Proc. Natl. Acad. Sci. USAVol. 83, pp. 9566-9570, December 1986Genetics

Molecular cloning, encoding sequence, and expression of vacciniavirus nucleic acid-dependent nucleoside triphosphatase geneJOSE F. RODRIGUEZ, JEFFREY S. KAHN, AND MARIANO ESTEBANDepartments of Biochemistry, Microbiology and Immunology, State University of New York Health Science Center, Brooklyn, New York 11203

Communicated by Chandler McC. Brooks, August 18, 1986

ABSTRACT A rabbit poxvirus genomic library containedwithin the expression vector Xgtll was screened with polyclo-nal antiserum prepared against vaccinia virus nucleic acid-dependent nucleoside triphosphatase (NTPase)-I enzyme. Fivepositive phage clones containing from 0.72- to 2.5-kilobase-pair(kbp) inserts expressed a P-galactosidase fusion protein thatwas reactive by immunoblotting with the NTPase-I antibody.Hybridization analysis allowed the location of this gene withinthe vaccinia HindIIID restriction fragment. From the knownnucleotide sequence of the 16-kbp vaccinia HindIlD fragment,we identified a region that contains a 1896-base open readingframe coding for a 631-amino acid protein. Analysis of thecomplete sequence revealed a highly basic protein, with hy-drophilic COOH and NH2 termini, various hydrophobic do-mains, and no significant homology to other known proteins.Translational studies demonstrate that NTPase-I belongs to alate class of viral genes. This protein is highly conserved amongOrthopoxviruses.

Although the large 185-kilobase (kb) genome ofvaccinia viruscodes for -200 polypeptides, only a few genes with biologicalfunction have been mapped. This includes thymidine kinase(1, 2), DNA polymerase (3, 4), one subunit of RNA guanyl-yltransferase (5), several RNA polymerase subunits (6), anda polypeptide with homology to epidermal growth factor(7-10).

It has long been known that vaccinia virus contains as partofthe core structure two nucleoside triphosphatase (NTPase)enzymes, which are different antigenically and dependent onnucleic acid for their activation (11-15). Although both aremonomeric enzymes of ":'68 kDa, NTPase-I hydrolyzed onlyATP or dATP, while NTPase-II hydrolyzed all four ribo- ordeoxyribonucleoside triphosphates (13, 14). Both enzymesproduce nucleoside diphosphates and Pi in stoichiometricamounts, and it is not yet known with what function ATPhydrolysis is coupled or the biological significance of theseenzymes. We have previously invoked a role of vacciniaNTPase enzymes to the impairment of interferon (IFN)action (16, 17). This is because in the so-called 2'-5'Asynthetase enzyme fractions from IFN-treated vaccinia vi-rus-infected cells, the synthesis of (2'-5')adenylyladenosineoligonucleotide [ppp(A2'p5')"A], collectively called (2'-5A),was inhibited as a result ofATP degradation. 2'-5'A is a potentinhibitor of protein synthesis that activates a specific RNaseL, which cleaves viral and cellular RNAs (18). Since vacciniavirus might alter the 2'-5A system through degradation ofATP and dephosphorylation of 2'-5A (16, 17), it was ofinterest to establish the role of vaccinia NTPase enzymes onblocking IFN action. To achieve this objective, we firstsought to identify the viral NTPase genes.We present here the molecular cloning, sequence data, and

expression ofthe gene coding for the vaccinia virus NTPase-Ienzyme. This was accomplished through the use of vaccinia

NTPase polyclonal antiserum in conjunction with a rabbitpoxvirus DNA library contained within the expression vectorXgtll.

MATERIALS AND METHODSIsolation and Characterization of NTPase Enzymes from

Vaccinia Virus. The procedures were as described (13).Purified viral cores (35 mg/ml) were treated with a solutionof0.3 M Tris HCl, pH 8.4/50mM dithiothreitol/0.1% sodiumdeoxycholate/0.25 M NaCl. DNA-free viral extracts wereprepared by absorption of viral DNA to DEAE-cellulosecolumns in the presence of high salt, the flow-through wascollected, diluted with buffer A [0.15 M Tris-HCl, pH8.4/0.1% Triton X-100/1 mM dithiothreitol/10% (vol/vol)glycerol/i mM EDTA], applied to a column of DEAE-cellulose equilibrated with buffer A containing 0.05 M NaCl.The ATPase activity collected in the flow-through wasapplied to a column of denatured calf thymus DNA, and thetwo viral ATPases eluted with a gradient of0.05-0.25M NaClin buffer A. Fractions were assayed for ATPase activity bythin-layer chromatography using polyethyleneimine-cellu-lose (PEI-cellulose) plates and 0.75 M potassium phosphate(pH 3.4) as solvent. Standard reactions (30 ,ul) were in 20mMHepes, pH 7.5/1 mM MgCl2/5 mM dithiothreitol/0.05%Nonidet P-40/3 ug of sonicated salmon sperm DNA/20 ,uMa- or y-32p, followed by incubation at 37°C for 60 min.Reactions were stopped by dilution with an equal volume of10% trichloroacetic acid, proteins were precipitated, acid-soluble material was collected after centrifugation for 2 minin an Eppendorf centrifuge, and 2-,u1 aliquots were run onPEI-cellulose plates. By definition, 1 unit ofenzyme will form1 nmol of ADP from ATP in 1 min. After 1251I labeling withBolton-Hunter reagent (ICN), the NTPase-I preparationshowed a major protein of ~'65 kDa.RIA of Vaccinia NTPase-I in Vivo and in Vitro. Rabbit

anti-NTPase-I antiserum was kindly provided by E. Paolettiand was prepared as described (15). For in vivo analysis,mouse L cells (107 cells in 100-mm dishes) were infected withpurified vaccinia virus (500 particles per cell), strain WR. Atvarious times postinfection, cell extracts were prepared in200 Al of buffer B [20 mM Tris HCl, pH 8.4/5 mM EDTA/phenylmethylsulfonyl fluoride (0.3 mg/ml)/1% NonidetP-40] containing 250 mM NaCl. Supernatant was collectedafter centrifugation in an Eppendorf centrifuge, mixed withone-half volume of DNA-cellulose (Pharmacia) equilibratedin buffer B, added to 700 Al of buffer B (without NaCl), andincubated for 1 hr at 4°C with rotation for protein binding tooccur. Thereafter, the DNA-cellulose was washed five timeswith 1 ml each time of buffer B and 50 mM NaCl andincubated for 1.5 hr at 4°C with NTPase-I antiserum (1:100dilution) in 200 ,ud of buffer B containing 50 mM NaCl and 1%bovine serum albumin. The DNA-cellulose was washed fivetimes with the same buffer containing 0.1% Triton X-100,incubated for 1.5 hr at 40C with 200 Al of 251I-labeled protein

Abbreviations: IFN, interferon; kbp, kilobase pair(s); kb, kilobase(s).

9566

The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement"in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 83 (1986) 9567

A (19) (3 x 105 cpm) in the same buffer, washed five times,and radioactivity in the pellet was counted. For in vitroanalysis, total RNA was extracted from virus-infected cellsand translated in the rabbit reticulocyte cell-free system (20),and NTPase-I activity was measured as described above.Antibody Screening of Kgtll Library and Protein Blotting.

A library prepared from randomly generated rabbit poxvirusDNA fragments contained within the expression vector Xgtll(6) was kindly provided by R. W. Moyer. Phages (1.2 x 106)were allowed to infect an overnight culture of Escherichiacoli Y1090 for 15 min at 370C and were plated at 2 x 105 in0.7% soft agar, incubated at 420C for 3 hr. Plates wereoverlaid with nitrocellulose paper presoaked in 10 mMisopropyl /3-D-thiogalactoside and incubated at 370C for 18 hr.Thereafter, filters were washed at room temperature inphosphate-buffered saline (PBS), blocked in PBS/0.5% bo-vine serum albumin/15% fetal calf serum for 1 hr, washedtwice for 10 min with a solution of 0.05% Tween 20 in PBS,and incubated overnight with 1:200 dilution of anti-NTPase-Iantiserum (21). Prior to its use in screening, the antiserumwas diluted 1:100 in PBS/10%o fetal calf serum and pread-sorbed with filters containing E. coli Y1090 cells infected withwild-type Xgtll phages. Immunopositive plaques were de-tected by secondary incubation with "25I-labeled protein Aand cloned.

Isolation of Recombinant Plasmids, Mapping, and DNASequence Analysis. Isolation of recombinant plasmid DNA,purification by CsCl density centrifugation, restriction sitemapping, and subcloning were carried out following standardprocedures (22). DNA sequence analysis was performed bythe chemical modification method ofMaxam and Gilbert (23).

RESULTSSpecificity of the Rabbit Antiserum for Vaccinia Virus

NTPase-I Enzyme. To provide evidence that antibodies bindspecifically to NTPase polypeptide, we sought to establishwhether such interaction removes the ATP hydrolytic activ-ity of the enzyme when the antigen-antibody complexes arebound to a solid support. The results are presented in Fig. 1A.When NTPase-I antiserum was bound to protein A-Sepha-rose beads, no hydrolytic activity was found in supernatant

A12 3 ~45 6 7 8 9 10

as expected from the removal of the enzyme by the boundantibody to the solid support (lanes 1-3); however, whennonimmunized serum was bound to the beads, the hydrolyticactivity was entirely found in the supernatant (lanes 4-6). Inthe absence of binding, purified NTPase-I hydrolized freey-labeled ATP (lane 7) to Pi (lanes 8-10).The specificity of the antiserum was further evaluated by

its ability to recognize a virion protein of -65 kDa in lysatesofpurified vaccinia, rabbitpox, and cowpox viruses (Fig. 1B).The findings of Fig. 1 establish that the antiserum inhibitsNTPase-I activity and also recognizes a 65-kDa polypeptide.

Cloning ofRabbit Poxvirus NTPase Gene. Recently, a rabbitpoxvirus DNA library in Xgtll has been constructed and usedto map two subunits of the viral RNA polymerase withspecific monoclonal antibodies (6). We sought to clone therabbit poxvirus NTPase-I gene from the genomic rabbitpoxvirus library by using the vaccinia NTPase-I antiserum.Immunopositive phage plaques were picked, cloned, re-screened several times, and cloned phages were isolated.Five of these phages were used to infect Y1089 E. coli cells,and the ,3-galactosidase fusion protein was identified bystaining and by immunoblotting using '251-labeled protein A(Fig. 2 A and B). There was no reactivity with lysates fromX wild-type infected cells, and as expected, only a single65-kDa polypeptide is immunoreactive in purified virions(Fig. 2C). Due to the specificity of the antiserum (Fig. 1), weconclude that we have cloned sequences from the rabbitpoxvirus NTPase-I gene.Mapping the NTPase-I Gene on Vaccinia DNA. Southern

blot hybridization analyses were first carried out with re-striction fragments of total vaccinia DNA, which was probedwith 32P-labeled DNA from recombinant phages. A repre-sentative example of blots hybridized with one such recom-binant is presented in Fig. 3A. A single band was found tohybridize, and from the known restriction maps of vacciniaand of rabbit poxvirus DNAs (24) this band maps withinHindIII fragment D for all recombinants. For finer mapping,HindIIID was digested with various restriction enzymes,blotted, and hybridized with 32P-labeled DNA from each ofthe recombinants (Fig. 3B). Based on hybridization data, wehave constructed a map in which the length and physicallocation of the insert is given (Fig. 3C). It is clear that all

B

NI 2 3 4 5 6 7 8

kDa

66-

sw

I

-... - - f,6

29 m

- ATP

1i -.

FIG. 1. Antibody specificity for vaccinia NTPase-I enzyme and for a 65-kDa virion-associated protein. (A) Immune and nonimmune rabbitanti-NTPase-I antiserum (5 ,ul each) was mixed with 200 ,ul of 50% protein A-Sepharose beads in PBS, incubated at 40C for 18 hr, beads werepelleted by centrifugation, resuspended in PBS containing 10% nonimmune serum, incubated 2 hr at 4°C, beads were pelleted, washed 10 timesin PBS, and beads were incubated with 50 units of purified NTPase-I at 4°C for 2 hr. Thereafter, beads were pelleted and ATPase activity wasassayed in supernatants by incubation with [y-32P]ATP at 370C for 5 min, 30 min, and 60 min, and each time point was analyzed by PEI-cellulose.(B) Purified NTPase-I (lanes 4 and 5), and 20 ,ug each of purified vaccinia (lanes 1 and 6), rabbitpox (lanes 2 and 7), and cowpox (lanes 3 and8) viruses were run on gels, immunoblots underwent reaction with anti-NTPase-I antiserum, and proteins were visualized by staining with amidoblack (Left) or after immunoperoxidase reaction (Right). Molecular size markers (lane M) indicated are bovine serum albumin (66 kDa), carbonicanhydrase (29 kDa), and cytochrome c (12.3 kDa).

Genetics: Rodriguez et al.

1. To *tog

Proc. Natl. Acad. Sci. USA 83 (1986)

B12 11 8 7 5

_-4;

-a -

_~~~~~~~~~~~~. ,

..n-IF ~ ~~~~~~~~~s

# ...HIf ,...IP4I~~~~~~~~~~. ._ *'5A

*Ii

Fii 4

FIG. 2. Immunoblot analysis of NTPase-I fu-sion protein. The phage clones (5, 7, 8, 11, and 12)and wild-type Xgtl1 were used to lysogenize bac-

_0 terial strain Y1089. Lysogens were grown at 320C,shifted to 420C, and then induced with isopropylf3-D-thiogalactoside. Lysogen proteins (40 jkg) werefractionated by electrophoresis through NaDod-S04/8% polyacrylamide gel and transferred to ni-trocellulose. (A) Coomassie blue stain of lysogenproteins from recombinant phages. (B) Reactivitywith NTPase-I antibody followed by treatment with125I-labeled protein A. (C) Coomassie blue stain(Left) and reactivity with NTPase-I antibody(Right) of polypeptides from purified vaccinia virus(lanes 1) and of lysogen proteins from Xgtll (wild-type) (lanes 2) infected cells.

independent recombinants have sequences that hybridizewithin the same region of vaccinia DNA.

Nucleotide Sequence and Deduced Amino Acid Sequence ofNTPase-I. Recently, the complete nucleotide sequence of theentire 16-kb HindIIID fragment of vaccinia virus DNA waselucidated (25). Knowing the specific region ofHindIIID thatcontains the NTPase-I gene, as well as the orientation of thegene in the genome, the appropriate DNA nucleotide se-quences were scanned for an open reading frame coding fora polypeptide of -65 kDa. Indeed, an open reading frameexists in a region spanning nucleotides 13,452 (first ATG) tonucleotide 11,556 of HindIIID and coding for a polypeptide

AHI X K S H X K S

!ws_ D __~_a

_

S~~~~~~...am_

B E+H+

HHHkh

of 71,113 Da (open reading frame 11). To confirm that theopen reading frame actually corresponded to the NTPase-Igene, we carried out sequence analysis of overlapping se-quences from the cloned inserts. Both the region of vacciniaDNA coding for the 71,113-Da protein and the rabbitpoxDNA in the expression vector were in the same reading frame(data not shown). We conclude that the open reading framebeginning at nucleotide 13,452 of HindIIID is the NTPase-Igene. The complete nucleotide sequence of the NTPase-Igene and of the corresponding amino acid sequence of theNTPase-I enzyme deduced by computer analysis is shown inFig. 4. It contains a 1896-base open reading frame coding for

BB E B E

HH H H H1Ulkb

39

1. '"

EtBr

,-..

1.9 -

IX'-

12

Vaccinia DNAD_ -HindIl

X \ BamHI

vv ,N

EcR

7--8

12'11

mRNA 0.5 kb

C

FIG. 3. Mapping vaccinia virusNTPase-I gene. Inserts from five recom-

* binant phages were digested with EcoRIand subcloned in pUC9 following stan-dard methodologies (22). The insertshad sizes of 2.5 kbp (Lrl2), 2.2 kbp(Lrll), 0.92 kb (Lr5), 0.78 kbp (Lr7),and 0.72 kbp (Lr8). (A) Blots of restrict-ed vaccinia DNA hybridized with nick-translated total vaccinia DNA (Left) orwith nick-translated 0.78-kb insert. Thefragment assignment (24) is given. (B)Ethidium bromide staining and blots ofpBR322 HindIIID vaccinia DNA frag-ment digested with various enzymesand hybridized with inserts of 2.5 (Lrl2)and 0.9 kbp (Lr5). B, BamHI; E, EcoRI;H, HindIII; K, Kpn I; S, Sal I; X, XhoI. (C) Physical map of the inserts. HpaII (o), or Alu I (r). There is an extraEcoRI site on vaccinia DNA (denotedby vv). D represents fragment D.

A12 Ii 8

C12

k I)a

1 2

kh

9568 Genetics: Rodriguez et al.

Genetics: Rodriguez et al. Proc. Natl. Acad. Sci. USA 83 (1986) 9569

a highly basic protein of 631 amino acids. The hydropathyplot is represented in Fig. 5. There are various hydrophobicsequences, ranging in size from 12 to 26 amino acids. TheNH2-terminal and COOH-terminal amino acids are hydro-philic. A computer search based on comparison of the aminoacid sequence with all other known ATPases and DNA-binding proteins failed to reveal significant homology. Clear-ly, vaccinia NTPase-I enzyme appears to have evolved froma different ancestor.

Regulated Expression of NTPase-I During Virus Infection.Vaccinia virus genes are coordinately regulated during in-

-30 0tAC ?tA MC TAC TMC TOTGSTTIT MA C ATAOta AtT ACT TAT CAC Tca TMA ATc ACT

Mt Sr3o 60

AM WCA CAC OCO QCC TATAs OAT ?AT OCA ITOS C AS ACT ACTaMT ATc CCT Ols GMLIe 9mg is Ale Ala yrTye aeM Tyr Ale Leu A.i Arg hr r AMe met ft0 V.1 01G

9 ~~~~~~~~~120ATc ATc 000 TOC LC csA cTa COC CTC aMO ST UT CM CAT mT OTA OCA AS 01 TMIet Not Oly Sr Aep Vel Vl Atr Lee Lye Aep Tyr OLe Nie Phm Vl Ale Arg VTl ike

150 1009A OSc ItA GLC Acs ATs CAT TCT C ITVA T IO CATGa ACO ONT OTC MOT AM ACALee Oly LoU Aep hr Met Ile ht Leu Lee LeG Whe Uie Glu he Ily Tel Oly Lye Th

210 240ATC ACT ACT TA TAT ATT CTC AMA CAT CIT AMO ST ATT AT ACc aaM TOO OCT ATT ATclet Thr Thr V.1 Tyr Ile Lou Le lie Lou Lye Asp Ile Tyr Thr Aen Tsp Ale II1 IIo

270 300IA ITO OTC AAM AMO OC ITO ATA GhA OAT CCT TOO AO AAC ACT ATA CTC ASA TAC OCT

Leu Lee Vel Lye Lie Ale Leu 11i Ole Aep Pge ry met Mae The I1 Le Arg Tyr Ale330 300

cCA Co ATA ACO aaM T TT AIT 2m AIT AMT TC SCT CMScaa AA SMSSA T AMPro Ole II The Lye Aey Cye I1I ike Ile a TV AspaM peys Asa Pho Arg Ma Lye

390 420ITT IT ACT aaT ATC Aaa ACT AT? AMT TCC AMO A0 AS eA TOC 00 AIT ATT OAT MWho Who 1he Asa I1e Lye Ihr Ile Aea 9g Lie hr Are Ile Cyi Val IleM1 ia p GIe

450 400TO CAT aaC TC AsICT MAA ICA ITA ATC AM SMA ST? M OAIC COT CCc ACT COT

Cyn lis Lea te Ile gerLLye Set Lee l1o LYe 01. Msly Lysie argA t. ghe Arg510 S40

KcA OM AT AMT m V ICT MO AC AIC aCA Ta AM AMC CAT aaM ATc AsTOT ITANg Vi Tyr Aea Who Lee tr Lye Ikr RIo Ale Leo Lye Me RIB Lie met IIe Cie Lee

S70 600TC OCT ACA OCT asc C AMT ACT e Caa GU TMC ACC ATO TO SIT aaC VIA CIA CSer Ale The Pgo Ilie T1 M a fer Vel G1a 01. ikekget Lou tel Msa Lee Leu Agg

630 660CCA Os 2= VI CaM CAC CMA TO Ca IT SO AM AM COT Csa OSIT T Ga AM GAPro Sly met Lee O1elislehr Lee Who Ole MsLye ALg Lee hi AM 010 Lie Ole

600 720A SIC ICC AM CTA OS OCo CsA TOT TOO TAC AU On aT aaC O ITT OCT AIT IT

Lee Tel mg Lye Lee Oly 01y Loe Cy$ ert Tyr I1e Te MAn eAsO Pho ser IIe Pbe750 700

CST SaC GA Sa 00 OCTWcA VOa IC OCTMOSMaaA OIL VIA ATO CS TAC 0MTATaM M hs 0C10 Gly lg Ala Mrcioe Ale Lie Lye Th 1I Lee met Ar Tyr Vi Lae

N10 640ATO TOO aaA aM Ca AG A AIT SAST CAM aa OCT AAA CTC OCT GM ATA aaA ACA 00Net leg LyS Lye Ole 01. 01. Ile ?yr Ole Lye Ale Lye Leu AleaGe IIs Tkr 01

670AaWaTCAV TT ACs AIT CT ASA CT A" OC ACT ACe ?IT ACO VC ST AOC ITT CCSt1i hr hr hoe Ar, Il. Lee Argo Ag let Ale hi hrhikeP hrhe MyohsN o PrO

930 060GU ASh CM ALT COT ST CC0 000 GU TC OCO CMA SO AlA GCA WCA CTA 'UT MRT SLTOme Arl Leae Ago Ase ie0 Ole Tyr Alea GasOl O IAle aA chr Lee acmae

000O 10g0II? MAA MT WAL VIA ASh ST ASR GA TIt TOT AMA OCT WCA IlL SAT ACC ITT MAA LOOPho Lye Lee Nor Loee Ar spM Arg Ole Pke Nor Lye Nor Ale Lee Myp hr Who Lie Lii

OS SaM CIA IO 05o C0 ST OCM LT OC OCCT SAt AsC OCT CsA m. ACT GSA IA AaaSly 010 Lou LS Sly Sly My a ac Ale Alea AsIMlghr Lou ike hr G01 Lee LIe

1110 1140OM AAA AMC 01 AAA Tm AsA sO LGU ItO 5GGL A TLcSQCA IcC CAT 00?AMA StOGle LYe r Vel Lye Who le AM Val Cye Lo 01y Ile Leo Ale Or lie Sly Lye Cyeii?1 1200CIA 01C mcT Sa CCL lIT SIT ALT Cho VO SA ATMIA M CIa taT STC AaaLee Vel ike 010 Pir PbO Vel Me Oe har Sly 1ie010 Ile L4 Leu L Tyr Who Li

1230 1h001C maT sOT AIC OCT AMV A cso T aICA ?ACTS ACA MA ST ACT AS ATOC Mc wCTel FM Sly I1. hr Mm Ie GIoOl sim r SrAtr hr Lye Ayp Tkr ar Ile Lye Ale

1200 132001 OCT 00 Tm aaC Caa SM OA aaC CT AMC O Saa TOC aLT AM Aca TOC OTA ITcValAle GlsikoaMe Oe Ol har s he MeaSly 010 Cie Ie* Lye hr Cie Vel ike

1350 1300T OCT aLGO OS asO SO SOTLIT AC TOc TIT ICA ASC MT ST AIC ITOLTC Vas sT

hor Sh Sh Gly Sly 51. Gly I1. hSeP WhoP ar 3leMsaA ipII Pe Ie Le aMP1410 1440

ATO acA TO aaC SO OC OT CIT co CAO AL OL Oc AScSOC LTcCOT CC aaM aLOmet hr Try Lea Os Ale hr Leo ar OU 1ie Tel Sly Arg All l1e Arg Le As Shr

1470 1500CAC SIT CIT7 ACT OCT CCL SMA COT ASR IAT OILAMC 010 CAC TIT LUt ATO OCT ASR VIAIle Tel Loe hr iro ire oie AL ArS Tyr Tel Ase Tel Eye ik Il3 not Ale Arq lee

153 lS6O~~~~~~~50OCT MT SOM TO CC? ACT OIL SC SM SC CU, ITT SM LIC AlT CMA OC MAA WCAMAAhr Aea Sly Not iro hr Tel Asm 010 AL Leo CIh Ile GIe leht L br Ly

1590031SAMGIT01 CM IoTO AS 010 ITT MA CAT WCA MA VIA SMA TOO LIT CAT OCM MVGO PeWho Ie 5GU Lee iNo Arg Val iNW Lye lie Thr hi Lee Gle Tery 310 lie Ale MeaSM MAA SAC TIC VOL CO AVO ShC MT SOG TOO OT TOO MAA LOC I TOOVICVO AS WCCOle LyesmWPho hrc ire 310 Ley Mea Oles SeGly Tery Lye Thr Lee Tel leg Aig Ale

1710 1740AIC SLT CIA TOO OC MC MAA Mt LIT LC MT? AMA CA LVI 50o 00T ACT MV LVI TOOIle Myp Lee hrt Ser Lee Lye Lee Ile hr Msa Lye Lee II. Ole Sly ThieAs ie1 Try,

1770 16000TAT = MI TOT ALIAS VIA ATO VOL LU MI? ASA OSh TT AM SOC OIL SAT 00? CSRTyrlohr aMe chr aMeLg Leu Net hrele M5As Ar, Sly ike Lie Sly Tel Amy Gly Are

1030 1160OTA TC ST OIL OLC SOT LAO UT CTA CAT SRT LA" COO S~C MT CCIC OTT ATA MAA AUAVsel Tyr Amy TelI Asy Sly Lee Iy Lee Ilie Amy lot ire Asy Lee ire Tel IIe Lys Ile

1900 1920

CCOORT0?MNAA VIA LIT TAT LIT ITOILL VOLW AIM AIC 010 LOT TM TiTf TO TM TGLej Myslyc Lis Le 0 TyC iIeI 10 ad

FIG. 4. Nucleotide sequence and deduced amino acid sequenceof vaccinisa virus NTPase- gene and gene product. Maxam-GilbertDNA sequence analysis of overlapping sequences from clonedinserts were carried out. The nucleotide sequence shown corre-sponds to the open reading frames ORE 11 and ORE 12 (25). An extraC has been found upstream from ORE 11 in ORE 12, putting thesetwo ORFs in-frame and, in turn, codings for a protein of 631 amianoacids. The nucleotides are numbered above the sequence in the 5' to3' direction; nucleotide 1 is the A of the ATG codon for the initiatormethionine. Amino acids are numbered beginning at the initiatormethionine.

i.003

Vaccinia NTPase-I

Residue

FIG. 5. Hydropathy plot of the NTPase-I enzyme. Hydropathyanalyses and homology comparisons were performed by using theInternational Biotechnologies sequence analysis programs. Thehydropathy program is based on the procedure of Kyte and Doolittle(26). Hydrophobic regions are above the x axis and hydrophilicregions are below it.

fection (27). From immunological studies, it has been inferredthat vaccinia NTPase enzymes are synthesized late in infec-tion (15). We have developed an RIA to measure NTP levelsin the intact virus-infected cell as well as in cell-free proteinsynthesizing systems. Typical results are presented in Fig. 6.It is clear that in virus-infected cells NTPase-I is increased atlate times postinfection (Fig. 6, in vivo). When the levels ofthe NTPase-I were measured in a cell-free system in responseto RNAs prepared at various times postinfection, a markedincrease was observed with late RNA (Fig. 6, in vitro). SinceNTPase-I was found to be increased both in vivo and in vitrowith samples prepared at late times postinfection, we con-clude that NTPase-I belongs to the late class of viral genes.

DISCUSSIONIn this report, we describe the cloning, mapping, encodingsequence, and expression ofthe vaccinia virus gene encoding

RIAIn vivo In vitro

30S

25C/I

x 20

C 15 120

10 8

5-40

HU 1 3 5 0 2 5Time postinfection, hr

FIG. 6. Vaccinia NTPase-I is expressed late in infection. Mea-surements by RIA of NTPase-I in the intact virus-infected cells (invivo) and in the rabbit reticulocyte cell-free system programmed withtotal RNA extracted from virus-infected cells (in vitro) were asdescribed. Values represent total radioactivity recovered in the pelletas antigen-antibody complex bound to DNA-cellulose after subtract-ing the radioactivity incorporated in samples from uninfected cells.The in vitro values given represent the amount of '25I incorporatedper constant amount of newly synthesized 35S-labeled proteins ateach time point. HU, infected cells treated with hydroxyurea andcollected 5 hr postinfection.

Proc. Natl. Acad. Sci. USA 83 (1986)

the nucleic acid-dependent NTPase-I enzyme. Several linesof evidence support this conclusion. (i) The polyclonal rabbitantiserum specifically reacted with purified NTPase-I en-zyme; (ii) the antiserum recognized a 65-kDa virion proteinin different strains of Orthopoxviruses; (iii) after screeningthe rabbit poxvirus DNA library with the antiserum, fivedifferent recombinants with overlapping inserts were iso-lated; (iv) the strand assignment, direction of transcription,and nucleotide sequence ofthe cloned inserts is placed withina 1896-base sequence in HindIIID fragment that codes for apolypeptide of 71,113 Da; (v) this polypeptide is highly basicand has ATPase activity. Two monomeric vaccinia NTPaseenzymes of 68 kDa have been previously isolated frompurified virions, and both can be distinguished by substratespecificities, nucleic acid dependence, and neutralizationwith specific antisera (13-15). Our results suggest that at theDNA level the two viral NTPase genes must be different,since hybridization analysis revealed only a single locus forthe NTPase-I gene.The physical location of the viral NTPase-I gene within

HindIIID DNA fragment is of interest. This 16-kilobase-pair(kbp) region has recently been sequenced and a number oftemperature-sensitive mutants have been mapped (25). Thesignificant features of neighboring sequences to the NTPase-Igene are as follows: (i) To the right of the NTPase-I gene(spanning HindIIID to HindIIIA), several other genes aretranscribed in the same leftward direction. These are a geneencoding a 35-kDa early protein of unknown function (28), agene encoding a 63- to 65-kDa late protein that confers theresistance-of vaccinia virus to rifampicin (29, 30), and twoother contiguous late genes encoding major (4b and 4a)core-associated polypeptides (31, 32). (ii) To the left of theNTPase-I gene, there are two open reading frames trans-cribed rightward, which could code for 28- and 24-kDapolypeptides (25). These findings stress the notion thatvaccinia virus genes are closely packed along the viral DNAand that transcription initiation and termination signals areclose or overlap with neighboring genes (6, 32-38). A con-served feature of late genes is the TAA sequence thatimmediately precedes the ATG translation initiation codon;the vaccinia NTPase-I gene does have this sequence.The role of vaccinia NTPase-I on virus multiplication and

in virus-host cell interactions is not known. Within the virionstructure, there are several enzymes that are ATP dependent.These include RNA polymerase, capping enzymes, poly(A)polymerase, and topoisomerase (27). Since energy-trans-ducing enzymes are involved in many biological processes,including transcription, DNA replication, DNA repair, andDNA recombination (39), the vaccinia NTPase enzyme couldplay a major role in virus multiplication. It is a well-knownphenomenon that in the course of vaccinia virus infectionthere are major morphological and biochemical changes inthe host cell (40). Biochemical changes associated withvaccinia virus ATPase activity are those found in IFN-treatedinfected cells. Vaccinia virus is relatively resistant to IFN inmany cell lines, and this resistance to IFN has been associ-ated with alterations of the 2'-5'A system (16, 17). Theavailability of the vaccinia NTPase gene and gene productwill undoubtedly provide the means to characterize thefunctional significance of this enzyme during virus-host cellinteractions.

Biochemical and sequence data indicate that there isprotein sequence homology between ATPases from widelyseparated species (41-43). However, by computer search nosignificant sequence homology has been found between thevaccinia enzyme with other known proteins. It appears that

the -vaccinia enzyme might have evolved from a quitedifferent ancestor.

We are particularly indebted to Dr. Enzo Paoletti (Albany, NY) forhis support and generosity in providing us with anti-NTP-I serum andpurified enzyme. We would like to express our gratitude to Dr. R. W.Moyer (Vanderbilt University, TN) for the gift of rabbitpox library,Dr. E. G. Niles (State University of New York, Buffalo, NY) formaking available the sequence of HindIIID fragment before publi-cation and for pointing out to us the extra C in open reading frame12, and to Dr. W. T. McAllister for computer facilities. We thankVictoria Jimenez for technical assistance and graphic arts. Thisinvestigation was supported by National Institutes of Health GrantsAl 16780 and CA 44262. J.F.R. was supported by a fellowship fromConsejo Superior de Investigaciones Cientificas, Spain.

1. Hruby, D. E. & Ball, L. A. (1982) J. Virol. 43, 403-409.2. Weir, J. P., Bajszar, G. & Moss, B. (1982) Proc. Natl. Acad. Sci. USA

79, 1210-1214.3. Traktman, P., Sridhar, P., Condit, R. C. & Roberts, B. E. (1984) J.

Virol. 49, 125-131.4. Jones, E. V. & Moss, B. (1984) J. Virol. 49, 72-77.5. Morgan, J. R., Cohen, L. K. & Roberts, B. (1984) J. Virol. 52, 206-214.6. Morrison, D. K., Carter, J. K. & Moyer, R. W. (1985) J. Virol. 55,

670-680.7. Venkatesan, S., Gershowitz, A. & Moss, B. (1982) J. Virol. 44, 637-646.8. Blomquist, M. C., Hunt, L. T. & Barker, W. C. (1984) Proc. Natl.

Acad. Sci. USA 81, 7363-7367.9. Brown, J. P., Twardzik, D. R., Marquardt, H. & Todaro, G. J. (1985)

Nature (London) 313, 491-492.10. Reisner, A. H. (1985) Nature (London) 313, 801-803.11. Munyon, W., Paoletti, E., Ospina, J. & Grace, J. T., Jr. (1968) J. Virol.

2, 167-172.12. Gold, P. & Dales, S. (1968) Proc. Natl. Acad. Sci. USA 60, 845-852.13. Paoletti, E., Rosemond-Hornbeak, H. & Moss, B. (1974) J. Biol. Chem.

10, 3273-3280.14. Paoletti, E. & Moss, B. (1974) J. Biol. Chem. 10, 3281-3286.15. Paoletti, E., Cooper, N. & Moss, B. (1974) J. Virol. 14, 578-586.16. Paez, E. & Esteban, M. (1984) Virology 134, 12-28.17. Paez, E. & Esteban, M. (1984) Virology 134, 29-39.18. Lengyel, P. (1982) Annu. Rev. Biochem. 51, 251-282.19. Kessler, S. W. (1975) J. Immunol. 115, 1617-1624.20. Esteban, M., Benavente, J. & Paez, E. (1984) Virology 134, 40-51.21. Rodriguez, J. F., Janeczko, R. & Esteban, M. (1985) J. Virol. 56,

482-488.22. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning:A

Laboratory Manual (Cold Spring Harbor Laboratory, Cold SpringHarbor, NY).

23. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, 499-560.24. Macket, M. & Archard, L. C. (1979) J. Gen. Virol. 45, 683-701.25. Niles, E. G., Condit, R. C., Caro, P., Davidson, K., Matusick, L. &

Seto, J. (1986) Virology 153, 96-112.26. Kyte, J. & Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132.27. Moss, B. (1985) in Virology, eds. Fields, B. N., Knipe, D. M., Chanock,

R. M., Melnick, J. L., Roizman, B. & Shope, R. E. (Raven, New York),pp. 685-704.

28. Tartaglia, J. & Paoletti, E. (1985) Virology 147, 394-404.29. Tartaglia, J., Piccini, A. & Paoletti, E. (1986) Virology 150, 45-54.30. Weinrich, S. L., Niles, E. G. & Hruby, D. E. (1985) J. Virol. 55,

450-457.31. Witteck, R., Richner, B. & Hiller, G. (1984) Nucleic Acids Res. 12,

4835-4848.32. Rosel, J. & Moss, B. (1985) J. Virol. 56, 830-838.33. Venkatesan, S., Baroudy, B. M. & Moss, B. (1981) Cell 25, 805-813.34. Bajszar, G., Wittek, R., Wier, J. & Moss, B. (1983) J. Virol. 45, 62-72.35. Bertholet, C.j Drillien, R. & Wittek, R. (1985) Proc. Natl. Acad. Sci.

USA 82, 2096-2100.36. Weir, J. P. & Moss, B. (1984) J. Virol. 51, 662-669.37. Plucienniczak, A., Schroeder, E., Zettimeissl, G. & Streeck, R. E.

(1985) Nucleic Acids Res. 13, 985-998.38. Hirt, P., Hiller, G. & Wittek, R. (1986) J. Virol. 58, 757-764.39. Kornberg, A. (1980) in DNA Replication, ed. Kornberg, A. (Freeman,

San Francisco), pp. 625-639.40. Bablanian, R. (1984) in Comprehensive Virology, eds. Fraenkel-Conrat,

H. & Wagner, R. R. (Plenum, New York), p. 391-429.41. Kanazawa, H. & Futai, M. (1982) Ann. N. Y. Acad. Sci. 402, 45-64.42. Schull, G. E., Schwartz, A. & Lingrel, J. B. (1985) Nature (London)

316, 691-695.43. Brandl, C. J., Green, N. M., Korczak, B. & McLennan, D. H. (1986)

Cell 44, 597-607.

9570 Genetics: Rodriguez et al.