) that reinitiation of SV40 RNA synthesis does not occur during the incubation of Sarkosyl extracts from SV40-infected cells. The sucrose gradient sedimentation profile of RNA products synthesized by the Sarkosyl nuclear extracts of FlGl-infected cells displayed a prominent peak with a sedimentation value of about 7 S similar to the profiles of RNA obtained by the Sarkosyl extract of cells infected with wild-type virus (Laub, Bratosin, Horowitz, and Aloni, submitted for publication). Evidence for the in vitro synthesis of RNA sequences homologous to the host DNA component of various substituted SV40 DNAs is shown in Table 1. In this experiment, the [3H]RNA product was depleted of viral sequences by three successive cycles of exhaustive hybridization to wild-type SV40 DNA on filters (Oren et al., 1976). It is evident from the data in Table 1 that the level of hybridization between the depleted [3H]RNA and DNA from the substituted virus stock used to infect the cells (“Homologous DNA”) is strikingly higher than the level of hybridization to wild-type SV40 DNA. The increased levels of hybridization to substituted SV40 DNA (as defined by the A/B ratio in Table 1) roughly parallels the proportion of host-substituted SV40 DNA in each stock. The cloned substituted SV40 DNA (F161F) in the F161 stock shares common host DNA sequences with some of those in the uncloned T8/777 and Rh 911 stocks; however, the substituted variants in the CV-l-P5 stock contain a completely different set of host sequences (Oren et al., 1976, 1978). We conclude,

TABLE 1 INVITRO TRANSCRIPTIONOFSUBSTITUTED SV40DNA

BY SARKOSYLNUCLEAREXTRACTS ~FINFEcTED CELLS~ Counts per minute hybridized to

Depleted [3H]RNA F161 T8l777 CVl-l-P5 Rh9111 Wild-type plaque-purified

Homologous Input DNA (cpm) (A)

Wild-type plaquepurified DNA (B)

Ratio A/B

7600 3500 5700 8000

3550 580 1650 885

160 55 175 315

22.2 10.5 9.4 2.8

5600

95

135

0.7

4 [3H]RNA was synthesized in vitro by Sarkosyl nuclear extracts prepared from BSC-1 cells infected with substituted SV40 stocks (see text for details of FI61, T8/777, CVl-l-P5, and Rh911 stocks) or with plaque-purified wild-type SV40. The in vitro [3H]RNA products were depleted of their wild-type SV40 sequences by three sequential steps of hybridization to 10 pg of plaque-purified wild-type SV40 DNA immobilized on nitrocellulose filters, as described under Materials and Methods. The unbound (“depleted”) [3H]RNAs were then tested for their capacity to hybridize back to DNA of the virus stock used for infection of the cells (“homologous DNA”) or to plaque-purified wildtype SV40 DNA as a control. The back-hybridization reactions were carried out at 68” for 20-24 hr in 4 x SSC containing 0.5% SDS. At the end of the incubation period, the Alters were washed and treated with 20 pgiml RNase for 1 hr at room temperature.

therefore, that the in vitro transcription, by Sarkosyl nuclear extracts, of host DNA sequences is not a peculiarity of one type of substituted SV40 variant. To determine if all parts of the F161F genome are transcribed in the in vitro system, the variant DNA was cleaved with endo *R H&(11 + III) (see Fig. 1). The capacity of [32P]RNA, synthesized in vitro by extracts of FlGl-infected cells, to hybridize with the different H&(11 + III) fragments of F161F DNA was determined by the gel-hybridization procedure of Southern (1975). The autoradiogram in Fig.

SUBSTITUTED

SV40 TRANSCRIPTION

87

Transcription of a Cloned Substituted SV.40 Variant in Intact Cells

FIG. 3. Fidelity of in vitro transcription of substituted SV40 F161F DNA. F161F DNA was digested with endonuclease R.Hin(II + III) as described in Oren et al. (19’78). The fragments were separated on a 1.4% agarose gel, transferred in situ to nitrocellulose filter paper (Southern, 1975), and hybridized with the following probes: (A) 32P-labeled F161F DNA; (B) 32P-wild-type SV40 DNA; (C) 3ZP-monkey BSC-1 cell DNA; (D) [azP]RNA synthesized in vitro by a Sarkosyl nuclear extract of BSC-1 cells infected with the F161 stock; (E) [32P]RNA synthesized in vitro from F161F DNA by E. coli polymerase. The [32P]DNAs were labeled by the “nick-repair” procedure of Maniatis et al. (1975) and hybridization was monitored by autoradiography.

3 shows first the results of control reactions with 32P-labeled F161F DNA (strip A), 32P-labeled wild-type SV40 DNA (strip B), and 32P-labeled monkey DNA (strip C), which confirm the presence of the six major Hin(I1 + III) fragment classes (strip A), the localization of the wild-type viral sequences in fragments 1, 2, and 3 (strip B), and the localization of the reiterated host DNA sequences in fragment 6 (strip C) (Oren et al., 1978). The hybridization reaction with [32P]RNA, synthesized in vitro by the Sarkosyl nuclear extracts (strip D) indicates that all segments of the F161F genome were transcribed. Strip E in Fig. 3 shows the pattern of hybridization with [32P]cRNA transcribed from intact F161F DNA by E. coli RNA polymerase. In this case, too, all parts of the F161F genome were transcribed, although some parts appear to be transcribed more abundantly than others.

The in vitro Sarkosyl nuclear extract system described above may not accurately reflect the controlled viral transcription process which occurs in intact cells. Accordingly, we next turned our attention to the possible transcription of substituted SV40 host DNA sequences in vivo. To achieve the most sensitive level of detection possible, a vast excess of unlabeled RNA from FlGl-infected monkey cells was hybridized in solution with highly radioactive cRNA probes synthesized, by E. coli RNA polymerase, either from intact F161F DNA or from Hin(I1 + III) fragments of F161F DNA. In the case of the cRNA probe synthesized from intact F161F DNA, the RNA was depleted of viral sequences by two successive hybridization steps to wildtype SV40 DNA, as described earlier. In the case of the cRNA probes synthesized from the Hin(I1 + III) cleavage products of F161F DNA, fragments 6 and 4 were chosen since they contain solely host DNA sequences either of the highly reiterated or nonreiterated type, respectively (see Fig. 1). The unlabeled cellular RNA was extracted at 48 hr postinfection and the hybridization reactions were monitored by the conversion of the labeled probes to a RNase resistant state, using control reactions with excess E. coli RNA to assess the level of self-annealing. Although F161F DNA is symmetrically transcribed by E. coli RNA polymerase, the low concentration of the probe in the above hybridization reactions resulted in only minor levels of self-annealing (lo-20%). The results of these experiments are shown in Figs. 4, 5, and 6. The hybridization reactions between the cellular RNAs and the f3H]cRNA probe directed against the host DNA components of F161F DNA are shown in Fig. 4. Unlabeled RNA extracted from FlGl-infected cells protects up to 70% of the [3H]cRNA probe. In contrast, RNA from wild-type SV40-infected cells or from mock-infected cells protects only lo-15% of the probe (after subtraction of the lo-20% self-annealing background determined in control reac-

88

HARTMAN ET AL.

tions with excess E. coZi RNA). Figure 4 also shows that the material in the RNA preparation made from FlGl-infected cells, which protects the [3H]cRNA, is sensitive to alkali degradation, indicating that the protection is due to RNA and not DNA. These results show, therefore, that RNA homologous to the host DNA components of F161F DNA is present in FlGl-infected cells. Such RNA sequences are either absent or present only at very low concentrations in uninfected cells and in cells infected with wild-type virus. To investigate the cellular compartmentalization of DNA sequences homologous to the host sequences in the F161F genome, unlabeled RNA was isolated separately from the nuclear and cytoplasmic fractions of FlGl-infected cells and hy-

0.6

1.2

2.4

012

0.24

0.40

RNA (mg/ml)

FIG. 5. Presence of RNA homologous to F161F host DNA sequences in the nuclear and cytoplasmic fractions of FlGl-infected cells. [$H]cRNA synthesized (by E. coli polymerase) from F161F DNA was depleted of viral sequences and hybridized in solution with excess cytoplasmic (panel A) and nuclear (panel B) RNAs isolated from cells infected with F161 virus (X), wildtype SV40 (0) or from mock-infected cells (0). The conditions of hybridization were as described for Fig. 4.

bridized with the F161F host [3H]cRNA probe, under the same conditions described for Fig. 4. The results in Fig. 5 show that transcripts of F161F host DNA sequences are present in both the nuclear and cytoplasmic fractions. The data in Fig. 5 indicate that more of the cytoplasmic RNA, compared to nuclear RNA, is required to protect 50% of the probe. This is expected since the major fraction of the cells’ total RNA is present in the cytoplasm rather RNA(mg/ml) than in the nucleus. FIG. 4. Transcription in viva of substituted SV40 To determine if both the reiterated and F161F host DNA sequences. [3H]cRNA was synnonreiterated cellular DNA sequences in thesized from F161F DNA by E. coli RNA polymerase. After depletion of viral sequences (see Ma- F161F DNA are transcribed, we performed hybridization reactions between total celluterials and Methods) the labeled probe was hybridized cells and in solution (3000 cpm or 3 x 10m4pg RNA per reaction lar RNA from FlGl-infected [3H]cRNA probes directed specifically volume of 150 ~1) with excess (0.5-400 pg RNA/150 ~1) unlabeled total cellular RNA from mock-infected against the reiterated type and against the cells (A) from cells infected with wild-type virus (A) nonreiterated type of F161F host DNA seor from cells infected with the F161 stock (X) which quences [Hin(II + III) fragments 6 and 4, contains the substituted F161F variant and wild-type respectively (see Fig. l)]. The results in helper virus. Hybridization was performed and moniFig. 6 provide clear evidence that both tored by RNase resistance as described under Materials and Methods. The self-annealing of the probe types of F161F host DNA sequences are (lo-20%) was measured in control reactions with the indeed transcribed in FlGl-infected cells. same excess of E. coli RNA and has been subtracted from the data points shown. The curve designated (0) represents the background level of RNase resistance obtained when FlGl-infected total cellular RNA was treated with alkali (18 hr at 37” in 0.3 N NaOH followed by neutralization with 0.3 N HCI and 40 nG4 Tris. HCl, pH 7.5) prior to hybridization.

Stability, Size, and Fate of the Fl61F Host DNA Transcripts in Coinfected Cells

To investigate the stability of the RNA transcribed from the host sequences in the F161F genome, the following experiment

SUBSTITUTED

SV40 TRANSCRIPTION

89

fected cell, then we expect the highest level of complementary RNA to be present in the in vi&o products of the transcription complex preparation (since processing is unlikely to occur during in vitro synthesis) compared to [3H]RNA from cells labeled in viva. We also might expect the level of F161F host DNA transcripts to be higher in the nuclear fraction relative to the cytoplasmic fraction of coinfected cells labeled in vivo.

06

12

1.8

24

RNA (mghnl)

FIG. 6. Evidence that F161F host DNA sequences of both the reiterated and nonreiterated types are transcribed in viva. F161F DNA was cleaved with endo*R H&(11 + III) and the DNA fragments which contain host DNA sequences of the reiterated and nonreiterated types were used, separately, as templates for the synthesis (by E. coli RNA polymerase) of t3H]cRNA. These two [3H]cRNA probes were then hybridized with excess unlabeled total RNA from FlGl-infected cells (O), wild-type SV40-infected cells (O), or mock-infected cells (X) under the conditions described in Fig. 4. Panel A shows the reactions with [3H]cRNA made from Hin(I1 + III) DNA fragment 6 (reiterated host sequences); panel B shows the reactions with [3H]cRNA from Hin(I1 + III) DNA fragment 4 (nonreiterated host sequences). See Fig. 1 and text for a description of the structure and H&(11 + III) cleavage pattern of F161F DNA.

was performed. C3H]RNA was isolated separately from the nuclear and cytoplasmic fractions of coinfected cells labeled with [3H]uridine from 45-48 hr postinfection. In parallel, transcription complexes were isolated from the nuclei of identically infected (unlabeled) cells and used to synthesize [3H]RNA in vitro, as described earlier. Each [3H]RNA preparation was then tested for its relative capacity to hybridize with the segment of the F161F genome which contains exclusively host DNA sequences (see below). If, as a consequence of posttranscriptional processing, the F161F host DNA transcripts are unstable in the coin-

For use in the hybridization tests, the F161F host DNA sequences were isolated by cleaving total F161F DNA with the restriction endonuclease BamI, HpaII, and Bgl I which generates six products corresponding to 11.4, 8.1, 7.7, 4.1, 4, and 3.8% of the unit-length genome (see Fig. 1). The largest of the cleavage products (the 11.4% fragment) is the BamI-HpaII cut which contains part of the nonreiterated host DNA sequences in Hin fragment 1, all of the nonreiterated host sequences of Hin 4, all of the reiterated host sequencesof Hin 6, and all of the host sequences of Hin 3. The results of the hybridization tests between the BamI-HpaII fragment and the different [3H]RNA preparations are summarized in Table 2. As can be seen in the final column, the in vitro [3H]RNA preparation, synthesized by transcription complexes, contained the highest proportion of sequences complementary to F161F host DNA (39.7% of the total virus-related sequences). In contrast, only 15.3% of the in vivo labeled nuclear virus-related RNA and 4.8% of the cytoplasmic virus-related RNA hybridized with excess levels of F161F host DNA immobilized on filters. In WT SV40-infected cells no host-substituted transcripts could be detected in any of the labeled RNA preparations, indicating again that the host-substituted transcripts are not transcribed from the cellular DNA. The failure to detect the host-substituted transcripts in uninfected or wild-type infected cells might conceivably arise from self-annealing of symmetrically transcribed RNA (Aloni, 1972). To explore this possibility, mock-infected BSC-1 cells, F161, or wild-type SV40 infected cells were labeled with [3H]uridine from 45-48 hr post-

90

HARTMAN ET AL. TABLE 2 DETECTION OF [3H]RNA HOMOLOGOUS TO F161F HOST SEQUENCESIN Fl61-INFECTED CELLP Counts per minute hybridized to filters containing

Source of [3H]RNA

Input (wn)

WT SV40 DNA

F161F host DNA*

Host-substituted RNA in total viral RNA sequences” (o/o)

FlGl-infected cells In vitro transcribed RNA Nuclear RNA Cytoplasmic RNA

10.0 x 103 13.8 x 106 3.5 x 106

1,050 14,640 7,310

690 2,640 340

39.7 15.3 4.4

WT SV40 infected cells In vitro transcribed RNA Nuclear RNA Cytoplasmic RNA

7.5 x 103 5.0 x 106 1.6 x lo6

680 24,580 28,280

10 10 30

1.4 0.0 0.1

u BSC-1 cells were infected with substituted F161 stock or with wild-type (WT) SV40 and labeled with [3H]uridine (42 Ci/mmol 0.1 mCi/ml) from 45-48 hr postinfection. r3H]RNA was synthesized in vitro using the Sarkosyl nuclear extract (see Materials and Methods), or extracted from the nuclear and cytoplasmic fractions. The hybridization reactions were performed under DNA excess conditions as described in Table 1. Each filter contained 2.5 pg of DNA. b F161F DNA was digested with end0.R ZZ@I, BarnI, and BglI as described by Oren et al. (1978). The digest was electrophoresed in a 1.4% agarose gel. The band corresponding to the host DNA fragment spanning the &a11 to the Bum1 cut (see text) was eluted by shaking in buffer containing 0.2 M NaCI, 10 mM Tris ‘HCI, pH 7.4, and 5 mJ4 EDTA for 18 hr at room temperature. c Calculated as: lcnm hvbridized to F161F host DNA/(cpm hybridized to WT SV40 DNA + cpm hybridized to F161F host DNAj]*x 100.

infection. Total cellular RNA was extracted, self-annealed, and treated with RNase. The RNase-resistant doublestranded RNAs, selected by chromatography on Sephadex G-100 columns (Aloni, 1972), were then denatured and hybridized with wild-type SV40 DNA and with the F161F host DNA segment. Labeled RNA of mock-infected or wild-type infected cells prepared in this way, again failed to hybridize with F161F host DNA. We noted, however, that the proportion of virus-related double-stranded RNA was higher in cells infected with F161 compared to cells infected with wild-type virus (data not shown). This latter finding suggests that the F161F genome is symmetrically transcribed. Similar observations have been reported previously (Laub and Aloni, 1975; Kuff et al., 1978). We next exploited the BamI-HpaII segment of F161F DNA in filter hybridization experiments to determine the size of

the host DNA transcripts in FlGl-infected cells. Total cellular RNA from FlGl-infected cells, labeled with [3H]uridine from 45-48 hr postinfection, was fractionated on sucrose gradients. Samples of each fraction were then hybridized to either wild-type SV40 DNA or F161F host DNA (BamIHpaII segment) on filters. The sedimentation profile of the wild-type SV40 RNA chains in panel B of Fig. ‘7 shows the expected distribution into two major peaks, corresponding to the 19 S and 16 S components (Weinberg et al., 1972). In contrast, the sedimentation profile of r3H]RNA species homologous to F161F host DNA appears to be more heterogeneous (Fig. 7, panel C). It is noteworthy that some of the RNA molecules transcribed from F161F host DNA are equal to or greater than half-genome size and must therefore arise from cotranscription of the host and viral sequences which are linked together in the F161F genome.

SUBSTITUTED I

I

A 50-

28s J

I

I

18s J

SV40 TRANSCRIPTION

91

fected cells, prompted us to examine their possible participation in protein synthesis. To this end, we analyzed the polyribosomal pattern of viral RNAs in F161 infected cells. The infected cells were labeled with [3H]uridine from 45-48 hr postinfection and the cytoplasmic extract was sedimented through a sucrose gradient. Each fraction of the gradient was assayed for total RNA content and for the presence of virus-related RNA as judged by hybridization to wild-type SV40 DNA or to F161F host DNA (BumI-HpaII segment) immobilized on filters. The results are given in Fig. 8. As shown in Fig. 8B, most, if not all, of the wild-type SV40 RNA synthesized during the 3-hr labeling period, is active in protein synthesis as judged by its predominant association with heavy polyribosomes. In contrast, the RNA complementary to F161F host DNA was found only in the disomal, monoribosomal, and lighter fractions, indicating a lack of involvement in protein synthesis. Treatment of the cytoplasmic extract with EDTA prior to centrifugation resulted in a shift of the labeled virus-related RNAs to the lighter part of the gradient (data not shown). DISCUSSION

The cloned substituted SV40 used in this study contains covalently linked host DNA sequences derived from both highly reiterFIG. 7. Sedimentation profile of in &JO transcripated and nonreiterated families in the tion products of F161 DNA. BSC-1 cells were infected monkey genome. Since the restriction with the F161 stock and labeled with [3H]uridine endonuclease map of this variant DNA was (0.5 mCii5 ml/lo’ cells) from 45-48 hr postinfection. Total cellular RNA was extracted and sedimented known (Oren et al., 19’78), it was possible to seek out the complementary RNA tranin a 15-30% sucrose gradient made up in SDS-buffer (Spine0 SW27.1 rotor, 18 hr, 25,000 rpm, 209 Panel scripts using hybridization probes directed A shows the profile of total [3H]RNA (5-~1 samples of against both classes of cellular DNA seeach fraction). Twenty-microliter samples of each frac- quences. Our results indicate that both the tion were then hybridized to wild-type SV40 DNA reiterated and nonreiterated types of cel(panel B) and 200~~1samples were hybridized to the lular DNA in the variant genome are in host DNA fragment cleaved from F161F DNA by fact transcribed in cells coinfected with treatment with end0.R HpaII, BumI, and BglI (see wild-type helper virus. text) (panel C). After incubation at 68” for 20 hr in Since cellular controls cannot operate 4 x SSC, 0.5% SDS, the filters were washed and during the in vitro incubation of transcriptreated with RNase (20 Fg/ml, 1 hr at room temperation complex preparations, a comparison of ture in 2 x SSC). the amounts of substituted SV40 RNA proThe presence and substantial size of RNA duced in the in vitro and in the intact cell transcripts complementary to F161F host systems provides one measure of transcript sequences in the cytoplasmic fraction of in- stability in vivo. The results in Table 2 IO 20 30 Fraction number

92

HARTMAN ET AL.

clearly show that the levels of substituted SV40 RNA (relative to wild-type SV40 RNA) generated in the in vitro system are more than double the levels detected in the nuclear fraction (and about nine times the level detected in the cytoplasmic fraction) of the coinfected cell. It should be recalled, in this connection, that under the conditions of the infection used, substituted SV40 DNA host sequences represent about 50% of the total virus-related DNA yield. The high yield of substituted SV40 RNA obtained during the in vitro elongation of transcription complex RNA chains indicates that the substituted SV40 DNA molecule is not at a disadvantage at the level of initiation. Consequently, it appears most likely that the reduced amounts of substituted SV40 RNA found in intact cells result from cellular processing controls which regulate the stability of the primary transcripts and/or the efficiency with which they are exported to the cytoplasm. A similar conclusion has been reached by Kuff et al. (1978). If post-transcriptional degradation is responsible for the comparatively low proportion of substituted SV40 transcripts in the cytoplasm of the coinfected cells, then some of our observations suggest that the degradation process occurs at a rather slow rate. The substituted SV40 RNA transcripts were readily detectable by hybridization between unlabeled cytoplasmic RNA from coinfected cells and the F161F host DNA cRNA probe (Fig. 5) indicating that the transcripts must be sufficiently stable to withstand exportation from nucleus to cytoplasm. Furthermore, the sedimentation profile of the substituted SV40 RNA transcripts, labeled for 3 hr, reveals the presence of large species with sedimentation coefficients in the range lo-20 S (Fig. 7, panel C). In addition to selective degradation, other forms of posttranscriptional processing may distinguish between wild-type and substituted SV40 RNA species. The lack of association with the heavier polysomal fractions involved in protein synthesis may well reflect aberrant chemical modifications of substituted SV40 RNA chains rather than nonspecific degradation.

I.0

A t

-a .B ?I6 .z

3

0, 0* c> (04 E I 8+

2 I

IS2 0 0

IO Fraction

20

30

40

0

number

FIG. 8. Polyribosomal profile of viral RNA from FlGl-infected cells. FlGl-Infected cells were labeled as in Fig. ‘7, washed with cold PBS, and lysed in a solution containing 25 mM NaCI, 25 n&J Tris.HCl, pH 7.4,5 mM MgCl,, 1% NP40,0.5% Na-deoxycholate, and 1 mg/ml tRNA. Nuclei were pelleted by centrifugation at 1500 rpm for 5 min, and the supernatant recentrifuged for 20 min at 10,000 rpm. The postmitochondrial supernatant was sedimented through a linear 15-45% (w/w) sucrose gradient in 25 m2MNaCl, 25 mJ4 Tris, pH 7.4, and 5 m&f MgCl,. Centrifugation was in a Spinco rotor SW27 at 2700 rpm, 3” for 4 hr. (A) Absorbance at 254 nm was measured in an ISCO model LJA-5 absorbance monitor equipped with a 130~1 flow cell (-). Aliquots of 25 ~1 from each fraction were precipitated with 10% TCA and counted (0). (B) Samples (100 ~1) of each fraction were hybridized to WT SV40 DNA (0) and 400 ~1 samples were hybridized to the F161F host DNA fragment (0) as in Fig. 7.

RNA from uninfected or SV40 wild-type infected monkey BSC-1 cells either failed to react or reacted to only minor extents with the hybridization probes complementary to the cellular DNA sequences in the F161 genome (Figs. 4-6). The extent of these reactions varied from experiment to experiment (compare, for example, the data in Figs. 4 and 5 with the data in Fig. 6). In addition, we were unable to detect RNA homologous to the F161F host DNA sequences in preparations, from both unin-

SUBSTITUTED

SV40 TRANSCRIPTION

fected and SV40 wild-type infected cells, which had been enriched for doublestranded RNA molecules. Nevertheless, we cannot exclude the possibility that some of the host DNA sequences incorporated into the SV40 genome may be transcribed in uninfected cells. The question of the transcription in normal cells of the highly reiterated African green monkey sequences is of interest since a related group of sequences of this type is frequently found in independent isolations of substituted SV40 DNA populations (Oren et al., 19’76; Rosenberg et al., 1977; Oren et al., 19’78). M. Singer and her co-workers (M. Rosenberg et al., 1977; H. Rosenberg et al., 1978) have demonstrated that the highly reiterated cellular DNA found in substituted SV40 DNA is derived from within a specific segment of the African green monkey genome (also known as the cr-component) which comprises a repeat unit of 172 base-pairs, reiterated 1.6 x lo6 times per diploid monkey genome. One hundred fifty-five of the one hundred seventy-two nucleotides in the African green monkey repeat-unit DNA are present in the DNA of a substituted SV40 variant similar to or identical to F161F (Rosenberg et al., 1977; Rosenberg et al., 1978). We have not been able to consistently detect the transcription of the cr-component sequences in uninfected cells of the BSC-1 line. We have also failed to detect RNA homologous to F161F host sequences in three lines of African green monkey CV-1 cells transformed by SV40 (J. R. Hartman, unpublished results). The a-component fraction of the African green monkey genome, like mouse satellite DNA (Flamm et al., 1969) may therefore be a class of repetitive eukaryotic DNA sequences which are rarely, if ever, expressed. ACKNOWLEDGMENTS We wish to thank M. Oren for helpful discussions and B. Danovitch, T. Koch, and E. Nabaa for their expert assistance. This work was partly supported by Public Health Service Research Grant CA14995, by Contract NO1 CP33220 from the National Cancer Institute, and by grants from the United StatesIsrael Binational Science Foundation (BSF) and the German Science Fund (GSF). E. W. is an Established Investigator of the Chief Scientist’s Bureau, Ministry of Health, Israel.

93

REFERENCES ALONI, Y. (1972). Extensive symmetrical transcription of SV40 DNA in virus yielding ceils. Proc. Nat. Acad. Sci. USA 69, 2402-2409.

ALONI, Y. (1974). Biogenesis and characterization of SV40 and polyoma RNAs in productively infected cells. Cold Spring Harbor Sym. Quant. Biol.

39, 165-178.

COX, R. F. (1973). Transcription of high-molecular weight RNA from hen-oviduct chromatin of bacterial and endogenous forms-B RNA polymerases. Eur. J. Biochem.

39, 49-60.

DAVOLI, D., GANEM, D., NUSSBAUM,A. L., FAREED, G. C., HOWLEY, P. M., KHOURY, G., and MARTIN, M. A. (19’77). Genome structures of reiteration mutants of SV40. Virology 77, 110-124. FERDINAND, F.-J., BROWN, M., and KHOURY, G. (1977). Synthesis and characterization of late lytic SV40 RNA from transcriptional complexes. Virology 78, 150-161. FLAMM, W. G., WALKER, P. M. B., and MCCALLUM, M. (1969). Some properties of the single strands isolated from the DNA of the nuclear satellite of the mouse (Mus musculus). J. Mol. Biol. 40, 423-443.

FRENKEL, N., LAVI, S., and WINOCOUR,E. (1974). The host DNA sequences in different populations of serially passaged SV40. Virology 60, 9-20. GARIGLIO, P., and MOUSSET,S. (1975). Isolation and partial characterization of a nuclear RNA polymerase-SV40 DNA complex. FEBS Lett. 56, 149-155.

GREEN, M. H., and BROOKS,T. L. (1975). Isolation of two forms of SV40 transcriptional complexes from infected monkey cells. INSERM 47, 33-42. HIRT, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol.

26, 365-369.

KUFF, E. L., FERDINAND, E.-J., and KHOURY, G. (1978). Transcription of host-substituted SV40 DNA in whole cells and extracts. J. Viral. 25, 28-36. LAUB, O., and ALONI, Y. (1975). Transcription of Simian virus 40. V. Regulation of Simian virus 40 gene expression. J. Viral. 16, 1171-1183. LAUB, O., and ALONI, Y. (1976). Transcription of SV40. Vl. SV40 DNA-RNA polymerase complex isolated from productively infected cells transcribed in vitro.

Virology

75, 346-354.

LAVI, S., and WINOCOUR,E. (1972). Acquisition of sequences homologous to host deoxyribonucleic acid by closed circular Simian virus 40 deoxyribonucleic acid. J. Viral. 9, 309-316. LEE, T. N. H., BROCKMAN,W. W., and NATHANS, D. (19’75).Evolutionary variants of SV40; Cloned substituted variants containing multiple initiation sites for DNA replications. Virology 66, 53-69. MANIATIS, T., JEFFREY, A., and KLEID, D. G. (1975).

94

HARTMAN ET AL.

Nucleotide sequence of the rightward operator of phage A. Proc. Nat. Acad. Sci. USA 72,1184-1188. OREN, M., KUFF, E. L., and WINOCOUR,E. (1976). The presence of common host sequences in different populations of substituted SV40 DNA. Virology 73,419-430. OREN, M., LAVI, S., and WINOCOUR,E. (1978). The structure of a cloned substituted SV40 genome.

repetitive monkey DNA found in defective Simian virus 40. Cell 11, 845-857. ROZENBLATT, S., LAVI, S., SINGER, M. F., and WINOCOUR, E. (1973). Acquisition of sequences homologous to host DNA by closed circular Simian virus 40 DNA. III. Host sequences. J. Viral. 12, 501-510. SHMOOKLER,R. J., Bus, J., and GREEN, M. H. (1974). ViTology 85, 404-421. Properties of the polyoma virus transcription comPENMAN, S. (1966). RNA metabolism in the HeLa plex obtained from mouse nuclei. Virology 57, cell nucleus. J. Mol. Biol. 17, 117-130. 122-127. RAO, G. R. K., and SINGER, M. F. (1977). Studies SOUTHERN, E. M. (1975), Detection of specific sequences among DNA fragments separated by gel on a defective variant of Simian virus 40 that is substituted with DNA sequences derived from monkey electrophoresis. J. Mol. Biol. 98, 503-517. DNA. II. Structure of the DNA. J. Biol. Chem. WEINBERG, R. A., WARNAAR, S. O., and WINOCOUR, E. (1972). Isolation and characterization of SV40 252, 5124-5134. ROSENBERG, H., SINGER, M., and ROSENBERG, RNA. J. Viral. 10, 193-201. M. (1978). Highly reiterated sequences of Simian. WINOCOUR,E., FRENKEL, N., LAVI, S., OSENHOLTS, Science ZOO, 394-402. M., and ROZENBLATT, S. (1974). Host substitution ROSENBERG, M., SEGAL, S., KUFF, E. L., and in SV40 and polyoma DNA. Cold Spring Harbor SINGER, M. F. (1977). The nucleotide sequence of Symp. f&ant. Biol. 39, 101-108.