Nucleotide sequence of the large double-stranded RNA segment of bacteriophage phi 6: genes specifying the viral replicase and transcriptase.

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JOURNALOFVIROLOGY,Apr. 1988, p. 1180-1185 0022-538X/88/041180-06$02.00/0

Nucleotide Sequence of

the

Large Double-Stranded

RNA

Segment of

Bacteriophage +6:

Genes

Specifying

the

Viral

Replicase

and

Transcriptase

LEONARD MINDICH,1* IRIS NEMHAUSER,"2PAULGOTTLIEB,' MARTINROMANTSCHUK,3 JACOB CARTON,' STEPHEN FRUCHT,1JEFFREY STRASSMAN,1 DENNIS H. BAMFORD,3 ANDNISSE KALKKINEN4

Department ofMicrobiology, the Public Health ResearchInstitute ofthe City of New York, Inc.,' and Department of Microbiology, New York University School of Medicine,2 New York, New York 10016, and Department of Genetics3 and

Recombinant DNA Laboratory,3 University of Helsinki, SF-OO100 Helsinki, Finland

Received 19 October1987/Accepted22November1987

Thegenomeof thelipid-containing bacteriophage

+6

containsthreesegmentsofdouble-stranded RNA. We

determined the nucleotidesequenceof cDNAderived from thelargest RNA segment (L). Thissegment specifies theprocapsid proteinsnecessaryfortranscription andreplication of the

+6

genome. Thecodingsequencesof thefour proteinsonthissegmentwereidentifiedonthe basisof size and the correlation ofpredictedN-terminal

amino acid sequences with those found through analysis of isolated proteins. This report completes the sfquenceanalysis of

+6.

This constitutes the firstcompletesequenceofadouble-stranded RNAgenomevirus. Bacteriophage +6 infects the plant pathogen

Pseudomo-nasphaseolicola HB1OY. Three separate pieces of

double-stranded RNA composeitsgenome,which is located inside apolyhedral nucleocapsid (26). The nucleocapsid has RNA

polymerase activity (20) and is covered byalipid-containing

membrane (26). The assembly pathway involvesthe forma-tion ofa polyhedral procapsid composed of four proteins,

P1,P2, P4, and P7, which is then filled withonecopyeach of thethreepieces of double-stranded RNApervirion (4, 16). These filled procapsids are then covered with a shell of

protein P8tobecomenucleocapsids, whicharesubsequently

enveloped within the lipid-containing membrane (15). This envelopment process is dependent upon the activity ofthe nonstructural protein P12 (17). The fourproteins that

con-stitutetheprocapsidarecoded for by the largest of the three

genomic segments,andtheirsynthesis begins early in infec-tion (25). Theprocapsid is responsible for genomic replica-tionandtranscription (24).

The study of the phage assembly processhas been facili-tatedby the cloning of cDNA of each genomicsegment(18). We recently determined the nucleotide sequence of the cDNA derived from the small RNA segment (12) and the middlesegment(P. Gottliebetal.,unpublished data). In this communication, we describe the nucleotide sequence

anal-ysis of cDNA derived from the large genomic segment designated L.

MATERIALSANDMETHODS

Bacterial strains, phage, and plasmids. Escherichia coli JM105 [A(lac pro) thi strA endA sbcB15 hsdR4 F' traD36 proABlacIq ZAM15]wasusedas ahost forM13cloning.E.

coliHB101 (hsd20,anrK- mK- strain) and MM294 (an rK-mK+ strain) were used as hosts for clones with plasmid

pBR322orpUC8 as avector. Phage M13 (mplO and mpll) replicative-form DNA (14) was used to clone cDNA frag-ments for the sequencing reaction. Plasmids pLMF72, pLMF306, pLMF308, and pLMF309arepBR322derivatives containing cDNA insertfragments ofsegment L(Fig. 1).

*Corresponding author.

Preparation of DNA. Purified single-stranded M13 phage DNA for thesequencing reaction templatewaspurifiedfrom

1.5-ml cultures by the procedure in the Amersham Corp. M13cloning andsequencing handbook. Largeplasmid

prep-arations(200-to500-ml overnight cultures)wereprepared by

the cleared-lysate method of Clewell (2), with subsequent CsClgradientcentrifugation. Restriction digests used endo-nucleasessupplied by Boehringer Mannheim Biochemicals, andthebuffer conditions werethose specified by this

man-ufacturer. Ligation reactions usedT4 DNA ligase supplied by Collaborative Research, Inc. Conditions were as

de-scribed in the M13 cloning and sequencing handbook. Procedures for 0.8% agarose and 5% polyacrylamide gel

electrophoresis have been previously described (13). Trans-formation of E. coli JM105 was as described in the M13

sequencing and cloning handbook. Transformation ofE.coli HB101 and MM294 has beenpreviously described (18).

Proteinpurification and amino acid sequencing. Unlabeled and[3H]leucine- and[3H]alanine-labeled 46h1sweregrown

and purified as described previously (1). Preparative and

analytical protein gel electrophoresis was performed as

previously described (1). Individual proteins electrophoreti-cally eluted from gel slices were free of contaminating materialasanalyzed by subsequent sodium dodecyl

sulfate-polyacrylamidegel electrophoresis.

Twosequencingstrategieswereusedtolocalize the amino terminus of the individual proteins upon the nucleotide sequence. First, unlabeled proteins (to which

14C

label had

been addedto facilitate protein localization within the

pre-parative sodium dodecyl sulfate-polyacrylamide gel) were

subjected to automated Edman degradation in a Beckman

890 Dsequencer and a0.1 MQuadril program. Theamino

acids were identified as their phenylthiohydantoin

deriva-tives by high-pressure liquid chromatography (Varian 5020 liquid chromatograph; UV-5 detector,269nm).

In the second method for radiosequence analysis, the eluted tritium-labeled proteins were degraded as described

above, together with 200 ,ug of apomyoglobin. Ninety

per-cent of each phenylthiohydantoin-derivatized sample was

counted by liquid scintillation, and the remaining 10% was

analyzed by high-pressure liquid chromatography, with the

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GENOMIC SEGMENT L OF +6 1181

scale(kbp)

segment L

6374 bp

1 2 3 4 5 6

Ene 7 ; gene 2; 1 gene 4ZE gen X

gone 7 gone2 gne4 gone1

fragment308

fragment309

fragment 306

fragment72

FIG. 1. Restriction map of the entire large genomic segment. Sites were obtained from restriction nucleasedigestion of the cloned cDNA fragments indicated. The illustration is aligned with the direction of transcription to the right. cDNA fragments used for determination of the nucleotide sequence are shown below the map. Kbp, Kilobase pairs; bp, base pairs.

knownsequence ofcarrierapomyoglobin used as a control for sequencerperformance.

DNA sequencing. Nucleotide sequence analysis was per-formed by the dideoxy chain termination methodof Sanger etal. (23). The sequence was determined for both strands. Fragments ofcDNAfor sequencingwerefirst cloned into the unique restriction site region of the replicative form of bacteriophageM13 DNA. Forthiswork, M13 vectors mplO and mpll were used (14). Our strategy involved the use of specific restriction fragmentsaswell as the use of unselected collections of restriction fragments generated by treatment with TaqI. Sequences were determined on over 550 frag-ments.Single-strandedDNAisolatedfrom mature M13 virus servedas atemplatefor dideoxy sequencing. The polymer-ase reaction was primed with a 17-nucleotide primer pur-chased from P-L Biochemicals, Inc., and chain extension waswiththeKlenow fragment of DNA polymerase I (Boehr-inger Mannheim Biochemicals). Labeling was performed with

[a-35S]dATP,

>600Ci/mmol (Amersham Corp.).

Dideoxy sequencing using dITP and reverse transcriptase. Some sections ofthe sequencedisplayed GC compressions. We resolved them by substituting dITP for dGTP in the reaction mixture (22). Chain extension reactionsusedavian myeloblastosis virus reverse transcriptase (Molecular Ge-netics Resources). In these reactions, the molar ratio of deoxy and dideoxy nucleotideswasaltered fromthat ofthe Klenowfragment-catalyzed reaction. Final reaction concen-trations were0.77 ,uM dATP to0.027 ,uM ddATP, 889 ,uM dCTP to819 ddCTP, 889 ,uM dTTPto 8.9 ,uM ddTTP, and 220 ,uM dITP to 0.35 ,uM ddGTP. The reaction buffer contained 33 mM Tris hydrochloride (pH 8.3), 6.7 mM

MgCl2,

46.7 mM KCI, and 20 mM mercaptoethanol. The

17-merprimerwaspresent at 0.3 ng perreaction. Thechain termination reactions were

catalyzed by

2.5 U of avian myeloblastosis virus reverse transcriptase at

42°C

for 20 min. These weresubsequentlychased withamixture of four deoxynucleotide triphosphatesat222 ,uMeach for an addi-tional 20min. Eachchain termination reaction contained2.5

,uCi

of

[a-355]dATP.

35S-labeled

DNA

fragments

were dena-tured and analyzed

by gel

electrophoresis

on 6 or 8% polyacrylamide

gels

(42.5 cm

long)

containing

8Murea, 89 mM Tris-borate (pH 8.3),and 2 mM EDTA.

Computer analysis. The computer

facility

of the Public Health Research Instituteofthe

City

ofNewYorkwasused.

It consists ofthe Vax 11/750 computer equipped with the multiuser UNIX operating system. Nucleic acid analysis programs were provided by the sequence analysis package of the Biomathematics Computation Laboratory, Depart-ment of Biochemistry and Biophysics,University of Califor-nia at San Francisco.

RESULTS

cDNA cloning of the 416 genome was described by Min-dich et al. (19). Plasmids were constructed that bore frag-ments of the L segment, and the protein reading frame positionswere estimated by restriction site analysis in con-junction with in vivo complementation studies, in vitro coupled transcription translation of plasmid DNA, and ref-erence to the cloning work of Revel et al. (21). Ultimately, the readingframe assignments were made by the identifica-tion ofthe N-terminal amino acid sequence of the isolated proteinsand comparing these with thesequences predicted fromthe nucleotide sequence of the cDNA.

The large genomic segment of416 contains 6,374 nucleo-tides andit has55.5%guanine-cytosine. Four reading frames werefound that correspond to the four proteins known to be coded forby this segment (Fig. 1 and 2). As with the other two genomic segments, there were appreciable noncoding sequences at the termini. Thetermini of the segment agree with the sequence found by Iba et al. (9) by direct sequenc-ing of the genomic RNA. The order of the genes is in agreement with thatfoundby Revel et al. (21) on the basis of theprotein synthesis competence of fragments on plasmids containingT7promoters. Thesalientresultsof the sequence analysisare asfollows.

Gene 7 is the first significant open reading frame in the segment.The readingframe starts with an AUG at position 458 and terminatesat position 940 with a UGA. Thereis a Shine-Dalgarno sequence preceding the initiation codon (Fig. 2). The calculated molecular mass ofP7 is 17.3 kilo-daltons. The amino-terminal methionine is partially absent on the basis of amino acid sequencing. The sequence pre-dictsanabsence ofcysteine, andthiswas confirmedby the lack of labeling ofP7 in vivo when

+16-infected

cells were exposed to radioactive cysteine (results not shown). P7 is predicted to have a high net negative charge, and this is consistent with the behavior of the protein in isoelectric focusing gels (17).

Gene 2 follows gene 7. The initiating codon begins at position943 with anAUG that overlaps the UGA termina-tion codon of gene 7. Gene 2 does not have a discernable Shine-Dalgarno sequence in the

vicinity

of the initiating codon. The reading frameends atposition 2937. The termi-nationcodonis UAA.ThecalculatedmolecularmassofP2 is 74.8 kilodaltons, and the N-terminal methionine is lost. The predicted amino acid composition indicates a basic protein, consistent with itsbehavior in isoelectricfocusing gels (17).

Gene4followsgene 2. It hasaShine-Dalgarnosequence, and the

initiating

codon is an AUG

beginning

at

position

2943. The reading frame terminates at position 3938. The termination codon is UAA. The calculated molecular mass ofP4 is 35.0

kilodaltons,

and the N-terminal methionine is lost.

Gene 1follows gene 4.Ithasa

Shine-Dalgarno

sequence, the initiating codon is an AUG

beginning

at

position 3951,

and thereadingframe terminatesat

position

6257 with UAA. Itisnotablethatgene1ends

only

117 base

pairs

from the end ofthe segment.This is much closer thanthosefor the small VOL.62, 1988

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[image:2.612.62.303.76.211.2]

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CMT CC CM ICTMC517 AC? ICC1WChCCS CC?Amh ICCe #0?71M 50aIC AM511 CMIWASICSCTMICCMTgM15CC 1644

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MAMOCIO A CCGMG ICACICAC7 AM?CACC? ?A ATOAM00SOCSC?CC?SAC?CCAGMCWGC SOTC CM T an7WWACMSI?CCC7,CC 204

Ho1Lye LeeGluSly.7zeeLysa es peleeg rTyW notLysVol Slylet3Wye3WuNi Gl 1lylly 7hzleel eeSlyIspIl 3Wuo Syc M&C ICQC?T COTGWAO 7C SOTCC ICCCAC T01CMOST1WTIACCScACCAICSCGA1W ACCCCICCM ICMCMCC C 010 21044

lSeeArWI Le1eg le olSlyleer SecSleSly ValSlyAsa7hz lop lee Set Sly 7A" Lee LeeSet,seclIeGiz Tyclee Val Sle

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IC?GMCTOMAW7 C0MIC? 011ICC WeMS10 CTCIOCTC AIT?OM 071a MTCM1aW C? CT?011C CMTC COC ICC1WM1 23923

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VOL.62, 1988 GENOMIC SEGMENT L OF ct6 1183

weanmWam KW1AMGC tanO SMC6?"a"aa aMGTIMtcranIIC66 CMGOTCATrammaTmGACTamCaM amCI"TSOT 3713

VOLNetV.1 V.1"As Pt. Met ValAapAla61.LysI1.61.TyrValPh.6ly6inValmetAlaseat 1hsVal61yAla It.LoaCya

ml 61?66CAmeOt.maMA ammmMvcmanAMamAAA ?COTA??TICM&CGOTmaGam cc?Caam amSC WVCmCAte M3

Al.Asp 61yAm.Va1setAUV1zEatPMaAre1hzA"Is. aLyeyAUVI. Ph.A."61yAl.AlaProLouAlaAt.Asp1hzlugNot amIMC£1 MITcaC61 AmCAmCA AMamCICcmaTCA?M Icam6 mC McrmC GMcogc1oem mca aem21 noGIL6OAC 3663 Pt. EazEa8tASPAn~IP.1hsSet MtLyeAla,LosAMp3s.hzSatIteAla,gatValAl.ft.OLa. Gla AtyStySatValAsp1h

81:Pt II

hc61? ccC M?m m CCCcCCcmca661 WIamC?WCTMaCia UaMO1A?AMA mC MC CIcAMLa"AM 1?mieaC661 3942 AmA"pA"Ass SWAla,proAtga" lymeame.no.S.zL.. IP1I tm" nLGD "A mA l ma am Coemcan mamCTcm mMcamWCm0GMma"a a cmChoCMaI= cGMaO Twoc1M 6 CCCOM CMmTCma 4673 SatIiAtyStybM1z 61.M&PMm& II. 6ly61.if LyeAs.61n,inseatVa1SlyW&aIn63Ai Pa. M6MAVWh UK

AOleh Ph.UazGIsAS,tMF1hzAl See61 V6616A sp61.oV.1alyLyeGIs Am II. AsTyrUt.V1i,S tb AU.rAsp IaPh.Ph. WC m cAMmaam emG TheMCmamaGm2aT=6A maacONMmcmawe TA?W an acma ma mc am Amcom 611 4363

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I

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I

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II

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I

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emTmama maAmaamamAcm166mcXam e maCGAtaMCA? 616mancmTC AMGmamc am amm=AaGAGMcAm 4573 61.Ila.SlyEat 1hzGle1.P.teIlEaatoHt4Alah31 I W Eat IS.VtI So Pt. Ph. LyeLaAspTPtGU n Ams 61. 1hz 1hz Tst

TTamc aemma: amm maCama"TCMatemTCmanmaam catA=mamca m emMA Ca IaChomcm amneamamcA 1663

AyaIt.6110y63.1zSMmAlaVal.s ESeEtUsl 61.Pal SmOAss V.1Us.1.1 Tyr 61GApIspua 61. Ph. AlaLye61. 31. 1m AMAMcma OMmcamo Ama mCCam ammaIancMT mcma Gwmcemcm emamtmacOCT aTC OMa amC manma 6163

amAssP 1hz Pt.ly V.1LyeLouAlAmYAmPrSm W61. Asp Ph.LAnAsp V.1 61. Pt. Asly tAS Asp SmHoSmls Ala1hzAlaAl

Al.PTyrt 6y~1Ala ValAlaRiAsPrGIVah St Ph.Val. 10alyeA asp1laV.1al lAl1l .VolSmy61. AspSlyr 1 Val?T Am I

amCamcACTmAGem ohAe"eaa amcThema m= mTcWAmc amc mc amAcm mcCO ham ccTMaGmAtemcmaa amTam 5"33

Sml AmlyAl.61.oMMt11hzrV.alSlyPhTyrSmA.1I.1 61. Asp Asp Tyt A.166 Asp AspAsPtoG.IEM V Al.MetIl Al. Alr

a AMc CCa emCCammCC TMt WA MCmGema=mcUAm maOG TMcmaAmmamAammA mMArCh MC TMaCOG616 Ate 6433

366AysPr IleGIyP1. AspuValSSet La.61.AlaAysp Ala Sm Amsp 166AlyeAspSmo Eat Ph.AmtTye Tyt Al.T.Pro EMI

catMT TM Am Gm CMMtam AM mammcGmaeeMOCmA= emCcA mc c?ACcAam ma cmarcmAa TICGUamC 531

SOleyAl.a V.1Al. be.AmaTyPt. 61.AV.1V.1 B.1Sm61.l61.1.SlyV.1y iVTrl Al1 1 GlyaSm LaIys Gla.V1 Tsp Amu V.1Asp hz61yLIa. AspIrt.Pt.AV1 SalyAyt11Am Al.Ea6.1ytyStI.asy C l I 1hz Pt. 61n Pot. Ia. 61. At. T. Ala TyVa GACam Cm AmCma GmaeWA aTe amAC C maCmCGA 1maCG mW AMCmT ma AmC maOMCmf am mTGCA tAw caT 5603 VamhLyePt. 31.61 Pt.IIt61. VatLa 1.Aly LyeVh.1l IleGlAlpLa. AmGtIshleVlRiAla 1* etuls 1. tsps PtAspGS.

I

SoaAlaSatyArt IalPhAl.iy61AspAl.SegyoatV1thAspLogiVaGlIet AmsLyAsg tyt1hzisAt1161Ala.1Le 1 Ph. 61, IalY

II 64

37

FIG.

2.Sequence

of cDN ApAsAp1tLaAlAl.geoAt.At.enLoz E6Thz t.AepuAspeAla thayet6Atfthep t Aspratd. ITteAlanAslat. o rdcso tefu n w

ormiddle

genomic

segVmen 1tsL.L..1hheSlcalaulyateadatoAlaVutlar,Ly 16.Et 1 h La.sA.infsctithanVas spmAsp 61.ml h

irlpocpi

ofPlis85.0 kildalt 1nsThemN-terminmamlmeticmca tinremcatins ma c mc mmca lm m mm c54

EaAalyAsSy. 3. spAs St e Ap a.s atSl taAmA16 S A.tAse,pro

apTspilaS

isaAloablohVh.1a srpts n withthe

basicityfou

La. 61.iseMlectriy166Lacetusin 1t76.Alp1.AarochzLe. apsidsmrAmtA166re EinthV.eAla o a p n o eah do

FIG.2Sequnceo cDNA f genmic sgmentL of 46 Th sequnce i thatof8thAnstand.siThe

transltoionpoductsy

of thefourknrown

withth

basictyfouDbyISoelectIcN

focsig

17.uroapidstedcattureintefr

fcasi

compoen

an

dodecahedrone

Thefourproteinscoded for by the largegenomicsegment cules each of P1 and P4, about 20 molecules of P2, and are

P1,

P2, P4, and P7. Theseproteinsaresynthesizedearly between 80 and 100 molecules ofP7. Although the results of

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1184 MINDICH ET AL.

this report change the predicted molecular weights for the proteins, the changes are not great enough to alter the expected stoichiometry oftheparticle significantly. Recent studies (10, 19) suggest thatP1 itselfcan forma dodecahe-dral structure. Itisnot knownwhether the active site(s)for polymerase activity is shared by different proteins or whether it is located within one ofthe four proteins ofthe procapsid; however, no RNApolymerase activity hasbeen demonstrated in any structure simpler than an RNA-filled procapsid.

Acomparison of the amino acidsequencesofproteins P1, P2, P4, andP7againstthe Dayhoff proteinsequencelibrary as of April 1987 showed no significant similarity to other proteins. However, a search for sequences suggestive of nucleotide-binding sites revealedthat P4containssequences highly similar to those found for a class of proteins that includesthemultiple drug resistance proteins oftumorcells, transportproteins ofbacteria, and UVrepair proteins (Fig. 3) (7,8, 27). Themeaning of this site inP4isnotclear. Since the

procapsid

is involved in RNApolymerization and pack-aging andmustitself be assembled, wecouldspeculate that the P4 site is involved in one of these processes. This sequence is, however, not characteristic of polymerases. Recentunpublished work inourlaboratory indicatesthat P4 has nucleotide triphosphate

phosphatase activity.

Iba et al. (9)showedthat thereisidentity among the ends ofthe threegenomicsegmentsfor17basesatthe3'end and for18basesatthe5'end,with theexception ofadifference in the secondposition. We have shown that the identity at the

3'

endismoreextensivebetweenthesmalland medium segments(Gottliebetal., unpublished data). We nowshow thatthis istrueforthe largesegment as well. Although the exact identity at the 3' end stops at nucleotide 18, there is overallsimilarity that continues until 80 nucleotides fromthe 3' end (Fig. 4). It appears reasonable that the regions of similarity should be involved in the regulation and mecha-nisms of transcription, replication, andgenome packaging.

During infection, production of proteins P1, P4,and P7is almostidenticalon amolar basis. ProteinP2 isproducedat about10% ofthe rateoftheothers (25).Nonsensemutations ingene 7 arecompletely polarontheproduction ofP2(11). Itappears that the mechanism of control ofthesynthesis of P2 is translational coupling. The same motif has been de-scribed fortwoothergenepairs in4)6. Production ofP12is dependentonribosome loadingongene8,whichis immedi-ately upstream, and production of P5 is dependent on ribosomeloadingongene 9 (12). Inbothofthesecases, the downstreamproductis synthesizedat about10% of the rate ofthe upstream product, and nonsense mutations in the upstream genes are completely polar on the production of the downstreamgene product. In the three cases, the

ribo-some-binding

site upstream from the amino acid initiating

RWPSEGIYSGVTALMGATGSGKSITLNE 11 III GLNLKVKSGQTVALVGNSGCGKSTTVQL NINLSIKQGEVIGIVGRSGSGKSTLTKL DLNFTLRAGETLGIVGESGSGKSQSRLR DINLDIHEGEFVVFVGPSGCGKSTLLRM GVSLQARAGDVISIIGSSGSGKSTFLRC NINLDIAKNQVTAFIGPSGCGKSTLLRT NINLVIPRDKLIVVTGLSGSGKSSLAFD P4 111-138 Mdr HylB OppD MalK HisP PstB UvrA-1 411-438 488-505 40-557 21--48 24--51 28--5 5 16--43

FIG. 3. Comparison ofaportion ofthe amino acidsequence of

protein P4 with those ofa groupof proteins withnucleotide-binding

sites. The sequences are derived from thegenes for the multiple

drug resistance protein (Mdr), hemolysintransport protein (HylB),

oligopeptide permease (OppD), andtransportproteins for maltose

(MalK), histidine (HisP), and phosphate(PstB) (7). Asequencefrom

anATP-dependent UV repair protein(UvrA-1) (5)is also shown.

A

L GUAAAAAAACUUUAUAUAG 1111111111111111 M GGAAAAAAACUUUAUAUAU 1I1111111111111111 s GGAAAAAAACUUUAUAUAA

B

L M S 3UCUUUUACCUGGAUUCUCUGUG UUUUCUACGUUGAGCUCCGUAUA

UCCAUAAGUCCUWAGAUWUCUAAGGCGAGACUCGCUWUGCGAGCGUCCAAUAGGACGGCCCCCUCGGGGGCUC UCUCUCU I11111 111111111 111111111111111111I II 1111111111 111111 11111111111111111

UAAAUAAGUCCUUAGAUUUCUAACGCGAGACUCGCUUUGCGAGCAUCCAAUAGGAUGGCCCCUUCGGGGGCUCUCUCUCU

1111 11111111 111111111111 1111 1111111111

UAAACAAGUCCUGUAUAAC-AAGGCGAGACUCACUAUGUGAGCGUCCAAUAGGACGGCCCCWCGGGGGCUCUCUCUCU

FIG. 4. Comparison ofthe 5' (A) and 3' (B) ends of the three genomic segments. Inallcases,the 5' endisattheleft.

codon is either absent or

spaced

far away. The

4)6

system also uses other means of control ofgene

expression.

The production ofproteins codedon the middle segmentdiffers among the genes, although eachgene hasitsown ribosome-binding site. It is likelythat the strength of the interaction between ribosomes and some of the messages determines the level of production of individual

proteins.

In

addition,

there is a dramaticchange in thepatternofsingle-stranded RNA synthesis during infection (3). This is certainly in-volved in the turn onof translation ofthelateproteins.

Anumber of viruses contain segmentedgenomes of dou-ble-stranded RNA. These include reovirus, rotavirus, blue tonguevirus,and

4)6.

Inaddition, anumberof

particles

that replicate double-stranded RNA have beendescribed, nota-bly, the killer particle in Saccharomyces cerevisiae (6). Although the replication of

4)6

RNA differs from most of these in that transcription of4)6 is by strand displacement, whereas the others use a conservative mechanism, we anticipatethat thereis considerablehomologyin theproteins ofthese particles. In particular, we expect to find thatthe replicaseproteinsarerelated.

The complete nucleotide sequence of the three genomic segments has beenentered into the GenBank database. The accession numbers are M17461 for segment L, M17462 for segmentM, andM12921for segmentS.

ACKNOWLEDGMENTS

Partsof this workweresupported by Public Health Servicegrant GM 34352 fromthe National Institutes of HealthtoL.M. I.N. was supported in part by National Research Service award 5 T32 AI-07180 from the National Institute of Allergy and Infectious Diseases. M.R. was supported in partby the Academy of Finland during histenureatthePublic Health Research Institute.

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18. Mindich, L., G. MacKenzie, J. Strassman, T. McGraw, S. Metzger,M.Romantschuk, andD.Bamford.1985. cDNA clon-ing of portions of the bacteriophage4)6genome. J. Bacteriol. 162:992-999.

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VOL.62, 1988