N-Tropic variants obtained after co-infection with N- and B-tropic murine leukemia viruses.

(1)

N-Tropic Variants

Obtained

After Co-Infection with N- and

B-Tropic Murine Leukemia

Viruses

NANCY HOPKINS,* PAULATRAKTMAN, AND KATHLEEN WHALEN

Center for CancerResearch,Massachusetts InstituteofTechnology, Cambridge,Massachusetts02139 Received forpublication 22September1975

Sc-i cells co-infected with small XC plaque-forming N-tropic and large XC

plaque-forming B-tropicmurine leukemia virusesproduced, in addition to

pa-rentaltypes, progeny withthephenotype, largeXCplaquemorphology,and

N-tropism. Thisphenotyperemained stablethroughendpointtitration andplaque

purification on NIH/3T3 cells and growthon BALB/3T3 cells. These N-tropic

viruses (XLP-N virus) grow to unusually high titer and make very large XC plaques.

Co-infection with RNAtumorviruses

carry-ingappropriate genetic markerscanyield

prog-enywithnovelphenotypes.Inthe avian system

three typesof interactions betweentwo

exoge-nously infecting viruses orbetween an

exoge-nous and an endogenous virus have been

de-scribed: phenotypic mixing, heterozygosis, and

geneticrecombination(4, 11,23-25, 27).Studies

with mammalian RNA tumor viruses have

been less extensive than those with avian

vi-ruses, in part because of a lack of suitable

markers. However, evidence for phenotypic

mixing and for recombination between

exoge-nous viruses (21, 26) orbetween anexogenous

and an endogenous virus (20) has been re-ported.

In this paper we describe the isolation of

viruses with novelphenotype after co-infection

ofSc-icells with N- andB-tropic murine

leuke-mia virusesderived fromBALB/c mice. The

N-or B-tropism of ecotropic murine leukemia

vi-ruses provides a natural genetic marker that

allowsastrongselection for viruses with either

hostrange (7, 13,14, 16).Recently,Hartley and

Rowe (5)described the isolation ofa mousecell

line, Sc-i, on which N- and B-tropic murine

leukemia viruses grow equally well. Hopkins

and Jolicoeur (8) andStephensonand Aaronson

(19) have recentlydescribedtheisolation of N-tropic murine leukemia viruses, derived from

BALB/c mice, thatmake XCplaques with

al-tered morphology. TheXCplaqueassay

devel-oped byRowe etal. (18) isbasedonthe

observa-tion (12)that whenmouse cellsproducing cer-tainmurine leukemia viruses come in contact

with XC cells(22), a ratcell line transformedby

an avian sarcoma virus, the XC cells fuse to

form syncytia.

We describe here the isolation of large XC

plaque-forming N-tropic murine leukemia

vi-ruses(XLP-N virus)fromamong the progeny of

Sc-i cellsco-infected with the small XC

plaque-forming N-tropic (SP-N) virus ofHopkinsand

Jolicoeur (8) and large XC plaque forming

B-tropic (LP-B) virus. Infectionby SP-NorLP-B

alone didnotyieldXLP-N virus. MATERIALS AND METHODS

Cells. BALB/3T3 (clone A31) cells (1) were ob-tained from Robert Pollack, Cold Spring Harbor; NIH/3T3 cells (9) were from Stuart Aaronson, Na-tional Institutes of Health; and Sc-1 (5) and XCcells were from Janet Hartley and Wallace Rowe, Na-tional Institutes of Health. Medium was Dulbecco modified Eagle medium with 10% calf serum (10% inactivated fetal calf serum was used for Sc-1 cells) and 50 ,ugof penicillin and 50,gof streptomycin per ml.

Viruses. SP-N virus is a clonal isolate derived by Hopkins and Jolicoeur (8) from a stock of the LP-N virus (WN 1802N, pool 1898) derived from BALB/c by Hartleyetal. (6). The LP-B virus used is a clonal isolatederived by Paul Jolicoeur from a stock of the LP-B virus (WN 1802 B) derived from BALB/c by Hartley et al. (6, 10).

XC assays.NIH/3T3cells orBALB/3T3cells were plated at 105 cells/60-mmdish. Thenext day they were infected with 0.2 ml of appropriately diluted viruscontainingpolybrene (8 fig/ml). All dilutions were plated in triplicate. After 2 h ofadsorption with frequent rocking ofthe plates, medium was replaced. The next day the medium was changed. Theday before the cells reached confluence the me-dium waschanged. The next day the medium was removed, and the cells were irradiated and overlaid with 106 XC cells as described (18). Medium was changedthe following day, and on day 3 after the addition of XC cells the plates were fixed and stained, either with Harris hematoxylin or cresyl violet acetate (1% aqueous solution).

Plaquepurification ofvirus. Virus was diluted sufficiently so that a number ofplates received 1 PFU. An XC assay was performed as described above except that the plates were not irradiated beforebeingoverlaid with XC cells. Onday 3 after addition of XC cellsa plaquewaslocated and cir-324

on November 10, 2019 by guest

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MLV PLAQUE VARIANTS 325

cled, and the cells from the circled area were re-moved with a cloning cylinder. The plate was then fixed and stained to confirm that no other plaques were present on the plate. The cells from the cloning were plated with 105 NIH/3T3 cells on a 60-mm dish. Four days later, culture fluids from this plate were collected and used to infect 105 NIH/3T3 cells on a 60-mm plate. When these cells had grown up their culture fluids were used as a source of plaque-puri-fied virus.

End point dilutions. NIH/3T3 cells (105) were plated on60-mm plates and infected the next day with 10-fold dilutions of virus. When the cells were confluenttheywere trypsinizedand transferredtoa 100-mmdish. At confluence they were split 1:5 and carried until reverse transcriptase assay (8) of the culture fluids showed a clear end point titer.

RESULTS

Properties of parental N- and B-tropic

vi-ruses. (i) Plaque morphology. XC plaques can

becharacterizedas tosize,sharpnessofoutline,

turbidity, and size and number of syncytia

withinthe plaque (18). For anyparticularvirus

these properties vary to some extent,depending

uponthecell lineemployedand the number of

cells present at the time of infection. Themore

cellspresent, the smallertheplaquesize. Iftoo

few cells are present at infection, the plaques

are large and diffuse and secondary plaques

appear.

Recently, we described the isolation ofan

N-tropic MLV that makes verysmallfuzzy-edged

plaques (SP-N virus) (8). This virus was

iso-lated from a stock of LP-N virusderived from

BALB/cbyHartley et al. (6) (WN 1802N, pool

1898). Aspreviouslydescribed, the XCplaques

made by SP-N are so tiny and indistinct on

NIH/3T3that theyare virtually impossible to

count accurately (Fig. 1 and 2). Furthermore, the endpoint titerof the virus is50 to100 times

higher than the (approximate) plaque titer.

Theplaque morphology ofSP-N appearstobe

stable since it has been retained through

re-peatedserial infectionsandendpointdilutions (8; unpublished data).

Thevisibility of SP-Nplaquesis sosensitive

to plating conditions that sometimes plaques

will bevisible on one but notanother

"dupli-cate" plate. Frequently plaques are seen on

someregionsofaplatebutnot

others,

presum-ably reflecting an uneven platingofthe NIH/

3T3 cells. SP-N plaques usually lack syncytia

altogether. Whensyncytiaarepresenttheyare

small andgenerallythereis not morethanone

perplaque. As we have shown previously (8),

SP-N virus is less infectious than LP-Nvirus.

Wedonotknowifthesmallplaquemorphology

ofSP-N is aresult ofitsreduced infectivityor

whether it is due to analteration inthe viral

proteins involved in the induction of XC syncy-tia.

The LP-B virus used in these experiments was a clonal isolate made by Paul Jolicoeur from the virus stock prepared by Hartley et al.

(6) from BALB/c mice (WN 1802 B). It forms

large plaques on BALB/3T3 cells (see Fig. 1). The syncytia in these plaques are larger and more numerous than those of SP-N or even of

LP-N plaques on NIH/3T3 cells. As stated

above, comparisons of plaque morphology on different cell lines must be made with reserva-tions.

(ii) Tropism. The preferential growth of

N-and B-tropic viruses on NIH N-and BALB cells, respectively, has been described extensively (7, 14, 16). The cellular restriction toward a virus

oftheopposite tropism isdetermined by a gene,

Fv-1, that acts internally to block viral growth (13, 14, 16). The stage of infection at which growth is blocked is not known. It is not known how many genetic markers on the virus

deter-mine its tropism, nor is it known if the viral

determinants of N- and B-tropism are allelic. Variations in the extent of restriction and in the ability of a virus to overcome the tropic restriction have been noted in different

labora-tories (3, 10, 15). However, different viruses

and cells have been employed in these studies.

Inour studies, we have used only NIH/3T3 cells

derived by Jainchill et al. (9) and BALB/3T3 (clone A31) cells derived by Stuart Aaronson

andGeorge Todaro (1). When XC plaque assays

are performed asdescribed above andplaques arescoredmacroscopically, then the N-and

B-tropic viruses derived from BALB/c mice

be-have as follows. N-tropic viruses form plaques onBALB/3T3cellsthatarecomparable insize

and morphology to those formed on NIH/3T3

cells;however, the titer of the virus is approxi-mately 100-fold lower on BALB/3T3 cells than

on NIH/3T3 cells. Tenfold dilutions of the

N-tropic virus result in 10-fold reductions in the

number of plaques observed on BALB/3T3 cells. These observations apply to LP-N virus (6, 8). It

is difficult to determine accurately the ratio

(PFU on NIH/PFU on BALB) for SP-N virus

and difficult to determine ifSP-N titrates on

BALB/c cells with a one- or two-hit pattern,

sinceSP-Nplaquesare sodifficultto countand

since their visibility and appearance varies

from assaytoassay andevenfromplatetoplate

within asingle assay. However, there is a

re-ductioninthe number ofSP-Nplaques

appear-ing on BALB/3T3 cells relative toNIH/3T3 (see

alsoHopkins and Jolicoeur [8]), andin assays

in which plaque visibility is optimal, this

re-duction appears to be 50- to 100-fold. The

B-tropic virus showsamuch greaterrestriction in

VOL.18,1976

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326 HOPKINS, TRAKTMAN, AND WHALEN

on

NIH / 3T3

In

SP-N

+

us

I"'

A)

eJ

I

'I 'I B

I

sP-:B

Ad I

.SP-N

and

C-B

c.k;x

-M'"X.-f

Si

r

MALts/

I

FIG. 1. XCplaquesonNIH13T3andBALB/3T3ofprogenyofSc-i cellsfromcross1singlyorco-infected with SP-N and LP-B. SP-N and LP-B mixed is acontrol showing thatifSP-N and LP-B aremixed in amountscomparabletothat emergingfromco-infectedSc-i cells(10-1 SP-Nplus10-1LP-B,first plate; 10'2 SP-Nplus10-1LP-B, secondplate) and the mixture is titeredonNIH/3T3, onedoes notobservethelarge plaques thatone seesamongthe progenyfromSc-icellsco-infectedwith SP-N and LP-B.Mock-infectedplates received 0.2 ml of medium containing polybrene (8y/ml).Todetermine the titer(PFUlml) ofvirusthe number ofplaques on aplateshould be counted andmultiplied bythe dilutionshown.

plaque formationonNIH/3T3 cells than does

N-tropic virus on BALB cells. B-tropic virus

stocks withatiter of 107PFU/ml show less than

10PFU/ml onNIH/3T3 cells. Ifoneplates the

virusonNIH/3T3atatotal dilutionof 101, only

anoccasionalsmall plaque isseen.

Co-infection of Sc-i cells with SP-N and LP-B viruses. (i) Plan of the experiment.

Plates (60mm)wereseeded with2x 105, 105, 5 X 104, 2.5 x 104, 104,5 x 103, and2.5 x 103Sc-i

cells in triplicate for infection the following day. The maximum multiplicity of infection ob-tainable was limited by the titer of the SP-N

stockemployed, whichwas5 x 104 (>5 x 104,

<105)infectious units/mlbyendpointtitration.

The LP-B virus was diluted to 105 PFU/ml.

Both viruses werethenbroughtto 8My of

poly-brenepermlby the addition of concentrated(80

y/ml) polybrene,andaplateofSc-i cellsateach

celldensitywastheninfected with SP-Nor

LP-B virus (0.1 ml of virus plus 0.1 ml of 8 y

polybreneperml)orco-infected with SP-Nand

LP-B(0.1mlofSP-Nplus0.1mlofLP-Bvirus).

Cellsplated at2 x 105arehenceforth referred

to as cross1, 105as cross2, etc.

No attempt was made to prevent further

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MLV PLAQUE VARIANTS 327

on

NIH/3T3

on

BALB/3T3

SP-NW''

LP-B

Soj 10-2

SP-N

+LP-N

N

-1-5

IU

1 0Ad

FIG. 2. XCplaques onNIHI3T3andBALB/3T3 cellsofprogenyfromthesecondNIHI3T3infection for

cross1.

rounds ofinfection. Rather, the cells were

al-lowedtogrow for varyingperiods oftimebefore

culture fluids were collected. This was

neces-sarytoaccumulateenoughcellstoproduce

rea-sonable quantities of virus. Further, we

as-sumed that once all the cells had become

in-fected there would benofurtherchangeinthe

amountand type of virusbeing produced. This

wasconfirmed forcross1, inwhichanalysis of

culturefluids taken3daysand 24days

postin-fectionyielded similar results.

(ii) Progeny ofSc-icells. The progeny of

Sc-1 cells infected with SP-N,LP-B, or SP-N plus

LP-Bwere titered on NIH/3T3 and BALB/3T3

cells todetermine theirtiterand tropism.

Fig-ure 1 shows theplaques produced on NIH/3T3

andBALB/3T3 by the progeny of

Sc-i

cells from

cross 1. Table 1A shows the titer on NIH/3T3 and BALB/3T3 cells of the progeny of singly

andco-infected cells from all seven crosses. As

expected, inallseven crosses

Sc-i

cellsinfected

with SP-N alone produced only SP-N virus,

whereas cells infected with LP-B alone

pro-ducedonlyLP-B virus.However, the seven

co-infections all yielded SP-N, LP-B, and also

vi-rusesthat madelargeXC plaques onNIH/3T3

cells. Toanswerthe question of whether these

plaquesweremadeby viruses derivedfrom

co-infection of

Sc-i

cells by SP-N and LP-B or

whethertheyaroseas aresult of co-infection of

the NIH/3T3 cellswithSP-N and LP-B atthe

timeof XC assay, SP-NandLP-Bprogenyfrom

Sc-i cells of cross 1 were mixed in amounts

comparable to those emerging from the

co-in-fected cells, and the mixture was titered on

NIH/3T3. Nolarge XC plaqueswere observed,

suggesting thatthe large plaques seen among

the progeny of the co-infected Sc-1 cells arose

from viruses present priortotheXCassay(Fig.

1). Henceforth, virusesresponsiblefor thelarge

XCplaquesonNIH/3T3cellsarereferredto as

XLP-N virus.

Inallseven crossesthetiterofvirusproduced

bythe co-infected Sc-1 cellswaslow relativeto

the titer of parental B-tropic virus produced

(see Tables 1A and2).However, the proportion

of XLP-N virus to LP-Bemerging from the

co-infected cells increased from2x 10-5 to 3 x 10-3

with increasing multiplicity of infection of the

Sc-i cells (Table 2). This frequency is not an

accurate measure of thefrequencywithwhich

XLP-Nviruses arisesince, asstatedabove, no

attempt was madeto prevent multiple rounds

of reinfection and since we have not yet

at-temptedto determine the actual percentage of

co-infected cells in each cross. Note that

be-cause of the low titer of SP-N virus it was

necessary to alterthe multiplicity of infection

of Sc-i cells by reducing the number of cells

inoculated rather thandilutingthe virus. It is

not clear towhatextentthe effective titer ofa virusdependsonthecelldensityatthetimeof VOL.18,1976

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J. VIROL. 328 HOPKINS, TRAKTMAN, AND WHALEN

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MLV PLAQUE VARIANTS 329 TABLE 2. Frequency of XLP-N progenya

Cross Approx MOI Titer LP-N/LP-Bamong

prog-of LP-B eny of co-infectedSc-1cells

1 0.05 2 x 10-5

2 0.1 1.1x 10-4

3 0.2 2.5x 10-4

4 0.4 3 x 10-4

5 1.0 9x10-4

6 2.0 3 x 103

7 4.0 2.9x 10-3

a Frequency is expressed as the ratio (XC titer of

large plaques on NIH/XC titer of large plaques on BALB) for the progeny of Sc-1 cells co-infectedwith SP-N and LP-B. MOI, Multiplicity of infection. infection. Therefore, the calculated multiplicity

ofinfection, and thus the calculated number of

co-infected Sc-1 cells, particularly for crosses

performed at very low cell densities, may be quite unreliable.

Two critical questionsnow arose concerning

the nature of the viruses responsible for the

large XC plaques appearing on NIH/3T3 cells.

(i) Would they breed true? (ii) Whatwastheir

tropism?To answer these questionsitwas

nec-essaryto trytoisolate XLP-N viruses.

Selection of XLP-N viruses. Wewere

reluc-tanttoattempttopurify theXLP-Nvirusesby

directly selecting the large plaques on NIH

cells,sincetherewasalargeexcessof SP-N and

LP-Bviruspresent atthe time ofinfection and

since plaque purification would have required

co-cultivation withXC cells. The lowfrequency

of XLP-N viruses made selection bymicrotiter

plating prohibitive. Thus, we sought to enrich

the titer of XLP-N virus relative to SP-N and LP-B.

Aspreviously reported (8), SP-N virusisonly

moderately infectious on NIH cells relative to

the LP-N virus ofHartleyet al. (6). As stated

above, B-tropic viruses are severely restricted

in growth on NIH cells. Since XLP-N viruses

made verylarge XCplaques onNIH/3T3 cells,

it seemed possible that ifthese viruses were

genetically stable they would have a growth

advantage over both the SP-N and LP-B

par-ents inNIHcells. In this case, serial passage of

the progeny of co-infected Sc-i cells through

NIHcells should enrich the proportion of

XLP-Nvirus.

NIHcells (5 x 105)wereplatedon each of 21 100-mmplates and infectedthenextdaywith 0.5 ml (diluted by 10% with concentrated

poly-brenetomake the solution8y ofpolybreneper

ml) oftheprogeny fromsingly andco-infected

Sc-1 cellsfrom the sevencrosses. Medium was

changed every24hand collectedonday3after

infection. Theseculture fluids were then used

for asecond infection ofNIH cells, which was

performed in the same way as the first

infec-tion. On day 3 postinfection culture fluids from

all 21 infections of the second NIH selection

were then titered on NIH/3T3 and BALB/3T3

cells(Table 1C). In addition, the culture fluids

from the first NIHselection of cross 1 (2 x 105

Sc-i cells) were titered (Table 1B). The XC

assay of progenyfrom the second NIHinfection

for cross 1 is shown in Fig. 2.

In all seven crosses, XLP-N virus was

re-coveredonly fromNIH-passaged progeny of the

co-infected Sc-i cells. The titer of this virus

increased rapidly with passage in NIH cells

(Table 1Band C). For example, in cross 1 in the

first NIHselection, the cells were infected with

approximately 50 PFU of large NIH

plaque-forming virus. Within three days the titer of

this virus had increased to 1.2 x 105 PFU/ml,

whereas the titer of parental LP-B had dropped

to 2.3 x 104PFU/ml. After the second infection

ofNIH/3T3 cells, the titer of XLP-N virus had

risen to 107i, whereas the titer of LP-B had

dropped sufficiently to allow one to observe the N-tropism of the XLP-N virus.

Sc-1 cells that had been infected with SP-N

alone yielded virus that retained the small

plaque morphology through NIH passage.

In four out of seven cases, after passage

through NIH/3T3 cells the progeny from Sc-i cells infected with LP-B alone acquired the

abilitytoplaqueonNIHcellsatthesametiter

as onBALBcells (see Table 1C andFig.2). The

gradual conversion of B-tropic murine

leuke-mia virus to NB-tropism by serial passage in

NIHcells inculturehas been observed

repeat-edly by Janet Hartley (see reference 12) and

presumably accounts for the results obtained. Interestingly, the plaques of this presumed

NB-tropicvirus aresmalleronNIHthanon BALB

cells and smalleronNIHcells than theplaques

made by the XLP-N viruses (Fig. 2). The

pre-sumed NB-tropic viruseswere unable to grow

rapidly on NIH cells, and after three serial

passages on NIH/3T3 never achieved titers

higher than5 x 103 (seeTable 1C).

Properties ofXLP-Nviruses. (i)Stability of

plaque morphology and tropism. To further

test the stability of the large plaque

morphol-ogyandN-tropism of the XLP-N viruses, virus

obtained from the secondNIHselection ofcross

1 (2 x 105 Sc-i cells) was passed once more

through NIH/3T3 cells andalsothrough BALB/

3T3 cells by the procedure described above.

Bothplaque morphology and tropismwere

re-tainedby passageineither hostcell(Table iD).

The culture fluids from thesecondNIH

selec-tionof the co-infected cellsfrom thiscrosswere

then titered by end point dilution. The end

pointtiter was 107(>107, <108) infectiousunits/

VOL.18,1976

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330 HOPKINS, TRAKTMAN, AND WHALEN

ml. Virus from this end point dilution (10-i)

remained large XC plaque forming and

N-tropic. This virus was plaque purified. The

plaque-purified virus formed large XC plaques andwas N-tropic. To furthertest thestability

of thesemarkers, theplaque-purifiedviruswas

passedthrough BALB/3T3 cells. A virus stock

withatiterof2x 107PFU/mlwasusedtoinfect

BALB/3T3 cellsat10-folddilutions from10-lto

10-4. Virus emerging from these infections

re-mained N-tropic and produced large XC

plaques.Figure3showsaschematicdiagramof the entire experiment from co-infection of Sc-1

cells through plaque purification ofa XLP-N

virus.

(ii) Plaquemorphology and titer. The

XLP-Nviruses obtainedfromthesestudiesgrow to

unusually high titers, at least 5- to 10-fold

higher than thoseobtainedwiththe LP-Nvirus

ofHartleyetal. (6).LP-Balsogrows tohigher

titersthan the LP-N of Hartleyetal. (6),andit

ispossible thatthis property was acquired by

the XLP-N viruses.

The plaquesmade by the XLP-Nviruses are

unusually large, and the syncytia within the

plaques are more numerous and considerably

larger than those observed for other N-tropic

variantderived from BALB/c mice.

DISCUSSION

Co-infection ofSc-icells withSP-Nand LP-B

viruses yields progeny XLP-N virus with the phenotype large XC plaque morphology and

N-tropism.Infection ofSc-icells withSP-Nor

LP-B alone does not yield XLP-N viruses. The

phenotype of XLP-N is stable through many

cycles ofgrowthon bothNIH/3T3 and BALB/

3T3 cells. XLP-Nviruses make largerplaques

andgrow tohighertitersthananyof the LP-N

STEP 1 STEP2

virusesderivedfrom BALB/c micethatwehave

studied previously(8).

A number of explanations for the origin of

XLP-Nviruses canbeconsidered. Probably the

simplest explanation for the results

summa-rized above is genetic recombination between

plaquemorphologyand tropism markers of

SP-Nand LP-Bviruses. Other explanationsmight

bephenotypic mixingorheterozygosis between

SP-NandLP-B. Wedonotknow whether these

mechanisms could generate viruses with the

XLP-N phenotype. Although they are not

ex-cluded as explanations, they seem somewhat

unlikely because ofthe stabilityof the XLP-N

phenotype andfailure tosegregateLP-B virus

aftergrowthof XLP-NonBALB/3T3 cells.

It isalsoconceivable that duringco-infection

LP-Bcausedageneticalterationinthegrowth

and plaque-forming properties of SP-N by a

mechanismotherthan recombinationor

heter-ozygosis.Hopkins and Jolicoeur (8) and Rappet

al. (17) have described the "conversion" of

weakly infectious XC N-tropic virus to LP-N

virus. Although infection ofSc-i or NIH cells

with SP-N under a variety of conditions has

never yielded either LP-N or XLP-N viruses,

andeventhoughnoneofourotherstudies with

N-tropic plaque variants derived from BALB/c

mice (8) or induced fromBALB/c cells in

cul-ture(unpublished data) hasyielded XLP-N

vi-ruses, we can not exclude the possibility that

co-infection with SP-N and LP-B facilitated

conversion of SP-N to this novel N-tropic

var-iant.

Nor we can exclude the possibility that the

XLP-N viruses we obtained resulted from

ge-neticexchange betweentheexogenously

infect-ing viruses and an endogenous virus of

Sc-i

cells. However,we cansay that if such an event

occurred, itapparently requires co-infection of

STEP3

Select XLP-N viruses from co-infected Purification of a XLP-N virus from cross 1 Sc-icellsby serialpassageof (A) prog- by end point dilution and plaque selection enythrough NIH cells. Singly infected onNIH/3T3.

Infect Sc-1 cells with: Sc-icell progeny serves as control.

SP-N -* Sc-i A. NIH B1\NIH C,

LP-B

SP-N + LP-B --.ii6 \ A 2\ .

.-..IH;

-+ Titer and select a

(at end point plaque on NIH/ dilution) 3T3

FIG. 3. Schematic diagram of the experiment from infection ofSc-i cells toplaque purification ofaXLP-N virus. XC assays of (A) progeny are shown inTable 1A and Fig. 1; (B) progeny are shown in Table 1B; (C) progeny are shown inTable 1C and Fig. 2.

----4 A B

C

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[image:7.509.63.452.490.628.2]

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VOL. 18, 1976

theSc-1 cells with both N- and B-tropic viruses

toproduce XLP-N.

Hopefully, further analysis of the XLP-N

vi-ruses will aid in determining the genotypic

basis for theirphenotype. Recently Buchhagen

et al. (2) succeeded in distinguishing the

pro-teins of N- and B-tropic viruses derived from

BALB/c mice. P. O'Donnell and E. Fleissner

(personalcommunication) have observed a

dif-ference intheability of these viruses to induce

G1X antigen upon infection.

(Gx

is anantigenic

determinant found on the envelope

glycopro-tein gp 69/71of some murine leukemia viruses.)

Analysis of the proteins and of the ability to

induce

Gx

of the parental and phenotypically

recombinant viruses from our studies may

provehelpful indetermining whether these

vi-ruses are geneticrecombinantsbetween N- and

B-tropic viruses. Acollection of such

recombi-nants might prove useful in defining the

pro-teinsand regions of the genomeinvolved in XC

plaque morphology, induction of

Gx

antigen,

and N-and B-tropism.

ACKNOWLEDGMENTS

Wethank Terry Grodzicker and Rex Risser for interest-ingdiscussions and David Botstein for acriticalreading of the manuscript.

Thiswork wassupportedby grantnumber 76-03 from the Health SciencesFundandPublic Health Service grant CA 14051from the National Cancer Institute.

LITERATURE CITED

1. Aaronson,S.A.,andG. J. Todaro. 1968.Development of3T3-like lines from BALB/c mouse embryo cul-tures:transformationsusceptibilitytoSV40. J.Cell Physiol. 72:141-148.

2. Buchhagen,D.L.,0. Stutman,and E. Fleissner. 1975. Chromatographicseparation andantigenicanalysis ofproteins of the oncornaviruses. IV. Biochemical typing ofmurine viral proteins. J. Virol. 15:1148-1157.

3. Declsve,A.,0.Niwa, E.Gelmann, andH.S.Kaplan. 1975.Replication kineticsof N-and B-tropicmurine leukemia viruses on permissive and non-permissive cellsinvitro.Virology 65:320-332.

4. Hanafusa, H., and T. Hanafusa. 1966. Determining factorinthecapacity of Roussarcomavirustoinduce tumorsin mammals. Proc. Natl. Acad. Sci. U.S.A. 55:532-538.

5. Hartley,J.W.,and W. P. Rowe.1975.Clonal cell lines from a fetal mouse embryo which lackhost-range restrictionsfor murine leukemia viruses. Virology 65:128-134.

6. Hartley, J. W.,W. P. Rowe, W. I.Capps, and R. J. Huebner. 1969. Isolation ofnaturally occurring

vi-rusesof the murine leukemia virusgroupintissue culture. J. Virol. 3:126-132.

7. Hartley,J.W.,W. P.Rowe,and R. J.Huebner.1970.

Host-range restrictions ofmurine leukemiaviruses inmouseembryocell cultures. J. Virol. 5:221-225.

8. Hopkins, N., and P. Jolicoeur. 1975. Variants of N-tropic leukemia virusderived fromBALB/cmice. J.

MLV PLAQUE VARIANTS 331 Virol. 16:991-999.

9. Jainchill,J. L., S. A. Aaronson, and G. J. Todaro. 1969. Murinesarcomaand leukemia viruses: assay using clonal lines ofcontact-inhibited mouse cells. J. Virol. 4:549-553.

10. Jolicoeur, P., and D. Baltimore, 1975. Effect of the Fv-1 locus on the titration of murine leukemia viruses. J. Virol. 16:1593-1598.

11. Kawai, S., and H. Hanafusa. 1971. Genetic recombina-tionwith avian tumor viruses. Virology 49-.37-44. 12. Klement, V., W. P. Rowe, J. W. Hartley, and W. E.

Pugh. 1969. Mixed culture cytopathogenicity: a new testfor growth ofmurine lukemia viruses in tissue culture. Proc. Natl. Acad. Sci. U.S.A. 63:753-758. 13. Lilly, F., and T. Pincus. 1973. Genetic control of

mu-rineviral leukemogenesis. Adv. Cancer Res. 17:231-277.

14. Pincus, T., J. W. Hartley, and W. P. Rowe. 1971. A major geneticlocus affecting resistance to infection with murine leukemia viruses. I. Tissue culture stud-ies of naturally occurring viruses. J. Exp. Med. 133:1219-1233.

15. Pincus, T., J. W. Hartley, and W. P. Rowe. 1975. A major geneticlocus affectingresistance toinfection withmurineleukemia viruses. IV. Dose-response re-lationships in Fv-1 sensitive and resistant cell cul-tures.Virology65:333-342.

16. Pincus, T., W. P. Rowe, and F. Lilly. 1971. A major geneticlocus affecting resistance to infection with murine leukemia viruses. II. Apparentidentityto a major locusdescribed for resistance toFriend murine leukemia virus. J. Exp. Med. 133:1234-1241. 17. Rapp, U. R., R. C.Nowinski, C. A. Reznikoff, and C.

Heidelberger. 1975. Endogenous oncornaviruses in chemically induced transformation. I. Transforma-tion independent of virus production. Virology 65:392-409.

18. Rowe, W. P., W. E. Pugh, and J. W. Hartley. 1970. Plaque assay techniques for murine leukemia vi-ruses.Virology42:1136-1139.

19. Stephenson, J. R., and S. A. Aaronson. 1972. Genetic factors influencing C-typeRNA virus induction. J. Exp.Med. 136:175-184.

20. Stephenson, J. R.,G.R.Anderson,S.R.Tronick,and S. A. Aaronson. 1974. Evidence for genetic recombi-nation between endogenous and exogenous mouse RNAtype-Cviruses.Cell 2:87-94.

21. Stephenson, J. R., S. R. Tronick, and S. A. Aaronson. 1974. Temperature-sensitivemutantsof murine leu-kemia virus. IV. Further physiological characteriza-tionand evidence for genetic recombination. J. Virol. 14:918-923.

22. Svoboda, J. 1960. Presence of chicken tumor virus in the

sarcomaof theadultratinnoculated afterbirth with Roussarcomavirus.Nature(London)186:980-981. 23. Vogt, P. K. 1967. Phenotypic mixing in the avian tumor

virusgroup. Virology32:708-717.

24. Vogt, P. K. 1971. Genetically stablereassortmentof markers during mixed infection with avian tumor

viruses.Virology46:947.

25. Weiss, R. A., W. S. Mason, and P. K. Vogt. 1973.

Genetic recombination betweenendogenous and

ex-ogenous avianRNAtumorviruses.Virology

52:525-535.

26. Wong, P. K. Y., and J. A. McCarter. 1973. Genetic studies oftemperature-sensitivemutantsof Moloney-murineleukemiavirus.Virology53:319-326.

27. Wyke, J. A., J. G. Bell, and J. A. Beamand. 1974.

Genetic recombination amongtemperature-sensitive mutantsof Rous sarcoma virus. ColdSpringHarbor Symp. Quant.Biol.39:897-905.

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