Expression and purification of the mouse mammary tumor virus gag-pro transframe protein p30 and characterization of its dUTPase activity.

(1)

Expression and Purification of the Mouse Mammary Tumor Virus

gag-pro

Transframe Protein p30 and

Characterization

of Its dUTPase Activity

BRITTA KOPPE,, LUIS MENENDEZ-ARIAS,* AND STEPHENOROSZLAN

Laboratory ofMolecular Virology and Carcinogenesis, ABL-Basic Research Program, NCI-Frederick Cancer Research and

Development

Center, Frederick,

Maryland

21702-1201

Received 16August 1993/Accepted5January 1994

The mouse mammary tumor virus gag-pro transframe protein (p30) contains the nucleocapsid protein domainderived from the3' end ofgag, fused to 154 residues encoded by the 5' region of the pro openreading frame. The DNA coding for p30 was cloned into the plasmid pALTER-1, and an additional nucleotide was inserted bysite-directed mutagenesis toallow theread-throughfrom the gag into the pro open reading frame. Theobtained insert was then cloned into pGEX-2T, aplasmid containing theglutathione S-transferase gene of Schistosoma japonicum and a nucleotide sequence encoding for a thrombin cleavage site. The chimeric protein (GST-p30)was isolated by affinitychromatographyon aglutathione-Sepharose 4Bcolumn, and after thrombin treatment, the excised p30 was further purified on a single-stranded DNA-agarose column. This protein showed dUTPase activity, with only negligible cleavage of dATP, dGTP, dCTP, dTTP, or UTP. Its apparentKmfor dUTP was 28 ,iM. The enzyme was inhibited by EDTA, but its effect could be reversed by

Mg2e

and other divalent cations. dUTPase activity was also detected in purified mouse mammary tumor virus, and p30 was the only protein recognized by antibodies directed towards the carboxyl-terminal sequence of the dUTPasecodingregion.

Aminoacidsequencecomparisonsamongproteinsencoded within the retrovirus genome allowed the identification of a novel class ofpolypeptides termed protease-like domains or pseudoproteases, on the basis oftheir similarity to the retro-viral aspartylprotease (20,26).Thesepseudoproteases, which were found only in certain retroviruses (nonprimate lentivi-rusesand type B and type Doncoviruses), appeared to have evolved by duplication of the retroviral protease gene and subsequent divergence (20). However, further analysis re-vealed a number of motifs in their primary structures which were also found in dUTPases encoded by herpesviruses and Escherichia coli (21).

dUTPases are enzymesthat hydrolyze dUTPto dUMP and

PPi.

They play an important role in nucleotide biosynthesis, since the hydrolysis of dUTP generates dUMP, required for the denovosynthesis ofthymidinetriphosphate, which is used as a substrate by DNA polymerases. In addition, dUTPases keep the intracellular concentration of dUTP at a low level, thusreducingtheincorporationofuracil intoDNA.dUTPases are ubiquitous enzymes and have been found in a variety of eukaryotic andprokaryotic organisms, including mammals(16,

42), insects (8), plants (28), and bacteria (4, 15), as well as

herpesviruses (41, 43) and poxviruses(3, 34).

In retroviruses, the dUTPase encoding region is located eitherinthepolgene,betweenthe reversetranscriptase

(RT)

and the integrase, as observed in lentiviruses (e.g., feline

immunodeficiency virus [FIV], equine infectious anemia virus

[EIAV],

caprine arthritisencephalitisvirus,and visnavirus),or

atthe5' end of the pro open readingframe (ORF),asin type B or D retroviruses. Virus-associated dUTPase activity has been found inpurified EIAVaswellasFIV. Inthelatter, the

*Correspondingauthor.

t Present address:MolecularImmunologyGroup,Nuffield

Depart-ment of Clinical Medicine, John Radcliffe Hospital, Headington, Oxford,UnitedKingdom.

dUTPasehas been expressedinbacteria andshown tocontain enzymatic activity (7). Its molecular mass was 15 kDa, as determined by sodium dodecyl sulfate (SDS)-polyacrylamide

gelelectrophoresis (PAGE). On the otherhand, preparations of Mason-Pfizer monkey virus (MPMV) and simian type D retrovirus SRV-1were also foundtocontain dUTPase activity (7). In thosevirusesaswell as in the mouse mammary tumor virus (MMTV), the dUTPase is encoded within the pro gene and arises from the proteolytic processing of the precursors Gag-Pro and Gag-Pro-Pol. In MMTV, the ribosomal frame-shift occurring at thegag-pro junction is responsible for the

expression ofa30-kDa transframeprotein,termedp30,which contains thenucleocapsid protein domain of Gag, fusedto154 aminoacid residues derived fromthe5' region of theproORF (13). However, its dUTPase activityhas not been reported so far.

Inthispaper, we report ontheMMTVdUTPase, whichwe haveidentifiedasthetransframeproteinp30.Thisproteinwas cloned and expressed with the pGEX-2T vector. pGEX-2T

contains the glutathioneS-transferase (GST) gene of Schisto-soma

japonicum

and a nucleotide sequence encoding for a

thrombin cleavage site and has been previously used for the

expression of other retroviral enzymes (22, 24). The recombi-nantp30wasfound tohave dUTPaseactivity, and its charac-terization is herein described.

MATERIALS AND METHODS

Viruses.TheC3H strain ofMMTVwasgrownin

Mm5mt/c,

cells andpurified bysucrose

gradient

ultracentrifugation

(23).

Moloney murine leukemia virus was obtained from culture supernatantsofchronicallyinfected NIH 3T3

cells,

maintained in Dulbecco's modified Eagle's medium

supplemented

with 10% calf serum, and

purified

as described above. EIAV (Wyoming strain) was grown in Cf2Th cells and

purified

as previouslyreported(32).Purified MMTV

(R-III/CrFK strain)

2313

on November 9, 2019 by guest

http://jvi.asm.org/

(2)

2314 KOPPE ET AL.

wassupplied bythe Biological Products Laboratory (Program

Resources Inc., National Cancer Institute-Frederick Cancer Research andDevelopment Center).

Plasmid construction. A747-bp DNAfragment containing

thecoding regionsof thenucleocapsidprotein (NC)and the5' end oftheproORFwassynthesized byPCR.Theplasmid p202 containing part ofthegag-pro-pol genes of MMTV (19) was

usedas atemplate.Twooligonucleotideswereusedasprimers:

(i) 5'CGCGGATCCGCAGCAGCCATGAGAGGA3', which contains the nucleotide sequence of the 5' end of the NC-coding region and a BamHI site, introduced in order to facilitate the following cloning steps, and (ii) 5'CGCGAAT TCAATGTACATGACTTGTIGA3', which contains the 3' end ofthe XcodingregionofproandanEcoRIsite. PCRwas

carriedoutwith the GeneAmpPCRReagentKitfrom Perkin-Elmer Cetus by following the manufacturer's instructions. A Perkin-Elmer 9600 thermalcyclerwasused for 30cycles.After 5 minof incubationat95°C,theinitialfivecycleswere94°C,30 s; 45°C, 30 s; and 72°C, 1 min, and then 25 cycles in which annealingwas done at 55°C. Program wasterminated with a

5-minincubationat72°C.TheobtainedDNAwasanalyzedon

a 1% agarose gel, electroeluted, purified over an Elutip-d

column (Schleicher&Schuell,Keene, N.H.), andcleaved with BamHI andEcoRI. The DNAfragment wasthencloned into the plasmid pALTER-1 (Promega), previously digested with both enzymes. DNA ligations were carried out overnight at

room temperature, with the DNA ligation kit from New England Biolabs (Beverly, Mass.).E. coliDH5otF' competent cells (GIBCO BRL) were transformed, andfreshly prepared

cultures containing the appropriate constructswere

superin-fectedwith thehelperphageR408 andused for theisolationof

a single-stranded DNA (ssDNA) template to be used in mutagenesis reactions.

Since expression ofp30 involves a ribosomal frameshift at

thegag-pro junction, site-directed mutagenesis was done in

ordertogenerateasingleORFcomprisingthe entirelengthof

p30.Thiswasdonewith theAlteredSitesinvitromutagenesis systemfromPromega,by followingthemanufacturer's instruc-tions. This system uses a phagemid (pALTER-1) which

con-tainstwo genes for antibiotic resistance. One ofthese genes,

fortetracycline resistance,isalways functional,whiletheother,

for ampicillin resistance, has been inactivated. During the mutagenesis reaction, ampicillinresistanceisrestoredby using

an oligonucleotide providedwith the kitwhich isannealed to

the ssDNA template at the same time as the mutagenic

oligonucleotide. In our constructs, thissynthetic

oligonucleo-tidewas5'CTGCCCCTTTACCAAGlYlTlTll lGA3',areverse

5' phosphorylated primer which corresponds to the overlap-ping regionofthegagandprogenes.Cytidine (underlined)was

inserted to make the natural frameshift. Annealing, mutant

strand synthesis,and ligationswere done asrecommended by

the manufacturer. After ligation, the incubation mixture was

usedtotransformarepair-minusstrainofE. coli (BMH71-18

mut S), and cells were grown in the presence of ampicillin.

DNAfromappropriatecolonieswasisolatedand digested with

BamHIandEcoRIinordertoisolatethep30-encoding insert, which was then cloned into the pGEX-2T vector (35). The insert was sequenced to ensure that no unwanted mutations had been introduced into the ORF during the PCR or the

mutagenesisreactions.DNAsequencingwasperformedby the

chain termination method (33), with the double-stranded

DNA sequencing system from GIBCO BRL and a set of appropriately spaced synthetic oligonucleotides which were

usedasprimers.

Protein expression and purification. Freshly prepared E.

coli cultures containing thedescribed plasmidwere grown at

37°C in 400 ml of Luria broth medium containing 50 jig of ampicillin per ml to an

A600

of 0.7to0.9. After induction with 1 mM isopropyl-3-D-thiogalactoside (GIBCO BRL) for 60 min, cells were harvested bycentrifugation at 4,000 x gfor 25 min at4°C. The cells were then resuspended in 30 ml oflysis

buffer(50mMTris[pH 8.0],0.1 MNaCl, 1 mMEDTA, 1 mM phenylmethylsulfonyl fluoride, 0.2 mM N-at-p-tosyl-L-lysine

chloromethyl ketone, 0.5 jig of leupeptin per ml, 1 jLg of aprotinin per ml), lysed by sonication, and centrifuged at

12,000 rpm for 10 min at4°Cwith a Sorvall SS-34 rotor. The supernatant was loaded onto aprepacked

glutathione-Sepha-rose 4B column (Pharmacia LKB) and washed with 50 mM Trisbuffer (pH8.0) until thenonspecifically absorbed material wasremoved. The column was then equilibratedwith thesame buffercontaining 25

jig

of RNaseAper ml andkeptat37°C for 30 min. Following this treatment, the columnwas extensively

washed andequilibrated in 50 mMTris-HCl (pH 8.3) contain-ing 250 mM NaCl, 2.5 mM CaCl2, and 0.5 U of bovine thrombin (Calbiochem, San Diego, Calif.) per ml. Thrombin cleavage was carried out at 37°C during 60 min. The protein

was eluted, and after addition of Triton X-100 to a final concentration of 0.1% (vol/vol), it was further purified on a ssDNA-agarose 2-ml column (GIBCO BRL). For such a purpose, the column was equilibrated at 4°C in 20 mM Tris-HCl buffer (pH 7.0) containing 0.1 mM EDTA and 0.1% Triton X-100.After the sample was loaded and the column was extensively washed with the equilibration buffer, proteins were eluted with a gradient of 0 to 1 M KCl in the same buffer. Fractions of 0.4 ml were collected, and the dUTPase activityof selected fractions was measured. Protein concentration esti-mates were obtained after acid hydrolysis, in evacuated and sealed tubes, in the presence of 5.7 N HCl for 24hat 105°C.

Protein sequence determination. The amino-terminal se-quence of the recombinant dUTPase was determined after SDS-PAGE and electrotransfer to a polyvinylidene difluoride-type membrane (ProBlott; Applied Biosystems, Foster City, Calif.) by following the previously described procedure (23). The electroblotting was done at 50V (100 to 170 mA) at4°C

for 60 min. Carboxyl-terminal sequencing was done by car-boxypeptidase digestion of the protein, which was previously immobilized on a polyvinylidene difluoride-type membrane (17a).

SDS-PAGE and immunoblotting. The purity of theprotein was assessed with 10% precast Tricine polyacrylamide gels (Novex, San Diego, Calif.). Western immunoblots (38) were carried out with a specific antiserum towards the carboxyl-terminal region of p30, obtained after immunization of rabbits with the high-performance liquid chromatography-purified synthetic peptide CIKEERGSEGFGSTSHV, coupled to key-hole limpet hemocyanin through its cysteine residue by reac-tion with m-maleimido-benzoyl-N-hydroxysuccinimide ester (Pierce, Rockport, Ill.), as previously described (5, 18). Detec-tion of antibody bound to the nitrocellulose membranes was done with protein G labeled with horseradish peroxidase (Bio-Rad), and the reaction was developed with enhanced chemiluminescence detection reagents and Hyperfilm-ECL (Amersham Corp.).

dUTPase assays. dUTPase activity was measured as de-scribed byWilliams and Parris (43). Briefly, assayswere done by adding to 50 ,ul of 0.1 M Tris-HCl buffer (pH 8.0), containing 0 to 0.2 mM dUTP, 4 mM 3-mercaptoethanol, 2 mM MgCl2, 4 mM p-nitrophenylphosphate, 0.2% (wt/vol) bovine serum albumin, 1 ,ul (1 ,uCi) of [5-3H]dUTP (15

Ci/mmol), and 49 ,ul of the enzyme solution at the appropriate dilution. In assays involving other nucleoside triphosphates, dUTP was excluded from the reaction mixture andsubstituted

J. VIROL.

on November 9, 2019 by guest

http://jvi.asm.org/

(3)

A

PstI

2580 P30

sssss

PstI

6736 2996 3741

gag

I

I pro I

POl

- insert

B

G

3260 G

_I

_{-~~~~~~~~~~~~~~~~~~~~~~~"}3300

DNA GCTGAAAATTCAAAAAACTTGTAAAGGGGCAGTCCCCTAGC

gag- ORF Ala Glu Asn Ser Lys ASn Leu *4*

pro-ORF *** Lys Phe Lys Lys Leu Val Lys Gly Gin Ser Pro Ser

p30

Ala Glu Asn Ser Lys ASn Leu Val Lys Gly Gin Ser Pro Ser

FIG. 1. Construction of clones expressing the MMTV gag-pro transframe protein p30. (A) The PstI MMTVgag-pol fragment is shown, indicating the region encodingp30. (B) Nucleotide sequence of the segment of MMTV DNA spanning the gag-pro overlap window with the corresponding amino acid sequence of the transframe protein p30.Arrows on the DNA sequence and theautoradiography indicate where a nucleotide (G) insertion has been introduced to generate a single ORF comprising the entire length ofp30.

by the corresponding nucleotide at the appropriate concentra-tion. Depending upon the experiment, 1 ,u (1 ,uCi) of

[8-3HJdATP

(17.1 Ci/mmol), [5,5'-3H]dCTP (21.5 Ci/mmol),

[8,5'- H]dGTP (25.7 Ci/mmol), [methyl-3H]dTTP (19 Ci/

mmol), or [5,6-3H]UTP (14.3 Ci/mmol) was added to the incubation mixture instead of the corresponding amount of

[5-3H]dUTP. All radioactive nucleotides were obtained from DuPont-NEN Research Products, except [5-3H]dUTP, which wasfromAmersham. Assays were carried out at 37°C for 2 to 20 min. A totalof 50

RI

of the reaction mixture was spotted on aDE81 ion-exchange paper disc(Whatman) and washed three times for 5 min(eachwash) in 1mMammoniumformate-4 M formicacid and then once for 3 min in95%ethanol. The discs werethendried and counted inaBeckman LS 5801 scintilla-tion counter. The reaction products were analyzed by thin-layerchromatography aspreviously described (1).

RESULTS

Bacterial expression and purification of the MMTV dUTPase (p30). In MMTV, the dUTPase encodingregion is locatedatthe 5' endof the pro gene and could beexpressedas partof the gag-pro transframeprotein (p30). Wehave

synthe-sizedby PCR theDNAencoding forp30, which includes the

nucleocapsid protein gene (derived from the gag ORF), and theputative dUTPase domain encoded in the pro ORF

(Fig.

1A). A nucleotide insertion was required to mimick the translational frameshift in the -1 direction, requiredforthe

synthesis of the entire p30. This was done by site-directed

mutagenesis and involved the insertion ofa G in the coding

strand (Fig. 1B).The nucleotide sequence of the whole insert wasfoundtobe identicalto thatreported byJacksetal. (17)

for the C3H strain, except for the nucleotide insertion indi-cated above. After mutagenesis, the insert was cloned into

pGEX-2T, a

prokaryotic expression

vectorcontaining thegst gene ofSchistosoma japonicum

(35)

and used for

expression

and purification of the MMTV gag-pro transframe

protein,

p30.

One hour after induction, cells were harvested, lysed, and centrifuged. The supernatants were then passed through a

glutathione-Sepharose 4B column. The chimeric protein was eluted with a buffer containing 5 mM glutathione. dUTPase assaysrevealed thatthis59-kDa chimeric protein (Fig. 2B) was

enzymatically active, was able to hydrolyze dUTP to dUMP and

PP1,

but failed to cleave other nucleotide triphosphates

(data not shown). The purified GST-containing proteins ob-tained from cultures expressing either GST alone (plasmid

pGEX-2T) or the chimeric protein GST-MMTV protease

(plasmid pPR207)(24) did not have dUTPase activity. The recombinant MMTV

p30

was purified after thrombin

cleavage of the chimeric protein, previously adsorbed to the

glutathione-agarose support. This protein showed dUTPase activity and was further purified by chromatography on ssDNA-agarose, since its nucleocapsid protein domain was expected tobind ssDNA. As shown inFig. 2A, the dUTPase activitywaseluted with aKCl gradient,and fractionsshowing theenzymatic activitywerefoundtocontaina30-kDa polypep-tide recognized by antibodies directed towards the carboxyl-terminal end of the putative dUTPase domain of the trans-frameproteinp30. SDS-PAGEof thepurified proteinis shown in Fig. 2B (lane 2), and the overall yield of the process is

approximately30to50 ,ug ofproteinper liter of culture (2g [wetweight]ofE.colicells).Thespecific activityof thepurified

dUTPase was 6,200 nmol per min and mg of enzyme, when measured in the presence of 0.2 mM dUTP.

The amino- andcarboxyl-terminalsequences of the recom-binant MMTV dUTPase are shown in

Fig.

3. These results were consistent with the primary structure

reported

for the MMTV

p30

isolated from

purified

virus

(13).

The

only

differ-ences were found at the amino

terminus,

since the recombi-nant dUTPase contains two extra amino acids

(Gly

and Ser)

notfound in the viral

p30

butintroducedas aconsequence of the cloning scheme. As

expected,

residues 3 to 18 of the recombinant dUTPasearealsoidenticaltoresidues 1to 16of the MMTV NC

protein

p14

(14). Together,

theresults of the

i

I~~~~~~~~~~~~~~~~~~~~~~~~~

on November 9, 2019 by guest

http://jvi.asm.org/

[image:3.612.74.567.83.301.2]

(4)

2316 KOPPE ET AL.

A

Fraction

B

13 15 17 19 21 23 25

kDa

M

1

2

97.4-VU

A.,.

46- :

30-21.5

14.3-

1.0,-2

_

0

*_

_X

4-'

0.5 C

0

Y

lO

Fraction

FIG. 2. Purificationof recombinant MMTVp30. (A)Chromatographic purificationofp30onanssDNA-agarosecolumn(bedvolume,2ml).

Theamountofproteinloadedonthegelwas25 pLg,andaliquotsof thecorrespondingfractionwereassayedfordUTPaseactivity (35 pl)andby

Westernblotanalysis (120,ul),witharabbitpolyclonalantibody specificforthecarboxyl-terminal regionofp30.(B)Purified recombinantp30 (lane 2)andthechimericproteinGST-p30 (lane 1),whicharecomparedwiththeRainbowproteinmolecularmassmarkers from Amersham(laneM).

Markers includemyosin (200kDa), phosphorylaseb(97.4kDa),bovineserumalbumin(69kDa),ovalbumin (46kDa),carbonicanhydrase (30 kDa), trypsininhibitor(21.5 kDa),andlysozyme (14.3 kDa).

protein sequence analysis of the purified dUTPase and

the DNA sequence data demonstrate that the recombinant p30 is identical to the p30 found in MMTV (C3H strain) particles.

Enzymatic propertiesof the recombinant MMTVdUTPase. The only nucleotide triphosphate which was cleaved by the recombinantp30 was dUTP, and nosignificant cleavage was

observed whendATP, dCTP, dGTP, dTTP,orUTPwasused

as a substrate in the assay mixture (Fig. 4A). Competition experiments were performed using unlabeled deoxyribo-nucleosidetriphosphates (dNTPs) to compete forcleavageof [3H]dUTPto[3H]dUMP(Fig. 4B).The resultsindicate that of the sixdNTPsused,onlydUTPwascapableofcompetingfor

cleavage of radiolabeled dUTP by the recombinant MMTV dUTPase. Initial velocity studies were performed at a fixed

magnesium concentration (1 mM) but with various

concentra-tions(5 to60F.M)ofdUTP.The apparentKmfortheMMTV

dUTPasewas28.0 ± 7.3 FM, and its Vmaxwas6,500 + 1,100

nmol per min and mg of enzyme. Divalent cations such as Mg2+, Mn2+, orCo2+ have asignificant stimulatory effecton

theactivity of thisretroviral dUTPase, and EDTA isastrong

inhibitor of the enzyme activity (Table 1). However, the

inhibitory effect of EDTA was reversed by divalent cations, such as Mg2+ or Zn2+. Though zinc is likely to bind the nucleocapsid protein domain ofp30, its additiontothe dUT-Pase assay cocktail results in a moderate increase of the

enzymeactivity. Asimilareffect has also beenreportedforthe

FIV dUTPase (40), which lacks the NC domain.In addition,

the presence of 1,10-phenanthroline (a zinc chelator) in the

assay doesnot have a significant effecton the MMTV

dUT-Pase activity, suggesting that the nucleocapsid moiety ofp30

exertsaminor influenceonits enzymaticactivity.

MMTV-associateddUTPase.ConversionofdUTPtodUMP

was almost complete (>90%), after 5 min ofincubation at

NH2-termina

1:

GLY-SER-ALA-ALA-ALA-MET-ARG-GLY-GLN-LYS-TYR-SER-THR-

PHE-VAL-LYS-GLN-THR-COOH-terminal: -VAL-HIS

FIG. 3. Amino- andcarboxyl-terminalsequences of the recombinant MMTV dUTPase. Underlined amino acids correspond to the identical amino- andcarboxyl-terminalsequencesof the viralp30,isolated from purified MMTV (13). The first two residues of the amino-terminal sequence derive from the thrombincleavagesite,located between GST and MMTVp30inthe chimeric precursor of therecombinant dUTPase.

kDa

46-

30-

21.5-0L

3 10

0 80

I-D 60

0

0 40

._

L-> 20 0 0

J. VIROL.

on November 9, 2019 by guest

http://jvi.asm.org/

[image:4.612.104.502.82.381.2]

(5)

MMTV dUTPase 2317

TABLE 1. Effects ofvarious divalent cations and chelators on the

activity of MMTV dUTPase"

Additive" Relative

activity"

None... 1.0

EDTA... 0

1,10-Phenanthroline... ... 0.9

Mg2+... 2.3

Mn2+... 1.9

o219...1.9 Z . .1...1.3

_

t;0l10]+TT

~~~~~~~~~~~~~~~~C

a 2...

().7

dUTP UTP dATP dGTP dCTP dTTP Cu2+... 1.1

B

a.80

0

00

0 dATP

c

.40

- dGTP

40

ALk dTTP -0-- dCTP

o 20 UTP

dUTP

0 II

0 0.1 0.2 0.3 0.4

Concentration of Unlabeled dNTP(mM)

FIG. 4. Substrate specificity of the recombinant MMTV dUTPase, p30. (A) Cleavage of different dNTPs todeoxyribonucleoside

mono-phosphate (dNMP), using recombinant p30. Incubationswerecarried outin thepresenceof5 pLM dNTP for 15minat37°C. Theamountof purified enzyme used in each assay was 75 ng. (B) Conversion of [5-3H]dUTP to [5-3H]dUMP in the presence of unlabeled dNTPs. Thesecompetition experimentsweredone inthepresenceof25to400 p.M unlabeled dNTP and 5,uM [5-3H]dUTP. Sampleswereincubated

at37°C for 5minafter addition of 75 ngofpurified dUTPase.

37°C, when dUTPase assays were performed with 15 p.g of detergent-disrupted purified MMTV in thepresence of 5 F.M

dUTP. However, when other nucleotides (dATP, dCTP, dGTP, dTTP, andUTP) were usedas substratesin the same

conditions, the cleavage was almost negligible (<4%). We

obtained similar resultswithEIAV, using dUTPand dATPas

substrates. However, the conversion of dUTP to dUMPwas

notobserved when theassays weredone in thepresenceof the

same amount of purified Moloney murine leukemia virus. EIAV and Moloney murine leukemia virus were used as

positive and negative controls, respectively, since dUTPase activity has been previously tested in purified preparations of those viruses (7).

Antibodies towards the carboxyl-terminal region of the putative dUTPase ofMMTVwereusedtodetectpolypeptides related tothisenzymeinpurifiedvirus. A 30-kDapolypeptide whichcomigrated with the purified recombinant p30wasfound

to be the only band recognized by the dUTPase-specific antibodies after Western blot detection, and smaller proteins corresponding totruncated versions ofp30werenotobserved

in these experiments (Fig. 5). Similar results were obtained with different isolates of the C3H strain, as well as other MMTV strains, such as R-III (data not shown). These data

"After thrombin cleavage, the MMTV dUTPase was extensively dialyzed

versus50 mMTris-HCI (pH 8.0). For these studies,MgCl,wasdeleted from the

standard reaction mixture and the dUTP concentration used was 0.1 mM.

Reaction mixtureswereincubatedat37°C for5to10min.

hThe chelators EDTA and 1,10-phenanthrolinewereaddedtofinal

concen-trations of 0.2mM, while 1 mMwasthe cation concentration in the incubation

assay.Divalentcation chloride saltswereused for this study.

' Relative activity referstothe values obtained in the absence of additive.The

standard deviation ofthemeasurementswasaround 10to 15CSc.

indicate that thegag-pro transframe protein p30 is the active

form of the MMTVdUTPase. In agreementwith this obser-vation, when disrupted viruswaschromatographedon

ssDNA-agarose under the conditions described for the recombinant

p30, the dUTPase remained boundtothe column unless 1 M

KCIwasaddedtothe elutionbuffer,asexpected, assuming that thenucleocapsid protein domain ispartof the active dUTPase in the virion.

DISCUSSION

Amino acid sequence comparisons showed that five

con-served motifs found in dUTPases from different sources (E.

coli, herpesvirus, vaccinia virus, etc.)werealsopresentinthe 5' region of the MMTVprogene(21). The determination of the

amino acid sequence of a 30-kDa polypeptide isolated from purifiedMMTVrevealed that thedUTPase-encoding region is expressed in fusion with the nucleocapsid protein domain derived from the3' end ofgag(13). In thispaper, we present

evidence indicating that the 30-kDa transframe protein of MMTV is a dUTPase. Our conclusion is supported by the

kDa 1 2

200

-

97.4-

69-

46-30 - _

21.5

14.3-FIG. 5. Western blot detection of dUTPase-containing

polypep-tides in MMTV. A total of100 ,ugofgradient-purifiedMMTV(C3H

strain) and 2 ,ugof recombinant p30wasapplied to lanes 1 and 2,

respectively. Blotswere developedwith a rabbitpolyclonal antibody specific for the carboxyl-terminal region of p30. Rainbow protein molecularmassmarkers from Amershamwere usedasreferences.

A

K~ri

0. 100

z

O 80 0~

Z 60

m

0

r 40

.2

> 20 0

0

:

VOL.68? 1994

on November 9, 2019 by guest

http://jvi.asm.org/

[image:5.612.66.306.82.392.2]

(6)

2318 KOPPE ET AL.

following

observations:

(i)

dUTPase assays showed that

gradi-ent-purified

MMTV contained the

enzymatic activity;

(ii)

Western blot

analysis

of the whole virus with antibodies directed towards the

carboxyl-terminal

region

of

p30,

which contains one of the five conserved motifs found in viral and bacterial

dUTPases,

suggests that

p30

is the

only protein

comprising

the entire dUTPase sequence found in mature

MMTV;

and

(iii)

the recombinant MMTV

p30

wasfound to

cleave dUTPtodUMP andPP1 butwasunabletocleave other nucleotide

triphosphates.

The MMTV dUTPasewasinhibited

by

EDTA,

though

this effect could be reversed

by

divalent

cations,

like magnesium or zinc, a characteristic which is

shared

by

other dUTPases.

However,

itsapparentKm

(28

,uM)

wasfoundtobe

relatively high

in

comparison

tothose

reported

for dUTPases isolated from

herpesviruses,

E.

coli,

Drosophila

melanogaster,

and severalmammalian tissues

(8, 16, 41, 43,

and

references

therein)

whoseKm values range from 1to 12

,uM.

Since the MMTV dUTPase is

expressed

in fusion with the

nucleocapsid

protein

domainderived fromgag,it is

expected

to

be associated to the viral RNA.

Despite

its

higher

Ki,,

the MMTVdUTPase would be closetoreverse

transcription sites,

where its

activity

would be

important

to prevent in situ the

incorporation

of dUMP into DNA.

The MMTV transframe

protein

p30

constitutesanovel type of retroviral

dUTPase, differing

from the

previously

character-ized FIV enzyme, since the latter is encoded within the

pol

geneand is

expressed

as a 15-kDa

polypeptide

(7, 40).

Type

B

and type Dretroviruses share averysimilar

genomic

organi-zation as well as a

high degree

of

similarity

among their

proteins.

The order of themature

proteins

in the

Gag

precur-sor of MMTV is

plO(MA)-pp2l-p3-p8-n-p27(CA)-pl4(NC)

(14),

and in MPMV

(a

type D

retrovirus),

the order is

plO(MA)-pp24-pl2-p27(CA)-pl4(NC)-p4 (12, 36).

The

struc-tureand

activity

of the

hypothetical

gag-protransframe

protein

ofMPMVhavenotbeendetermined. This

protein

is

expected

to be a dUTPase and could be similar to MMTV

p30.

According

tothe

prediction

ofHatfieldet al.

(11),

the

frame-shift

signal

is locatedwithin the NC

coding region

of MPMV.

Therefore,

the additional maturation site found between

p14(NC)

and

p4

inthe MPMV

Gag

precursorwouldbe absent

in the

corresponding

transframe

protein.

At present, we lack an

explanation

for

why

dUTPase is

expressed

in some retroviruses and not in

others,

such as

human

immunodeficiency

virus. The

specific

function of dUTPase in the retrovirus life

cycle

is still unclear. Recently,

Threadgill

et al.

reported

that the deletion of the dUTPase

gene in EIAV had noeffecton

replication

when theviruswas grown onfetal

equine kidney

cells. However,when grownon their natural host cells

(macrophages),

replication

wasverylow

compared

with that ofthe wild type

(37).

In certain viruses,

suchas

herpes simplex

virus type

1,

dUTPaseappearstohave

an

important

role in

pathogenesis (29),

since

dUTPase-defi-cient mutants were attenuated for neuroinvasiveness,

neuro-toxicity,

andreactivationfrom

latency. Therefore,

itseemsthat

dUTPase is

required

forvirusreplication in certaincells.

In

addition,

the retroviraldUTPases couldplayanimportant

role in

reducing

mutation levels as previously suggested (7). dUTPase

activity

not

only

lowers the dUTP concentration in

the

cell,

but

supplies dUMP,

thesubstrate for the thymidylate

synthetase,

increasing

cellulardTTPpools. HighdUTP levels,

which may result from either the lack of dUTPase or the

inhibition ofthe

thymidylate synthetase

(9, 10), result in the

misincorporation

of uracil in the DNA and could contributeto

high

mutationrates dueto

hyperrecombination

or accumula-tion of

point

mutations. The presence of dUTPase in some retroviruses could be

advantageous by

preventingmutationsin

their genome. This phenomenon might be related to the fidelity of their RTs, known to be rather lowcompared with DNApolymerases (2),since the retroviral enzymesaredevoid ofproofreading mechanisms. Most of the studies on thefidelity

of the RTs have been carriedoutwith enzymes from retrovi-ruseslacking the dUTPase gene, suchasthe avian myeloblas-tosis virus, murine leukemia virus, or human immunodefi-ciency virus type 1 (27, 30, 31). One could speculate that dUTPase serves in retroviruses to keep dUTP levels low, thereby safeguarding efficientreversetranscription by enzymes with high fidelity. Retroviruses with high mutation rates as a result of lowfidelity of their RTs may be less dependenton dUTPase activity, since their RT may be able to continue strand extension despite nucleotide misincorporations. How-ever,thedegreeandnatureofenvgenevariation in EIAV are remarkably similar to those in human immunodeficiency virus type 1 (25), and this observation correlates well with dataon thefidelityof the EIAVRT,whichwasfoundtobe similar to that of humanimmunodeficiencyvirus type 1 RT (6). On the other hand, fidelity varies also depending on the particular nucleotidetriphosphate. Thoughit is known thatprokaryotic DNApolymerases can use dUTP as a substrate withkinetics similar to those for dTTP (39), there are no reports on the ratesofmisincorporationof uracil into DNAby retroviral RTs. Further studies will be required todetermine the role ofthe dUTPaseduringthe virus lifecycleand thepossible evolution-aryadvantagesof itspresencein certain subsets ofretroviruses.

ACKNOWLEDGMENTS

We expressourgratitudetoTerryCopelandand Pat Wesdockfor

peptidesynthesisandantibodyproduction, Cathy HixsonandSuzanne

Spechtfor amino acidanalysis,Young Kim for amino acid sequencing, MarilynPowersforoligonucleotide synthesis, DougStevensforhelp withvirus production, and Deanna Gotte for assistance with PCR

cloning and DNA sequencing. We also thank Cathy Hixson for determinationofcarboxyl-terminalsequences.

This research was sponsored in part by the National Cancer

Institute, DHHS,undercontractN01-CO-74101 withABL. REFERENCES

1. Beardsley,G.P.,and H.T. Abelson.1980. A thin-layer chromato-graphic method for separation of thymidine and deoxyuridine nucleotides. Anal.Biochem.105:311-318.

2. Bebenek, K.,and T. A. Kunkel. 1993. The fidelity of retroviral

reversetranscriptases,p.85-102. In A. M. Skalka and S. P. Goff

(ed.), Reverse transcriptase. Cold Spring Harbor Laboratory Press,ColdSpring Harbor,N.Y.

3. Broyles,S. S.1993. Vaccinia virus encodes afunctional dUTPase.

Virology 195:863-865.

4. Cedergren-Zeppezauer, E. S., G. Larsson, P. 0. Nyman, Z. Dauter,and K.S. Wilson. 1992. Crystalstructureof a dUTPase. Nature (London)355:740-743.

5. Chamberlain,N.R., L. DeOgny, C. Slaughter, J. D. Radolf, and M. V.Norgard. 1989.Acylationof the47-kilodaltonmajor mem-brane immunogen ofTreponema pallidum determines its

hydro-phobicity.Infect. Immun.57:2878-2885.

6. DeVico, A.,R. C.Montelaro, R. C. Gallo, and M. G.

Sarngadha-ran. 1991. Purification and partial characterization of equine infectious anemia virus reverse transcriptase. Virology 185:387-394.

7. Elder, J. H.,D. L.Lerner, C. S. Hasselkus-Light, D. J.Fontenot, E.Hunter,P.A.Luciw,R.C. Montelaro, and T. R. Phillips. 1992. Distinct subsets of retroviruses encode dUTPase. J. Virol. 66: 1791-1794.

8. Giroir,L.E., and W.A.Deutsch.1987. Drosophila deoxyuridine

triphosphatase. Purification andcharacterization. J. Biol. Chem. 262:130-134.

9. Goulian, M., B. Bleile, and B. Y. Tseng. 1980. The effect of J. VIROL.

on November 9, 2019 by guest

http://jvi.asm.org/

(7)

methotrexate on levels of dUTP in animal cells. J. Biol. Chem. 255:10630-10637.

10. Goulian, M., B. Bleile, and B. Y. Tseng. 1980. Methotrexate-induced misincorporation of uracil into DNA. Proc. Natl. Acad. Sci. USA77:1956-1960.

11. Hatfield, D. L., J. G. Levin, A. Rein, and S. Oroszlan. 1992. Translational suppression in retroviral gene expression. Adv.

Virus Res. 41:193-239.

12. Henderson, L.E.,R.Sowder, G. Smythers,R. E.Benveniste,and

S. Oroszlan. 1985. Purification and N-terminal amino acid

se-quence comparisons of structural proteins from retrovirus-D/ Washington and Mason-Pfizer monkey virus. J. Virol. 55:778-787. 13. Hizi, A., L. E. Henderson, T. D. Copeland, R. C. Sowder, C.V.

Hixson, and S. Oroszlan. 1987. Characterization ofmouse

mam-mary tumorvirus gag-pro gene products and the ribosomal frame-shift site by protein sequencing. Proc. Natl. Acad. Sci. USA 84:7041-7045.

14. Hizi, A., L. E. Henderson, T. D. Copeland, R. C. Sowder, H. C. Krutzsch, and S. Oroszlan. 1989. Analysis of gagproteins from

mouse mammary tumorvirus. J. Virol. 63:2543-2549.

15. Hoffmann, I., J. Widstrom, M. Zeppezauer, and P. O. Nyman. 1987.Overproduction and large-scalepreparationofdeoxyuridine triphosphate nucleotidohydrolase from Escherichlia coli. Eur. J.

Biochem. 164:45-51.

16. Hokari, S.,andY.Sakagishi. 1987. Purification and characteriza-tion ofdeoxyuridine triphosphate nucleotidohydrolase from ane-micratspleen:atrimercompositionof the enzymeprotein.Arch. Biochem. Biophys. 253:350-356.

17. Jacks,T.,K. Townsley,H. E. Varmus,andJ. Majors. 1987.Two

efficient ribosomalframeshiftingevents are requiredforsynthesis of mouse mammary tumor virusgag-related polyproteins. Proc. Natl.Acad. Sci. USA 84:4298-4302.

l7a.Jahan, M., C. V. Hixson, S. Oroszlan, and T. D. Copeland. Unpublisheddata.

18. Lerner, R.A., N. Green, H.Alexander, F.-T. Liu, J. G. Sutcliffe,

and T. M. Shinnick. 1981. Chemically synthesized peptides pre-dicted from the nucleotide sequence of the hepatitis B virus genomeelicit antibodiesreactive with the nativeenvelopeprotein

ofDane particles. Proc.NatI.Acad. Sci. USA78:3403-3407. 19. Majors, J. E.,and H. E. Varmus. 1981.Nucleotide sequences at

host-proviraljunctionsformousemammarytumourvirus. Nature (London) 289:253-258.

20. McClure, M. A., M. S. Johnson, and R. F. Doolittle. 1987. Relocation ofa protease-likegene segment between two

retrovi-ruses. Proc. Natl. Acad. Sci. USA 84:2693-2697.

21. McGeoch,D.J. 1990. Protein sequencecomparisonsshowthat the 'pseudoproteases' encodedbypoxvirusesandcertain retroviruses belong to the deoxyuridine triphosphatase family. Nucleic Acids

Res. 18:4105-4110.

22. Menendez-Arias, L., D. Gotte, and S. Oroszlan. 1993. Moloney

murine leukemia virus protease: bacterialexpression and charac-terization of the purified enzyme.Virology196:557-563. 23. Menendez-Arias, L.,C.Risco,and S.Oroszlan. 1992.Isolationand

characterization of ot,-macroglobulin-protease complexes from purified mouse mammary tumorvirus and culture supernatants fromvirus-infected cell lines. J. Biol. Chem. 267:11392-11398. 24. Menendez-Arias, L., M.Young, andS. Oroszlan. 1992.

Purifica-tion and characterization of the mouse mammary tumor virus proteaseexpressedin Escherichia coli.J. Biol. Chem. 267:24134-24139.

25. Payne,S.L.,F.-D.Fang,C.-P.Liu,B. R.Dhruva,P.Rwambo,C.J. Issel, andR. C. Montelaro. 1987.Antigenicvariation and

lentivi-rus persistence: variations in envelope gene sequences during EIAVinfection resemblechangesreportedforsequential isolates

of HIV. Virology 161:321-33 1.

26. Power,M.D.,P. A.Marx,M. L.Bryant,M. B.Gardner,P.J. Barr,

and P. A. Luciw. 1986. Nucleotide sequence of SRV-1,a typeD

simian acquired immunedeficiency syndromeretrovirus. Science

231:1567-1572.

27. Preston,B.D.,B.J.Poiesz,and L. A. Loeb.1988.FidelityofHIV-1

reversetranscriptase. Science 242:1168-1171.

28. Pri-Hadash, A., D.Hareven,and E. Lifschitz. 1992.A meristem-related gene from tobacco encodesadUTPase:analysisof expres-sion invegetativeandfloral meristems.Plant Cell 4:149-159. 29. Pyles, R. B., N. M. Sawtell, and R.L. Thompson. 1992. Herpes

simplexvirus type 1 dUTPasemutants are attenuated for

neuro-virulence, neuroinvasiveness, and reactivation from latency. J. Virol.66:6706-6713.

30. Ricchetti, M., and H. Buc. 1990. Reverse transcriptases and

genomic variability: the accuracyof DNA replication is enzyme specificand sequencedependent. EMBOJ. 9:1583-1593. 31. Roberts, J. D., K.Bebenek,and T. A.Kunkel. 1988.The accuracy

ofreversetranscriptasefromHIV-1. Science 242:1171-1173.

32. Roberts, M. M., and S. Oroszlan. 1989. The preparation and biochemical characterization of intactcapsidsofequineinfectious anemia virus. Biochem.Biophys. Res.Commun. 160:486-494. 33. Sanger, F.,S.Nicklen,and A. R.Coulson.1977. DNAsequencing

with chain-terminating inhibitors. Proc. Natl. Acad. Sci. USA 74:5463-5467.

34. Slabaugh,M. B., andN. A. Roseman. 1989. Retroviral protease-like gene in thevaccinia virus genome. Proc.NatI.Acad. Sci. USA 86:4152-4155.

35. Smith,D.B.,and K. S.Johnson. 1988.Single-steppurificationof

polypeptidesexpressedinEscherichiacoliasfusions with glutathi-one S-transferase. Gene67:31-40.

36. Sonigo, P., C. Barker, E. Hunter, and S. Wain-Hobson. 1986.

Nucleotide sequence of Mason-Pfizermonkeyvirus: an immuno-suppressiveD-type retrovirus. Cell 45:375-385.

37. Threadgill,D.S.,W. K.Steagall,M. T.Flaherty,F.J.Fuller,S. T.

Perry, K. E. Rushlow, S. F.J. Le Grice,and S. L. Payne. 1993. Characterization of equine infectious anemia virus dUTPase:

growth properties of a dUTPase-deficient mutant. J. Virol. 67:

2592-2600.

38. Towbin, H., T. Staehelin, andJ. Gordon. 1979.

Electrophoretic

transfer of proteins from

polyacrylamide gels

to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA76:4350-4354.

39. Tye, B.-K., J. Chien, I. R. Lehman, B. K. Duncan, and H. R. Warner. 1978. Uracil incorporation: a source of

pulse-labeled

DNAfragments in thereplicationof theEscherichiacoli

chromo-some.Proc. Natl.Acad. Sci. USA 75:233-237.

40. Wagaman,P.C.,C. S.Hasselkus-Light,M.Henson,D.L.Lerner,

T. R. Phillips, and J. H. Elder. 1993. Molecular

cloning

and characterizationof

deoxyuridine

triphosphatase

fromfeline

immu-nodeficiencyvirus(FIV). Virology196:451-457.

41. Williams, M. V. 1984. Deoxyuridine triphosphate

nucleotidohy-drolase induced by herpes simplex virustype 1. Purification and characterization of induced enzyme. J. Biol. Chem. 259:10080-10084.

42. Williams, M. V., and Y. Cheng. 1979. Human

deoxyuridine

triphosphate nucleotidohydrolase. Purification and characteriza-tion of the deoxyuridine

triphosphate

nucleotidohydrolase

from

acute

lymphocytic

leukemia. J. Biol. Chem. 254:2897-2901.

43. Williams, M. V., and D. S. Parris. 1987. Characterization of a

herpes simplex virus type 2

deoxyuridine

triphosphate

nucle-otidohydrolase andmappingofagene

conferring

type

specificity

for the enzyme. Virology156:282-292.