Structural and antigenic analysis of the nucleic acid-binding proteins of bovine and feline leukemia viruses.

Vol. 46, No. 1 JOURNALOFVIROLOGY,Apr. 1983,p.177-186

0022-538X/83/040177-10$02.00/0

CopyrightC1983, American SocietyforMicrobiology

Structural and Antigenic Analysis of the Nucleic Acid-Binding

Proteins of Bovine and

Feline Leukemia Viruses

M.ALICEMORGAN, TERRY D. COPELAND,ANDS.OROSZLAN*

Biological Carcinogenesis Program, National Cancer Institute-Frederick CancerResearchFacility,

Frederick,

Received 9 September 1982/Accepted6January 1983

The nucleicacid-binding proteins of bovine leukemia virus (BLV) and feline leukemia virus (FeLV) were isolated in ahigh state of purity with chloroform-methanol extractionfollowed by reversed-phase liquid chromatography. Selective solubilization and purity of BLV p12 and FeLV plOwas confirmed by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The compositions and molec-ular weightsweredetermined by amino acid analysis. An abundance of lysine and arginine residues along with their size identifies both BLV p12 and FeLV plOas small basic proteins similar to well-defined type C viral nucleoproteins. NH2-terminal degradation by the semiautomated Edman method provided the se-quence of the first 40 amino acids for both proteins. The putative nucleic acid binding site found in severaltypeC viralnucleoproteinswascontained within this

sequence, withthe most homology centered aroundan eight-amino acid region involving sevenidentical residues andonesubstitution. Antiseraweredeveloped in rabbits, and specificity and titers were determined by electroblotting and immunoautoradiography. By this technique,animmunological cross-reactionwas foundbetween BLV p12 and FeLVplO. The sharedantigenic determinantmost

likely exists in the highly conserved eight-amino acid region. Although this sequenceis alsohighly conserved in the nucleic acid-binding proteins of murine leukemiaviruses, the shared antigenic determinant isnotfound in these orany other type C viruses tested. It is suggested that substitution of arginine (BLV pl2/FeLV p10)tolysine (murine leukemia virus p10) is sufficienttoelicitachange inantibody specificity.

Bovine leukemia virus (BLV) and feline

leu-kemia virus (FeLV) are non-genetically

trans-mittedexogenousretroviruses showntobe

asso-ciated withdiseasein theirrespective hosts (4,

5).

BLV is regarded as the causative agent of

enzootic bovine leukosis, a lymphosarcoma,

found in domesticated cattlewidespreadin both

North America and Europe.Itinfects20%of all

dairy cattle and isfound in 60%o of all herds in

theUnited States.It canbetransmitted

horizon-tally through cell-to-cell contact or insect

vec-tors and vertically by congenital means (6).

Recently infectious BLV was demonstrated in

themilkofdairycows(7).Itisalso infectiousin

otherspecies includingsheep, goat,pig (18), and

chimpanzee(32).The gag geneproductsarethe

internal structuralproteins;p24,themajor

struc-tural protein (8); p15, probably a major

phos-phorylatedcomponent(31);p12,thenucleic

acid-binding protein (NBP) (17); and p1O, which is

less well characterized. The precursor to gag

geneproductsisa-65,000-daltonprotein(Pr65)

whichwasshownto containp24, p15, p12,and

plOby trypticpeptide mapping (9).BLV has not

been readily classified into one of the

well-defined groups of retroviruses. For example,

antiserum to BLV p24 failed to detect

cross-reactivity with the internal protein of FeLV,

Rauscher murine leukemia virus (R-MuLV),

foamy-likebovine syncytiavirus, Mason-Pfizer

monkey virus (8, 20), oravian oncornaviruses

(19).

FeLV is alsoanaturally occurring infectious

retrovirus which causes leukemia and

lympho-sarcomaincats and isresponsible for one-third ofallcancerdeaths. Theprimarymodeof virus

transmission is horizontalthrough saliva,

infect-ing most free-roaming pet cats. Pathogenic

forms of leukemiacanbeinducedby inoculation

ofcats with FeLV (5). FeLV canalso grow in human embryonic lungcells, dog kidney cells,

and pig embryo cells (15) and can produce

lymphosarcoma indogs (28). FeLV strainscan

bedivided intosubgroups A, B,andCbased on

theirpatterns of attachment toreceptorsatthe

hostcell membrane. The gaggene-encoded

in-ternal proteins are p15, p12, p27, andplO(15),

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178 MORGAN, COPELAND, AND OROSZLAN

whicharesimilarin propertiestotheproteins of

MuLV. Based on protein sequence homology

and immunological relatedness with MuLV,

FeLVis classifiedas typeC, subgroup I (23).

The only BLV protein partially sequenced is

p24,which is foundtocontainsequence

homolo-gy with type C virus p30 structural proteins,

especially with FeLVp27, suggesting thatBLV

p24 and FeLV p27 are evolutionarily related

proteins (22). BLV p24 has recently been found

(25)tohavestructural homology with the

analo-gous24,000-molecular-weight proteinfroma

vi-rus associated with human adult T-cell

leuke-mia-lymphoma and designated HTLV (26).

Several linesof evidence indicate that the NBPs

of the virionmaybeamongthemostconserved

gaggeneproducts ofretroviruses. Asthe

RNA-associated internal core proteins of the virus,

theymayhaveanimportant functionin

replica-tion and assembly and theymaybeunder high

evolutionary constraint. As a continuation of

ourstudies onthe BLV-FeLVrelationship, we

sequenced and immunologically characterized

the NBPs of these viruses. Theprimary

struc-ture data of BLV p12 and FeLVplO show the

presence ofahighly conserved segment in the

putative nucleic acid binding site region. This

andthe results of immunologicalanalysiswhich

indicate cross-reaction permit the delineation of

the shared antigenic determinant.

MATERIALS AND METHODS

Virus. BLVwas grownin fetal lamb kidney cells (33),and theRickard strain(A, B)of FeLVwasgrown in feline lymphoblasts (27). Viruses were purified by sucrose density gradient centrifugation according to standardproceduresandobtainedfrom theViral Re-sources Laboratoryof theNational Cancer Institute-Frederick Cancer ResearchFacility, Frederick,Md.

Chloroform-methanol extraction. NBPs of BLV and FeLVwerepartiallypurified bychloroform-methanol

extraction (21). Chloroform-methanol (2:1, vol/vol) wasaddedtothevirussuspensioninthepresenceof low-ionic-strength buffer, 0.01 MTris-hydrochloride, pH 7.4)-0.001 MEDTA-0.05 M NaCl. Aftervigorous agitation for 2 min, the phases were separated by

centrifugation at 2,000 rpm for 20 min at 4°C. The phosphoproteinand RNApartitionedtotheaqueous

phase,thevirallipidspartitionedtotheorganic phase, and theremainingviralproteinswerecontained in the interphase.To solubilize BLVp12and FeLVplO,the interphase was resuspended in high-ionic-strength buffer, 0.01 M Tris-hydrochloride (pH 7.4)-0.001 M EDTA-1.0 MNaCl,and theextractionwasrepeated

with the NBP contained in the high-ionic-strength

aqueousphase.

RPLC. Further purification was performed by re-versed-phase liquid chromatography(RPLC)on a Wa-ters> BondapakC18 column(13).The NBPcontained in theaqueousphasefromhigh-ionic-strength chloro-form-methanol extraction was lyophilized and then resuspended in 6 Mguanidine-HClandadjustedtoa pH of2.0withl1otrifluoroacetic acid. A 0to 30%o

gradientof acetonitrile in 0.05% trifluoroacetic acidat pH 2.0 wasusedtoelute the NBP from the columnby reduction of the hydrophobic interactions. Proteins wererecovered bylyophilization.

Reductionandcarboxanidomethylation. Proteinwas reduced in the presence of6Mguanidine-HCI-0.1 M NaHCO3 (pH 8.5) buffer with 0.17 M dithiothreitol.

Sampleswereflushed with N2 and incubatedatroom temperature for 3 h. Reducedproteinswere

carbox-amidomethylated with 0.4 M iodoacetamide (room temperature;13to14h).

SDS-PAGE.Sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), according to the formulation of Laemmli(17), wasusedtodetermine

purity of the NBPs and approximate molecular weights.

Amino acid analysis.Thecompositionand molecular weightof the NBPs weredetermined by amino acid

analysis on a DurrumD500 amino acid analyzeras

previouslydescribed (11). Therawdatawereanalyzed byaprogramrunontheNational Institutes of Health DEC 10 computer which selectsamolecularweightfor the protein based in suchaway as to minimize the departure fromintegralvaluesforallresidues (3).

NH2-terminal sequence analysis. NH2-terminal se-quence analysis by semiautomated Edman degrada-tionwasperformedwith thespinning-cupliquid phase system (24) on a Beckman 890C sequencer in the presenceof Polybrene (30). RPLC on a Waters

phenyl-alkylcolumnwas usedtoidentify andquantitate the PTH derivativesofaminoacids(10).

Antisera.AntiseratothepurifiedNBPs were devel-oped in rabbits. The initialinjectionwasmade

intra-dermallyin thebackfootpadsand subcutaneously in four sitesonthehindof the rabbit with 150 ,ug of NBP in phosphate-buffered saline mixed with an equal volume of Freund complete adjuvant. The rabbits wereboostedsubcutaneouslyapproximatelyevery10

days with 50 Fg of purified protein each time in

phosphate-bufferedsalinemixed withanequalvolume

of Freund incomplete adjuvant. Ten days after the secondboost,thefirsttestbleed of 10 ml from theear veinwasobtained. The rabbitswererepeatedly boost-ed and then blboost-edaccordingtothis schedule. Animals immunized with BLVp12andFeLV plOwerebledout

byheart punctureafter 130 and 182days, respectively.

Anti-BLV p24guinea pig serum andanti-FeLV p27 rabbitserum wereavailable frompreviousstudies(8). Electroblotting and lmmunoautoradlography. The

specificities of the antiseraweredeterminedby elec-troblotting and immunoautoradiography (29). BLV and FeLV wereelectrophoresed on7.5to20%o

SDS-polyacrylamideslabgels.Afterelectrophoresisthegel

was equilibrated in transfer buffer (0.04 M sodium phosphate, pH 6.5). Aminobenzyloxymethyl paper was converted to the active diazobenzyloxymethyl form(DBM paper)bytreatmentwithfreshly prepared

4mMNaNO2 in 1.17MHCI for 30 minoniceandthen washed with water and equilibrated with transfer buffer. Virus was transferred from the gel to DBM paperelectrophoreticallyfor 2h at25 V.Remaining active groupsonthepaperwereblockedafter incuba-tionat roomtemperature for2h with1% bovine serum albumin in transferbuffer. The paper was rinsed in waterandcutvertically into 0.5-cm-wide strips (repre-sentingalane ofseparated viral proteins from SDS-PAGE) whichwererotatedat37°C in rabbit antiserum J. VIROL.

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ANALYSIS OF NBPs OF BLV AND FeLV 179

2.0

A B

1.0!-C

A30

-0 10 20 30 40 0 10 20 30 40 0 40 50 60

Time (Min)

FIG. 1. RPLC of(A)BLVp12, (B) FeLVplO, and (C)reduced andcarboxamidomethylatedBLVp12.

diluted in 0.05 MTris-hydrochloride(pH7.4)-0.005M EDTA-0.15 M NaCl-0.25%gelatin-0.05% Nonidet P-40(TENG-N) for15 to16 h. Thestripswererinsed in water and incubated first at 37°C for 2 h in four changes ofTENG-Non ashakingwaterbathand then for 2h in125I-labeledproteinAat40,000to50,000cpm perml of TENG-N.Labelwasremovedandthestrips

werewashed with four changes of 0.05 M Tris-hydro-chloride(pH7.4)-0.005M EDTA-1.0 M NaCl-0.25% gelatin-0.4% Sarkosyl (TENG-S)for 2 hat37C. The stripswererinsed in water, airdried,andexposedto flashedX-rayfilm withanintensifyingscreenfor 15to 16h.

DOTtests.Antibody titersweredeterminedby direct-ly spotting DBM paper strips with 1 Fg ofpurified

NBP in0.04M sodiumphosphate buffer, pH6.5. With this methodanexact amountofproteinwasboundto DBMpaper.Reaction with serial dilutions ofantisera,

2'"I-labeledprotein A, and autoradiographywere

car-riedoutasdescribedabove.

RESULTS

Purification of BLV and FeLV NBPs. The

NBPs of BLV and FeLV were purified by chloroform-methanolextraction andRPLC. The proteins partitioned to the high-ionic-strength aqueous phase during extraction with chloro-form-methanol.Anapproximate purity of >95%

afterchloroform-methanolextractionwas deter-mined by SDS-PAGE. RPLC was used to re-move possible contaminating protein

compo-nentsand RNA andallow for desalting and rapid concentration ofprotein bylyophilizationfroma volatile solvent. RPLC profiles of the extracted proteins from BLV, FeLV, and reduced and carboxamidomethylated protein from BLV are shown inFig. 1A, B, and C, respectively. Both nucleoproteins elutedat25%acetonitrileasthe majorcomponentof theextraction. The elution

patterns of BLV and FeLV NBPs indicated multiple protein peaks (three) eluting within a narrow range of acetonitrile gradient. When BLV p12 was reduced and carboxamidometh-ylated the elutionpatternshowedasingle major

peak, suggesting that the apparent chromato-graphic heterogeneitymayhavebeen duetothe formationof inter-orintramolecular S-S bonds.

The purity of the proteins and approximate molecular weights were determined by SDS-PAGE.Purified NBP fromBLVis demonstrated inFig. 2A. InoursystemithasanMrof-8,000 comparedwith standard proteins. Previous no-menclature has identified this protein as BLV p12 (4). We will use the same designation. Figure 2B confirms the purity of FeLVp1O, the U

e

0

a A0 4c

20 ' c 00

00

l0

VOL.46,1983

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180 MORGAN, COPELAND, AND OROSZLAN

-4 ..-

-.; ,_, * t W .S,,.1

..._{r b _}L_{b b} * E

* E

_ r

*- Fo *9 E [ 's... .FR

.. s. ::.

*'- 1!o _

7 _L

*

* s

-.o. _

... ...

...,,X,4.

_

< . ... i:

.L

--@- _

FeLVp10 FeLV

FIG. 2. SDS-PAGE of BLV virus and purified BLV p12 (A) and of FeLV virus and purified FeLV plO (B).

Standard proteins (molecular weights in parentheses) are phosphorylase b (94,000), bovine serum albumin (67,000), ovalbumin(45,000),carbonicanhydrase(29,000), soybean trypsininhibitor(21,000),lysozyme (14,300), andaprotinin (6,400).

NBPfrom FeLV, which hasamolecularweight

of-6,000.

Amino acid composition. The aminoacid

com-positional data of BLV p12 and FeLV p1O,

based on 24-,48-, and 72-hhydrolysis,aregiven

in Table 1. The total number of amino acids

TABLE 1. Aminoacidcomposition ofBLV

p12

BLVp12 FeLVplO

Aminoacid Residues Nearest

ReiusNearest

per

ineger

Asp 3.72 4 6.09 6

Thr 2.90 3 1.81 2

Ser 2.33 2 1.93 2

Glu (2.1) 2 5.29 5

Pro 14.83 15 3.70 4

Gly 5.82 6 3.23 3

Ala 3.08 3 3.25 3

Val 1.26 1 1.99 2

Met 0.71 1 0.00 0

Ile 1.03 1 0.94 1

Leu 2.04 2 3.59 4

Tyr 0.82 1 1.02 1

Phe 0.00 0 0.00 0

His 2.18 2 0.99 1

Lys 7.36 7 8.78 9

Arg 4.82 5 5.76 6

Cys (not determined) Trp (notdetermined)

Molwt 6,034 5,652

minus cysteine and tryptophan is 55 for BLV

p12 compared with 49 forFeLV p1O. Note the

abundance of basic amino acids, lysine and

arginine, in bothproteins. The numberof

resi-dues ofserine,alanine, isoleucine, andtyrosine

areidentical forbothproteinsas istheabsence

ofphenylalanine. BLV p12 has a high proline

content notsharedbyFeLVp1O.The

computer-assisted molecularweightsfrom thecomposition

(withoutcysteineand tryptophan) are 6,034for

BLVp12 and5,652for FeLVp1O.

NH2-terminal sequence. TheNH2-terminal

se-quenceasdeterminedbysemiautomated Edman

degradation of BLV p12 is shown in Fig. 3A.

Assignmentswerebased on theoccurrenceof a

newlyarising peakateachcycle compared with

standardPTH amino acids. In asingle

microse-quenceanalysis of4nmolofBLVp12,

unambig-uousassignmentsweremadefor residues1to 40

except cysteines in positions 24, 27, and 37,

leucineresidue 28(which was lost), and

threo-nine 39. Cysteine residues cannot be identified

after Edmandegradationoftheunmodified

pro-tein duetotheirinstability. Thecysteines were

determined by degrading purified peptides

de-rived by endoproteinase

Lys-C

re-duced and carboxamidomethylated BLV p12.

These two peptides, one representing the

frag-ment of residues 18to 29 and theother

repre-sentingthatofresidues 30 to40,wereavailable

from other studies. Sequence analysis of the

peptides confirmed the initialresults of Edman

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VOL. 46, 1983

BLV p12

Val-His-Thr-Pro-Gly-Pro-Lys-Met-Pro-Gly-Pro-Arg-Gl n-Pro-Al a-Pro-Lys-Arg-Pro-Pro a 2.9 1.5 3.6 3.2 1.9 2.0 3.1 1.4 2.7 1.8 1.3 3.0 1.6 1.7 1.5 1.3 2.1 2.1 0.9 1.9

b 4. 3 4. 7 5.3

ANALYSIS OF NBPs OF BLV AND FeLV 181

10 15 20

21 25 30 35 40

Pro-Gly-Pro-Cys-Tyr-Arg-Cys-Leu-Lys-Gl u-Gly-Hi s-Trp-Al a-Arg-As p-Cys-Pro-Thr-Lys

a 0.2 0.3 0.4 0.2 0.2 0.1 0.2 0.1

b 6.8 6.2 4.5 2.9 7.5 6.2 2.3 4.1 1.1 17 14 17 8.9 19 17 16 6.2 3.1 2.9 2.5

FeLV plO

1 5 10 15 20

Ala-Thr-Val-Val-Al a-Gln-Asn-Arg-Asp-Lys-Asp-Arg-Gl u-Gl u-Lys-Lys-Leu-Gly-Asp-Gln a 6.8 4.7 9.0 5.6 5.7 3.4 3.0 3.4 3.3 4.8 2.9 2.4 3.7 3.3 3.2 5.1 2.1 1.3 2.1 2.0

21 25 30 35 40

Arg-Lys-Ile-Pro-Leu-Gly-Lys-Asp-Gln-Cys-Ala-Tyr-Cys-Lys-Glu-Lys-Gly-His-Trp-Val a 2.2 4.1 1.6 1.9 1.0 1.3 1.1 1.1 1.0 0.7 1.5 0.7 1.3 0.5 0.2 0.2 0.5

b 25 10 16 9.8 8.9 8.6 11 15 4.2 4.4

FIG. 3. NH2-terminalamino acid sequence of BLVpl2and FeLVplO. Numbers belowresiduesareyieldsin nanomoles ofPIHamino acid derivatives as obtained by degrading the unmodified proteins (rows a) and

fragments (rows b). Inthe initialanalysis ofBLV p12the assignmentsfor residues 21 to 30werebased on qualitative data due to instrument malfunction which didnotallow accurate quantitation. Inputs for Edman

degradationwere asfollows: BLVp12-intactprotein,4nmol;fragment18to29,21nmol; fragment30to40,24 nmol; FeLVp10-intact protein,12nmol;fragment23to27,33nmol;fragnent28to34, 18nmol.

degradationandalso allowedassignments to be

made atpositions 28 and 39 asshown.

Figure3B shows thesequencefor FeLV p1O.

The first40 amino acidresidues, except cysteine

residues 30 and 33 and lysine 27, were

deter-mined by a single microsequencing analysis of

12nmol ofprotein. Assignmentsof the cysteines

and lysine residue 27 were made as previously

discussed for BLV p12, using Lys-C cleavage

fragments

S. Oroszlan, manuscript in preparation). BLV

p12and FeLVplONH2-terminal sequences are

comparedforhomologywith each other and the

aminoacid sequenceofthe NBPfromR-MuLV

(12) in Fig. 4.

Immunological

and FeLV p1O. Antisera specific for the NBPs

were developed in rabbits. The immunization

5 10 15 20 25 30 35 40

FeLV plO A T V V A Q N R D K D R E E K K L G D Q R K I P L G K )Q C A Y C K E K G H W V R D C P K BLV p12 V H T P G P K M P G P R Q P A P * * * K R P P P G P * * * C Y R C L K E G H W A R D C P T R-MuLV plO A T V V S G Q R Q * D

R.Q

FIG. 4. NH2-terminal sequences of BLVp12, FeLVp1O, and R-MuLVplOarecompared insingle-letter aminoacid code:A,alanine;T,threonine;V, valine; Q, glutamine;N,asparagine; R,arginine; D,aspartic acid; K,lysine; E, glutamic acid; L, leucine; G, glycine, I, isoleucine; P, proline;Y, tyrosine; H, histidine; W,

tryptophan; M, methionine; S, serine; C, cysteine. Any residue identical witharesidue fromeither of the other

proteinsis underlined. Gaps are indicated by an asterisk. The sequenceof FeLV plObeyondresidue 40 istaken fromotherstudies(Copelandetal.,unpublisheddata). The sequence of R-MuLV plO is takenfrom Hendersonet al.(12).

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182 MORGAN, COPELAND, AND OROSZLAN

16

12

0

x

4

6

X, 4

0

x

-F

2

BLV p12 Immunization

20 60 100

Time (days)

140 180

FeLV plO Immunization

20 60 100 140 180

Time (days)

FIG. 5. Immunization schedules and titers of anti-BLVp12rabbit serum andanti-FeLV plO rabbit serum.

Symbols:0,testbleed; l , injection.

schedules with the resulting antibody titers for

BLV p12 and FeLV plO are shown in Fig. 5. Arrowsindicatedayswheninjections(a total of

nine for each rabbit) were made, and circles

indicate the titers oftest bleed and bleed-out

sera.Eachserum wastitratedby

immunoautora-diography, usingtheDOTtest. Thehighesttiters

obtainedfor anti-BLVp12serum, and FeLVplO

antiserumwere16,200and5,800. Atotalof550

jig

rabbit. Immunization with BLV p12 was

com-pletedin 130daysand thatwithFeLVplOwas

completedin 182days. Inspiteoftherelatively

large doses ofantigensandlong periodof

immu-nization. theresultingantiserawerehighly

spe-cific. This reflects onthehigh stateofantigenic

purity oftheRPLC-purifiedproteins.

Figure 6 shows an actual titrationpattern of

anti-BLVp12 and anti-FeLV plO rabbit seraas

obtained by the DOT test. All test bleeds and

bleed-out serawere titrated by this procedure,

butonlyresultsoftwobleeds(oneearlyand one

later) from each rabbit immunized with the

NBPs areshown. Test bleed1of anti-BLVp12

rabbitserumtitratedat1,800 withanincreaseto

16,200at testbleed5, which didnotreactwhen

tested against BLV p24. Test bleed 2 of

anti-FeLVplO rabbitserumtitratedat1,800 withan

increase to 5,400 in test bleed 7, which was

negative when spotting FeLV p27. Prebleeds

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ANALYSIS OF NBPs OF BLV AND FeLV 183

Anti BLV p12

TB#1 TB#5 TB#5

Reciprocal of

Antiserum Dilutions

200

600

1,800

4:

0:

.0

A*

.w

TB#2

Anti FeLV p10

TB#7 TB#7

I

*_

5,400

16,200

t

t

1

BLV p12 BLV p12 BLV p24

Antigen

t

t

t

FeLV p10 FeLV p10 FeLV p27

Antigen

FIG. 6. Titration of anti-BLVp12rabbitserum and anti-FeLV plOrabbit serum by NBPspottingof DBM paper. TB, Test bleed.

taken before immunization of eitherrabbitwere

negative.

Specificity of antiseratoBLVp12 and FeLV

plO wasfurther studied by

immunoautoradiog-raphy involving the electrotransfer of

SDS-PAGE-separated viral proteins to DBM paper

(Fig. 7). In lanes 1 to 3 FeLV proteins were

transferred and stained with appropriate

anti-sera.Lane1shows thereaction with anti-FeLV

p27 rabbit serum. Lane 2 wasstained with

anti-FeLVplOrabbitserumwhich reacted with plO

only. Thesameprotein wasalsorecognized by

antiserum toBLVp12asshown in lane 3. BLV

proteins were transferred in lanes 4 to 6 and

stained withappropriate antisera. Lane 4shows

the reaction of BLV with anti-BLV p24guinea

pig serum. Lane 5 was stained with anti-BLV

p12rabbitserum.Rabbit antiserumtoFeLVplO

also cross-reacted withBLVp12. Both antisera

to BLV and FeLV NBPs reacted specifically

withBLV, recognizingp12only. Various other

retrovirusesweretestedwith anti-BLVp12and

anti-FeLV plO rabbit sera after virus proteins

weretransferredto DBM paper. Table 2

summa-rizes these results.Asexpected, anti-FeLVplO

serum reacted in an interspecies fashion with

other mammalian type C virus NPBs but not

with Rous avian sarcoma virus p12. The anti-BLVp12serum,however, didnotrecognizeany

type C virus testedexcept FeLV. Homologous

andheterologous titers (numbers in parentheses)

for both seraarealsogivenin Table 2.

DISCUSSION

Chloroform-methanol extraction and RPLC in

combination are effective methods in which to

purifyto ahighstateof puritythe NBPsofBLV

and FeLV. The purity of BLV p12 and FeLV

plO was confirmed by SDS-PAGE. In spite of theapparenthomogeneity in thissystem,RPLC

patterns indicatedmultipleprotein peakseluting

withina narrow rangeofanacetonitrilegradient.

This heterogeneity of

chloroform-methanol-ex-tracted BLV p12was showntobe dueto

disul-fide cross-links between cysteine residues. The

reduced and carboxamidomethylated protein

elutedas asingle peak.Itis assumed that similar

intra- or intermolecular cysteine interactions

may be responsible for the chromatographic

heterogeneity of FeLVp1O. Itis clear from the

partial sequence data that both proteins have

morethanonecysteine.

Theaminoacidcompositiondataindicate that

both NBPs containanabundanceofbasic amino

acids. Noaminoacidappeared withhigher

resi-due number than lysine plus arginine in either

proteinexceptthe 15residues ofprolinefound in

BLVp12. This unusually highfigureforproline

in BLVp12 isnotshared in thecompositionsof

FeLVplOandof other known NBPsoftypeC

viruses. Recently, the primary structure of the

NBP (p1O) of R-MuLV was reported (12).

R-MuLV plOisabasicproteincontaining5lysine,

9arginine, and 2 histidine residues,atotalof 16

basic amino acid residues of 56 in thecomplete

protein. Gel filtration andelectrophoretic

analy-sis hadoriginally estimatedthemolecularweight

to be between 7,000 and 10,000. The exact

molecularweight is reportedas6,347. The

mo-lecular weights calculated from composition

(without cysteineand tryptophan) forBLV p12

(6,034) andforFeLVplO(5,652)arealso smaller

thanmolecularweights determinedby gel

elec-trophoresis. Results indicatethat BLVp12and

FeLV plO are similar in size and composition, VOL.46,1983

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184 MORGAN, COPELAND, AND OROSZLAN

FeLV LV

po27.

;F

F

FIG. 7. Specificity of anti-BLV p12and anti plO rabbit sera by immunoautoradiography. was transferred to DBM paper (lanes 1 to

stained with anti-FeLV p27 rabbitserumataI

dilution (lane 1), anti-FeLV plO rabbit serur

1:6,000 dilution (lane 2), and anti-BLV p12

serumata1:100 dilution(lane 3).BLVwastran! toDBMpaper (lanes4to6)and stained withant p24 guinea pigserumata1:2,000dilution(lane4 BLVp12 rabbitserumata1:10,000dilution(I

and anti-FeLV plO rabbit serum ata 1:100 d

(lane6).Arrowsindicate positionofproteinafte PAGE.

exceptfor proline in BLV p12 (see above), structurally defined MuLV viral NBP.

The alignments of FeLV p1O, BLV pl

R-MuLVplOareshowninFig.4.BLVpl

FeLVplONH2-terminalsequences dono

tain much homology in the first 40 residi

the proteins. The most homology betwee

proteins centers around a common regioi

contained in R-MuLV p1O. This conserv

gion has been expressed as aset of thre

residuesspacedatN, N + 3,andN+ 13

Gly-HissequenceatN+ 7andN+8, wh

isthepositionin theamino acidsequence

firstCys residue ofthe set(cysteineinpc 30 in the alignment). Involvement of ty:

andlysine residues in nucleic acidbinding;

tyofproteins has been indicated with stud

the chemical modification of R-MuL%

(L. E. Henderson, C. W. Long, and S. (

lan, Fed. Proc. 39:1606, 1980) and the

binding protein coded for by gene 5 of

riophage fd(2).Tyrosinehas also beenimj

ed intheability ofgene32protein from bac

phage T4 to bind to single-stranded DN

The conserved region of FeLV plO co

bothtyrosineandlysineresidues andaG

sequenceattheappropriate positions. Th

teines and theGly-His sequenceof BLVp12 in

positions 31 and 32 align with that in FeLVplO

and R-MuLV p1O. The homologous region of

BLV p12 alsocontains tyrosine and lysine. Due

to their sequence homology with a

well-identi-fied NBP inaregion consideredtobeimportant

fornucleic acid binding, BLVp12 and FeLVplO

aredeterminedtobe the NBPs from the

respec-tive viruses.

The results from immunological studies

indi-cate an authentic cross-reaction between BLV

p2;4

p12

p1O.

Therefore,

p12

FeLV plO mustshare acommonantigenic site

within theirstructure. AntiseratoBLV p12 and

FeLV plO were tested in

immunoautoradiog-_p% raphy with other retroviruses. Anti-FeLV plO

serumreactedwith the NBPs ofall mammalian

butnottheaviantypeC viruses tested (Table 2).

Anti-BLV p12 serum does not react and thus

doesnotshare antigenic determinantswith any

v-FeLV of the other viruses testedexceptFeLV. These

FeLV results also show that antigenic determinants

3) and shared between feline plO andmurine plOare

1:6,000 not present in BLV p12. The comparison of

m at a NH2-terminal sequences (first 40 residues) of

febrred

proteins

(see

Fig.

4)

si-BLV

homologous

region

plO

1), anti- BLV p12 not shared by R-MuLV

p1O,

ane5), couldreadilyaccountfor the

FeLV/BLV-specif-lilution ic antigenic cross-reaction(seeabove).

rSDS- The amino acid sequence of BLV p12 and

FeLV plO shows a highly homologous region

beginning with Gly-His, positions 37 and 38 in

tothe the alignment ofFig. 6. Within this

eight-resi-due-long homologous region there is only a

2,and

12and

t con- TABLE 2. Reaction of anti-FeLVplOand anti-BLV

ues of p12rabbitserawith the NBPs of various retroviruses

,n the aftertransferof separated viral proteins from

SDS-n also PAGEtoDBMpaper

ed

re-e Cys

anda

ereN of the

sition rosine

activi-lieson

plO

)rosz- DNA-

bacte- plicat-

cterio-A (1). ntains ly-His

e

cys-Reaction with antiserumto:

Virus' FeLV

plOb BLVpl2C

BLV +(400)d + (16,200)

FeLV +(5,400) + (1,600)

M-MuLV +(1,600)

-RD-114 +

R-ASV

-a Besides BLV and FeLV, the following viruses weretested: Moloney-MuLV, catendogenous type C virus (RD-114), and Rous avian sarcoma virus (R-ASV).

bAntiserum was used at 1:6,000 dilution in the homologous system and 1:100 in the heterologous systems.

c Antiserum dilutionswere1:10,000in the homolo-gous systemand1:100in theheterologous systems.

d The numbers in parentheses represent the titers of bleed-outseraasdeterminedbytheDOTtest.

J. VIROL.

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VOL. 46,1983

single amino acid difference between FeLV and

BLV. Alanine of BLV p12 is substituted for

valine in FeLV p10. In this position R-MuLV plO has alanine, like BLV p12. C-terminal to this

residue, both bovine p12 and feline plO have

arginine whereas the murine plO has lysine.

Since the complete amino acid

sequence

p12 (Copeland et al., manuscript inpreparation)

and the nearly complete sequence of FeLV plO

(T.

Copeland

al., unpublished data)

show ahigher degree of homology in the other

parts of the molecules, the above eight-residue

segment is the mostlikely candidate to represent

the shared antigenic site responsible for BLV

pl2/FeLVplO cross-reaction readily detectable

on denatured proteins. The Ala-Val exchange

may not be considered significant enough to

altertheimmunogenic response and subsequent

antigen-antibody complexing. Although arginine

andlysine are both basic amino acids, theyare

stericallydifferent. This difference could explain

why anti-BLV p12 serum reacts with FeLVplO

but not with the murinep10.Chemical

modifica-tion of arginine andlysine residues and the use

ofsyntheticpeptideswillaidthe accurate

defini-tion of the immunogenic determinant shared by

BLV andFeLV.

ACKNOWLEDGMENTS

Wegratefullyacknowledge theexcellent technical assist-anceofCatherine V.HixsonandYoung Kim. We also thank Donald C. Fish andcolleaguesforassistance with immuniza-tions.

This research was sponsored by thePublicHealthService National CancerInstitute under contractN01-CO-75380with LittonBionetics.

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