Evidence for helper independent murine sarcoma virus

VIROLOGY

66,

268-28-I (1973)

Evidence I. Segregation

for

Helper

Independent

of Replication-Defective

Unizlersily

Sarcoma

Virus

and Transformation-Defective

J. I<. BALL, J. A. McCARTER, Ca.ncer Research Laboratory,

Murine

AND

S. 1.1.SUKDERLAND

of K’esteru Ontario,

Accepted August

Viruses

London,

Ontario,

Canada

S, 1973

In the conventional focus assay for murine sarcoma virus (Hartley and Rowe, 1966), the formation of a focus involves repeated rounds of infection, and, as is shown in this report, the possibility of alterations in the genome of the virus is t,hereby increased. Mult.iple rounds of infection were avoided by infecting cells in suspension, plating them sparsely, and allowing them to grow into colonies. SC cells were added to detect which colonies were producing leukemia virus. When cells were infected with the Moloney sarcoma-leukemia virus (M-MuSV(MuLV)), four t.ypes of colonies were seen: (1) morphologically normal with synrytia (XC+) or (2j without syncytia (XC-), (3) morphologically transformed with no syncgt.ia, (4) transformed with syncytia. The proportions infected by MuSV (transformed cells) or by MuLV (XC+) conformed t.o Poisson’s dist,ribution, and this allowed the calculation of the tit.ers of MuSV and MuLV. Clones of chronically infected cells could readily be isolat,ed. A clone of transformed cells called G8 was derived from JLS-V9 cells infected with M-MuSV(MuLV). The cells produced no MuLV detectable by cocultivation with SC cells, but they did produce sarcoma virus detected by the production of sarcomas in mice and morphological transformation of several lines of mouse cells in culture. The virus had a density of 1.16 g/cm3. The kinetics of focus formation were one-hit when assayed by the conventional assay. Virus picked from most (32/38) of these foci consisted of a mixture of sarcoma virus and leukemia virus but some (4i’38) foci were found that produced sarcoma virus alone (presumably “competent” sarcoma virus, i.e., helper-independent). The presumed “competent” sarcoma virus was carried through 4 successive passages and each time, most of the foci were found to contain both M&V and MuLV, but some produced MuSV only. In contrast., the original, chronically infected G8 cells did not release detectable MuLV t,hrough more than 30 passages. Leukemia virus or defective sarcoma virus segregated from the competent MuSV with low and equal frequencies only when new mouse cells were inferted. Examination of the individual cells within foci formed by spread of viral infecbion showed that. some cells produced only competent virus; other cells from the fori produced MuSV and MuLV; others were transformed nonproducers containing defertive hluSV t.hat could be rescued by superinfection wit,h MuLV; and still others were transformed but MuSV could not, be rescued from them. No evidenre was found for the presence of a helper virus in excess of the concentration of sarcoma virus and rompetence appears to be a property of the virion itself. The data suggest that the competent MuSV is similar to the helper-independent strain of the Kous avian sarcoma virus. It. is not known why t,his virus is negative in the XC assay, although the MuLV that segregates from it is positive in this assay. In the search for a helper virus, a form of MuSV was found that did not, morphologically transform the cells it infected, nor was it produced by them, but, both transformation and release of bIuSV appeared on superinfection with hIuLV. 268 Coppight All rights

@ 1973 by Academic Preasu, Inc. of reproduction in any form reserved.

MURINE

SARCOMA

INTRODUCTION

Maloney murine sarcoma-leukemia virus complex (RI-I\IuSV(RL-RluLV) induces foci of t,ransformed cells in cultures of mouse embryo cells (Hartley and Rowe, 1966). The titrat,lon pattern of the virus was two-hit but could be convert’ed to one-hit by the addition of exogeneous RIuLV. These findings were interpreted to mean that RluSV was defective for replication in the absence of RluLV and that foci arose in monolayers of mouse embryo cells by viral spread. Subsequent studies (Aaronson and Rowe, 1970) showed. that RIuSV can infect and transform Balb/c 3T3 cells in the absence of MuLV. Therefore, foci in cell monolayers can be formed either by viral spread (dependent upon coinfection with MuLV) or by cell division (independent of coinfection with MuLV). The rate of cell division of such RIuSV taansformed cells would determine the time required to form a visible focus. It, was found (Aaronson and Rowe, 1970) that early developing foci (3 days post infection) could be attributed to viral spread, resulting in a t,wo-hit titration pattern. Late-arising foci (7 days post infection) consisted of those which arose as a result of viral spread and others which resulted from cell division. Under these latter cxpcrimental conditions a one-hit titration pattern n-as obtained. The one-hit titration pattern found for RI-MuSV(nImuLv) in. monolayers of rat cells (Parkman et al. 1970) could also be accounted for by showing that these foci arose primarily by cell division (.$aronson, 1971). Therefore, in order to detect all RIuS1’infected cells it was necessary to devise an assay in which the detection of all transformed cells should be independent of ?tIuLV Such an assay has been briefly referred to bJ Aaronson as a ‘Wue transformation” assa) (baronson et al., 1970). As reported in the present paper, such an assay has been dcveloped and has bwn made the basis for a simultaneous assay for both RIuSV and

n,IuLv. In addition to t,he species of RIuSV described above, another type of transforming virion has also been described (Fischingcr and O’Connor, 1970). Certain preparations of iu-nIkx'(lu-RhLv) were found to con-

VIRUS

269

tain a high proportion of MuSV which appeared to be competent for both transformation and replication. The evidence for this act’ivitg was indirect, being obt#ained from virus titration st,udies. Since this act,ivity could be removed from extracts of tumors induced by RI-RIuSV(M-RIuLV) b\ physical methods it was concluded t#hat,such a compet8ent MuSV was in fact an interviral aggregate of RIuSV and RIuLV. Additional t.ypes of RIuSV transformed cells have also been described: M-RIuSV(RIMuLV) transformed cells (S+L-) which produce viral particles demonstrating 110 MuSV or RIuLV activity (Bassin et al., 1971) and two RI-RluSV(M-RIuLV) transformed clones derived from normal rat kidney (NRK) cells, one of which released RIuSV t,hat t,ransformed NRK cells lvith one-hit tkration kinetics but was not t,ransmissible and the second clonewhich released noninfectious virus (Somers and Kit, 1971). We now report that among the viral progeny of a C~OIW 0f nI-nhsv(nI-nhw) chronically transformed cells (referred t,o as GS) arc 2 new species of nId3v: (1) nIusv, which in bhe absence of MuLV is competent for replication and transformation [we refer to this species as replicat,ion positivetransformat,ion positive (R+T+)] and (2) RIuSV, which can infect and persist in t,he cell t,hrough many cell divisions but can transform and replicate only when such cells are superinfe&ed with exogenous RIuLV (i.e., replication negative-transformation negative (R-T-). B? analogy replication negative-transformatlori positive nhsv would be reprcsent,ed as (R-T+) and T\IuLV as (R+T-) . MATERIALS

SND

METHODS

Cell cultures alwl medium. JLS-V9 cells (Wright, et al., 1967) were obtained from Dr. S. Chandra (Chas. Pfizer and Co., Inc., Nagwood, New Jersey). TB cells were derivrd from a mixed culture of fetal thymus and bone marrow from CFW/D mice (Ball et al., 1964) and were established in culture according to the procedures of Wright et al. (1967). Cloned lines of TB cells were used in all experiments. 3T3/FL cells were ltindl> supplied b? Dr. R. H. Bassin and maintained as described (Bassin et al., 1971) and

3T3 cells (Todaro a,nd Green. 1963) wre ohtained from the American Type Culture Collection and were cloned heforr use. 3C cells were kindly provided by Dr. J. 1Y. Hartley. All cells were nlaintained by routine trypsinizat#ion and subculturing every Z-1 days. All cell cultures were seeded at 2 to 5 X lo5 cells in 75 cm flasks obtained from Falcon Plastics (Los Angeles, California j . The medium used for growth and in viral assays (except for 3T3/‘FL cells) was Eagle’s h&ma1 Essent,ial medium containing 10 ‘7. heat-inactivated fetal calf serum (Grand Island Biological Co., Grand Island, New Pork) to which penicillin, streptomycin, and mycostatin wre added. .A113T3 cells were maintained in hIcCoy’s 5h medium cmtaining antibi&cs but no mycostatin. All cells were routinely passaged about once per morkh through the anti-PPLO agent, Tylocine (Grand Island Biological Co.). All incubations were carrird out at 37” in a humidified atmosphere of J 5, CO, and 95 “c air. Ch.ronically tramforwled G8 c1on.e. The RIoloney hIuLV(hIuSV) transformed clone was derived from JLS-V9 cells and is referred to as GS. The JLS-V9 cells were seeded as single cells and 6-8 hours after plating were infected wilth Maloney MuLV (AIuSV) diluted in medium cowaining DE-YE-Dextran (Vogt, 1967). -1 transformed colony of cells which showed no ?tIuLV activit#g as detected by the XC cells (Klement et al., 1969) Iv-as picked, and the cells were distributed in 0.005-ml portions to 21 Falcon microtest dish wells (Falcon Plastics, Los Angeles, California). After growth for several days, the contents of t,he well GS were transferred to a 2.5~ml Falcon tissue culture bottle. For t’he first 2-3 passages, the cells remained rounded up. However, on subsequent passages as the cells reached confluence a high proportion of t.he cells flatt’ened out and assumed a normal morphology. T’imses. hl-hiluSV(M-MuLV) was kindl) supplied by Dr. J. B. Roloney as a 1 g,/ml extract from tumor tissue from Balb/c mice. This was used to prepare a viral harvest on TB or 3T3 cells. Cells were infected at a m.o.i. of 1.0 and 1 X lo6 cells seeded in a 75

cm? Fal[~)~t flask. X (i-daj, tissur cultuw harvest \\as the source of M-~IuS1’(~1us;t~l in all experiments. For pwparation of viral st,ocks from the GY chronically transformed cells, the cells were growl for 3 days and t.hen 2 to 3 24hr t,issue culture harvests were nade. 311 viral harvests wre either filterrId through 0.22 pm or 0.45 pm RIillipore filters before use, or some were sonicated for E-20 see using a Branson Model W 140 Sonicator before filtration. Rloloney RIuLV was obtained by infecting JLS-V9 41s as a single cell suspension in a spinner flask wiith a viral preparation of a tissue culture harvest of RIoloney RIuLV (MuSV) and picking a clone of cells showing liC activity but no transformation. The XC cells were eliminated by exposure to ant,i XC serum raised in mice. T’iral i,~jections. RIuLV was assayed using XC cells and a modification (Wong et al., 1973) of the original method (Klement et al., 1969). Cklls .were removed from T-75 flasks using trypsin (0.25 % in isotonic saline citrate) before t,he cultures became confluent and were transferred in complet#e medium t,o spinner flasks maintained at 37°C. After 4 hr of continuous stirring, the concentration of cells was measured and adjusted to 1 X lo6 cells per ml in complete medium. Virus was diluted in medium lacking serum and containing 25 pg per ml DEAE-dextran (Vogt, 1967). Diluted virus (1 .O ml) was mixed lvith 1.0 ml of cell suspension in sterile disposable plastic tubes (12 X 75 mm, Falcon Plastics) and shaken at 37” for 40 min. The suspension was then diluted with complet#e medium, and 4 ml containing t,he appropriat’e number of cells were transferred to 60 mm dishes (Falcon Plastics). Focus a.ssayfor Muxb’. In this assay focus formation is dependent on viral spread (Hartley and Rowe, 1966). Cells lvere infected in suspension as described above and 2 X IO” infected cells were plated. The plat,es xvere fixed wilth 70 % h,‘IeOH when confluent (3 days post infection), stained with Giemsa and the number of foci determined. The tit#er is expressed as focus-forming units rer milliliter (FFUjml). IQectious ceders assay for MI&T’ productiole. 10’105 transformed cells were irradi-

nhmj

MURINE;SARCOMA

ated with gamma rays from a 6oCo source (5000 rads) Gammacell 220 (Atomic Energy of Canada) and mixed Lvith a single-cell suspension containing 2 X lo6 normal TB cells in the presence of 2 pg/ml Polybrene (Toyoshima and Vogt, 1969) (Aldrich Chemical Co., Milwaukee, Wisconsin). The mixed cell suspension was plated in a 60-mm dish, and t,ransformation was assessed on day 3 post p1atin.g. Sin2dtan.eou.s JILT’-IIISV assay. Cells were infected in suspension as described above and plated at 500 per plate. Cultures were incubated at 37” for 4 days. The medium was t,hen removed and 5 X lo5 XC cells in 4.0 ml of medium were added. After a further 1 day at 37”, the medium was removed and the cells were fixed with 70’;) methanol and st#ained with Giemsa. Four different types of cell clones could be identified: (1) morphologically transformed, large, round, highly refractile cells infected by hIuSV only.; (2) morphologically t,ransformed clones surrounded by XC syncytia indicating infection by RIuSV and RIuLV; (3) clones of cells with normal morphology surrounded by XC syncybia indicating infection by MuLV and (4) clones of cells with normal morphology w&h no surrounding XC syncytia. The proportion of colonies infected with RIuSV only, with hIuLV only, and wilth both RIuSV and hIuLV was measured. Bv Poisson’s formula, the fraction of cells notinfected (by either hIuSV or MuLV) = e-=, where s = multiplicit,y of infection (m.o.i.). The viral titers were obtained by multiplying the m.o.i. by the number of cells exposed (106) and by the dilution factor. In this assay all RIuSV infected and transformed cells should be det#ected whether or not the cells are producing virus (i.e., in this assay RIuSV det,ection is independent of viral spread). The titer is expressed as infectious unit,s per ml (IU/ml). CZordn.g. All cloning was done by either plating cells at low numbers, 10 per 60 mm dish and overlaying with 0.7 % agar [purified agar (Difco)] within 24 hr of plat,ing or by plating 2 X 10” cells per 60 mm dish and overlaying with 0.7 % agar 6-8 hr post plating. Clones of cells were picked with or without the use of 0.15% trypsin following removal

271

VIRUS

of the overlaying agar using a sterile Pasteur pipet with a diameter of 1.1-5 mm. Incorporation of uridine-3H. The test cells (106) were seeded in T-75 flasks (Falcon Plastics); between 8 and 10 bottles were used for each cell line examined. When the cell monolayers were nearly confluent (3-4 days after seeding) the medium was removed and replaced with medium containing 5% heatinact,ivated fetal calf serum and 25 &i of uridine-53-H (5 mCi/O.O43 mg, New England Nuclear Corp.) per milliliter of cult,ure medium. Virus was harvested either 4 or 16 hours post labeling and centrifuged at 1000 g for 10 min to remove cells. The supernat#ant was then spun at 30,000 g for 2 hr to pellet the virus. The viral pellet was then resuspended in TNE buffer (0.01 JI Tris, 0.1 A1 NaCl, 0.001 dl EDTA, pH 7.4) and the suspension was layered on a 10 to 65 % linear sucrose gradient,. The gradients were centrifuged in a SW 40 rot,or for I8 hr at 35,000 rpm. Gradient, fractions were collected, treated with NCS tissue solubilizer (Amersham,/Searle) and 5 ml of scintillation fluid (BBOT and toluene) was added. The levels of radioactivity and buoyant density of each fraction were determined. RESULTS

Si~multaneous assay for JIuST’ and ilf IAT' The data obtained in 2 separate experiments using different harvests of h,I-RluSV(RI-RIuLV) are given in Table 1. As can be seen, t)he fraction of cells infected with either virus alone was dependent on the viral dilution according to Poisson’s disbribution. Some clones \vere bot#h transformed and surrounded by syncytia, and the observed proport,ion of such clones corresponded closely with that predicted from the separate titers of RI&V and MuLV, thus indicating that such clones arose from cells coinfected with both viruses and that both viral funct,ions were expressed independently. This assay should detect all cells infect,ed and transformed by RIuSV so that the titer for RIuSV obtained in this assay would always be higher than that obtained in an assay where RIuSV detectsion is dependent on viral spread even though an optimal m.o.i. of exogeneous RIuLV is present. We consistent,ly find that t,he t#iter of h,IuSV in prepara-

272

BALL,

Expt. No.

Virus dilution

Fraction MUSV

1 (106)”

2 (5 x

105‘)

l/5 l/10 l/20 l/JO 1;‘20 1 i-lo 1 @O 19’160

hIcCrll~TE:R

0.86 0.82 0.63 0.45 0.65 0.51 0.35 0.20

infected

ANI)

WNI)EI:LANI)

Fraction nIuS\‘(hIuLVJ

MuLV

Calculated

Found

0.30 0.27 0.12 0.17 O.-ii 0.31 0.29 0.18

0.26 0.22 0.07 0.07 0.30 0.16 0.10 0.04

0.29 0.22 0.11 0.12 0.33 0.20 0.15 0.06

Titer MUSV” 9 1.7 1.9 2 2 1.i 3.0 3.4

x x x x x x x x

106 10; 10’ 10’ 107 10’ 10’ 10’

MuLVd 1.i 2.1 2.-l 7.2 1.2 0.8 2.5 2.9

x x x X x x x x

106 106 106 lo6 10’ 107 lo7 10’

(1Infection was carried out as described in Materials and Methods. The data for each virus dilution for each experiment was derived from 4 plat,es with a total of 600-800 clones per dilution being screened. 6 Number of cells infected. c Focus-forming units per milliliter. d Infectious units/ml.

of M-RIuSV(hI-MuLV) assayd by the simultaneous assay is equal to or higher than that of the RluLV.

TBBLE 2 THE EFFECT OF SSERIAL P.ISS.\CE OF G8 CIZLLS ON THE TITER OF Mu% (R+T+) AND MuLV

Characteristics

Passage level

tions

of G8 Cells

The origin of this clone of chronically transformed cells is described in Materials and RI&hods. For the initial few passages the cells remained rounded up. However, on subsequent passages a large proportion assumed a more normal-appearing morphology as t,he cells became confluent. As noted in Table 2 the RIuSV(R+T+) titers in tissue culture harvest of G8 cells remained constant during 30 passages (subculturing at 34-day intervals). In order t’o examine GS cells for the production of hIuLV they were plated at 1000 cells per 60-mm dish (plating efficiency 807) and grown for 34 days to allow them to form colonies. XC cells (5 X 105) weret,henadded and the plates were fixed and stained 24 hr later. No XC positive colonies were detected in any of t,he plates. In addition, G8 cells were recloned b! plating 10 cells per 60 mm dish. The medium was replaced with medium containing 0.7 % agar 2-1hr after plating. Twenty such clones were obtained and all were shown ho be producing MuSV by t,he infectious centers assay. No RIuLV could be detected when the cloned cells were exposed to XC cells as described above. No RIuLV has ever been detected by this met,hod in the examinat,ion

5 10 15 20 30

Titer Mu%’ (R+T+)a

MuLVb

1.3 x 4.4 x 2.4 x 3.i X 1.3 x

O/iooO O/4000 O/4000 O/4000 O/WOO

103 103 10” lo3 10”

D Assayed under condit,ions where focus formation was dependent on viral spread. Expressed as ffu/ml. b Assayed by plating loo0 G8 cells per 60 mm dish, and allowed to grow for-l days at which time 5 X lo5 XC cells were added. Plates were fixed, stained and screened for XC syncytia 2-1 hr later. Four plates were scored per determination.

of more than 10’ cells over a period of 2 years. Properties of T,‘iral Progeny of G8 Cells Cell free tissue culture harvests of G8 cells (0.05 ml) induced sarcomas at t,he site of injection in newborn CFW/D (Ball et al., 1964) and NIH Swiss (NIH, Bethesda, Maryland) mice. Twenty of 20 mice injected developed sarcomas at the injection site 8-10 days post injection. The G8 cells also produced particles with a buoyant density of 1.16 g/cm3 when fhe

MURINE

SARCOMA

chronically infected cells were grown in the presence of uridine-3H (Robinson et al., 1965). In vitro transformation was demonstrated on cloned JLS-V9, TB, 3T3/FL, and 3T3 cells (Todaro and Green, 1963). Titration patterns of different filtered preparations of viral harvests from CT8cells are shown in Fig. 1. Similar results were obtained using sonicated preparations of virus. The virus was assayed by infecting TB cells in suspension and plating 2 X lo5 cells per 60-mm tissue culture dish. Confluent monolayers were formed by 3 days post infection at which time the plates were fixed and stained. Titration patterns of different preparations of M-MuSV(RI-NuLV) (3 day harvests from 3T3/FL cells) carried out’ in the same manner are included for comparison. The t,itration pat,tern for the MuSV produced by GS cells is clearly one-hit over the whole

dilution range. On the other hand, the titration patterns for the R-I-MuSV(M-MuLV) clearly show two-hit titration kinetics. The two-hit tit,ration pattern for M-MuSV(MMuLV) could be converted to one-hit kinetics by the addition of optimal amounts of M-MuLV (m.o.i. = 0.1). E$ect. of Coinjection with, Exoger1.ou.sMuLT and G8 Virus In the experiments just described the viral harvests from G8 cells were assayed under conditions where focus format,ion was dependent on viral spread so that any MuSV requiring MuLV for expression in such an assay would not have been detected. Therefore, the viral harvest from G8 cells was tit,rated in the presence of optimal concentrations of M-MuLV (m.o.i. = 0.1). The results are shown in Table 3. Four different viral harvests from G8 cells or clones of G8 cells B.

A.

w-

ii \ 28

Ia

2i3

VIRUS

-ti

’ (6)

131

(31

LL w tlIC

VIRUS

DILUTION

Frc. 1. (A) Titration curves for 2 different filtered viral harvests from G8 cells in the absence of exogenous MuLV (O-O, harvest No. 1; 0-0, harvest No. 2) and in the presence (X-X) of optimal concentrations of MuLV (m.o.i. 0.1). (B) Titration curves for 3 different filtered 3-day viral harvests (O--O, No. 1; O----O, No. 2; n----O, No. 3) from M-MuSV. MuLV in the absence of exogenous MuLV and in the presence (X---X) of optimal concentrations of MuLV (m.o.i. = 0.1). The numbers in parentheses refer to the total number of plates scored for foci for each dilution.

which is clt~ff~ctive for rcylication sfmce of ,\IuLI’.

showed increased levc+ of RluSV n-hcbn titrated in t,he presence of MuLV. For 3 viral preparations t#he titer increased lo-fold and for a fourth a loo-fold increase in RIuS\T tit,er was found. One-hit kinet,ics were obtained for all praparat.ions assayed both in t#he presence and absence of MuLV. These data indicate that viral harvests from GS cells cont,ain both RIuSV competent for replication and transformation and ?tluSV TABLE

TB cells were infected in suspension with varying dilutions of t#he viral harvest, from G8 cells or from clones of transformed cells hhat had been infectsed with viral harvests from GS cells. The cells were infected and t,he assay carried out as described in Materials and Methods under “simultaneous BIuSV-RluLV assay.” The titration of a sample of RI-RIuSV(1\1-RLuLV) assayed under t.he same conditions is included for comparison. The data are given in Table 4 together with the titers of the same viral harvesk obtained under conditions where the detection of transformat#ion is dependent on viral spread (assay carried out as described in Materials and RIethods under “Focus Xssay”j. As can be seen the h,IuSV t,it,er is approximately IOO-fold higher when assayed under conditions n-here the det,ection

3

EFFECT OF ADDED imIULV ON THE: FOrUsFORMING TITER OF VIK.LL HARYESTS FKOM (8 C:)NCI~:NTRITION 0F USING THE 0PTrhr.k~ M-MuLV (m.0.i. = O.lj MuSV titer”

Source of virus

fM-hIuLV

-M-MULV G8-4-6 G8-6 C;8-30 G8-84

1 1 8 7

x x x x

2 2 s 4

10’ 103 103 10”

in t,lic ab-

x 106 x 10’ x 10” x 10’

a average t,iters derived from 6 to 9 plates per determination and expressed as focus-forming units per ml. TABLE

4

A COMPARISON OF MuSV TITERS OBT.LINP:D DNDER CONDITIONS WHERE THE DETECTION TRANSFORMATION Is DEPENDENT ON OR INDEPENDENT OF VIR.LL SPRF,ID Titer of MuSV

Virus preparation

Dependent on spread Dilution M-Mu%’

G8-6 GIj-30

G8-84

u FFU, * Total

(MuLV)

l;u) l/60 l/80 1,:lOO 1,‘20 l;60 1,‘20 l/40 li80 1;‘320 l/640 l/10 1,‘20 1.‘40 1,‘xo

forus-forming unit.s. number of plates included

(6)b (6) (6) (6) (3) (3) (6) (12) (9) (6) (6, (6, (6) (31 (3)

1 8 6 4 1.2 2 8 8 8.4 8 7.6

in average

x x X x x x x x x x x

Titer MuLV (IU,/ml) Independent

FFU” ,ml 10’ 10’ lo3 10” 10’ 104 103 103 103 103 103

tit,er.

OF

Dilution

of spread FFU (‘ml 2 x 105 4 x 105 6 X lo5

1,‘lO i’21) l/20 (12) l/40 (9)

1.5 x 106 2.4 x lo6 4.8 x 106

1120 (3j

3.6 x

lo6

0

1120 (6)

1.3 x

106

0

l/l0

3.4 x

106

0

(6)

MURINE

SARCOMA

of transformation is not dependent on viral spread. Furthermore, since the assay can be used simultaneously for both RIuSV and &IuLV it permitted us to see if any cells infected by the RIuSV derived from GS cells were also producing RIuLV. As noted in Table 4 no MuLV was detected when cells were infected wilth harvests from GS cells, but XIuLV was detected when cells were infected nith a preparat,ion of RI-RIuSV(RIMULV) . Retention, of MuST’(R+T+) Activity from G8 Cells through Successive Cycles of Ijlfection Although the evidence indicated that G8 cells do not produce RIuLV, it was found that when viral harvests from GS cells were allowed to undergo a number of successive rounds of replication RIuLV could be detected. Since the evidence indicated (AaronSOJJet al. 1970) that early arising foci resulted from viral spread it, was necessary to examine a number of early arising foci induced by GS viral progeny t’o see whether the MuSV(R+T+-) activity was retained. Cells were infected and platsed for the focus assay as described in Materials and Methods. Following cell attachment, 6-S hr post plating, the medium was replaced with medium containing 0.7 ‘% agar. Three days after infection foci were picked using 0.12 ‘“0 trypsin. Some of the clones of cells that gren were transformed and others were normal in morphology. In order to eliminate the clones with normal morphology all plat#es were repicked to yield a preparation composed of transformed cells only. Each transformed clone wa,s th.en assayed for MuSV product,ion by the infectious centers assay and for RIuLV b\- plat,ing 1000 cells per transformed clone, allowing the cells to grow t,o form visible colonies and t,hen exposing them t.0 XC cells. The result,s are given in Table 5 and an outline of t#he method used to serially passage the virus given in Fig. 2. As can be seen for 2 experiments, the transformed cells were, in almost all instances, virus producers. In experiment 1 (Table 5), 32 of 38 clones were found to produce both RIuLV and RIuSV but 4 transformed clones produced

275

VIRUS TABLE

5

VIRAL PROGENY FROM TRANSFORMED CELLS DERIVED FROM Assaw FOR MuSV DEPENDENT oiv VIRAL SPRKID Erpt. NO.

1 2

m.o.i.

0.005 0.074l.B

Number Viral production clones picked hIuS MUSK. NP* hfuL1 3s 33

-I 6

32 23

2 4

ReSCUe

0 0

* Transformed, nonviral producer clones as det.ermined by response to SC cells, infectious centers assay and 3H-uridine incorporation.

MuSV only. The viral harvest from one of the 4 clones was selected and used to infect TB cells in an identical experiment,. A total of 33 foci of transformed cells were picked, grown up, repicked and assayed for viral production. The results are given in Table 5, experiment 2. Of 33 foci picked, 6 produced RIuSV only and 23 produced both RIuSV and

nhw.

Psocluction of Heterogeneous AIu.SV Species by Transfolmed Cells with.& Single Foci In order to see whebher the high proportion of foci producing RluSV and RIuLV resulted from the successive rounds of infect,ion by the virus during recruit’ment of normal cells during focus formation, it was necessaq t,o isolate a number of individual transformed cells from each of many foci and to determine t#he type of virus being produced. The viral harvest from one of the RIuLV negative RIuSV positive t,ransformed clones (experiment 2, Table 5) was used to infect cells with an m.o.i. of 0.0001 (based on t#he number of FFU per ml as determined in the focus assay) and 2 X lo5 cells were plated per 60-mm Falcon petri dish. Foci were detect’ed 4 days post plating, and the transformed cells from 11 individual foci were picked ,without the use of trypsin, diluted, and plated at low numbers. As soon as clones of transformed cells were detected (48 hr post plating) the medium was replaced with medium containing 0.7%’ agar. Clones of transformed cells were picked 3-5 days later, grown up and assayed for viral production. The results are given in Table 7. The data clearly indicate that not all foci

BALL,

276

McCARTER a Tirrue

SND HUNDERLBND Cellr

Culture

1

Supernatant

(filtered) 1 TB cells J 38 Foci picked and tranrfomed clones repickad 2 produced MuSV(hLV) 2 were non-virur producerr r 4 produced WSV only (R%+; ti-) 4 Tissue culture aupernatant frcm 1 hSV only producer (filtered)

V

TB cells 1 33 Foci

TB x311* pickad and transformed produced MuSV(HuLV) were non-virus producers produced MuSV, only (w;

clones

J 20 Foci picked (not repicked)

repicked (All

R+T’)

produced

lbSV@hLV)

Tissue culture rupernatant from 1 kkGV only producer (filtered) TB cells 11 Foci picked and cells of each focus grown up as individual clones 22 clones 10 produced MuSV(h1.V) 4 were non-virus producers only MuSV(ih+) C 8 produced FIG. 2. Diagram of the procedures used in the serial passage of virus harvests from G8 cells. of transformed cells produced in the cell monolayers are homogeneous with respect to their viral progeny. While foci 2 and 7 appear to be homogeneous, two other foci clearly were not. The apparent homogeneity may have been due to the small number of transformed cells recovered and examined. However, the viral heterogeneity among the transformed cells derived from the clones from foci numbers 9 and 11 was clearly demonstrated. Because of the low m.o.i. used in &is experiment (0.0.5-0.003 as deter-

mined by the simultaneous assay), it must be concluded that, the viral heterogeneity demonskated in cert#ain clones arose as a result of segregation. The viral progeny from one of the clones producing MuSV(R+T+) only (Table 6, clone 124, focus No. 2) n-as used t#o infect new cells using the simultaneous assay. Of 3010 transformed clones examined only 473 were producing MuLV indicating a segregation rate for MuLV of approximately 15 % . ,4 low number (9 of 54) of transformed nonproducer clones were also

MURINE TABLE VIRUS PRODUCTION CLONES ISOLATED BY MuSV(R+T+) Focus FORKATION SPRE.~D

TABLE

7

8 9

11

123 124 125 111 126 127 128 130 112 113 114 115 132 116 117 118 119 120 121 122 131 133

MSV (R+T+) MPV (R+T+) MSV(R+T+j MSV-MLVb MSV-MLV MSV-MLV MSV-MLV NP NP NP MSV-h1LV MSV-MLV NP MSV (R+T’ MSV (R+T’ MSV-MLV MSV-MLV MSV (R+T’ MSV (R+T’ MSV-MLV MSV(R+T+j MSV-MLV

VIR.4L

Clone No.

+MuLVa

4 .9 x 105 I.5 x 105 3 .2 x 105 I.4 x 105 1 .o x 105 i.5 X 10’

1.i X lo2 5.4 X lo2 ‘.5 x 10” s.0 x 10’

1.1 x 104 1.1 x 10’ x 105 1.1 x 105

11.3

a m.0.i. = 0.1. b In all cases where a clone produced both hISV and MLV, 100~, of t.he clones produced both MuSV and MuLV.

found following infection with viral harvest from clone 124 indicating that RIuSV(R+T+) segregated RluSV(R-Tf) with an equal frequency to that found for the segregation of MuLV. Some of the transformed MuSV producing clones (RluLV negative) were picked, grown to large numbers and the tissue culture harvests from several such clones were selected for further study. When these viral preparations were assayed it was found that the majority of the clones were now producing both MuSV(R+T+) and MuSV(R-T+) but no MuLV. The data, that the given in Table 7 indicate MuSV(R-T+~I was present in loo-to lOOOfold excess. One clone (352) was found to

347 348 351 349 352

7

PROGENY ARISING AFTER CELLS \vITH MuSV(R+T+)a

INFECTION ONLY&

Titer (FFU/ml)C -MuLV

Titer (ffu/ml)

-nmv 2

277

VIRUS

6

BY INDIVIDUAL TRANSFORMED FROM SINGLE FOCI INDUCED UNDER CONDITIONS WHERE Was DEPENDENT ON VIRAL

virus production

FOCUS NO.

SARCOMA

0.5 5 4 4 6

x x x x X

+MuLVd 102 103 102 10’ lo2

2.8 1.6 5 1.2 8

X x x x X

lo3 105 103 106 lo2

OF

Titere (IU/mU ND 6 x lo6 4 x 10’ 1 x 107 ND

a Clone 124-focus no. 2 from Table 7 was the source of virus. *Each titer is the average derived from 6-9 plates from 2 separate experiments. c Assayed using the focus assay as described in Materials and Methods. d m.0.i. = 0.1. e Assayed using t.he Simult,aneous Assay as described in Materials and Methods. ND, not determined.

produce low levels of MuSV(R+T+) only. RSuSV(R-Tf) was detected both by the focus assay (using an optimal m.0.i. for MuLV) and the simultaneous assay. The majority of transformed clones mere not producing RIuSV (assayed b? t,he infectious centers assays as described m Materials and Methods). RIuSV could be rescued from such nonproducer transformed clones by superinfection with RluLV. Therefore, the data indicat,e that it is possible to retain this R+T+ activity through 4 successive cycles of infect,ion under conditions where double infections of single cells were unlikely (m.o.i. 0.14.01 as determined in the simultaneous assay). Attempts to Detect a Helper Wus Attempts were made to see whether G8 cells were producing MuLV which was not detected by the XC assay but which might account for the apparent competence of the MuSV. TB cells were infect.ed with filtered viral harvests from G8 cells (m.o.i. = 0.1 with respect to FFUjml) as described in Materials and Methods. After 3 days of growth a number of clones with normal. morphology were selected for further studies. The cells were infect,ed with a preparation of AI-RIuLV of known titer. The rationale was that such cells with normal morphology,

278

BALL.

McCARTER

if they were in fact producing a species of RIuLV which could not be detected by the XC assay would be refractory to superinfection with RI-RIuLV because of viral interference (Sarma et al., 1967). In experiment 1, clones lrith normal morphology mere picked from plates originally seeded wiit,h 100 infected cells, whereas in experiment 2. clones wiith normal morphology were picked from plates seeded with only 10 infect’ed cells per 60-mm dish and were overlayered with 0.7 0; agar 6-8 hr post plating. Twentyone normal clones were infected with RIRIuLV, and the results are given in Table 8. There was no significant viral interference when such cells were infected with RI-RIuLV. To further check this possibility the supernatant from a clont exhibiting maximum TABLE

8

THE EFFECT OF iWMuLV INFECTION ON CLONES OF CELLS WITH NoRnf.kL ~IORPHOLOGP AND NEG.YTIVE FOR RluLV PRODUCTION DERIVED FROM TB CELLS INFECTED \VITH A VIRlL H.IRVEST FROM G8-6a CELLS -

Ez&. Nb.

1

2

Cell clone NO

M M s M M M M S s s 8 M B B B B B B B B B

2 4-1 4-2 8 9 13 15 16 19 20 21 28 1 10 13 14 15 16 17 19 20

Infection

2.4 2.0 17.0 2.0 13.0 2.2 6.2 20.0 6.2 7.2 9.2 5.2 3.0 7.0 10.0 1.0 4.0 3.0 4.0 4.4 5.0

with

MuLV

6.8 6.8 10.0 6.8 10.0 6.8 6.8 10.0 10.0 10.0 10.0 6.8 20.0 10.0 10.0 10.0 10.0 10.0 7.0 7.0 i.0

Ratio

1.25 0.41 0 0.45 l.i5 0.45 0.93 2.11 0.08 0.59 1.31 0.91 0 0.38 0.25 0.88 0.05 0.36 0.05 0.26 0.21

of

Rescue

NI) + ND + + + + + + ND + + 0 + + + + + + + +

a G8-6 (Table 9) was used as source of virus. b By infectious centers assay (see Materials and Methods). ND, not determined.

AND

SUNDERLAND

reduction in MuLV titer (clone 1, experiment, 2) was used to infect normal cells in the presence of Polybrene. The “infected” cells were passaged 3 times and were then used to assa>- a preparation of MuLV of known titer. No interference to infection by RI-?tIuL\’ was detecbable. ,%lso the titer of RIuSV (viral progeny of clone 124, Table 6) on clone 1 cells was identical to the tit,er obtained on normal TB cells. Furthermore, exposure of 3 of the clones listed in Table S (clones RI1.5, Bl and B16) t,o uridine-3H (see Materials and Methods) did not result in the detectsion of particles with a buoyant, densit,) of 1.16 g,/cm3. Detect.ioll of Additional

Mu.ST’ Species

However, the significant, observation made in t#he above experiment was that RI-RIuLV infect,ion of 19 of the 21 clones resulted in the appearance of a significant fraction of cells w&h a transformed morphology, and these cells were shown to be producing hIuSV by the infect,ious centers assay and b\- the focus assay. It would appear therefore that t,he viral harvest from G8 cells contains a species of RIuSV which will infect and persist in cells (some have been carried for at least 23 passages) without altering their normal morphology. One of the clones described in Table 8 (clone 15) has been recloned, and subclones with normal morphology were isolated. No virus particles with a buoyant density of 1.16 g/cm3 were detected when large numbers of clone 15 cells were grown in the presence of uridineJH. All subclones became transformed and released RluSV on infection with MuLV. We refer to the iXuSV species responsible for this activity as replication negative-transformation negative (R-T-). The Isolation of 116uLT~Negahe Transformed Cells Producing MuSV with Diflering Properties Viral harvests from a number of clones of transformed cells (Fig. 2) were assayed for the 3 species of MuSV so far described; (1) MuSV(R+T+), (2) MuSV(R-T+), and (3) RluSV( R-T-). MuSV (R+T+) was measured by the focus assay as described in Materials and Methods. MuSV(R-Tf) was measured by 2 different assays: (1) by coinfecbing cells

MURINE

SARCOMA

with viral harvests from RSuSV(R+T+) cells plus exogenous M-MuLV (using an optimal m.o.i. of 0.1) and using the focus assay or (2) by the simultaneous assay as described in Materials and Methods. MuSV(R-T-) was measured by the simultaneous assay with the addit,ion of exogenous MuLV (m.o.i. 2.0). The difference in MuSV bit,ers in the presence and absence of MuLV resulted from the transformation of those clones infected with lUuSV(R-T-) but, not expressing a transformed morphology in the absence of MuLV. As a result of t#hese assays, it, was found (Table 9) that, of the 13 MuSV-positive MuLV-negative kansformed clones, 2 were producing all 3 MuSV viral activities and the other 11 yielded only MuSV (R+T+) and RIuSV (R-T+). The latter species was present in lOO- to 1000-fold excess. In the 2 clones producing all 3 NuSV species the R-Tspecies was present in an approximat,ely lofold excess. The viral harvest from one of these 2 clones (GS-6) was used for the isolat,ion of R-T-- clones as described in Table S.

opment and careful examination of the assays used to characterize these viruses. A detailed description and validation of the modification of the XC plaque assay for MuLV has been published (Wong et al., 1973). This assay was also the basis for the simultaneous assay for MuSV and RluLV used in the present communicat,ion. This assay, in which the detection of transformed cells is dependent only on cell division should permit the detection of all cells infected and transformed by RluSV whether or not the infection was productive (Aaronson et al., 1970). The t’iters of RIuSV one obtains wilth this assay are higher than when the same viral preparations are assayed using the conventional focus assay (Hartley and Rowe, 1966). Similar findings have been reported by (Aaronson et al., 1970). The assay for RluSV developed by Hartley and Rowe (1966) is based on the detection of foci of transformed cells which arise in cell monolayers as a result of spread of viral infect,ion (and not by cell division). The formation of a focus as a result of cells being infected with defective RI&V must therefore involve rescue of RIuSV from the nonproducer transformed cells, together with spread of the rescued virus to previously uninfect,ed cells. Therefore, the titer of

DISCUSSION

The initial isolation- of a clone of morphologically transformed cells producing a number of species of RluSV with differing biological properties necessitated t#he develTABLE ISOLATION OF MuLV

Trans-

9

NEGATIVE TRANSFORMED CELLS PRODUCING SPECIES OF MuSV

DIFFERING

formed cell line

279

VIRUS

PROPERTIES WITH RESPECT TO TRANSFORMATION AND Type of MuSV assay

-

Dependent on spread - MULV

VII~ION~ WITH

REPLIC.LTION

Characteristic of MuSV

Independent of spread

+MuLV

- MULV

+MuLV

81-l 30-2 36-3

5.0 X 10za 1.5 X 10” -4.7 X 103

5.0 x 101 1.5 X 10” 3.1 X 101

6.0 X 105 1.6 x lo6 1.7 x 106

5.0 x 106 2.2 X 106 2.0 x 106

R+T+. R+T+: R+T+:

R-T+

36-l 36-4 32-5 30-5 44-3 50-5 84-7 84-9 G-30 G-6

2.0 Y3.0 ,5.0 .3.0 5.0 0.7 1.0 1.0 .Y.O 2.0

4.4 1.5 5.0 1.0 3.0 1.3 1.2 1.0 8.0 2.0

1.2 1.6 1.2 1.3 1.1 2.2 5.4 4.2 1.3 3.6

1.6 1.6 1.6 1.2 1.0 5.2 6.8 3.8 2.1 1.0

R+T+ : R+T+; R+T+. R+T+: R+T+: R+T+j R+T+; R+T+. R+T+: R+T+:

R-T+ R-T+ It-T+ It-T+ R-T+ R-T+ R-T+ R-T+ R-T+ R-T+.

X X x x x x X x x x

102 102 10” 102 103 102 102 103 103 10”

x x x x X x X x x x

10’ 104 101 104 10’ 10” 104 106 10’ 106

x x X X x x X X x x

106 lo5 105 106 10” 106 105 106 106 10”

X X X x X

10” lo5 lo5 10” 10” X lo5 X lo5 x 106 x 106 x 10’

0 Titer in focus-forming units per milliliter. Each titer is the average derived separate experiments. Variat,ion between plates and experiments was negligible.

R-T+.

R-T-

R-T+’

R-T-

from 8 plates from 2

250

BALL,

McCARTEK

MuSV obtained using such an assay will depend on having MuSV-infected, MuLVinfect)ed, and normal cells near enough to one anot,her so that viral spread can occur. There is a sharp optimal m.o.i. for MuLV in such assays; an m.o.i. greater than 0.1 results in a decrease in the MuSV t,iter presumably as a result of infectsing too large a proportion of cells with MuLV and consequent interference with infection by MuSV (Sarma et al., 1967). The MuSV biters obtained in the simultaneous assay are always higher than those obtained using the focus assay. Lower efficiency of the focus assay has been reported for bot#h MuSV (Aaronson, 1970) and RSV (Rubin, 1960). We consistently find that, preparations of M-MuSV(MMuLV) assayed by the simultaneous assay cont,ain MuSV, t,he titer of which is always equal to or great,er (lo-fold) than that of MuLV (Tables 1 and 4). The notion t’hat MuLV is always present in excess in preparations of M-MuSV(M-MuLV) may be in error. The data of Table 1 indicate that t,he simultaneous assay does give a reliable measure of the titers of both MuSV and MuLV. Virtually identical results were obtained for 2 separabe experiments using different viral harvest#s of RI-MuSV(M-MuLV). The proportion of cells infected with either virus was found to vary with the multiplicity of infect,ion in accordance wirith Poisson’s distribution. The tit#ers of virus calculated from the proportion of cells not infected were highly reproducible. Spread of virus from infected colony to uninfected colony, had it occurred to a significant extent,, would have resulted in a departure from adherence to Poisson’s distribut,ion. Some clones were both kansformed and surrounded with XC syncytia and the observed proportion of such clones corresponded closely with that predicted from the separate t#iters of the MuSV and MuLV. These data strongly support the fact t’hat such clones arose from cells coinfected with bot,h viruses and that both viral functions were independently expressed. Furthermore, these procedures permit one to infect single cells wilth a high probability that a cell will be infected with one infectious unit, of virus and not more. Finally, the procedures used in this assay provide for much greater

AND SUNDERLANI)

accuracy in cloning bot,h virus and virus infected cells than assays based on the use of monolayers for viral detection. The data in Tables 2 and 4 indicak that, G8 cells are not producing MuLV which will induce XC syncytia formation nor were JLSVS cells from which G8 cells were derived as a clone. We have screened approximately 1 X 10’ G8 cells (or cells from subclones of G8) and no clone was found to be producing MuLV by this criterion. Neither was MuLV detected when viral harvests of G8 cells were assayed using the XC assay. Since the titer of MuSV(R+T+) in such viral preparations is only 1-5 X lo3 FFU per ml, and since the is 15”c, any segregation rate for RIuLV arising by segregation was below the limits of d&&ion by the XC assay. The limit of detectability of MuLV in t.he XC assay (Wong et. ab., 1973) is 1 X lo3 III/ml. Millipore-filt.ered (0.45 pm) or sonicated and filt,ered t,issue culture harvests of t.he viral progeny of G8 cells resulted in a onehit titrat,ion patt,ern when asayed under condit8ions where the det,ection of transformat,ion was dependent on viral spread. Under identical conditions (i.e., in the same experiment) preparations of RIuLV) gave the characteristic two-hit8 titration pattern (Fig. 1) as previously reported (Hartley and Rowe, 1966; O’Connor and Fischinger, 1965). Although the majority of foci arising in cell monolayers are the result of combined infection by MuSV(R-T+) and RfuLV (Tables 5 and T), some foci were also found to be producing AIuSV(R+T+). This finding, together with the one-hit titration pattern (Fig. 1) found for the viral progeny of G8 cells does not necessarily mean that this RIuSV is competent for replication in t.hr absence of MuLV. It is possible to account for t,hese observations in a number of ways: (1) t,he viral harvests contain interviral aggregates of MuSV(MuLV) not removed by filtering or sonication, and in infection of cells wilth these aggregates t,he expression of MuLV is suppressed by MuSV; (2) G8 cells produce an excess of RIuLV not detectable by the XC assay, and this 1IuLV can fun&ion as a helper virus for the replication of RIuSV; (3)

nhw

nI-nhsv(w

MURINE

SARCOMA

TB cells function as helper cells (Weiss, 1973); (4) the MuSV produced by G8 cells is competent for both transformation and replication in the absence of MuLV. The Jirst possibility can be ruled out. The isolation of MuSV-producing MuLV-negative clones permitted us to prepare aggregates of MuSV and MuLV (O’Connor and Fischinger, 1969) with viral preparations of known titers, where the number of infectious units per ml of MuLV and MuSV (as determined by simultaneous assay) were equal (1 .O X lo6 infectious units per ml). Filtration (0.45 pm Millipore filter) removed all viral activity. However, when t’he unfiltered aggregate preparation was assayed under similar conditions, all infected clones were both transformed and surrounded bv XC syncytia. Further, the titers of both viruses recovered were virtually identical with t,he titers of each of the viruses used to prepare the aggregat,e. The average titer obtained in 2 separate experiments (expressed as infectious units per ml) for MuSV was 0.9 X lo6 (range 0.8 X lo6 t,o 1.0 X 106) and for MuLV was 1.1 X IO6 (1.0 X lo6 to 1.2 X 106).The presence of MuSV in the aggregat,e did not interfere wit,h the detectability of RIuLV in the XC assay. Other experiments showed that when cells are coinfected with MuLV and viral progeny from G8, the titer of the MuLV as detected in the simultaneous assay was identical with that of the MuLV used in the coinfecti.on preparations. Thus if MuLV is being produced by GS cells, the presence of MuSV should not have interfered with its detection. The secod possibility is that either G8 cells or TB cells produce an excess of non-XC syncytia-inducing MuLV which functions as a helper. This is also unlikely. Electron micrographic studies of TB cells have never revealed any viral particles. Also in many studies TB cells have been exposed to uridine-5-3H and no particles with a buoyant density of 11.16 g/cm3 have ever been detected. Viral interference studies (Sarma et al., 1967) also indicated that, G8 cells were not producing a non-XC registering MuLV. No viral interference was found when clones of cells with normal morphology, obtained

VIRUS

281

following infection with MuSV(R+T+) at a high m.o.i. were superinfected with MuSV(R+T+) or MuLV of known titers (Table 8). Quantitative data resulting from the use of the two assays used in this work rule out t,he presence of excess helper virus as accounting for the apparent competence of G8 virus. When the viral harvest from G8 cells was assayed for MuSV using the focus assay (see Materials and Methods), the titer of MuSV was 2 X lo4 FFU per ml and the kinetics were one hit. If the foci resulted from the interaction of defective MuSV and helper virus, then, to explain the one-hit kinetics, the helper virus must have been present in excess of the defective MuSV. It can be calculated, using the fact tha.t an m.o.i. of 0.1 for MuLV is required to convert two-hit to one-hit kinetics (Fig. l), that the titer of a helper virus should have been of the order of 8 X lo6 infectious unit,s per ml, i.e., 400-fold greater than the titer of mlsv. However, when the same viral harvest was assayed by the simultaneous assay (see Materials and Methods), the titer of MuSV was found to be 2 X lo5 infectious units per ml. Furthermore, by picking transformed clones and examining them in the infectious centers assay, it was found that only 1: 10 of them produced MuSV; the remaining 9 : 10 were transformed nonproducers from which MuSV could be rescued by superinfection with MuLV. The titer of ‘lcornpetent” MuSV was therefore 2 X lo* infectious units per ml in agreement wilth the value obtained from the focus assay. The conclusion reached from the data obtained using the focus assay was that helper virus, if present, in order to convert all the foci of infection by sarcoma virus into productive centers of infection, must have been present in 4OO-fold excess over defective sarcoma virus. But the conclusion from the simultaneous assay was that defective sarcoma virus was present in lofold excess over “compet#ent” virus capable of a productive infection. These conclusions are obviouslv incompatible with one another, and this, toget,her with the fact that both assays gave the .same value, 2 X lo* units per ml, for comptt,ent MuSV is a

strong argunlent for denying the role of a helper virus in conferring apparent, competence on the MuSV. This argument is based on clone G8 which produces both defectjive MuSV and competent RIuSV. Other clonesi.e., 124-produce only competent, MuSV when assayed in the focus assay in the presence of optimal amounts of exogenous RluLV (Table 6). It could be argued that the competence of clowtl viral isolates derives from the presence of a large excess of helper virus sufiicient to convert all centers infected by defective sarcoma virus t,o product’ive centers. When the viral progeny from clone 124 were used to infect new TB cells (using t,he simultaneous assay) and the transformed clones picked and assessed for viral production it was found that 9 clones of 54 picked were nonproducers from which RIuSV could be rescued by superinfecbion &.h RIuLV. This frequency of production of RIuSV(R-T+) is virt,ually identical with that found for the segregation of RuLV from M&V(RfTf). The fact that there were nonproducer clones must mean t#hat there could not have been an excess of helper virus present among the viral progeny of clone 124. The third possibility, that TB cells are functioning as helper cells (i.e., complementation of defective viral functions by cellular functions) is also unlikely. If the one-hit titrat,iott pattern observed resulted because TB cells were functioning as helper cells, then the titration kinetics for MMuSV(bI-RIuLV) should also have been one hit, and it, clearly was two hit, (Fig. 1). Furthermore, for all preparations of G8 virus assaved in the simult,aneous assa.y, the majo&y (90%) of the transfortned clones were nonproducers from which MuSV could be rescued by superinfection wilth MuLV. It is clear that the cells do not act to supplant the helper function of RluLV. All our available data strongly support’ the idea that a species of MuSV competent for replicabion and transformation exists. There is no evidence, therefore, t’hat apparent competence arises from other than the nature of the genome of the sarcoma virion itself; i.e., it is a helper-independent sarcoma virus. Presumably, the reason why it has

not been discovtwcl befurc is because of its tcndenc2, not to breed true, i.cs., to sogregatc AluLV or defective RIuSV whenever it, irife& a murine cell. Is RIuSV(R+T+) the murine coutttwpnrt, of the helper-iiidepenclcnt avian sarcoma viruses, or does it only appear to be so? The behavior of both the avian and murinc viruses is consist,ent wilth t,hat of a single infectious particle that. contains genetic information for both t,ransforming and nont8ransforming virus, but how is t,his information arranged wiithitt t,he particle? Is the genome homozygous (contains a single, &able genetic complement), heterozygous (contains unstable, genet#ic determinants derived from t\vo parenbal virions-Weiss et al., 1973; Martin and Weiss, 1973), or heteropolyploid (cont,aitts two or more complete genet#ic complements within a single particle)? So far as MuSV(R+T+) is concerned we have demonstrat.ed that its behavior is different from that of an interviral aggregate of MuSV and RIuLV as already discussed. We would expect’ a cell infected with a heterozggote or heteropolyploid t,o produce AIuSV and RuLV as if it were doubly infected. We have demonstrat,ed that MuSV and MuLV can be assayed in t#he presence of one another without mutual interference and that cells chronically infect.ed witah RIuSV(R+T+) (G8 and 134 cells for example) when cocultured wilth XC cells do not behave as if they were doubly infected. We do not kno\t why MuSV(R+T+) does not produce XC syncytia, but we suggest that it may be that cannot infect XC cells. When virus produced by clones GS or 124 is used to infect TB cells, segregation of MuLV and RluSV(R-T+) occurs, but not necessarily in the same cells; i.e., some cell clones were found that, produced MuSV and RluLV but others were found t#hat produced high levels of MuSV(R’IU) wilth lower levels of MuSV(R+T+) unaccompanied by detectable RIuLV. All t#hese findings are consistent with the interpretation that MuSV(R+T+) does not consist of heterozygous or heteropolyploid particles, but until more is known about the nature of the genome, we cannot rule out

nhsv

MURINE

SARCOMA

thab such particles account for our observations. Studies on the nature of the RNA of avian tumor viruses indicate that the genome is segmented and that the segment size of RSV is greater than that of the nontransforming virus (Duesberg and Vogt, 1970). The above data, together \l;ith the observat#ion t,hat nontransforming viruses segregate from RSV (Vogt, 1971), have led to the hypothesis that nontransforming viruses are deletion products of RSV. By analogy with the above, it might be expected that the RNA subunit size of MuSV(R+T+) should be larger t,han t,he genome subunit size of MuLV. Evidence in support of this has recently been obtained in this laboratory (In preparation). The species of MuSV(R-T-) which we detected has not, to our knowledge been previously described. This MuSV was able to infect and persist in the cell for at least 23 passages (subcultured every 33 days), but did not induce any detectable morphological changes in the infected cell. The infected cells produce no viral particles, and the presence of the MuSV genome becomes apparent only when the infected cells are superinfected with MuLV. SuperinfecCon with MuLV resulted in morphological changes as detected by the rounding and more refract,ile properties of the cells and the rescued MuSV was detected using t’he infectious centers assay. This species of MuSV(R-T--) either lacks some genetic information required for transformation and such information can be supplied by the MuLV or RIuLV fun&ions ts alow the expression OF such informat’ion present but not expressed in the MuSV(R-T-). This MuSV(R-T-) was readily detectable among the viral progeny of G8 cells, and was present in excess (IO-fold) of the other MuSV species. However, after successive clonings of MuSV(R+T+) an MuSV(R-T-) \vas not det,ected. This latt,er observation indicates that, the RLuSV(R-T-) is a viral function, not a property of the infected cells (Trager and Rubin, 1966). Since we have been able to demonstrate MuSV(R-T-) activity directly in extracts of M-MuSV(M-MULV)induced tumors (unpublished), me assume

283

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that all the G&type viruses would be present in all preparations of M-MuSV (M-MuLV). The heterogeneous nature of individual foci found when MuSV(R+T+) (at low m.o.i.) was assayed on cell monolayers indicates that caution must be exercised in using this technique to clone purify virus, or for the isolation of clones of virus-infected cells. The availability of clones of chronically transformed cells producing infectious MuSV in t,he absence of any detectable MuLV should aid in genetic and biochemical studies directed toward characterizing and contrast’ing the genomes of MuSV and MuLV. Such studies are underway. ACKNOWLEDGMENTS The authors wish to thank Barbara Choo and Deanne Harvey for expert technical assistance and Pat Blair for the uridine-3H incorporation studies. This work was supported by the Nat,ional Cancer Institute of Canada and the Medical Research Council of Canada. S.S. is supported by a Studentship from t.he Medical Research Council. REFERENCES AARONSON, S. A. (1971). Isolation of a rat-t.ropic helper virus from M-MSV-0 stocks. Vz’ro2ogy 44, 2!&36. AARONSON, S. A., and ROWE, W. P. (1970). Nonproducer clones of murine sarcoma virus transformed Balb/3T3 cells. Virology 42, 9-19. AARONSON, S. A., JAINCHILL, J. L., and TOD~RO, G. J. (1970). Murine sarcoma virus transformation of Balb/3T3 cells: Lack of dependence on murine leukemia virus. Proc. iVat. Acad. Sci. U.S. 66, 1236-1243. B*LL, J. K., HUH, T. Y., and MCC.4RTER, J. A. (1964). On the stat,istical dist,ribut,ion of epidermal papil1omat.a in mice. hit. J. Ca~ncer 18, 12@123. BBSSIN, R. H., PHILLIPS, L. A., KR.~MER, M. J., H~AP~L.~, D. K., PEEBLES, P. T., NOMUR~, S., and FISCHINGER, P. J. (1971). Transformation of mouse 3T3 cells by murine sarcoma virus: Release of virus-like particles in the absence of replicating murine leukemia helper virus. Proc. Xat. Acad. Sci. U.S. 68, 1520-1524. FISCHINGER, P. J., and O’CONNOR, T. E. (1970). Replication of defective and competent, forms of murine sarcoma virus in mouse cell cultures. ViroZogy 41, 233-243. H.~RTLEY, J. W., and ROWE, W. P. (1966j. Produc-

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