Covalently linked cell and SV40-specific sequences in an RNA from productively infected cells

VIROLOGY

60,

Covalently

558-566 (1972)

Linked

Cell from

‘SH,\IUEL Department

and

SV40-Specific

Productively

Sequences

Infected

ROZENBLATT

AND

an RNA

Cells

ERNEST

of Genetics, Weizmann Institute

in

WINOCOUR

of Science, Rehovot, Israel

Accepted August 8, 1972 High molecular weight simian virus 40 (SV40) specific RNA molecules, whose size exceeds that of unit length virus DNA by severalfold, are present in the nuclei of productively infected monkey BS-C-1 cells. Hybridization experiments with highly purified preparations of the large viral RNA molecules show that they contain hostspecific sequences covalently linked to virus-specific sequences. The results suggest that the proportion of host-specific sequences exceeds that of viral sequences in this class of viral RNA molecules and that they arise from the cotranscription of integrated viral DNA and adjacent cellular DNA during the lytic cycle of infection. INTRODUCTION

A high molecular weight (HMW) viral RNA, whose size exceeds that of a single st’rand of viral DNA, has been detected in the nuclei of cells productively infected with polyoma virus (Acheson et al., 1971), and SV40 (Tonegawa et al., 1970; Jaenisch, 1972; Weinberg el al., 1972). Previous reports have indicated that the HMW SV40 RNA contains nonviral sequencesin addition to viral sequences (Jaenisch, 1972; Weinberg et al., 1972); however, the origin of the nonviral sequenceswas not determined. In the present report, we show that SV40 HMW RNA, isolated from infected monkey BS-C-1 cells, contains viral sequencescovalently linked to sequenceswhich are complementary to cellular DNA. Closed circular SV40 DNA, in which a part of the viral genome has been deleted and replaced by covalently linked cell DNA sequences(called substituted molecules) are produced in monkey BS-C-l cells infected under certain conditions (Aloni et al., 1969; Lavi and Winocour, 1972; Tai et al., 1972). When the cells are infected with plaquepurified viral stocks, no substituted viral DNA progeny can be detected. On the other hand, when the cells are infected with serially passaged viral stocks, the progeny cont’ains 558 Copyright All rights

@ 1972 by Academic Press. Inc. of reproduction in any form reserved.

substituted molecules in a proportion which depends upon the multiplicity of infection and the passage history of the virus (Lavi and Winocour, 1972). Although closed circular substituted virus DNA molecules probably arise initially from t’he integration of virus DNA followed by the excision of linked celluIar and viral DNA sequences, the HMW viral RNA containing host-specific sequencescould be generated by the continuous transcription of free substituted viral DNA molecules, rather tha.n by the transcription of virus DNA integrated into the cellular genome. We therefore studied the presence and nature of the HMW viral RNA not only in cells infected under conditions where substituted viral DNA is synthesized, but also in cells infected with plaque-purified virus under conditions where no detectable level of free viral DNA molecules homoIogous to ceil DNA appear in the progeny. We conclude from these studies that at least some of the HMW viral RNA results from the cotranscription of integrated viral DNA and adjacent cellular DNA. MATERIALS

AND

METHODS

SDS Buffer, pH 7.4 (Penman, 1966) contains 0.01 M Tris .HCI, 0.001 M EDTA, 0.1 M NaCl, and 0.5 % w/v Bu$ers

and reagents.

SV40

RNA

CONTAINING

sodium dodecyl sulfate (SDS). Lysis Buffer is the same as SDS Buffer, but contains 1% SDS and 300 pg/ml polyvinyl sulfate. Hybridization Buffer (Weinberg, el al., 1972) contains 50% (v/v) formamide, 0.75 M NaCl, 0.5 % w/v SDS, and 0.5 M Tris.HCl (final pH adjusted to 7.4). Elution Buffer contains 90 volumes of 100% formamide, 9 volumes of distilled water, and 1 volume of SDS Buffer (final pH adjusted to 8.4). TKM Buffer contains 0.05 M Tris.HCI (pH 6.7) 0.025 M KC1 and 0.0025 M MgCIZ . Dimethyl sulfoxide (DMSO), deuterated DMSO, and formamide were obtained from Fluka AG, Switzerland (purist grade). SSC is 0.15 21/;rNaCl, 0.015 M sodium cit’rate (Marmur, 1961). Cells. The BS-C-1 line of African green monkey kidney cells (Hopps et al., 1963) was obtained from Flow Laboratories. The cells were cultured in 90-mm diamet,er plastic petri dishes in Eagle’s medium (EM) with a 4-fold concentration of amino acids and vitamins, supplemented with 10 % calf serum. After virus infection, the concentration of serum was reduced to 2 %. virus. Two stocks of SV40, strain 777 (Gerber, 1962) were used. The plaquepurified stock was produced by infecting BS-C-1 cells at a very low mult’iplicity of infection (an average of 10 PFU per 4 X lo6 cells) wit’h a single-plaque isolate which had been previously subjected to 2 sequential plaque-purification procedures. None of the closed circular viral DNA molecules extracted from this plaque purified virus stock, or from cells infected at 100 PFU/cell with this virus, hybridized detectably with BS-C-1 cell DNA (Lavi and Winocour, 1972). The serially passaged virus stock was produced by infecting BS-C-1 cells at a multiplicity of 0.2 PFU/cell with nonplaque-purified strain 777 virus which has been maintained for several years in this laboratory by serial passage at multiplicities in the range 0.1 to 1 PFU/cell. In cells infected at 100 PFU/cell with the serially passaged virus, about 20 % of the closed circular viral DNA progeny molecules contain sequences homologous to cell DNA (Lavi and Winocour, 1972). Infection and labeling of cells. BS-C-l cultures (4 X lo6 cells per culture) were infected with 1 ml of plaque-purified or serially pas-

HOST

SEQUENCES

559

saged virus (4 X 10s PFU/ml in both cases) in EM wit,h 2% calf serum. After a 2-hr period at 37” for virus absorption, 7 ml of fresh medium were added to each culture. The infected cells were labeled with 3Huridine (500 &Ji/2ml medium per culture, 29 Ci/mmole; The Radiochemical Centre, Amersham, England) from 38 hr to 44 hr post-infection. RNA extraction. At the end of the labeling period, the cultures were washed 3 times with phosphate-buffered saline (PBS) (Dulbecco and Vogt, 1954). To the cell sheet in each plate, were added 2 ml of PBS containing 0.1% Nonidet P-40 (Eorun et al., 1967). After 10 min on ice, the cytoplasmic lysate was carefully removed and centrifuged at’ low speed at 4°C; polyvinyl sulfate (300 pg/ml) and SDS (final concentration of 0.5 %) were added to the supernatant,, which was then extracted for cytoplasmic RNA. The cell nuclei, which remain attached to the surface of the plastic petri dish after the Nonidet P-40 t’reatment, were lysed by adding 2 ml per plate of lysis Buffer; nuclear RNA was extracted from this fraction. To extract RNA from whole cells, the cells were lysed directly by adding 2 ml of Lysis Buffer per ccl1 sheet. RNA was extracted by a modification of procedures reported by Weinberg et al. (1972). The lysates were extracted 3 times with phenol-chloroform-isoamyl alcohol and 4 times with chloroform-isoamyl alcohol (Penman, 1966) at room temperature. LiCl was added to the aqueous phase to a final concentration of 2 ill and the solution was left overnight at 4°C. About 60% of the 3H-RNA precipitated (>90 % of the DNA and all the 4 S RNA remained in solution) and was collected by centrifugation (Serval RC2, 13,000 rpm, 30 min). The precipitate was resuspended in SDS Buffer, and reextracted once with phenol and 4 t*imes with chloroform-isoamyl alcohol. RNA was again precipitated from the aqueous phase by the addition of LiCl, collected by centrifugation, and either stored under alcohol at -20°C or resuspended in Hybridizat’ion Buffer and centrifuged at 1500 g for 30 min at room temperature to remove insoluble material. Selection of virus-specific RNA. Virusspecific RNA was selected by preparat,ive

,560

ROZENBLATT

hybridization to SV40 DNA in 50% formamide at 37” without RNase treatment (Weinberg et al., 1972). The SV40 DNA was prepared exclusively from the plaquepurified virus stock and displayed no detectable homology to cell DNA. The procedures for virus purification, isolation of closed circular viral DNA by sedimentation in alkaline CsCl solutions, and immobilization of denat’ured viral DNA on nitrocellulose membrane filters, have been described elsewhere (Lavi and Winocour, 1972). The hybridization reaction contained the 3HRNA suspended in 1 ml of Hybridization Buffer, one filter with 10 pg of immobilized SV40 DNA, and one blank filter. After incubation at 37” for 22 hr in a water-bath shaker, the filters were removed, washed under suction with 10 ml of Hybridization Buffer, and incubated for a further hour at .37” in 10 ml of fresh Hybridization Buffer. Finally, each filter was washed on both sides with 50 ml of 1 X SSC (containing 0.5% SDS) under suction. To recover the hybridized RNA, the filters were incubated for 1 hr at’ 37” in 1 ml of Elution Buffer. Approximately 70 % of the 3H-RNA was eluted from t’he filter by this procedure. The RNA was precipitated out of the Elution Buffer by the addition of 10 pg/ml of E. coli tRNA, NaCl to 0.3 M, and 2 volumes of ethanol. A variable amount of unlabeled, singlestranded SV40 DNA was eluted, together with 3H-SV40 RNA, from the filters treated with Elution Buffer. Hence, prior to the rehybridization tests, the 3H-RNA preparations were treated with DNase and filtered through nitrocellulose membrane filters as -described below. Fractionation of virus-specific RNA. The virus-specific RNA was fractionated according to chain length in deuterated DMSO:sucrose gradients (Strauss et al, 1968; Sedat and Sinsheimer, 1970). The RNA precipitated from the hybridization Elution Buffer was resuspended in 20 ~1 of a solution containing 10d3 M EDTA and 0.5% SDS; 200 ~1 of 100 % DMSO were then added. The solution was layered on top of 10 ml of a .gradient (O-10 % sucrose, 10 % deuterat,ed DMSO-99 % deuterated DMSO) formed by mixing, in a gradient maker, 5 ml of a soluhion containing 10 % dueterated DMSO and

AND

WINOCOUR

90 % DMSO with 5 ml of a solution containing 10 % w/w sucrose, 99% deuterated DMSO, and 1% 10e3 M EDTA in water. The gradient was centrifuged at 35,000 rpm for 23 hr at 26” in the Spinco SW 41 rotor. Fractions were collected from the bottom of the tube and the amount of 3H-RNA in a Noth volume of each fraction was determined after precipit’ation with 10 % trichloroacetic acid. The 3H-RNA was precipitated out, of select’ed pooled fractions (Fig. 1) by the addition of 2 volumes of ethanol and 10 pg/ml Escherichia coli tRNA, resuspended in TKM-Buffer, and treated with 50 pg/ml of DNase I (electrophoretically pure, Worthington Biochemical Corporation) for 1 hr at room temperature. The DNase was removed by extraction with phenol-chloroformisoamyl alcohol. The 3H-RNA was then precipitated with ethanol, resuspended in 6 X SSC, and slowly filtered, twice, through Millipore nitrocellulose membrane filters prior to the rehybridization test’s. Rehybridization procedure. The selected 3H-RNA species were resuspended in 1 ml of 3 X SSC containing 0.1% SDS and incubated at 65” for 48 hr with a filter containing 10 pg of plaque-purified SV40 DNA or 50 pg of BS-C-1 cell DNA, or 50 pg of E. coli DNA, or with a blank filter. The BS-C-1 cell DNA and E. coli DNA were prepared according to Aloni et al. (1969). At the end of the reaction, the filters were washed 4 times with 2 X SSC. One filter of each duplicate reaction was dried, counted to determine the amount of 3H-RNA bound, rinsed in toluene to remove the scintillation chemicals, treated with RNase in 2 X SSC (50 pg/ml, 1 hr at room temperature), washed again 4 times with 2 X SSC, and recounted. The second filter of each duplicate reaction was treated directly with RNase before counting. The same levels of RNase-resist,ant 3H-RNA bound to t’he filter were obtained by both methods. RESULTS

Isolation of XV40 RNA from Productively Infected Cells Since host-cell RNA metabolism continues unabated after SV40 infect,ion, the virusspecific RNA species were selected by pre-

SV40

RNA

CONTAINING

parative hybridization to plaque-purified SV40 DNA, using a formamide hybridization and elution procedure in which RNA degradat’ion is minimal (Weinberg et al., 1972). The plaque-purified SV40 DNA used in this and all subsequent hybridization reactions showed no detectable homology to host cell DNA (Lavi and Winocour, 1972). The yields of viral RNA obtained from the cells are shown in Table 1. Approximately 3 % of the total RNA labeled 3844 hours postinfection was hybridized to and recovered from the filter-bound SV40 DXA. This value is 30- to 100-fold greater than the amount, of radioactive RNA bound to and recovered from blank filters. RNA extracted from uninfected cells, labeled with 3H-uridine for G hr, did not, hybridize detectably to plaquepurified SV40 DNA (Table 1). TABLE SELFCTION

OF

HYHRIDIZATION

HOST

“: 0

al-

I

1

RNA TO PL.KXJGPURTFIIW SV40 DNAa ~V~@SPECIFIC

561

SEQUENCES

-l

_I

25c

HY

Virus used to infect cells Prep- 3H-cpm in- Percentage of inaraput to put hybridized to tion hybridiand &ted from zation filters containing reaction SV40 No DNA (X 10-C DNA Plaque-purified

~Serially

passaged

Mock-infected

1 2 3 4 5 6 7 8

37 42 119 58 36 53 312 14

2.82 3.48 2.77 3.14 2.78 3.11 2.14 0.03

0.035 0.098 0.043 0.065 0.044 0.035 0.029 0.019

0 Groups of 10-27 BS-C-l cell cultures were infected at a multiplicity of lOOPFU/cell with either a plaque purified or serially passaged stock of SV40 and labeled with 3H-uridine from 38-44 hr post-infection (or for a 6-hr period in the mockinfected control). See Materials and Methods for the procedures used to extract 3H-RNA, to select virus-specific species by hybridization to plaquepurified SV40 DNA in formamide, and to recover the hybridized RNA. No RNase treatment was used in t,his hybridization-selection procedure. The 3H-RNAs were extracted from the cells’ cytoplasmic fraction in preparations 1 and 4, from the nuclear fraction in preparations 2 and 5, and from whole cells in preparations 3, 6, 7, and 8.

0

IO FRACTION

20

30

NUMBER

FIN. 1. Sedimentat,ion pattern of virus-specific RNA in a deuterated DMSO sucrose gradient. The aHlabeled viral RNA species (filled circles) selected by formamide hybridization to plaquepurified SV40 DNA, were centrifuged in a deuterated DMSO-sucrose gradient (O-10% sucrose, 10% deuterated DMSO-99% deuterated DMSO) as described in Materials and Methods. Sedimentation is from right to left. The 3 peaks of the 3aP-labeled uninfected cell RNA denote the positions of 4 S, 18 S, and 28 S RNA species, respectively. The acid-insoluble radioactivity in $icth volume of each fraction was determined. The viral RNA was isolated from the cytoplasmic fraction (panel A), and from the nuclear fraction (panel B) of cells infected with plaque-purified virus; or from whole cells (panel C) inferted with serially passaged virus. For the low molecular weight (LMW) viral RNA species, fractions 18-21 of the cytoplasmic viral RNA (panel A) were pooled. For the high molecular weight (HMW) viral RNA species, fractions 1-15 of the nuclear viral RNA (panel B) or whole cell viral RNA (panel C) were pooled.

ROZENBLATT

562

AND WINOCOUR

plexes and RNA secondary structure are disrupted in this type of gradient. Figure 1 shows the sediment’ation pattern of virusspecific RNA from the cytoplasmic (panel A) and nuclear fractions (panel B) of cells infected with plaque-purified SV40. Most of the viral RNA from the cytoplasmic fraction sediments together with 18 S rRNA and will be referred to in this paper as low molecular weight (LMW) viral RNA (other experiments show that the cytoplasmic LMW viral RNA can be resolved into two components which sediment with values of 16 S and 19 S in sucrose-SDS gradients (Weinberg et al., 1972). The sediment,ation pattern of the nuclear viral RNA (Fig. 1B) indicates that about 45 % of this RNA is highly heterogeneous and sediments faster than 28 S rRNA. Taking the molecular weight of t’he 28 S rRNA species as 1.9 X lo6 daltons (McConkey and Hopkins, 1969), and on the basis of the relationship between molecular weight and sedimentation in DMSO found by Strauss et al. (1968), we estimate the size range of the HMW SV40 RNA speciesto be from 1.9 X lo6 daltons to 10 X lo6 daltons. The bulk of the HMW viral RN-4 is thus considerably larger than a single strand of SV40 DNA (approximately 1.7 X lo6 daltons). The proportion of HMW to LMW viral RNA species in cells infected with serially passagedvirus was virtually identical

Fractionation of SV40 RNA Species The viral RNA preparations listed in Table 1 were centrifuged through a deuterated DMSO-sucrose gradient. Since DMSO acts as a strong denaturing agent for RNA (Strauss et al., 1968), hydrogen-bonded comTABLE

2

OF HMW SV40 RNA IN CELLS INFECTED WITH PLAQUE-PURIFIED OR SERIALLY PASSAGED VIRUS”

PROPORTION

Virus

used to infect

cells

RNA

extracted

from

PeWent&f sv40

RNAb

Plaque purified Serially

Nuclear fraction Whole cells Nuclear fraction Whole cells Whole cells

passaged

44.6~ 30.4 43.7 24.7 30.w

0 Viral RNA was selected as described in Table 1 and fractionated in deuterated DMSO-sucrose gradients. b Cpm of the 3H-RNA fractions sedimenting faster than the 28 S ribosomal RNA marker (fractions l-15, Fig. 1) expressed as a percentage of the total cpm of 3H-SV40 RNA recovered from the gradient. c The sedimentation pattern of this viral RNA is shown in Fig. 1B. d The sedimentation pattern of this viral RNA is shown in Fig. 1C. TABLE HYBRIDIZATION Virus used to infect cells

Type

of S$r

OF SELECTED selected

SPECIES

Serially passaged

3 SV40 RNA TO Hosr .IND VIRAL

aH-cpm input

Percentage

hybridization

SV40 DNA -RNase

Plaque purified

OF

LMW (cytoplasmic) HMW (nuclear) HMW (whole cell) LMW (cytoplasmic) HMW (nuclear) HMW (whole cell)

21,700 18,400 29,600 19,706 21,006 17,000

44.7 21.3 16.5 45.0 20.5 17.6

+RNase

37.9 7.0 5.9 40.0 7.1 6.7

BS-C-1 -RNase fRNCi%

1.2 3.0 2.8 1.1 2.9 2.6

-RNase

0.3 1.2 1.3 1.4 4.6 4.6

+RNase

0.2 0.7 1.0 1.0 3.1 3.4

DNA

to:b DNA

E. coli DNA -RNase +RNW!

1.5 1.7 1.3 1.4 1.5 1.4

-RNase

NTc 0.06 0.08 NT 0.10 0.09

+RNase

NT 0.08 0.03 NT 0.07 0.02

0 The SV40 RNA species were selected by formamide hybridization to plaque-purified SV40 DNA (Table 1) followed by sedimentation in deuterated DMSO-sucrose gradients (Fig. 1, Table 2). See legend to Fig. 1 for details on the HMW and LMW species of viral RNA. b The hybridization reactions (in duplicate) were carried out at 65°C for 48 hr in 3 X SSC containing 0.1% SDS (see Materials and Methods). The levels of 3H-RNA bound to blank filters (0.1% without RNase, 0.01% with RNase) have been subtracted. c NT = not tested.

SV40 RNA

HYBRIDIZATION

OF INTACT

Type of SV40 RNA

AND

CONTAINING

FRAGMENTED

“H-cpm input

TABLE HMW

SEQUENCES

4 SV40 RNA

AFTER

563

A SECOND

Percentage hybridization SV40 DNA - RiYase

HMW HMW after second selectionb HMW after limited alkali degradation and second selection”

HOST

SELECTION

to?

BS-C-1 cell DNA

+RSase

- RNase

+ RNase

STEP

E. coli DNA - RNase

+ RSase

18,151 16,796

22.7 21.3

G.9 7.6

4.0 4.5

3.1 3.6

NT 0.2

NTe 0.2

11,894

48.5

39.8

1.6

0.3

0.3

0.2

(1The HMW SV40 RNA was isolated from whole cells infected with serially passaged virus, purified by formamide hybridization to plaque-purified SV40 DNA (first selection step) and fractionated in a deuterated DMSO-sucrose gradient, as described in Table 1 (preparation 7) and Fig. 1C (pooled fractions 1-15). b A sample of the HMW SV40 RNA was subjected to a second selection step by formamide hybridization (no RNase) to plaque-purified SV40 DNA. The percentage of HMW SV40 RNA which hybridized to SV40 DNA in this step was ll.l%, of which 79% was recovered by the elution procedure (0.08y0 was bound t,o the blank filter control). c Another sample of the HMW SV40 RNA was first converted to low molecular weight, (ca. 13 S) fragments by limited alkali degradation (see Fig. 2) and then subjected to a second selection step by formamide hybridization (no RNase) to plaque purified SV40 DNA (13$& hybridized to SV40 DNA of which 83cx was recovered by the elution procedure; 0.06% was bound to the blank filter). d The conditions of hybridization (at 65°C in 3 X SSC) and RNase treatment are described in Materials and Methods. The values of aH-RNA bound to blank control filters (0.1% without RNase, 0.03% with RNase) have been subtracted. e Not t,ested.

to t’hat found in cells infected with plaquepurified virus (Table 2). For the rehybridizat,ion tests to viral and cellular DNAs described below, the HMW species (isolated either from the nuclear fraction or from whole cells) sedimenting faster than the 28s rRNA marker were pooled; the pool of LMW viral RNA specieswas derived from the cytoplasmic fractions only (Fig. 1A). More than 96 % of the RNA in these pools was sensitive to RSase treatment (50 pg/ml in 2 X SSC, 1 hr at room temperature) and none of the RNA was converted to a RNaseresistant form after self-annealing (in 3 X SSC, 45 hr at 65’C). The Presence of SequencesComplem,entary to Cellular DNA in the HMW Viral RNA Species Evidence that the HMW viral RNA speciescont,ain sequencescomplementary to cellular DNA was first obtained from rehyexperiments in which the bridizat,ion

capacity of the viral RNA t’o form a RNaseresistant complex with cellular and viral DNA was examined. It will be noted from the data in Table 3 that the HMW viral RNA, from cells infected either with plaque purified or serially passagedvirus, hybridized significantly with host DNA; the amount of RNA bound by the host cell DNA was lo- to lOO-fold greater than the amount bound by E. coli DNA. However, the HMW viral RNA induced by serially passagedvirus hybridized back to host DNA with an efficiency which was 3- to 4-fold higher than that of the HMW viral RNA induced by plaquepurified virus. The level of hybridization between host cell DNA and the cytoplamnic LMW viral RNA species was lower than that obtained with HMW speciesand it was not studied further. In all cases examined, the RNase resistance of the hybrid complex between HMW SV40 RNA and host DNA was significantly higher (by a factor of about 2) than that of the complex formed with

564

ROZENBLATT

1% 0

IO FRACTION

20

AND

30

NUMBER

FIG. 2. Sucrose gradient sedimentation of HMW SV40 RNA after limited alkali degradation. A sample of BMW SV40 RNA (pooled fractions 1-15 in Fig. 1C) was treated with 0.05 M Na&OI , pH 11, for 3 min at 8O”C, fast-cooled, neutralized, and sedimented through a 15-307o sucrose gradient containing 0.1% SDS (closed circles; SW 41 rotor, 30,000 rpm, 16 hr, 20°C). Sedimentation is from right to left. The 32P-labeled 4 S, 18 S, and 28 S RNA species (open circles) were obtained from uninfected cells. The 3H-viral RNA fractions denoted by a bar, in the 13 S region of the gradient, were pooled and used in the hybridization experiment described in Table 4.

SV40 DNA, suggesting t’hat the majority of the sequences in the HMW RNA are hostspecific rather than virus-specific. We also found, in agreement with previous results (Weinberg et al., 1972) that the capacity of the HMW viral RNA to hybridize back to viral DNA was lower t)han that of the cytoplasmic LMW species (by a factor of 5 to 6 in Table 3). Additional evidence that the viral and host specific sequences are located in the same HMW RNA molecule is presented in Table 4. In this experiment, HMW SV40 RNA, initially selected by hybridization to plaque-purified SV40 DNA and fractionated in a deuterated DMSO-sucrose gradient as described above, was divided into 3 samples. One sample was converted to low molecular weight (ca. 13 S) fragments by limited alkali digestion (see Fig. 2), subjected to an additional selection step by formamide hybridization to plaque-purified SV40 DNA, and t,hen tested for its ability to hybridize with host DNA. The second sample was subjected to the additional selection step, without prior fragmentation, and tested for its ability to

WINOCOUR

hybridize with host DNA. The third sample was tested directly for its capacity to hybridize with host DNA. If the cell-specific and virus-specific sequences were initially linked together in the same molecule, the selection step by hybridization to plaquepurified SV40 DNA should remove the cellspecific sequences from the population of fragmented molecules. This prediction is fully confirmed by the results shown in Table 4. The HMW RNA which was first converted to low molecular weight fragments, and then selected by formamide hybridization to SV40 DNA, lost, essentially all its capacity to form a RNase-resistant complex with host cell DNA. The additional selection step by itself did not reduce the capacity of the intact HMW viral RNA to hybridize with host DNA. As also expected, the population of alkali-degraded RNA fragmenm, after selection by hybridization to SV40 DNA, rehybridized back to SV40 DNA with high efficiency and the resulting hybrid complexes were highly resistant to RNase. DISCUSSION

The nuclei of monkey cells productively infected with SV40 contain a HMW virusspecific RNA which is several times larger t,han a single strand of t’he virus DNA. The high sedimentation rate of this viral RNA species cannot be due to intermolecular or intramolecular hydrogen-bonded complexes since its sedimentation is unaffected by denaturing conditions. The data in Tables 3 and 4 clearly establish that the HMW viral RNA contains host-specific sequences covalently linked to virus-specific sequences and suggest that the host-specific sequences predominate. Since the infection of monkey BS-C-1 cells with serially passaged SV40 stocks leads to the production of substituted viral DNA molecules, it might be argued that HMW viral RNA molecules containing linked cellspecific sequences arise from the continuous, uninterrupted transcription of this special class of circular viral DNA molecules in which a segment of the viral genome has been replaced by cellular DNA. However, two observations indicate t,hat the occurrence of HMW viral RNA cont’aining host-specific sequences cannot be fully explained on the basis of this argument. First, the

SV40 RNA

CONTAINING

HMW RNA was found in cells infected with plaque-purified virus (Fig. lB, Table 3) under conditions where the synthesis of subst(ituted viral DNA progeny cannot’ bc ,detected (Lavi and Winocour, 1972) and the proport’ion of HMW to LMW viral RNA species in such cells was the same as that, found in cells infected with serially passaged virus (Table 2); second, whereas the proportion of cell-specific sequencesin the HMW RNA appears to be greater than the proportion of virus-specific sequences(Table 3), the reverse is found in the majority of closed circular subst’ituted viral DNA molecules (Tai el al., 1972; unpublished experiments of $S. Lavi and E. Winocour). We propose, therefore, that, at least some of the HMW RNA in t,he productively infected monkey cells arises from the cotranscription of integrated viral DNA and adjacent cellular DNA. In SV40-transformed cells, which have been reported to contain chromosomally integrat’ed viral DNA (Sambrook et al., 1968) a similar HMW viral RNA complementary t’o both the viral and cellular ,genomeshas been found (Wall and Darnell, 1971). The results reported in this communication supply further evidence that integration-like events between the cellular and viral genomes can also occur during the productive cycle of infection (Lavi and Winocour, 1972; Tai et al., 1972; Ralph and Colter, 1972; Hirai and Defendi, 1972). The HMW RNA from BS-C-l cells infected with serially passagedvirus appea,rsto contain a greater variety of host sequences since it hybridizes to host DNA more efficiently than the HMW RNA from cells infected with plaque-purified virus (Table 3). We have suggestedpreviously (Lavi and Winocour, 1972) that the substituted SV40 DNA molecules, which are present in serially passaged SV40 stocks, integrate at a wide variety of sites in the cellular genome; in contrast, the integration sites for plaque purified (nonsubstituted) viral DNA molecules may be much more restricted. We would further suggest, therefore, that the comparatively higher level of hybridization between host DNA and the HMW RNA induced by serially passaged virus results from the cotranscription of cell DNA and virus DNA at a wide variety of integration

HOST

SEQUENCES

565

sites. We are currently testing this proposal by competition hybridization experiments between host-specific sequencesisolated from the HMW RNA induced by plaque-purified virus and those isolated from the HMW RNA induced by serially passagtd virus. Competition hybridization experiments between uninfected ccl1 RNA and the cellular RNA sequences isolated from the HMW viral RNA molecules may also provide information on the int,eresting possibility that SV40 infection induces, as a result of virus DNA integrat,ion, the t,ranscription of ccllular genes which arc normally silent in uninfected cells. ACKNOWLEIXiMENTS This work was supported in part by grant DRG-1061-A from the I)amon Kunyon Memorial Fund for Cancer Research. We are indebted to Dr. Maxine Singer for her helpful suggestions during the writing of the manuscript. We also thank B. Uanovitch for his expert) technical assistance. REFFRENCES J ACHESON, N. H., BUETTI, IS., SCHKI~IZER,K., and WEIL, It. (1971). Transcription of the polyoma genome: synthesis and cleavage of giant late polyoma-specific RNA. Proc. IV&. Acad. Sci. U.S.A. 68, 2231-2235. ALONI, Y., WINOCOUH, E., SACHS, I,., and TORTEN, J. (1969). Hybridization between SV40 UNA and cellular DNA’s. J. Mol. Bid. 44, 335-345. BORUN, T. W., SCHMIFF, M. I)., and ROBBINS, E. (1967). Preparation of mammalian polyrihosomes with the detergent Nonidet P-40. Riochim.

Biophys.

Acta

1.49, 302-304.

DULBECCO, R., snd VOG’F, M. (1954). Plaque formation and isolation of pure lines of poliomyelites viruses. J. Exp. Med. 99, 167-182. GERBER, P. (1962). An infectious deoxyribonucleic acid derived from vacuolating virus (SV40). Virology

16, 96-97.

HIKAI, K., and DIGFEN~I, V. (1972). Integration of simian virus 40 deoxyribonucleic acid into the deoxyribonucleic acid of permissive monkey kidney cells. J. Viral. 9, 705-707. HOPPS, H. I<., BERNHFXM, B. S., NILZLAK, A., TJIO, J. H., and SMMXCL, J. E. (1963). Biologic characterist,ics of a cont,inuous kidney cell line derived from t,he African green monkey. .I. Immunol. 91, 41ti-424. JAENISCH, R. (1972). Eviderlce for SV40 specific RNA containing virus and host specific sequences. X’ature (I~oMZOI~) New Biol. 235, 46-47. L.IVI, S., and WINO~UI~, E. (1972). Acquisition of

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