Infectious proviral DNA in human cells infected with transformation-defective type C viruses

VIROLOGY

70, 301-312 (1976)

Infectious

Proviral

DNA in Human Cells Infected Defective Type C Viruses’

with Transformation-

M. 0. NICOLSON,Z F. HARIRJZ H. M. KREMPIN,2 R. M. MCALLISTER,’ AND R. V. GILDEN:’ Department of Pediatrics, University Angeles, P. 0. Box 54700, Terminal

of Southern California School of Medicine; ‘Childrens Hospital Annex, Los Angeles, California 90054; and ‘iFlow Laboratories Rockville, Maryland 20852 Accepted November

of Los Inc.,

1, 1975

Human rhabdomyosarcoma cells (RD) productively infected with the transformationdefective feline virus, RD-114, contain viral-specific DNA integrated into the chromosomal DNA which specifies complete information for progeny virus formation. This cellular DNA is infectious for permissive cells of human and canine origin, giving rise to progeny virus identical in assessed biochemical and biological properties to the parental virus. The host range for transfection with RD-114 DNA is similar to that of the intact virus. Although progeny virus could first be detected by reverse transcriptase assay at 2 to 4 weeks after transfection, virus is actually released at 3 days post-transfection, as shown by assay of culture media on sensitive indicator cells. The minimum dose of RD-114 DNA required for one infectious event in 2 x 10” RD cells is 0.10 to 0.20 pg. This gives a calculated efficiency of infection of 1 infectious unit per 5 x lo4 to 5 x lo5 viral gene copies, if there are 2 or 20 copies per infected cell, respectively. RD cells, productively infected with the transformation-defective mammalian viruses FeLV and GaLV, also contain viral-specific DNA, which gives rise to the respective virus of origin upon application to permissive RD cells.

evidence that integrated proviral nucleoIt is now widely accepted that a DNA tide sequences contain information for type C virus expression was first provided intermediate is involved in the replication of RNA type C viruses through the agency by Hill and Hillova, who productively of an RNA-directed DNA polymerase, transformed chicken embryo fibroblasts with DNA extracted from Prague (Pr)coded for, at least partially, by the virus. Biochemical evidence confirms the exist- RSV transformed nonproductive rat XC ence of double-stranded proviral DNA, co- cells (Hill and Hillova, 1971; Hill and Hilvalently integrated into the host DNA of lova, 1972). Other laboratories have concells both productively and nonproduc- firmed these original experiments (Svoboda et al., 1972; Levy and Kazan, 1974). tively infected with type C viruses (Fujinaga et al., 1973; Baluda, 1972; Hare1 et al., Chinese hamster embryo cells nonproductively infected with Schmidt Rupin (SRI1972; Hill and Hillova, 1971; Benveniste and Todaro, 1974; Kang and Temin, 1974). RSV (Svoboda et al., 1972) and chicken cells productively infected with the same Although these represent the acquisition of new DNA sequences complementary to virus (Montagnier and Vigier, 1972) also yielded DNAs infectious for chicken fibrothe viral RNA of origin, direct biological blasts. DNA of hamster cells transformed by a ts mutant of SR-RSV induced in chick I This work was supported by Contract PH 43-68-1030 within the Special Virus-Cancer Proembryo cells progeny virus possessing the gram of the National Cancer Institute, NIH, PHS. same temperature-sensitive characterisINTRODUCTION

301 Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

302

NICOLSON

tics as the parental virus (Hill and Hillova, 1972). Although in each of these cases, virus released from the transfected chicken cells possessedthe subgroup characteristics of the original transforming virus, a potential complication is the ubiquitous presence of endogenous leukosis virus nucleotide sequences in the recipient chicken cells, and hence, the possibility of some participation in transfection. The same is true of nontransforming proviral DNA from productively infected chicken cells which also gives rise to progeny with subgroup antigenicity of the parent rather than of subgroup E as might be expected from expression of the endogenous chicken leukosis genome (Hill et al., 1974). The most conclusive evidence for transfection of complete C-type virus information in cells lacking endogenous viral nucleotide sequences is the infectivity of RSV proviral DNA (from productively infected chicken fibroblasts) for Muscovy duck cells (Cooper and Temin, 1974). Two attempts to infect duck cells with XC DNA, however, were unsuccessful (Svoboda et al., 1973; Levy et al., 1974). Recent evidence has extended the range of avian infectious proviral information to include cells infected with the avian reticuloendotheliosis (Cooper and Temin, 1974) and myeloblastosis (Fourcade et al., 1974) viruses. The infectivity of mammalian type C proviral information has received less attention. One report describes the transformation of 3T3 cells with nuclear DNA of MSV-transformed nonproducer cells of both mouse and hamster, with release of progeny MSV only by those cells also infected with Rauscher leukemia virus (Karpas and Milstein, 1973). More recently, mink fibroblast cells were transfected with DNA of Kirsten sarcoma virus-transformed BALB/c mouse cells, giving rise to an endogenous xenotropic mouse virus as well as Kirsten sarcoma virus (Scolnick and Bumgarner, 1975). The experiments described in this paper establish the presence of infectious proviral DNA in human cells infected with the mammalian transformation-defective viruses RD-114, feline leukemia virus (FeLV) and gibbon ape lymphosarcoma virus (GaLV). The pro-

ET AL.

viral DNAs can transfect and give rise to progeny virus in mammalian cells which have not been shown to possess endogenous viral functions potentially capable of complementing incomplete proviruses. We further show that the transfection host range of RD-114 DNA is similar to that for the intact virus, that the bulk of infectious provirus is associated with cellular chromosomal DNA, and that the efficiency of transfection is similar to that reported for avian proviral DNAs. MATERIALS

AND

METHODS

Chemicals DEAE-dextran, MW = 2 x 106, came from Sigma Chemical Company; electrophoretically purified DNAse I and RNAse A were from Worthington Biochemical Corp.; Pronase B and &bromodeoxyuridine were supplied by Calbiochem; CsCl, optical grade, came from Harshaw Chemical Company and [Me-3HlTTP and [53Hluridine were purchased from New England Nuclear Corp. Cells RD is a cultured human rhabdomyosarcoma line (McAllister et al., 1969). The D17 dog osteosarcoma line was obtained from J. Riggs, the rhesus kidney cells from W. Nelson-Rees, the feline embryo fibroblasts (FEF) from S. Rasheed, and WI-38 cells from Flow Laboratories. The human carcinoma lines KB and HeLa were obtained from Dr. W. Nelson-Rees; the human sarcoma lines TE-85 and TE-418 have been described (McAllister et al., 1975). The KC cell line was obtained from Dr. C. Long. RD-114 cells and RD-FeLV cells are RD cells productively infected with the respective viruses (McAllister et al., 1973), as are the RD-GaLV (Kawakami et al., 1972) cells. NRK is a line of normal rat kidney cells and B-7 is a cloned line of NRK transformed and nonproductively infected with KiMSV (Klement et al., 1971). All cells were grown in Eagle’s minimal essential medium (MEM) supplemented with 10% heat-inactivated fetal calf serum (Flow Laboratories) and penicillin and streptomycin. Derivation of cell clones by agar

INFECTED

HUMAN

CELL

suspension culture has been described (MacPherson and Montagnier, 1964). DNA Isolation DNA was extracted from confluent cultures (nuclei or whole cells) of RD-114, RDFeLV, or RD-GaLV cells or from fetal cat liver and spleen. Cells were lysed with 0.4% sodium dodecyl sulfate in 0.1 M NaCl, 0.05 M Tris, 0.01 M EDTA (pH 8.2; DNA buffer), and incubated at 37” for 18 hr with 100 wglml pronase (self-digested, 2 hr at 37”). The solution was extracted twice at room temperature with an equal volume of chlorofornnisoamyl alcohol (24:1), precipitated with 2 vol of ethanol, and redissolved in 0.01 M NaCl, 1.0 mM EDTA, and 0.01 M Tris (pH 7.2). Boiled RNAse was added to give 100 pglml, and the solution was incubated for 18 hr at 37”. Pronase was added to give 50 Fglml, and the incubation was continued for at least 12 hr. The NaCl concentration was adjusted to 0.2 M, and the DNA was extracted three times with chloroform:isoamyl alcohol. DNA was precipitated from solution with 2 vol of ethanol, redissolved in NTE (0.1 M NaCl, 0.01 M Tris, 1 mM EDTA, pH 8.2) and dialyzed against NTE before use. In some cases, the DNA was further subjected to equilibrium density gradient centrifugation in CsCl solutions. In one instance, RD-114 DNA was extracted by the method of Gross-Bellard et al. (but substituting pronase for proteinase K) in order to minimize shear degradation during extraction (Gross-Bellard et al., 1973). DNA concentrations were determined by absorbance at 260 nm or by the Burton modification of the diphenylamine reaction (Burton, 1956). Nuclear DNA was extracted by the same methods from nuclei prepared by the technique of Penman (1966). The procedure of Hirt (1967) was used to separate chromosomal from extrachromosomal DNA. Sedimentation of Native and Denatured DNA Isolated DNAs were subjected to velocity sedimentation in neutral sucrose solutions made in NTE, pH 8.2, or in NTE containing 1.0 M NaCl. Linear 5 to 20%

C-TYPE

303

PROVIRUS

sucrose gradients were centrifuged in an SW27 rotor at 4”, 22,000 rpm for 12 hr, or at 15”, 10,000 r-pm, for 36 hr. Ribosomal 18 S RNA and adenovirus-2 DNA (31 S) were added as sedimentation markers. CsCl Equilibrium Centrifugation

Density Gradient

DNA was mixed with saturated CsCl in 0.02 M Tris, pH 8.0, to give a starting density of 1.695 g/cm’ and centrifuged at 20” in an SW27 rotor at 22,500 rpm for 66 hr. DNA fractions recovered from gradients were dialyzed against NTE and in some cases precipitated with 2 vol of ethanol and redissolved in NTE. Infection

of Cells with DNA

Cells. Cells (2.5 to 4.0 x 10”) were seeded into T-75 Falcon plastic flasks and incubated at 37” for 24 hr before use. In each experiment, two flasks of cells were trypsin dispersed and counted at the time of the experiment and the cell counts were averaged to provide an estimate of DNA dose per cell. DNA treatment. The cells were treated with DNA by one of three techniques: (1) Twenty-four hour cell cultures were incubated for an additional 24 hr with normal growth medium containing 10ea M bromodeoxyuridine (BrdU). The BrdU medium was removed, and cells were treated with DEAE-dextran (D-dex) and DNA as described in (2). (2) BrdU-treated or 24-hr untreated cultures were rinsed with MEM, overlaid with 2 ml of D-dex (100 pgl ml) and left at room temperature for 30 min. D-dex was removed and 2.0 ml of DNA solution in 0.01 M Tris, pH 7.4, 0.15 M NaCl was added. After 60 min at 37”, the DNA solution was removed and replaced with 10 ml of normal growth medium. Cultures received fresh medium 24 hr later and three times weekly thereafter. They were passaged at approximately loday intervals. (3) Cells were treated by the method of Graham and Van der Eb (1973). The DNA solutions, diluted to give the DNA concentrations indicated for each experiment, contained 0.137 M NaCl, 0.005 M KCl, 0.7 mM Na,HPO,, 0.0056 M dextrose, 0.025 M Tris, 0.125 M CaCl,, at a

304

NICOLSON

final pH of 7.1. The CaCl, was added last, and the solution was mixed vigorously and allowed to stand at room temperature for 30 min. The medium was removed from 24hr cell cultures, and each was overlaid with 2.0 ml of the DNA solution and kept at room temperature for 20 min. Ten milliliters of normal growth medium was added to each and incubation was continued at 37” for 7 hr. The DNA-containing medium was removed at 7 hr and replaced with 10 ml of normal growth medium. Thereafter, the cultures were handled as described under (2). Culture media were assayed at 2to 3-week intervals for detection of viral reverse transcriptase CRT), and the cultures were maintained up to a total of 12 weeks. Assay of the cells for the appropriate P3bantigen was carried out on all cultures at 12 weeks or, in some cases, when the cultures were positive for RT.

ET AL.

approximate molecular weight range of 3 to 10 X 106. Subsequent preparations varied from an average of 22 to more than 50 S in neutral sucrose sedimentation, depending on control of mechanical shear during extraction. The molecular size range of the unsheared DNA was confirmed by measurements made in the electron microscope after spreading by ammonium acetate. RD-114 DNA extracted by the method of Gross-Bellard et al. (19731had sedimentation coefficients in neutral sucrose of 40 to more than 75 S. When RD and D-17 cell cultures were treated with whole cell, CsCl-banded RD114 DNA (16 to 26 S), virus release as measured by the RT assay was first detected at 4 (RD cells) or at 2 weeks (D-17 cells). Combined results of three separate experiments are shown in Table 1, tabulated at 12 weeks. For both RD and D-17 cells, the calcium phosphate-DNA comVirus Assays plex method was the most uniformly effecThe host-range studies, virus assay by tive for transfection, inducing 100% virusmeasurement of reverse transcriptase (RT) producing cultures. D-dex, either alone or activity, virus group specific (P,,) anti- in combination with BrdU treatment was gens, and antisera and neutralization as- less efficient at high DNA concentrations. says have been described (Filbert et al., The use of BrdU was based on a report by 1974). Methods for murine sarcoma virus Svoboda et al. (1973) that pretreatment of rescue and virus interference (Gilden et chicken fibroblasts with BrdU enhanced al., 1972), syncytium induction on KC cells their sensitivity to transfection with XC (Rand and Long, 1972), and for inhibition DNA, although the mechanism of action by specific antibody of viral polymerase was unclear. In no case did transfection activity (Long et al., 1973) also have been occur in the absence of any cell treatment, described. [5-3HlUridine-labeling, iso- and all activity was abolished by prior pycnic centrifugation of virus, and the ex- digestion of the DNA with deoxyribonucletraction and velocity sedimentation of ase. There was no grossly detectable differviral RNA have been described (Klement ences in either time of appearance or titer et al., 1973). of progeny virus detected, relative to the amount of DNA applied within the 15 to Electron Microscopic Studies 150 pg/culture range. The group specific Published methods were used for meas- antigen (P,,,) of RD-114 was detected in all urements of viral RNA and cellular DNA cell cultures positive for DNA polymerase. by electron microscopy (Kung et al., 19741 Calf thymus, salmon sperm, or RD DNAs and for visualization of C-type viruses did not induce the release of detectable (McAllister et al., 1971). virus or the appearance of viral P,, antigen. More precise characterization of the RESULTS transfection progeny virus (designated TDNA extracted from RD-114 cells 114) showed that [3H]uridine-labeled virus banded sharply at a density of 1.698 g/cm” banded sharply at a density of 1.155 g/cm3 in CsCl gradients. The first DNA prepara- in linear 15 to 55% sucrose gradients, and tion sedimented in neutral sucrose gra- contained RNA which sedimented at 50 to dients in the region of 16 to 26 S, or an 60 S in 10 to 30% linear sucrose gradients.

INFECTED

HUMAN

CELL TABLE

DETECTION

OF

REVERSE

DNA cell origin

RD-114d RD-114 RD-114 RD-114 RD-114 RD-114 RD-114 RD-114; DNA&’ RD-114; DNAsed RD-114, DNAsed RD Calf thymus Salmon sperm

TRANSCRIPTA~E

Cell pretreatment”

Ca-P Ca-P D-dex D-dex B + D-dex B + D-dex None Ca-P Ca-P D-dex Ca-P Ca-P Ca-P Ca-P

C-TYPE 1

AND RD-114 Pa0 ANTIGEN WHOLE CELL DNA

DNA dose @g/culture)

150 15 150 15 150 15 150 150 15 150 15 15 15 -

305

PROVIRUS

IN CULTURES

RT positive cultures/ cultures treatedb

TRANSFECTED

WITH RD-114

Identity of P,, antigen RT positive cultures’

RD

D-17

RD

D-17

8/8 212 216 212 212

616 NT l/2

RD-114 RD-114 RD-114 RD-114 RD-114 RD-114 None None None None None None None None

RD-114

212 o/4 O/8 o/2 O/6 o/4 o/4 o/2 o/2

212 l/2 212 012 NT NT NT o/2 NT NT o/2

in

RD-114 RD-114 RD-114 RD-114 None None None

fl Cell pretreatment described in methods. Ca-P, calcium phosphate; D-dex, DEAE-dextran; B + D-dex, BrdU followed by DEAE-dextran. b Cultures were considered positive if 1 ml of medium incorporated ZlOOO cpm of [Me-:‘H]TMP (background of 50 to 200 cpm subtracted) per 60 min of incubation. ’ The correlation between RT positivity and detection of P,, antigen was 100%. All cultures positive for P,,, antigen had titers of 28, the reciprocal of the highest dilution giving a 3 to 4+ complement fixation with antibody to RD-114 P,,. ‘( RD-114 DNA preparation 1, CsCl banded, sedimentation coefficient 16 to 26 S. ” Concentrated DNA stock solution incubated at 37” for 60 min with 50 pg/ml DNAse, 6 m&f MgCl,.

By electron microscopy, this RNA had a molecular weight of 5 x lo6 and possessed the secondary structural feature characteristic of RD-114 viral RNA subunits (Kung et al., 1974). Sucrose-banded T-114 virus contained active RNA-directed DNA polymerase, which was completely inhibited by antibody to RD-114 RT. Examination of T-114 cells in the electron microscope revealed budding particles with the typical morphology of type C virus. In further characterization studies of T114 virus, it induced a P,, cross-reactive with antisera to P,, of RD-114 virus and baboon virus but not with antisera to P30)s of other mammalian type C viruses (Fig. 1). The virus replicated in RD, D-17, and rhesus kidney cells but not in cat cells (Table 2). The virus did not transform WI38 or rhesus kidney cells. It was neutralized by antisera to RD-114 virus (Table 3); it interfered with focus formation by MSV (RD-114) but not by MSV (GaLV) (Table 4). Finally, T-114 virus had the capacity to

rescue MSV from B-7 cells and to induce syncytia in KC cells. Thus, in all measurable respects, the progeny virus derived by transfection of RD cells with RD-114 proviral DNA was identical to the parental RD-114. The host range for transfection with RD114 whole-cell DNA is similar to the host range for RD-114 virus (data not shown). Thus, both RD-114 DNA and the intact virus are capable of infecting and replicating in certain human and dog cells, not in cat cells (RD-114 is a xenotropic cat virus), mouse cells, or in rat cells (Rasheed et al., 1973). These results do not preclude the possibility that proviral DNA may enter and even replicate in nonpermissive cells, but progeny virus produced from an initial small number of cells would be unable to infect the remainder, and hence, would be undetectable by the RT or P3,, assays. The RT assay was capable of detecting progeny virus formation by RD cells, at the earliest, 4 weeks after transfection

306

NICOLSON

FIG. 1. Outer wells contain 0.5-1.0 mg/ml T-114 virus was prepared by pelleting fluid original volume. Center wells contain guinea Viruses: Fe, feline; Ra, rat; Mu, mouse; BKD,

ET AL.

of indicated, purified virus disrupted by 0.5% Triton X-100. from a virus-producing culture and resuspending in l/200 pig antiserum to purified P,, of RD-114 or baboon virus (B). baboon virus on dog kidney cells. TABLE

2

HOST RANGE OF T-114 VIRUS COMPARED TO THAT OF RD-114 VIRUS AND FELV Cell assay system

RD D-17 Rhesus kidney Cat embryo fibroblasts

T-114 virus

RD-114 virus

FeLV

RTQ

RD-114 P,Ob

RT

RD-114 P3R

RT

10-4” 10-S 10-I None

10-a 10-S 10-l None

10-4 10-S 10-Z None

10-4 10-S 10-Z None

10-Z 10-S 10-l 10-d

F;,LY 30 10-Z 10-S 10-I 10-Z

o Reverse transcriptase activity. b.r RD-114 or FeLV P,, antigens detected by complement fixation test using antibody to electrofocus purified P,, protein of RD-114 (b) virus or FeLV cc). ‘I Highest virus dilution causing induction of RT and RD-114 or FeLV P,, antigen 21 days after infection of cell assay system. None of the uninfected control cell cultures had detectable RT activity or detectable P,, antigens.

with RD-114 DNA. However, when filtered culture fluids from transfected RD cells were seeded onto indicator RD cells at daily intervals up to 3 weeks post-transfection, and the indicator cells were cultured for 12 weeks, the results are as shown in Fig. 2. The earliest detectable release of infectious progeny virus by this technique appears to be 3 days after the initial transfection, but the quantity of virus produced on Days 3 and 4 is apparently very low and requires 12 weeks for the indicator cell cultures to register as positive by RT. These results raised the question of initial proviral infectivity of the applied

DNA; what proportion of the 2 to 4 x lo6 cells so treated receive complete proviral information and are able to transcribe this to progeny virus? This question was approached initially by attempting to derive a large number of cell clones from an RD culture transfected with 15 pg of RD-114 DNA. Twenty-four hours after DNA treatment the culture was dispersed and the cells were plated according to the agar suspension culture technique, lo4 cells/lOcm petri dish. After 21 days, 150 individual colonies were isolated, grown up, and assayed for RT and for RD-114 P,, at 8 weeks after initial transfection. All cultures were

INFECTED TABLE

HUMAN

CELL

3

VIRUS NEUTRALIZATION OF T-114 VIRUS T-114 virus incubated

RT

with”

(cpm)”

Titer RD114 P,, antigen’ 4

2649

Eagles medium with 10% fetal bovine serum Rabbit RD-114 antiserum 1:lO 1:20 1:40 1:80

0 26 741 1329


n Tissue culture fluid pool diluted lo-’ from T-114 culture was incubated with equal amounts of respective dilutions of antiserum or medium for 1 hr at room temperature before inoculation of RD cells. RT and P:,,, assays were carried out 14 days after infection. b [“HITMP cpm incorporated per milliliter of culture medium per 60 min. c See Table 1, footnote c. TABLE

C-TYPE

307

PROVIRUS

at lo”, lo”, 104, and lo3 cells/culture vessel, 10 of each dilution. These cultures were assayed at 8 weeks for RT and RD-114 P,,,. The results plotted in Fig. 3 show that the 50% infectivity level lies very close to lo4 cells, that is, approximately one in every lo4 cells treated with RD-114 DNA under these conditions is initially replicating progeny virus. RD cells also could be transfected with DNA extracted from RD-FeLV and RDGaLV cells. The transfected cells released virus into the culture medium as detected by RT assay, and, when tested by complement fixation, contained the P:,,,antigen of the respective FeLV or GaLV virus (Table 5). It was of interest to establish the nuclear location of the RD-114 proviral DNA and its potential integration into the cellular chromosomal DNA. DNA extracted

4

T-114 VIRUS INTERFERENCE WITH MSV (RD-114) BUT Nor MSV (GaLV) Focus FORMATION ON WI-38 CELLS WI-38 cells used for assay”

Uninfected T-114 infected GAL infected

Focus formation by challenge virus MSV (RD114)”

MSV (GaLVjb

Diffuse focus formation No foci

Diffuse focus formation Diffuse focus formation No foci

Diffuse focus formation

” WI-38 cell cultures (1.5 x IO” cells/250 ml flask) infected with 1.2 ml of undiluted T-114 pool (titer lo4 RT inducing units per 1.2 ml in RD cells) or 1.2 ml of GaLV virus pool (titer lo’ RT inducing units per 1.2 ml in RD cells). Fourteen days later, 5-cm dishes were seeded with 0.4 ml of challenge virus. h Dilutions of up to 10m3cause diffuse focus formation on WI-38 cells 7 days after infection. For interference assays challenge virus was used undiluted and at 10-l dilution.

uniformly negative for virus production, implying that fewer than one in 150 cells initially support progeny virus formation. This was examined further by a cell dilution technique, whereby RD cells, 24 hr after transfection with 15 pg of RD-114 DNA, were dispersed, pooled, and plated

L

&+.!/

I

,,/,,

1

,/

,,,,

1 2 3 ‘I 5 6 7 6 3 IO II I2 13 Ii 1: I6 ,7 18 c,rire Medim 01 Tronsfected Ceils toys CaSlOyedRn-Tmnrtectia”

FIG. 2. Twenty-four hour cultures of RD cells (3 10” cells per T-75 flask) were transfected with 15 Kg of RD-114 DNA by the calcium phosphate method as described in Methods. At daily intervals following transfection, the culture fluids were withdrawn (10 ml) filtered through 0.45pm filters, and each 10 ml was seeded onto one RD indicator culture. The transfected fluids were removed from indicator cultures after 24 hr and replaced with growth medium. Indicator cells were subcultured when necessary, and assayed for virus production at 8 and 12 weeks, by RT and RD-114 P,, assays. Cultures positive for RT also contained detectable RD-114 P:,,. x

308

ET AL.

NICOLSON TABLE TRANSFECTION

DNA dose (pg/culturel

5

OF RD CELLS WITH RD-FeLV

Cell pretreatment*

AND RD-GaLV

Cultures

positive

DNA”

PROVIRAL

for R~Naffl~r~~~

after treatment

RD-FeLV RT 150 15 150 15 150 15 150 15 150 DNAsed’ 15 DNAsed

Ca-P Ca-P D-dex D-dex B + D-dex B + D-dex None None Ca-P Ca-P

213 213 213 313 o/3 l/3 013 o/3 o/3 o/3

with

RD-GaLV

Identity

P,,

RT

FeLV FeLV FeLV FeLV None FeLV None None None None

TABLE

PsO

GaLV GaLV

3/3 313

n All results tabulated at 12 weeks after initial DNA application. b See Table 1, footnote a. r See Table 1, footnote b. d Only cultures positive for RT contained detectable P,, antigen. All cultures positive titers of 2 8, those positive for GaLV P,, had titers of 4 (see Table 1, footnote cl. p See Table, 1, footnote e.

TRANSFECTION

Identity

for FeLV P,, had

6

OF RD CELLS WITH NUCLEAR

RD-114

DNA” Cultures positive for B..ml$res 150.0

I06I05104 I03I02 Cell Number plated 24hrs

after Tronsfection

FIG. 3. RD cells were transfected with 15 pg of RD-114 DNA by the calcium phosphate technique as described in Methods. At 24 hr the cultures were dispersed, and the cells were pooled and plated at lo”, lo”, lo*, and IO3 cells per culture vessel, 10 for each dilution. The cultures were grown up and assayed at 8 weeks for RT and RD-114 P,,. Cultures positive for RT also contained detectable RD-114 S”.

from isolated nuclei of RD-114 cells (average MW 22 x lo6 by velocity sedimentation) was applied to RD cells, using the calcium-phosphate technique. Table 6 shows that 100% of the RD cultures so treated were transfected, and that as little

15.0 1.5 0.15 150.0 DNAsedd 15.0 DNAsed a All cultures Results tabulated * See Table 1, c See Table 1, d See Table 1,

28 28 28 28
313 313 3/3 3/3 o/3 o/3 treated with at 12 weeks. footnote b. footnote c. footnote e.

Titer RD-114 P,, detected in celk?


calcium

phosphate.

as 0.15 pg of DNA (per approximately 2 x 10” cells) was capable of eliciting this reactivity was desponse. Transfecting stroyed after incubation with DNAse. To explore further its association with chromosomal DNA, proviral DNA was isolated from RD-114 cells by two methods designed to minimize shear degradation. DNA extracted from nuclei by the technique of Gross-Bellard et al. (1973) was fractionated by velocity sedimentation as

INFECTED

HUMAN

CELL

outlined in Methods (5 to 20% sucrose in 1.0 M NaCl, 30 pg DNA/17-ml gradient, 10,000 rpm, 36 hr). Ninety-three percent of the DNA sedimented faster than the 31 S marker, and three size classes were tested for infectivity in RD cultures. DNA was also extracted by the Hirt technique (1967) and separated into pellet and supernatant fractions, each of which was also tested for infectivity. The results of these experiments are shown in Table 7. Under the conditions used, the RD-114 provirus is associated with the high molecular weight DNA. At approximately 10 pg/culture, the three DNA sedimentation fractions were equally as infectious as the unfractionated DNA. Ninety-five percent of the cell DNA was recovered in the Hirt pellet and 5% was recovered in the supernatant fraction. When compared for infectivity at 1 pgl culture, 100% of cultures receiving the pellet fraction were transfected, whereas only one out of three was positive of those receiving the supernatant DNA, suggesting that there is no enrichment of provirus in extra chromosomal DNA. These experiments do not rule out the possibility of some nonspecific trapping of low molecular weight proviral DNA by high molecular weight DNA, but do suggest an associaTABLE TRANSFECTION DNA fraction

Unfractionated 40-55 s 55-70 s > 70 s Hirt pellet”

Hirt supernatant”

7

OF RD CELLS WITH HIGH WEIGHT RD-114 DNA DNA dose ( Fgi culture)”

10.0 6.7 10.9 9.7 10.0 1.0 0.1 1.0 0.1

MOLECULAR

RT positive cultures~lcultures treated

3/3 313 3/3 3/3 313 3/3 o/3 l/3 o/3

C-TYPE

309

PROVIRUS

tion between chromosomal DNA and the bulk of the provirus. The minimum quantity of RD-114 DNA required to infect RD cell cultures under our standard conditions varied within a narrow range depending upon the origin (nuclear or whole cell) and extraction techniques employed. In general, the quantitative endpoint ranged between 0.1 and 0.2 pg of DNA required to induce one infectious event in approximately 2 x 10” RD cells. As shown in Table 6, 0.15 pg of nuclear DNA induced virus production in 100% of the cultures so inoculated, whereas Table 8 shows the microgram end-point for whole-cell DNA to lie between 0.1 and 0.2 pg/culture. Furthermore, addition of carrier salmon sperm DNA at a constant 10 pg/ml did not improve the sensitivity of transfection (Table 8). DISCUSSION

These experiments provide evidence that human rhabdomyosarcoma cells, productively infected with the transformation-defective mammalian type C virus, RD-114, contain proviral DNA in the infected cell nucleus, closely associated with the chromosomal DNA. A single exposure to DNA extracted from these infected cells or from RD cells infected with FeLV or GaLV, can induce the formation of fully infectious progeny virus in RD cells and in TABLE TRANSFECTION

RD-114 RD-114 RD-114 RD-114 RD-114 RD-114 None RD-114 None

(’ See Table 6, footnote a. * See Table 1, footnote b. ” See Table 1, footnote c. ” Both pellet andsupernatant fractions were subjected to equilibrium density gradient centrifugation in CsCl before testing for infectivity.

RD-114 DNA/ culture” (pg)

1.0

0.5 0.2 0.1 0.05 0.02

8

EFFICIENCY OF RD-114 DNA FOR RD CELLS

WHOLE-CELL

Percentage of cultures positive for RT’ and RD-114 P,,,’ at 12 weeks RD-114 DNA alone

RD-114 DNA plus salmon sperm DNA (10 pg/ml)

100 100

67 67 33 0 0 0

67 0 0 0

a All cultures treated with calcium phosphate. Transfection technique described in Methods. b See Table 1, footnote b. c See Table 1, footnote c.

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some instances in D-17 cells. The biological and biochemical identity of the progeny to the original parental virus is evidence for the complete nature of the proviral information and is in agreement with the results of transfection experiments with avian sarcoma virus (Montagnier and Vigier, 1972; Svoboda et al., 1972; Hill and Hillova, 1972; Cooper and Temin, 1974). Confirmation that the infectious molecules were DNA was provided by the loss of activity upon digestion with DNAse, resistance to 18 hr of incubation with high concentrations of RNase, by cosedimentation of the infectivity with chromosomal DNA and by cobanding with doublestranded cellular DNA in CsCl equilibrium density gradients. The progeny virus did not acquire any transforming capacity during the transfection process in RD cells, nor did the cells provide any measurable helper function inasmuch as pretreatment with BrdU did not improve the efficiency of transfection and, further, progeny virus was produced from human RD and dog D-17 cells with equal efficiency. In all experiments to date, the presence of specific P,, antigen in the transfected cells is correlated with production of whole virus as assayed by RT activity in the culture medium. This suggests, but does not confirm, that partial transcription/translation of integrated viral genes does not occur in transfected cells, or that if partial viral DNA copies are integrated in cell DNA that they are not transcribed. Nuclear RD-114 DNA was at least as efficient as whole cell DNA in transfection and within a wide size range (8 to more than 100 x 10” daltons) there was no apparent correlation between infectivity and DNA molecular weight. Although the host range of RD-114 provirus was similar to that of the intact virus, we cannot specify whether restriction is due to the inability of nonpermissive cells to take up, integrate, and transcribe proviral DNA since the restriction in horizontal spread of early progeny virus in nonpermissive cells would inhibit our ability to detect virus by the RT or P,, assays. The efficiency of transfection can be cal-

ET AL.

culated from the minimum quantity of provirus-containing cellular DNA required to induce progeny virus, which, in the case of RD-114 DNA in RD cells, is 0.1 to 0.2 pg per 2 x 10” cells. Based on an average of either 20 (Fujinaga et al., 1973) or 2 (Benveniste and Todaro, 1974) viral gene copies per infected RD-114 cell, the approximate specific infectivities are one infectious event per 5 X lo5 or per 5 x lo4 viral gene copies, respectively. Although on this basis transfection seems remarkably inefficient, the calculated range is in surprisingly close agreement with efficiency data from other laboratories studying avian transforming proviral DNA in chicken cells (Hill et al., 1974; Svoboda et al., 1973; Cooper and Temin, 1974). In conclusion, we have shown that the DNA of a human cell line productively infected with transformation-defective mammalian C-type viruses can induce progeny virus production in permissive cells. Further, we have discussed the location of the proviral DNA, the transfection, host range, and the efficiency of transfection, and we have raised some questions regarding the nature of host-cell control of transfection. ACKNOWLEDGMENTS The authors wish to thank Robert Rongey for electron microscope pictures, Hsien Jien Kung for DNA length measurements by electron microscopy, and Jean Filbert and Mary Peer for expert technical assistance. They also wish to thank Dr. Norman Davidson for helpful discussions and comments. REFERENCES M. A. (1972). Widespread presence in chickens of DNA complementary to the RNA genome of avian leukosis viruses. Proc. Nut. Acad. Sci. USA 69, 576-580. BENVENISTE, R. E., and TODARO, G. J. (19741. Multiple divergent copies of endogenous C-type virogenes in mammalian cells. Nature (London) 252, 170-173. BURTON, K. (1956). Study of conditions and mechanisms of diphenylamine reaction for colorometric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-23. COOPER, G. M., and TEMIN, H. M. (1974). Infectious Rous sarcoma virus and reticuloendotheliosis virus DNAs. J. Viral. 14, 1132-1141. BALUDA,

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FILBERT, J. E., MCALLISTER, R. M., NICOLSON, M.O., and GILDEN, R. V. (1974). RD-114 virus infectivity assay by measurements of DNA polymerase activity and virus group specific antigen. Proc. Sot. Exp. Biol. Med. 145, 366-370. FOURCADE, A., HUYNH, T., and LACOUR, F. (1974). Transfection of chicken embryo cells with DNA extracted from avian virus-producing neoplastic cells. J. Virol. 14, 407-411. FUJINAGA, K., RANKIN, A., YAMAZAKI, H., SEKIKAWA, K., BRAGDON, J., and GREEN, M. (1973). RD-114 virus: Analysis of viral gene sequences in feline and human cells by DNA-DNA reassociation kinetics and RNA-DNA hybridization. Virology 56, 484-495. GILDEN, R. V., LEE, Y. K., and LONG, C. (1972). Rescue of the murine sarcoma virus genome from nonproducer cells by the RD-114 type-C virus. Znt. J. Cancer 10, 458-462. GRAHAM, F. L., and VAN DER EB, A. J. (1973). A new technique for the assay of infectivity of human adenovirus-5 DNA. Virology 52, 456-67. GROSS-BELLARD, M., OUDET, P., and CWAMBON, P. (1973). Isolation of high molecular weight DNA from mammalian cells. Eur. J. Biochem. 36, 3238. HAREL, L., HAREL, J., and FREZOULS, G. (1972). DNA copies of viral RNA in rat cells transformed by Rouse sarcoma virus (RSV). Biochem. Biophys. Res. Comm. 48, 796-801. HILL, M., and HILLOVA, J. (1971). Virologie. Production virale dans les fibroblastes de Poule traites par I’acide desoxyribonucleique de cellules XC de Rat transformees par le virus de Rous. C. R. Acad. Sci. Paris 272, 3094-3097. HILL, M., and HILLOVA, J. (1972a). Virus recovery in chicken cells tested with Rous sarcoma cell DNA. Nature New Biol. 237, 35-39. HILL, M., and HILLOVA, J. (1972b). Recovery of the temperature-sensitive mutant of Rous sarcoma virus from chicken cells exposed to DNA extracted from hamster cells transformed by the mutant. Virology 49, 309-313. HILL, M., HILLOVA, J., DANTCHEV, D., MARIAGE, R., and GOUBIN, G. (1974). Infectious viral DNA in Rous sarcoma virus (RSV) transformed nonproducer and producer animal cells. Cold Spring Hurbor Symp. Quant. Biol. 39, 1015-1026. HIRT, B. (1967). Selective extraction of polyoma DNA from infected mouse cell cultures. J. Mol. Biol. 26, 365-369. KAWAKAMI, T. G., HUFF, S. E., BUCKLEY, P. M., DUNGWORTH, D. C., SNYDER, S. P., and GILDEN, R. V. (1972). Type C virus associated with gibbon lymphosarcoma. Nature New Biol. 235, 170-171. KANG, C.-Y., and TEMIN, H. M. (1974). Reticuloendotheliosis virus nucleic acid sequences in cellular DNA. J. Viral. 14, 1179-1188.

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KARPAS, A., and MILSTEIN, G. (1973). Recovery of the genome of murine sarcoma virus (MSV) after infection of cells with nuclear DNA from MSV transformed non-virus producing cells. Eur. J. Cancer 9, 295-299. KLEMENT, V., NICOLSON, M. O., and HUEBNER, R. J. (1971). Rescue of the genome of focus forming virus from rat non-productive lines of B’-bromodeoxyuridine. Nature New Biol. 234, 12-14. KLEMENT, V., NICOLSON, M. O., NELSON-REES, W.. GILDEN, R. V., OROSZLAN, S., RONGEY, R. W., and GARDNER, M. B. (1973). Spontaneous production of a C-type RNA virus in rat tissue culture lines. Znt. J. Cancer 12, 654-666. KUNG, H., BAILEY, J. M., DAVIDSON, N., NICOLSON, M. O., and MCALLISTER, R. M. (1974). Structure and molecular length of the large subunits of RD114 viral RNA. J. Viral. 14, 170-173. LEVY, J., and KAZAN, P. (1974). The importance of DNA size for successful transfection of chicken embryo fibroblasts. Virology 61, 297-302. LONG, C., SACKS, R., NORVELL, J., HUEBNER, V., HATANAKA, M., and GILDEN, R. V. (1973). Specificity of antibody to the RD-114 viral polymerase. Nature New Biol. 241, 147-149. MACPHERSON, I., and MONTAGNIER, L. (1964). Agar suspension culture for the selective assay of cells transformed by polyoma virus. Virology 23, 291294. MCALLISTER, R. M., MELNYK, J., FINKLESTEIN, J. Z., ADAMS, E. C., JR., and GARDNER, M. B. (1969). Cultivation in vitro of cells derived from a human rhabdomyosarcoma. Cancer 24, 520-526. MCALLISTER, R. M., NELSON-REES, W. A., JOHNSON, E. Y., RONGEY, R. W., and GARDNER, M. B. (1971). Disseminated rhabdomyosarcomas formed in kittens by cultured human rhabdomyosarcoma cells. J. Nat. Cancer Inst. 47, 603-611. MCALLISTER, R. M., NICOLSON, M., GARDNER, M. B., RASHEED, S., RONGEY, R. W., HARDY, W. D., JR., and GILDEN, R. V. (1973). RD-114 virus compared with feline and murine type-C viruses released from RD cells. Nature New Biol. 242, 75-78. MCALLISTER, R. M., NELSON-REES, W. A., PEER, M., LAUG, W. E., ISAACS, H., JR., GILDEN, R. V., RONGEY, R. W., and GARDNER, M. B. (1975). Childhood sarcomas and lymphomas: Charactenzation of new cell lines and search for type-C virus. Cancer 36, 1804-1814. MONTAGNIER, L., and VIGIER, P. (1972). Virologie. Un intermediaire ADN infectieux et transformant du virus du sarcome de Rous dans les cellules de Poule transformees par ce virus. C. R. Acad. Sci. Paris, 274, 1977-1980. PENMAN, S. (1966). RNA metabolism in the HeLa cell nucleus. J. Mol. Biol. 17, 117-130. RAND, K. H., and LONG, C. (1972). Syncytial assay for the putative human C-type virus, RD-114, uti-

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Folia biologica, Prha 18, 149-153. SVOBODA,J., HLOZANEK, I., MACH, O., MICHLOVA, A., RIMAN, J., and UR~ANKOVA,M. (1973). Transfection of chicken fibroblasts with single exposure to DNA from virogenic mammalian cells. J. Gen. Viral. 21, 47-55. VARMUS, H. E., GUNTAKA, R. V., DENG, C. T. and BISHOP, J. M. (19741. Synthesis, structure, and function of avian sarcoma virus-specific DNA in permissive and non-permissive cells. Cold Spring Harbor

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VARMUS, H. E., VOGT, P. K., and BISHOP, J. M. (1973). Integration of Rous sarcoma virus-specific DNA following infection of permissive and nonpermissive hosts. PNAS 70, 3067-71.