Comparative analysis of the genomes of feline leukemia viruses.

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NOTES

Comparative Analysis of the Genomes of Feline Leukemia

Viruses

ZEDAF. ROSENBERG,"2FINN S. PEDERSEN,2" AND WILLIAM A. HASELTINE'2*

Departmentof Microbiology,HarvardSchoolof PublicHealth,'andSidney Farber CancerInstitute,

CharlesA.DanaCancerCenter,2Boston, Massachusetts, 02115, and Department of MolecularBiology,

University of Aarhus,Aarhus,Denmark3

The genomesofseveralstrains of felineleukemiavirus (FeLV) werecompared

by two-dimensional polyacrylamide gel electrophoresis of the large RNase

T,-resistant oligonucleotides ofthe 70S RNA. Differences between each strain of

FeLV tested were detected by this method. We estimate that the degree of

sequenceidentitybetween the viruses is: FeLV A(Glasgow-1)toFeLV B

(Snyder-Theilen),52%;FeLV A (Glasgow-1) toFeLV C (Sarma), 66%; FeLV B

(Snyder-Theilen) toFeLV C (Sarma),37%. The fingerprints of two independent isolates

ofFeLV strainsofsubgroupA(Glasgow-1andRickard) weredetectablydifferent.

We conclude that the RNase

T,

oligonucleotide fingerprint pattern provides a usefultool for identification of FeLVstrains.

Feline leukemiaviruses (FeLV's) are a hori-zontally transmittedgroupof type Cretroviruses

that causelymphoma,

lymphoblastic leukemia,

fibrosarcoma, and

aplastic

anemia incats

(1, 2).

This class of viruses ishorizontally transmitted,

as demonstrated by epidemiological studies of multiple-cathouseholds(3)andbyexperimental

infections under laboratory conditions (4, 5).

TheFeLV's have been classified into three

dis-tinctsubgroups,A (FeLV A), B (FeLV B), and

C (FeLV C), on the basis of

type-specific

neu-tralizationandinterferencetests (14, 15).Tests ofhomology by nucleic acid

hybridization

show

that these viruses are closely related. At least

85%of the

complementary

DNApreparedfrom

anyone of thesubgroups willanneal toany of

theothersubgroups. Moreover,themelting

tem-perature of the hybrids formed between the

complementaryDNA ofonesubgroup and the

RNA of another is within 1°C of that of the

homologoushybrids (10).

Although the FeLV's appeartobe veryclosely

relatedbyhybridization techniques, they differ

markedlyin their abilitytoinfectheterologous

celllines.SubgroupAvirusesreplicateprimarily

in feline cells, whereas viruses ofsubgroups B

andCreplicate additionallyin humanand mink cells (8, 16). These viruses also differ in their

naturalpatternsofoccurrence. All field isolates

of FeLV contain FeLV A either alone or in a

mixture ofsubgroups (8, 15). FeLV C is rarely

isolated innature and hasneverbeendetected

in the absence ofother FeLV subgroups(7). In diseased cats under natural conditions, no ob-served correlation exists between the FeLV

sub-group isolated and the type of disease that is

produced(7).

The differences in the biology of the FeLV's

motivatedus toinvestigatethe structure ofthe

viral genomesusing a method capable of

detect-ing differences between closely related viruses.

For thisstudy, we used a high-resolution two-dimensional RNA fingerprinting technique

which permits direct comparison of

approxi-mately 15% of the viral genome (11, 12).

Ge-nomic 70S RNAwasprepared fromvirus strains

propagated in fibroblasts. The fingerprints of

three prototype strains, FeLV A (Glasgow-1),

FeLV B(Snyder-Theilen),andFeLVC (Sarma),

areshowninFig. 1.

About60largeRNase

T1-resistant

oligonucle-otideswereidentified in thefingerprintof each

virus.Theuniqueoligonucleotides areidentified

asthosespecies that migrate slowly in the

sec-ond dimension and that appear to be present in

unimolar amounts asjudged by the amount of

radioactivity in the gel corresponding to the

individualoligonucleotides.Ourprevious studies

indicate that the labeling methods used here

resultinuniformlabelingof theoligonucleotides

(11, 12). Thecomplexity of thefingerprint

pat-ternsfor these threevirusesis that anticipated

ifeach isolatewere apuresinglespecies andnot

amixtureoftwo or morevirusstrains.

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NOTES 543

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-4

:w vl

*M

*o*O

A

ow I

B

C

FIG. 1. Autoradiograms of the two-dimensional

RNA fingerprints ofFeLVsubgroups. Viruses and

cellswereprovided by0.Jarrett. FeLVA

(Glasgow-1)wasisolatedfromacatwith spontaneous

alimen-tary lymphosarcoma (9). FeLV B (Snyder-Theilen)

waspurified from amixture of subgroupsAand B

presentasthe helperviruses in the Snyder-Theilen

strain ofFeSV (14). FeLV C(Sarma) waspurified

fromamixtureof subgroupsA and C isolatedfrom

FL-237 cells (14). Viruswas harvestedfrom

super-natant culturefluidscollectedfrom confluentroller bottlesonce ortwicedailyand concentratedby

ultra-centrifugation.Viruspelletswerethensuspendedand

pelleted through30%sucrosein TNE (50mMTris,

pH 7.5, 100 mMNaCl, 1 mM EDTA), followed by

Inspection ofFig. 1 revealssome similarities

and somedifferencesinthe fingerprint patterns ofeachstrain. To obtainqualitative estimates of thesimilarityinthefingerprints, equalamounts of3P-labeled oligonucleotidesderivedfrom each

virus were mixedinpairs and then analyzed by

two-dimensional gel electrophoresis. To

deter-mine thenumber ofunique oligonucleotides that

hadthesame electrophoretic mobility, the fin-gerprintpattern ofthe mixture was then com-pared withindependent

fingerprints

of each vi-rus.

Aschematicrepresentation ofonesuch

exper-imentis picturedinFig. 2Afor FeLV A

(Glas-gow-1) and FeLV B (Snyder-Theilen). The S unique

oligonucleotides

that have thesame mo-* bility in the two virus strains are indicated as solid circles, those that areunique to FeLV A (Glasgow-1) are indicated by open circles, and * those thatare

unique

toFeLVB

(Snyder-Thei-len) are indicated

by

shaded circles. Of the 61 unique

oligonucleotides

of FeLVA

(Glasgow-i),

32(52%) comigrate with those ofFeLV B

(Sny-der-Theilen),

and32of the53

unique

oligonucle-otides of FeLV B

(Snyder-Theilen) comigrate

withthoseof FeLVA

(Glasgow-i).

The extent of comigration permits a rough

estimation of the sequence

identity

of the two viral strains (Table 1). The estimate assumes that the sequence

identity

of the

oligonucleo-tides is

representative

ofthe sequence

identity

of the entiregenome.Calculationsbased on

co-migrationmayslightly overestimate the degree

of sequence identity, since

oligonucleotides

whichcomigratemayhave thesamebase

com-digestionwithproteinase K (E.M. Biochemicals). 70S

viral RNA-containing fractions,pooledfromalinear

15 to30%sucrosegradientin TNEplus 0.2% sodium

dodecyl sulfate,werepassedover an

oligodeoxythy-midylic acid-cellulose column (Collaborative

Re-search, T-3). Thepolyadenylic acid-containing 70S

RNAwaseluted with10mMTris-hydrochloride(pH

7.5),precipitated twice with ethanol, and suspended

in 20 mMTris (pH 8.0)-2 mM EDTA. In vitro

32p

labelingofviral RNA wasperformed according to

themethodof Pedersen and Haseltine (11, 12). A

0.2-to 0.4-pg sample ofviral RNA was digested with

RNaseT, (Calbiochem)and bacterialalkaline

phos-phatase(RNasefree; WorthingtonBiochemicalCo.).

The digested RNA was subsequently labeled with

[y-32P]ATP

and polynucleotide kinase (P.L.

Bio-chemicals). They-32P-labeled oligonucleotides were

electrophoresedin a first-dimension 10%

polyacryl-amidegelcontaining6M ureaand25mM citric acid

(pH3.5), followed by electrophoresis in a

second-dimension 22.5%polyacrylamide gel containing 50

mM Tris,50mM boricacid, and1 mM EDTA (pH

8.3). Thegelwasthenautoradiographed.(A) FeLVA

(Glasgow-i);(B)FeLVB(Snyder-Theilen); (C)FeLV

C(Sarma).

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544 NOTES

0

00

2nd

1iSt

0 0

3S

0 0

0 2nd

A

0

ist

B

,.:,*

,*S

0

o00

o

e

o

0

.

o

0 lb@0

0 0

*

0

c0

0

cP@

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,

0

S 0

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ceD

FIG. 2. Comparison ofthegenomes ofFeLV.Equalamountsoflabeled RNaseT,-resistant oligonucleotides

fromtwodifferentstrainsweremixedtogetherimmediately before electrophoresisin thefirstdimension.

Two-dimensionalgelelectrophoresiswasperformedaspreviouslydescribed. (A)Schematicrepresentationofthe

mixtureofFeLV A (Glasgow-i)and FeLV B (Snyder-Theilen). RNaseT1-resistantoligonucleotides: (open

circles) found onlyinFeLVA; (shadedcircles), found onlyinFeLVB; (black circles),commonto both FeLV

A and FeLVB. (B)Schematicrepresentation ofthe mixtureofFeLV A (Glasgow-i)andFeLV C(Sarma).

RNase T1-resistant oligonucleotides: (open circles) found onlyin FeLVC; (shadedcircles), found only in

FeLVA;(black circles),commontoboth FeLVA and FeLVC.(C)Schematicrepresentationofthemixtureof

FeLV B (Snyder-Theilen) and FeLV C(Sarma). RNase T1-resistantoligonucleotides: (open circles) found onlyin FeL VC; (shaded circles)found onlyinFeLVB; (blackcircles)commontobothFeLV B and FeLV C.

2nd

L1st

c

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TABLE 1. Calculatedsequence identities of the FeLV's

No. (%) showingsequence identity with viral genome"

Viralgenome FeLV A

(Glasgow-i) FeLV B FeLV C FeLV A (Rickard)

FeLVA(Glasgow-1) 59/59(100) 32/61 (52) 37/58(64) 46/60(76)

FeLV B(Snyder-Theilen) 32/53(60) 55/55 (100) 21/56(37) ND

FeLVC(Sarma) 37/56(66) 21/56 (37) 56/56(100) ND

FeLV A(Rickard) 46/59(78) ND ND 60/60(100)

aNumberof RNase T,-resistant oligonucleotides found in common between the two viral genomes/total

number ofuniqueoligonucleotides.The number of unique oligonucleotides scored for a particular viral strain

variedslightlyfromexperiment toexperimentasaresult of the degree of resolution of each fingerprint. The

numbers inparenthesesarethe percentages of sequenceidentity. ND, Not done.

* 0

P.:s

oll

41

0 C* 0 .

0

0- Wc1

S-0.1

position butnotnecessarily thesamesequence. On the other hand, this calculation maybe an underestimate, since the mobility of an oligo-nucleotidecanbealteredbyachange inasingle nucleotide.

Similar analysiswasdone onmixtures of 32P-labeledoligonucleotides ofFeLV A

(Glasgow-i)

and FeLV C

(Sarma)

(Fig. 2B) andofFeLV B (Snyder-Theilen) and FeLV C (Sarma) (Fig. 2C). The number of comigrating oligonucleo-tideswasdeterminedasdescribed above and is listed in Table 1 for eachexperiment. We

esti-matethat64 to66%of thesequencesofFeLV A

(Glasgow-i)

and FeLV C

(Sarma)

areidentical.

ThegenomesofFeLV B (Snyder-Theilen) and

FeLV C (Sarma) are more distantly related. Only about one-third (37%) of the unique oligo-nucleotides have the same electrophoretic

mo-bility.

To determine whether the fingerprint of an FeLV strainwasaffected by thespecies of fibro-blastsonwhich the viruswasgrown,the finger-print of FeLV B

(Snyder-Theilen)

grown on feline fibroblastswascompared with that of the samevirusgrown oncanine fibroblasts. No dif-ferences in the

fingerprints

of the two prepara-tions were detected in this experiment (not shown).

Thepreceding experiments demonstrate that viruses of thedifferent

subgroups

canbe

distin-guished

from one another by

analysis

of the unique large RNase

T1-resistant

oligonucleo-tides. Todetermine whether viruses of thesame subgroup could also be

distinguished

from each otherby thismethod,the

fingerprint

of the viral RNA ofFeLV A(Glasgow-1)was

compared

with thatofanothersubgroupA

virus,

FeLVA

(Rick-ard) (Fig. 3). We estimate that the degree of

sequence identity between thesetwo strains is

0

C

FIG. 3. Comparison ofthe two subgroupA virus

genomes.FeLVA (Rickard)wasisolatedfromF422 cells, a spontaneous lymphoidcelllineprovided by M. Essex(13). (A)FeLVA (Glasgow-1); (B)FeLVA (Rickard); (C)Schematicdiagram. RNase

T,-resist-ant oligonucleotides; (open circles) found only in FeLV A (Rickard); (shaded circles) found only in FeLVA(Glasgow-1);(black circles)commontoboth. B

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546 NOTES

76 to 78% (Table 1). It is possible that the

background spots on theautoradiogram of the

fingerprint of FeLV A (Rickard) represent

se-quencesfromasecondviral orsubviral

compo-nent.

The extent of sequence identity detected

among the FeLV strains was anticipated from

previousstudiesofnucleicacidhomology. These

studiesindicated an overallhomology ofabout

85% among all FeLVstrainstested. This

homol-ogy is consistent with the degree of sequence

identityrepeatedhere, with thepossible

excep-tion of therelatively lowdegreeofidentity,37%,

found between FeLV B (Snyder-Theilen) and

FeLVC (Sarma).

Theresults presented here demonstrate that

afingerprint of the 70SgenomicRNAprovides

auseful method foridentifyinganFeLV strain.

No two FeLV strains tested had the same

fin-gerprint,includingtwoindependentsubgroup A isolates.Thefingerprints presentedhereshould provide a standard for future FeLV

identifica-tion. The subgroup type of each of the isolates

used here wasconfirmed in blindtests of

sub-groupspecificity by OswaldJarrett.

Asanexampleof theutilityof this method for virological studies of FeLV, we have used the RNAfingerprinting techniquetocorrectly

iden-tifythe FeLVhelpercomponentof a mixture of

FeLV and defective feline sarcoma virus

ge-nomesasbelonging to subgroup B. The helper

virus had been previouslythought to belong to

subgroup C. We have alsofingerprinted avirus describedassubgroup B and detectedasecond subgroup A viral component in the analysis.

Thisviruswassubsequentlytyped as two viruses

with both subgroup A and subgroup B

specific-ities.(Apreliminaryreportof thisworkhasbeen

published[6]).

We thankMyronEssex,Oswald Jarrett,and William Hardy for helpful discussions and Robert Crowther for technical assistance.

W.H. isthe recipientof an American Cancer Society Fac-ulty Research Award. F.S.P. is a Fellow of the Leukemia Society of America, Inc. and the Danish Natural Science ResearchCouncil. Z.F.R. issupportedby Public Health Ser-vicegrant AID 0014 from theNational InstitutesofHealth.

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2.Essex,M. 1975. Horizontallyandvertically transmitted oncornaviruses ofcats.Adv. Cancer Res.21:175-248. 3. Essex,M.,S. M.Cotter,A. H. Sliski,W. D.Hardy,

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9. Jarrett,O., andP. H.Russell.1978.Differentialgrowth and transmissionin catsof felineleukemia viruses of subgroupsAandB. Int.J.Cancer21:466-472. 10. Levin, R., S.K. Ruscetti, W. P. Parks, and E. M.

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