Molecular cloning and partial sequencing of hepatitis A viral cDNA.

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0022-538X/85/050247-09$02.00/0

Molecular Cloning and Partial

Sequencing

of Hepatitis A

Viral

cDNA

DAVID L. LINEMEYER,l* JOHN G. MENKE,' ANTONIA MARTIN-GALLARDO,' JOSEPH V. HUGHES,2

ALEXANDER YOUNG,' AND SUDHA W. MITRA'

DepartmentofBiochemical Genetics' and Department of Virology and CellBiology,2 Merck Sharp & Dohme Research

Laboratories,

Merck

Institute

for

Therapeutic

Research

Rahway,

New

Jersey

07065

Received 19October 1984/Accepted 8 January 1985

HepatitisA viruswaspurified from infected monkey kidney cellcultures, and the viral RNA wasused to

synthesizedouble-strandedcDNA.ThiscDNAwascloned either after insertion intoaplasmid-primed synthesis

system or after insertion into the PstI site of pBR322. The resulting clones were mapped by restriction

endonuclease analysis and by cross hybridization of the viral inserts to generate a composite map which representedatleast97% of the viralgenome,lackingca.220bases fromthe 5'endof thegenome.Theclones were verified tobe hepatitisA virus specific basedon their positive hybridization to viral RNAand tototal hepatitisAvirus-infected cellularRNAfromaheterologousmarmosethost system. The nucleotidesequenceof 3,054 base pairs of cDNA homologoustothe5'half of the viralgenomewasdetermined,andanopenreading frame of 854 consecutive coding tripletswasidentified. Inaddition,sequenceswhich encode the VP-1 and VP-3 viral structural proteins werelocated in the nucleotidesequence.

Hepatitis A virus (HAV) is a small RNA virus which

generallycausessubclinicalliver disease butcan cause acute

hepatitis (24). Type A hepatitis outbreaks have been

attrib-uted to contaminated food and water, and the disease is

transmitted predominately byafecal-oral route asthevirus

is shed infecalsamples ofinfected individuals (6). Thevirus is perpetuated either by poor environmental and personal

hygiene,asis prevalent inlower-socioeconomic-levelgroups,

orbya reservoirof nonepidemic cases which areclinically

unrecognized.Thevirus is oftenspread byyoungchildren in

daycare centers oramongmalehomosexualsandisreported

toberesponsible for20to40% of sporadic hepatitiscasesin

urban adults(7).

The incidence of hepatitis A is decreasing in developed

societies, creating larger numbers of unprotected

individu-als. Prophylaxis relies on passive immunization with

im-mune serumglobulin. All HAV virions regardless oforigin

or strain are

immunologically indistinguishable;

thus, there

is only one serotype (8). Development of active vaccines

with killed or live attenuated HAV to protect against the

reservoir of subclinical nonepidemic cases has been made

possible by in vitro cultivation of the virus (21, 22).

How-ever, an alternative antigen for active vaccination against HAVwould be desirable.

It is only recently that HAV has been characterized in

sufficient detail to allow its classification as a picornavirus

with the characteristics of the enterovirus group (9). The

virion has no envelope, is 27 nm in diameter, and has a

bouyant density of 1.34 g/ml in CsCl (27). The particles

contain a single-stranded RNA genome with a

polyadenyl-ated[poly(A)] tractlocatedatthe 3' end (5, 29);assuch, the

genomecanfunctionas amessenger to supportthesynthesis of viral proteins (16). By analogy to other picornaviruses, the genome should contain a large open reading frame for translation of a polyprotein which is proteolytically proc-essed into the virus proteins (25). As with other

picorna-viruses, four structural proteins have been described with

molecular weights of 32,000 to 33,000 (VP-1), 26,000 to

*Correspondingauthor.

29,000(VP-2),22,000to27,000 (VP-3), and 10,000to14,000 (VP-4). VP-1 appearsto be the major surfacecomponentof

thevirus and contains atleastoneneutralization epitopefor

HAV(4, 10).

In an effort to produce a subunit vaccine to HAV by recombinant DNAtechnology,wereporthere the molecular

cloningof the HAV genome. Twopreviousreports ofHAV

cloning have been described. Von der Helm et al. (31)

described cDNA clones of up to 1,000 base pairs (bp)

synthesized by using an oligodeoxythymidylic acid

[oligo(dT)], primer and RNA extracted from HAV purified

fromfecalmaterial. These clones were shown to hybridize

to

32P-labeled,

HAV-specific RNA derived from

HAV-in-fected PLC/PRF5 cells and to express a product which

reacted ina radioimmunoassay.

By using virus extracted from infected marmoset livers,

Ticehurst et al. (30) reported cloning HAV cDNAs which

represented atleast99% ofthe viralgenome. These clones

were shown to hybridize to RNA from HAV-infected Afri-can green monkey kidney cells but not to RNA from

uninfected cells. In addition, the nucleotide sequence of

some500 basesfromthe 3' endofthe genomewasreported. In this paper we report the molecular cloning of HAV cDNA

representing

at least 97% ofthe viral genome. Also

presentedis the nucleotide sequenceof3,054bp fromthe 5'

half of the genome and the localization of the sequences which encode the VP-1 and VP-3viral structural proteins.

MATERIALSANDMETHODS

GrowthandpurificationofHAV. LLC-MK2 cells(a

mon-key kidney cellline)were infected withthe CR326 strain of

HAV(21)at amultiplicityof0.1 to0.5 andweremaintained

at35°C in Eagle minimal essential medium (Earle salts; Flow

Laboratories, Inc.) with 0.2% fetal calf serum and 2 mM

glutamine for 21 to 28 days. The infected cells were then

washed and scraped in phosphate-buffered saline and pel-leted. The cells were suspended in lysis buffer (10 mM

Tris-hydrochloride [pH7.5], 10 mM NaCl, 1.5 mM

MgC92,

1% NonidetP-40),subjectedto two cyclesofsonication for 20 s, and incubated on ice for 10 min. Cell debris was

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removed by centrifugation at 10,000 x g for 20 min. The supernatant was removed and sodium-Sarkosyl (SLS) was

addedto0.5%. Aftera30-minincubationat37°Candabrief

sonication, theviral supernatantwaspelleted througha20%

sucrose shelf in 0.1% SLS and TNE (10 mM

Tris-hydro-chlroide [pH 7.4], 150 mM NaCl, 1 mMEDTA) for20 h at

100,000 x g. After centrifugation, the viral pellet was

sus-pended in0.5% SLS inTNE,sonicated,andincubated for30

minat37°C.Viruswasbanded on 25 to45% preformedlinear

cesium chloride gradients by centrifugation for60 h.

Frac-tions were collected and assayed for their refractive index

and forhepatitis Aviral antigenby radioimmunoassay.The

majorviral peaks were pooled, diluted 20-fold in TNE with

0.1% SLS,and pelleted.

RNA extraction. The CsCl gradient-purified virus was

suspended in TNE containing 20 mM EDTA and 1% SLS.

The suspension was extracted with an equal volume of

phenol saturated with 0.1 M Tris-hydrochloride (pH 7.4) at

65°C. The aqueous layer of the extraction was further

extracted twice with equal volumes ofCHCl3-isoamyl alco-hol(24:1) and precipitated with 0.2 M sodium acetate (pH 5.5) and 2 volumes of ethanol at -20°C. The ethanol

precipitatewas collected by centrifugation and dissolved in

sterile distilled water.

Total cellular RNAs from uninfected and HAV-infected

LLC-MK2 cells or marmoset liver cells were extracted as

describedpreviously (3).

Synthesis and cloning of 3' cDNA. The initial HAV cDNA

cloneswere prepared byamodification ofthe procedure of

Okayama and Berg (20). Briefly, the viral RNA was

an-nealed to oligo(dT)-tailed plasmid pBR322-simian virus 40

(map units 0.71 to 0.86), and cDNA was synthesized by

avianmyeloblastosis virus(AMV) reversetranscriptasewith

theoligo(dT) tail as primer. This first strand of cDNA was

oligodeoxycytidylic acid [oligo(dC)]-tailed by usingterminal

transferase, restricted with HindIII, and annealed to an

oligodeoxyguanylic acid [oligo(dG)]-tailed linker pBR322-simianvirus40 (map units 0.19 to 0.32). Inacombined triple

reaction, the RNA was removed by RNase H, the second

cDNA strand wassynthesized byDNApolymeraseKlenow

fragment with the linker as primer, and the cDNA was

ligated with T4DNAligase. Thisreaction mixturewasthen

transformed into Escherichia coli HB101. The resulting

cloneswereselectedby resistancetoampicillinand screened

by hybridizationto a

32P-labeled

HAV cDNApreparedfrom

the purified viral RNA by using reverse transcriptase and

calfthymusDNAfragments asprimer.

Primer extension cDNA synthesis and cloning. Arestriction

fragment of one of the initial HAV cDNA clones was

purified by electroelution (17) from an agarose gel and

denatured by boiling for 10 min and quick cooling in ice

water. The denatured fragment was usedas a primer after

annealingorhybridization tothe viral RNA. Forannealing,

160 ngofprimerwasaddedto ca. 1

Rg

ofpurifiedHAVRNA in 20 ,ulof water, heated to 70°C for 10min, and cooled to

37°C. For thehybridization, 140 ngofprimerwasaddedto1

,ugofpurifiedHAV RNAin80%formamide-0.4MNaCl-0.01

M PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid [pH

6.4])-2 mM EDTA and incubated at 47°C for 3.5 h. The

formamide was removed byfour sequential ethanol

precip-itations as previously described (15). The first strand of

cDNA was then synthesized with 1.5 U of AMV reverse

transcriptase per ,ul in the presence of50 mM

Tris-hydro-chloride (pH 8.0)-0.34 mM dCTP-1 mM dGTP-1 mM

dATP-1 mM dTTP-10 mM 2-mercaptoethanol-0.8 ,uCi of

[a-32P]dCTP-0.7

UofRNasinfor30 minat42°C.Thesecond

strand was synthesized by using the same triple enzyme

reactiondescribed aboveforthe initialclones. The

double-stranded cDNAwasoligo(dC)-tailed by usingterminal

trans-ferase, annealed to pBR322 which was oligo(dG)-tailed at

thePstI site, andtransformed into competent E. coli RR-1

(17). Theresultingclones were selected for

tetracycline-re-sistant, ampicillin-sensitive growthand screened forpositive

hybridizationtothe 32P-labeled cDNAprobe prepared from

HAV RNA.

Analysis of cDNA clones. Toensurethat thecDNA clones

were generated from HAV genetic information, selected

clones were labeled with

[cx-32P]dCTP

by nick translation

(17) and hybridized to uninfected and HAV-infected

LLC-MK2 cellular RNAor marmosetliver RNAboundto nitro-cellulose membrane filters (29). In addition, hybridizations

were performed with RNA extracted from virus purified

from the livers of infected marmosets or from infected LLC-MK2 cells.

The inserts of the initial clones isolated after plasmid

priming were analyzed by restriction enzyme digestions

after releasefromthe vector byPstI-PvuII double-enzyme

cleavage.

The clones isolated after

primer

extension were

analyzed after release from the vectorwithPstI digestion.

The clones were organized on a genomic map by analysis

with multiple restriction enzyme digestions and Southern

blot

analysis

(28) with various cloned32P-labeled inserts as probes.

Analysis of nucleotide sequence. Nucleotide sequencing

was performed with clones T28-18, T28-71, T28-77, and

T28-94 by the procedure of Maxam and Gilbert (18). In

addition,

Sau3A and TaqI fragments ofclone T28-77 were

subcloned into phage M13 (19) and sequenced

by

the

did-eoxynucleotide

chain termination procedure described

by

Sanger

et al. (26).

Also,

in afew areasof this 5' half ofthe genome,

oligonucleotide

primerswere synthesizedand used

for direct

sequencing

ofthe

purified

viral RNA(32).

Enzyme and radioisotope sources. Restriction endonucle-ases were obtained from Bethesda Research

Laboratories,

NewEngland

Biolabs,

orInternational

Biotechnologies,

Inc.

AMV reverse transcriptase was obtained from Life Sci-ences, Inc.

Polynucleotide kinase,

terminal

deoxynucleoti-dyl

transferase,

andribonucleaseHwerefromP-L

Biochem-icals,

Inc.The sourceof RNasinwasfrom

Biotec,

Inc. The

T4DNAligaseand DNApolymeraseKlenow

fragment

were

obtained from P-L

Biochemicals,

Inc., Collaborative

Re-search, Inc.,and New England BioLabs. Bacterial alkaline

phosphatasewasfromBethesda ResearchLaboratories.The

[a-32P]dCTP

and

[y-32P]ATP

radioisotopes

were obtained from Amersham Corp.

RESULTS

Plasmid-primed cDNA synthesis and cloning. The initial

clonesof HAV cDNA were obtained

by

using

a systemof

plasmid-primed cDNA

synthesis

which has been described

(20). Viral RNA was extractedfrom

purified

viral

particles

grown in LLC-MK2 cell cultures as detailed above. The HAVRNA

migrated

in

methylmercury-agarose

gels

at asize

slightly largerthanRNA from the

closely

(Fig. 1). Also,

possibly

duetothe slower

growth

and lower

titers of HAV, it was not

possible

to isolate HAV RNA withoutasignificantamountofdegradationasshown

by

the

smearof RNA below the band of

full-length

RNAin lane 1. This viral RNA(500 ng)wasannealedtothe

oligo(dT)-tailed

plasmid (1,400 ng) derived from

pBR322-simian

virus 40

(map units 0.71 to

0.86),

and the first strand ofcDNA was

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synthesized by reverse transcriptase in the presence of

[32P]dCTP

with theRNA astemplate and theoligo(dT) tailas

primer. The cDNA was then tailed with dCMP, and the

second strandofcDNA was synthesized by DNA polymer-ase I with an oligo(dG)-tailed linker derived from

pBR322-simian virus 40 (map units 0.19 to 0.32) as primer after

removal ofthe RNAby RNase H treatment.

The first strand of cDNA ranged in size from 0.5 to 2.5

kilobases(kb) byalkaline-agarosegelanalysis(Fig. 2). With

the same conditions ofsynthesis, it was possible to obtain cDNA aslarge as 4kb from poliovirusRNA(Fig. 2, lane 3).

Synthesis of cDNA with different concentrations of KCI

(lanes 1 and 2) or by previous denaturation of the RNA

template with heat or treatment with methylmercury (data notshown) did not significantly increase the size of cDNA

obtained. Since at least 50% ofthe viral RNA was

appar-ently full-length,the reasonlongercDNAproductswere not

generated is not known atthis time.

After ligation, the cDNA was transformed into E. coli

HB101,and the clones were selected byampicillin-resistant

growthyieldingca. 104 transformants perjxgof cDNA. The

cloneswere screened subsequently by colony hybridization

to a representative HAV-specific probe prepared by the

synthesis of

[ot-32P]dCTP-labeled

cDNA from HAV RNA

with calf thymus DNA fragments as primer. Restriction

1

2

3

FIG. 1. Methylmercuric hydroxide-agarose gel electrophoresis of RNA extracted from purified virus particles. Viral RNA was

extracted frompurified HAV particles,asdescribedin thetext,and electrophoresedat60Vthrougha1%agarosegel containing 10 mM methylmercurichydroxide (17). To visualize the RNA, the gelwas

treated with0.2M ammoniumacetatecontaining 1 ,ug of ethidium

bromide perml and irradiated with UVlight. The RNA analyzed

was viral RNA from HAV (lane 1), RNA from human embryo

fibroblasts(lane 2), and viral RNA from poliovirustypeI(lane 3;a

giftfrom V. Racaniello).

FIG. 2. Alkaline-agarose gel analysis ofviral cDNA. Plasmid-primed cDNA synthesis was performed as described in the text. Aftersynthesis of the first strand of cDNA, asamplewasethanol precipitated, suspended inwater,anddigested with PvuIItorelease thevector. The mixturewas electrophoresedon a1% agarosegel

containing 30 mMNaOH and 2mM EDTA. Thesynthesisof HAV

cDNAwasinthepresenceof 100 mMKCI (lane 1)or150 mMKCI (lane 2). Also shown is cDNAsynthesized from poliovirus RNA in the presence of 100 mM KCI (lane 3). The location of X-HindIII

fragments migratingin thesamegelarenotedontherightin kb.

endonuclease analysis of the hybridization-positive clones revealed a family of cloned cDNAs which all appeared to

havethesame terminus,presumably originatingfrom the 3' poly(A)tractof the RNAgenome,and extended for various lengths (datanotshown). Thelargest HAV insert obtained

was2.3 kb in length, from clone a18.

Hybridizationof cloned cDNAtoHAV RNA. Toverify the authenticityof theclones,afew cloneswereusedtoprepare

32P-labeledprobes bynicktranslation(17),and theseprobes

werethen hybridized by Northern analysistouninfectedand HAV-infected cellular RNAs as well as to viral RNA. An exampleof thehybridizationwithaprobe preparedfromone

of theclones is shown inFig.3. Theclonehybridizedwellto

RNAof viruspurifiedfrom either HAV-infected LLC-MK2 cells (lanes 1 and 2) or livercells of HAV-infected marmo-sets (lane 7). In all cases the cloned DNA hybridized to a

band of 7.5 to 8 kbas well as smallerdegraded RNA. The clone also hybridized to total cellular RNA extracted from eitherHAV-infectedLLC-MK2 cells(lane 4)orlivercells of

HAV-infected marmosets (lane 6). Importantly, there was nodetectable hybridizationofthe cloned cDNAto uninfec-tedLLC-MK2 total cellular RNA(lane 3)ortototalcellular RNA from livers of uninfected marmosets (lane 5). In the lastcases,althoughthetotalcellular RNApreparationswere largely degraded, the HAV specificity of the hybridization was obvious, indicatingtheauthenticity of the clones.

Additional cDNA synthesis and cloning. Restriction

en-zyme analysis of the largest clone, a18, indicated the

pres-1

2

3 -4.3

-2.2

-0.6

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1234

56 7

FIG. 3. Hybridization of cloned cDNAto various RNA prepa-rations. RNA preparations were electrophoresed through a 1% agarose gel containing 10 mM methylmercuric hydroxide as de-scribed in the Fig. 1 legend. The gel was then soaked in three

changesof 10 mM sodiumphosphate (pH6.8)for 10mineach,and the RNA was transferred to a nitrocellulose membrane as previ-ously described (29). The membrane filter was hybridized with a

12P-labeled probe prepared by nick translation (17) ofa 3' HAV cDNAclone. The clone used, a12, is homologoustoclone a18but lacksca. 300bpatthe 5'end. The RNApreparations hybridized by

theprobeincludetwodifferentpreparationsof RNA extracted from viruspurifiedfrom HA V-infected LLC-MK2 cells(lanes1and2)or

from viruspurifiedfrom the livers of HAV-infectedmarmosets(lane 7). Total cellular RNAs analyzedwerefrom uninfected LLC-MK2 cells (lane 3), HAV-infected LLC-MK2 cells (lane 4), liver cells fromuninfected marmosets (lane 5),and liver cells fromn HAV-in-fectedmarmosets(lane6). Viral RNAswere runat20 ng,and total cellular RNAswere runat1 ~Lg.

enceoftwoPvulI sitesneartheextreme5' endof this clone

(see Fig. 5), which when cleaved generated a 280-bp DNA fragment. This fragment was purified by electroelution (17)

from a 1.5% agarose gel ofPvuII-digested clone a18 DNA and used toclonefurther HAV cDNA sequencesbyprimer extension. Thepurified fragmentwaseither annealedtoviral RNAbyheatingto90'C and slowcoolingorwashybridized

tothe RNAat 420C in thepresence of80% formamide(15).

Thefirst strand of cDNAwassynthesized bytheaddition of

reverse transcriptase. Thesynthesis of the second strand of cDNA was accomplished by the addition of RNase H to

degrade the RNA, DNA polymerase I to catalyze the synthesis, and DNA ligase to join internal pieces of the second strand. The cDNAwassizefractionatedon asucrose

gradient, and two pools of cDNA were collected, a larger

poolwith cDNAranginginsize from less than 0.5toca.8kb and asmallerpoolof sizes less than 2.5 kb as shown inFig. 4. The cDNA in thepoolenriched for larger-sized products

wasclonedbytheaddition ofpoly(dCMP)tails,annealingto

poly(dG)-tailedPstI-cleaved pBR322,andtransformation of

E. coli RR-1. Positive clones wereselected by

tetracycline-resistant, ampicillin-sensitive growth and screened by

hy-bridization to the representative HAV cDNA probeused in the initial cloning. Of 220 tetracycline-resistant clones, 37 hybridized to the HAV cDNA probe. However, only fourof these hybridized to a probe preparedby nick translation of the 280-bpfragment primer, indicating that the majority of the clones were generated by nonspecific priming of the AMV reverse transcriptase. The clones contained inserts ranging in size from ca. 0.3 to 4.2 kb. As with the initial clones, the authenticity of a number of these clones was examined by preparing radiolabeled, nick-translatedprobes from the cloned inserts and hybridizing them to RNA. Amongtheclones analyzedwere T28-77 and T31-2 (seeFig. 5). All ofthe clones examined were found to hybridize to HAV-infected cellular RNA but not to uninfected cellular RNA (data not shown) verifying the HAV specificity of these clones.

Since themajority of the clones seemed to be generatedby nonspecific priming of thereversetranscriptase, the synthe-sis of cDNAfrom HAV RNA was examined in the absence ofprimer orafterannealing an unrelated primer. cDNA of almost genome length was synthesized in the absence of primer (Fig. 4, lane 3). The size and yield of this cDNAwas evenlarger than that generated by cDNA synthesis primed with a 340-bp fragment of clone T28-71 (lane 4) or with a

375-bp fragment of pBR322 DNA (lane 5). Indeed the

initiation of synthesis of these latter three cDNAs was

shown to berandomby hybridizing these [32P]cDNAs tothe cloned HAV cDNAs cleaved with restriction enzymes in

1

2 9.6-

6.5-4.3

2.2 1.4-3 4

5 -9.6

6.5

4.3

2.2

1.4

0.6

FIG. 4. Methylmercuric hydroxide-agarose gel electrophoresis of HAV cDNA prepared by primer extension. Double-stranded

cDNAwassynthesized from purified HAV RNAby usingaDNA

fragment primerasdescribed inthetextandwaselectrophoresedas

described in the legendto Fig. 1. The280-bp fragment generated fromthe 5'endof clonea18bydigestion ofthe insertwithPvuIlwas

usedasprimer, andtheresultingcDNAwassize fractionatedon a

5to25%sucrosegradient containing10 mMTris-hydrochloride (pH 8.0),50 mMNaCl,and 5 mM EDTA inanSW50.1rotor at190,000

xg for 6 hat 4°C. Pools were made of the faster (lane 1) and slower

(lane 2)migratingcDNA. In aseparateexperiment, double-stranded

cDNAwas preparedwith no DNA primer (lane 3), a340-bp

frag-mentgenerated fromthe 5'endof cloneT28-71 by BamHI-HincII digestion (lane 4),or a375-bp EcoRI-BamHI digestion fragment of pBR322 (lane 5).Twiceasmuchreactionsamplewasloaded inlanes 4and 5asin lane 3. Thelocations ofX-HindIIIand4X174-HaeIII

DNAfragments migratingin thegelsarenotedatthesidesin kb.

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8 7 6 5 4 3 2 1 0 3'

I I I I I i K B

VP4 VP2 VP3 VP1

.. . .~~~~~~~~~~~~~~~

POLIO RNR

VP3 VP1

Hc B P B Bg H B Hc

Pv Hc X NNPv

a HRV RNR

Hc PHc Bg BBg

',' ,' ,i'§ HRV cDNR

Pv Pv H E Pv

Ho Bg BBg

PvPv H E Pv

Hf Hc P

', * ', T32-1

Hf T Pv

B Hc Hc

II ',i, T28-1 8

X NNPv B Hc

', 'i,,

T28-123

X NNPv B P B BgH B

. III --Jr T28-94

Pv Hc X

Hc B P B Bg H

III I I T28-7 1

Pv Hc

B HcHcB P B BgH B Hc

,

IS

l,

lII

| | , l,

X,,,T28-77

Pv Pv Hc XNNPv

0 1000 2000 3000

DNR

Sequence *

FIG. 5. Schematic representation of restriction endonuclease cleavage sites onHAV cDNAclones. The viral RNAfrompoliovirus is shownforcomparisonwith the 3' poly(A)attheright (N)andthe locationofthe sequenceswhich encode the structuralproteins (12).The viral RNA from HAV is shown with the 3' poly(A) at the right and thelocation ofthe sequenceswhichencode theVP-1andVP-3proteins

(see text). Below the HAV RNA is shownacomposite restrictionmapofthe HAVcDNA, and thelocations of the cDNA inserts of the various clonesdescribed. The oligo(dG)-oligo(dC)tails at the ends of the cloned insertsare notshown. The lineat thebottom shows the location oftheregion ofcDNAwhich has beensequenced along with the basesequence numbers. Restrictionenzyme sitesidentified are

BamHI (B),BgIII(Bg), EcoRI (E), HindIII (H),HincIl (Hc),Hinfl(Hf), PstI (P), PvuII (Pv), NcoI (N), TaqI (T), and XbaI (X).

Southern analyses (28; data not shown). Similar results of

nonspecific primingwerereported by Kupperetal. with foot

and mouth disease virus (FMDV) when cDNA synthesis

originating from various regions covering about 75% of the

genome were found after

priming

with oligo(dT) (14). Mapping of the cloned cDNAs. Restriction maps were

prepared by using a number ofthe larger clones. By

com-paring these maps and by examining patterns of cross

hybridization with labeled inserts and

restriction-enzyme-cleaved DNAfrom otherclones inSouthern analyses (data

not shown), thepositions of the cloned inserts were aligned onthe map(Fig. 5). Asexpected, clonea18mappedto one end ofthe genome since it wasgenerated by the

plasmid-primed cDNA synthesis which required a poly(A) stretch

likethat present at the 3' end ofthe viral genome (30) for

priming.

Atthe

opposite

endofthe map, itwas foundthat

Hc BPv P

_1 11 1I

B

clone T28-77 had a 550-base inverted repeat at its 5' end

which was probably generated by a "snap-back"

synthe-sized

by

reverse transcriptase

during

first-strand

synthesis.

Not including these repeated sequences, the clones

over-lapped to form a map of 7.3 kb; clones a18, T31-2, and

T28-77 were sufficientto producethe entiremap.

Partial nucleotidesequence of the HAV genome. The DNA sequencesof clones T28-71,T28-77, andT28-94 were

deter-mined by the Maxam and Gilbert technique of

chemical

sequencing

(18).Inaddition,M13cloneswereprepared from

fragments of clone T28-77 and were sequenced by the

dideoxynucleotide

termination method (26). All ofthe

re-gions were sequenced two or three times to ensure the

correctness of the sequence. In a few cases the sequence

was verified by direct sequencing of the viral RNA from

specific synthesized oligonucleotide primers (32). The

se-Bg Hc H

lI I l

B X HcN N

I1

II

Pv

1000

I~~~~~~~~~. ... I...v

I~~ II

2000 3000

2 OOO

OOO.

3

.I.

<4-4

-FIG. 6. Strategy of sequencingHAVcDNA.This schematicrepresentation shows the locations, directions, andextentsofDNAsequence

information obtainedby the Maxam and Gilbert technique (18) (solid arrows), the Sanger technique (26) with M13 clones of T28-77 (dotted arrows), anddirectRNAsequencing(dashed arrows). Also shownarethe locationof restrictionenzymecleavage sitesasdescribed inthe legendtoFig.5. For the locationof the sequenced regiononthe viralgenome, seeFig. 5.

HRV

cLDNR

Clo n e s

0

5'/

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252 LINEMEYER ET AL.

0 *0 27 00%4

CTC TCC CCTTGC CCT AGG CTC TGG CCG TTG CGC CCG GCG GGT CAA CTC CAT GAT

L-j

0000** ** 81 108

TAG CAT GGA GCT GTA GGA GTC TAA ATT GGG GAC GCA GAT GTGTOGGAC GTC ACC

L~~~~~~~~~J~~L.

*

LA~~~~~~5

* &J2

TTG CAG TGT AAA CTT GGC TCT GTCTTCC ACAAGGGT AGG

to t

. 89 ... . 6

CTA CGGGTG ALA CCT CTT AGG CTA ATA CTT CTA TGA AGA GAT GCT TTG GATGAL

* 243 ** 270

GCA ACAGCG GCG GAT ATT GGT GAG TTG TTA AGA CAAAA$CCA TTCAACGCC GGA

297 to. too 324 CGACTGOCTCTC ATCCAGTOG ATG CATTGA GTG OATTGATTG TCA 000 CTG TCT

L-J

000@00 ~~~~~~351**

CTA gGTTT; ATCTCA GAC CTC TCT GTG CTTAGOGCA AAC ACC ATTTOOCCTTfA

... 405 *** 432

ATGGGATCCTOT GAG AGOGG0 TCC CTC CATTGA CAG CTGGACTOTTCTTTG 000

* . 459 s.. 486

CCT TAT GTG GTG TTT 0CC TCTGAG GTA CTC AGOGGC ATTTAGOTTTTT CCT CAT

513 540

TCT TAA ACA ATA ATG AAT ATG TCC AAA CAA CGA ATT TTC CAG ACT GTC 000 AGT Thr Ile MET Asn MET Ser Lys Gln Gly Ile Phe Gln Thr Val Gly Ser

L-J Lwj

567 594

GGCCTTGACCAC ATC CTG TCT TTG GCAOATATTGAOGAAG GA CMATG ATTCAG

Gly Lou Asp H1S Ile Lou Ser Leu Ala Asp Ile Glu Glu GluGlnMETIleGln

621 648

TCCGTT OTT AGOACT GCA GTG ACTGOT OCTTCT TAT TTT ACT TCT GTGGAC CAA Ser ValVal Arg Thr Ala Val ThrGly Ala Ser Tyr PheThr Ser Val AspGln

675 702

TCT TCAGTT CAT ACTOCT GAO OTTGGC TTA CAT CAA ATT GAA CCC TTG AAA ACC Ser SerVal His Thr Ala GluValGly LeuHis GlnIle Glu Pro Leu LysThr

729 756

TCTOTTGATAAACCT AOTTCT AAG AAG ACTCAG000GAO AAG TTT TTC CTG ATT Ser ValAsp Lys ProSer Ser Lys LysThr Gln Gly Glu Lys Phe PheLeu Ile

783 810

CAT TCT OCT OAT TGOCTC ACT ACA CATOCTCTA TTT CATGAAOTTGCA AAA TTG

H1i Ser AlaAsp Trp Lou Thr Thr His Ala Leu Phe His GluVal Ala Lys Leu

837 864

GAC GTGGTG AAA TTA TTG TAT AATGAO CAGTTT GCC GTC CAAGOTTTG TTG AGA Asp Val Val Lys Leu Leu Tyr AsnGlu Gln Phe Ala ValGlnGly LeuLeu Arg

891 918

TACCAC ACA TATOCA AGATTTGGC ATTGAOATT CAA OTT CAG ATAAATCCC ACA

Tyr His Thr Tyr AlaArgPheGly Ile Glu IleGlnValGln IleAsn Pro Thr

945 972

CCCTTT CAG CAAGG0000 CTA ATTTOT OCTATGOTTCCT ALTGAC CAA AGT TAT Pro PheGln GlnGlyGly Leu Ile Cys AlaMET Val Pro Ser AspGln SerTyr

999 1026

GOT TCG ATAGCA TCC TTG ACTOTTTATCCT CATGOTTTG TTAAAT TGC AAC ATT

Gly Ser Ile Ala Ser Leu Thr Val Tyr Pro HisGly LeuLeu AsnCys Asn Ile

1053 1080

AAC MT GTGOTT AOAATAAAGOTTCCA TTTATT TAT ACT AOAGOT OCT TAT CAC

AanAsn Val ValArg Ile Lys Val Pro Phe Ile TyrThr Arg Gly AlaTyr His

1107 1134

TTT AAGOATCCACAGTATCCAOTTTOO GAATTA ACAATC AOAOTTTOOTCAGAO PheLys Asp ProGln Tyr Pro Val TrpGlu Leu Thr IleArg ValTrpSerGlu

1161 1188

TTOAAT ATTGOAACAGOA ACT TCAOCTTAC ACTTCA CTTAAT OTTTTAOCTAGO Leu Asn IleGlyThrGly Thr Ser Ala TyrThr Ser Leu AsnVal LeuAla Arg

1215 1242

TTT ACAOAT TTO GAOTTA CAT GOA TTAACTCCT CTT TCT ACA CAG ATG ATGAOA

Phe Thr Asp Lou Glu LouHisGly Leu Thr Pro Leu SerThr GlnMET METArg

---_eVP 3

1269 1296

AATGAATTTAGAOTTALTACT ACTGAA AATOTT GTAAATTTO TCGAATTATGAA

AsnGlu Phe ArgVal Ser Thr ThrGlu Asn Val ValLAn Leu Ser Asn TyrGlu

1323 1350

OATGCAAGOGCAAAA ATG TCT TTT OCTTTGOATCAGGAA OAT TOG AAM TCTOAT Asp Ala Arg AlaLys METOar Phe Ala LeuAspGln Glu AspTrp Lys Ser Asp

1377 1404

CCTTCCCAAGOTGOTGOAATTAAAATTACT CAT TTT ACT ACC TOG ACA TCCATT Pro SerGlnGly Gly Gly IleLys Ile Thr His PheThr Thr TrpThr Ser Ile

1431 1458

CCA ACC TTAOCTOCTCAG TTTCCA TTC AATOCTTCAOAT TCG OTTGOA CAA CAA

Pro Thr Leu Ala AlaGlnPhe Pro PheAsnAla Oar Asp Oar ValGlyGlnGln

1485 1512

ATTAAAOTTATT CCA GTGGACCCA TAT TTT TTC CAG ATO ACAAAC ACC AATCCT Ile LysVal Ile ProVal AspProTyr Phe PheGln MET Thr Asn Thr AsnPro

1539 1566

OATCAAAAGTOTATAACTGCC TTG OCTTCTATTTOTCAG ATG TTT TGC TTT TOG

Asp Gln Lys Cys Ile Thr AlaLeu Ala Ser IleCysGln METPhe Cys Phe Trp

--.-*- *@@@ *@@@*@--~~~~... ... ..

1593 1620

AGOGGAGATCTTGTTTTTOATTTTCAG GTTTTTCCAACC AAATAT CATTCAGOT ArgGlyAspLouValPhe AspPhe Gln Val Phe Pro ThrLys Tyr His SerGly

1647 1674

AGO TTGTTG TTTTGCTTTOTTCCT 000MTGAG TTGATAOAT OTTACTGGAATC Arg Leu LeuPhe Cys Phe Val ProGly Asn Glu Leu IleAsp Val ThrGly Ile

1701 1728

ACATTAAAA CAGGCAACC ACTOCTCCTTOTGCA GTGATGGACATT ACAGOAGTG

Thr Leu LysGlnAla Thr Thr Ala ProCys AlaValMETAsp Ile Thr Gly Val

1755 1782

CAGTCA ACCTTOAOA TTTCOT OTT CCT TOOATTTCTOAT ACA CCC TATCGAGTO

Gln Ser Thr LeuArg PheArg Val Pro Trp IleSOr Asp Thr Pro Tyr ArgVal

1809 1836

AATAGO TAC ACG AAO TCAGCACATCAAAAAOGT GAOTAT ACTGCCATT000AAG

Asn Arg Tyr Thr LysSer Ala His GlnLysGly GluTyr Thr Ala Ile GlyLys

1863 1890

CTTATT GTOTAT TOT TAT AATAGOCTO ACT TCT CCT TCT AATOTT OCT TCT CAT Leu Ile Val Tyr CysTyr AsnArg Leu.Thr Ser ProSer Asn Val Ala SOrHis

1917 1944

OTTAOA OTTAATOTTTAT CTTTCAGCAATTAATTTGGAA TOTTTTOCTCCT CTT Val Arg Val AsnVal Tyr LeuSer Ala Ile Asn Leu GluCys PheAla ProLeu

1971 1998

TAT CAT OCT ATG GAT OTTACCACACAGOTTGOAOAT OATTCAGGA GOTTTT TCA TyrHis Ala MET Asp Val Thr Thr Gln Val Gly Asp AspSer GlyGly PheSer

2025O'VP - 2052

ACA ACAOTT TCGACAGAGCAGAATOTTCCTOATCCC CAAOTTGOTATAACA ACT ThrThr Val OarThr Glu GlnAsn Val Pro Asp ProGln ValGly IleThr Thr

2079 2106

ATGAAG GACCTO AAA 000AAAGCCAATALG OGA AAG ATGOAT OTT TCAGOAGTO

METLys Asp Leu Lys Gly Lys Ala Asn Arg Gly Lys MET AspVal SerGlyVal

2133 2160

CAAGCA CCT GTGGOAOCTATC ACA ACA ATT GAGOATCCAGCATTAGCAAAGAAA

Gln Ala Pro Val Gly Ala Ile Thr Thr Ile Glu Asp Pro AlaLeu Ala Lys Lys

2187 2214

GTA CCTGAA ACG TTT CCT GAA TTGAAOCCTGCAGAO TCTAOACAT ACATCAOAT

Val ProGlu Thr Phe Pro Glu Leu Lys ProGly GluSOrArg His Thr SOrAsp

2241 2268

CACATG TCT ATT TAT AAA TTCATGOGAAGO TCT CAT TTT TTGTOTACTTTTACC His MET Ser Ile Tyr LysPhe METGly ArgSer HisPhe LouCys Thr Phe Thr

2295 2322

TTC AAT TCAAAT AAT AAA GAGTAC ACA TTTCCAATA ACCTTGTCT TCG ACTTCT

Phe Asn SOrAsn Asn Lys Glu Tyr Thr Phe Pro Ile Thr LouSer OarThrSer

2349 2376

AAT CCT CCT CATGOTTTA CCA TCA ACA TTAAGO TGOTTC TTCAATCTOTTTCAG Asn Pro ProHis Gly Leu Pro Ser Thr Leu Arg TrpPhe Phe Asn Leu PheGln

2403 2430

TTG TAT AOAGOACCATTGOATTTGACA ATT ATC ATC ACAOGAOCTACTqATGTG Leu Tyr OrgGly Pro LeuAsp Leu Thr Ile Ile IleThr GlyAla Thr Asp Val

2457 2484

OATGOAATG0CC TOG TTT ACTCCA GTAGOCCTTOCTOTTGAC ACC CCATOGGTG Asp Gly METAla Trp Phe Thr ProValGly Leu Ala Val Asp Thr Pro Trp Val

2511 2538

GAAAAOGAATCAOCTTTG TCT ATTOATTATAAA ACT0CCCTTOGA OCTOTTAOA

Glu Lys GluOar Ala LeuSer Ile AspTyr Lys Thr Ala LouGly Ala Val Arg

2565 2592

TTTAATACAAOL AOL ACA000 AAC ATT CAG ATTAOATTG CCATGOTATTCT TAT Phe AsnThr Arg ArgThr Gly AsnIleGln Ile Arg LeuPro TrpTyr Ser Tyr

2619 2646

TTA TATOCT GTOTCTOGAGCACTO OATGGC TTGCGA OAT AAGACAOATTCTACA Leu TyrAla Val SerGlyAla Loeu AspGlyLouGly Asp Lys Thr AspSOr Thr

2673 2700

TTTGOATTGOTTTCC ATA CAG ATT GCA AAT TACAAC CAC TCTOATGAA TAT TTG PheGly Leu Val Ser IleGln Ile Ala Asn Tyr Asn HisSOrAsp GluTyr Leu

2727 2754

TCCTTT ALT TOTTATTTG TCT GTC ACA CAA CAA TCA GAO TTC TAT TTT CCTAOA

SOr Phe SerCys Tyr LouSer Val ThrGlnGlnSOrGluPheTyr Phe ProArg

2781 2808

OCTCCA TTAMATTCAAATOCTATG TTGTCC ACTGAGTCTATOATGALT AOAATT Ala ProLouAsnSOr AsnAla MET Leu Ser Thr Glu SerMETMETSerArg Ile

2835 2862

GCAOCTGOAGACTTGGAGTCA TCAGTOOAT OATCCTAOATCAGAOGAAGACAGA Ala AlaGlyAsp LouGluSerSOr ValAspAspProArgSer GluGluAsp Arg

2889 2916

AOATTTGAO ALTCATATAGAATOT AGOAAACCA TAT AAAGAA TTG AOATTG GAG

ArgPheGluSOrHis IleGluCys Arg Lys Pro TyrLys Glu LeuArgLou Glu

2943 2970

OTT000 AAA CA AOACTT AAA TATOCTCAGGAAGAGTTG TCA AATGAAGTGCTT Val Gly LysGlnArgLeuLysTyr Ala GlnGluGlu LeuSer AsnGlu Val Leu

2997 3024

CCA CCT CCTAGO AAAATGAAGG00 TTA TTTTCACAA GCCAAA ATT TCTCTT TTT

Pro Pro ProArg LysMET LysGlyLeu PheSer GlnAlaLys IleSerLeu Phe

3051 TAT ACTGAOGAACATGAAATA ATGAAA TTT TyrThr Glu Glu HisGlu IleMETLysPhe

J. VIROL.

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quencingstrategyisshowninFig.6,and the DNAsequence

corresponding to the region ofthe HAV genome believed,

by analogytopoliovirus, toencode the structural proteins is

presented in Fig. 7. As found with poliovirus (12, 23) and with FMDV (1, 11), there wasalarge openreading frame for

translation beginningnearthe 5' end. In the DNA sequence

ofHAV, the open reading frame started atDNA sequence base 493 with Thr-Ile-Met-Asn-Met and continued until the end ofthe currently identified sequence, an open reading frame of 2,562 bases. A repeat of 30 bases wasfound in clone T28-77 beginning at position 1934,- but this was determined

tobecaused by areading mistake of thereverse

transcript-ase since this repeat was not present in the overlapping clones T28-71andT28-94 nor was it seenbydirect

sequenc-ing ofthe viral RNA. In addition to the sequence ofthe

structural gene region, the 3' end of clone al8 was se-quenced and found to contain a poly(A) tract (data not shown).

A

32P-labeled

oligonucleotide primer was prepared which was 18 bases in length and homologous to aunique region near the extreme 5' end of the sequenced genome. The sequence homologous to the primer was GGACTGGCTC TCATCCAG and was located from bases 271 to 288 in the HAVnucleotide sequence(Fig. 7). Theprimerwasannealed to HAV RNA, and cDNA was synthesized by reverse

transcriptase in the presenceofactinomycinD(42p.g/ml)to

prevent snap-back. The size ofthis product analyzed by

electrophoresisthrough apolyacrylamide gel was520 bases

(Fig. 8)indicatingthat the 5'end of the genome was ca. 220

bases from the first base in the DNA sequence presented

here.

Correlationof amino acid sequence data. Inpoliovirusmost ofthe protease cleavages occur atGln-Gly pairs toprocess

thepolyprotein intothe individual mature peptides(12, 23).

Other cleavages occur at Asn-Ser or Tyr-Gly pairs. An

analysis of the proposed open reading frame of the HAV

sequence demonstrated none of the above pairs in the regions predicted for protein processing. Thus, it was be-lieved the protease cleavage sites in HAV were different from those in poliovirus. To locate the position of the sequenceswhichencodedvarious structuralproteins, amino acid sequence data was obtained from acrylamide-gel-puri-fied VP-1 and VP-3 peptides. The N-terminal amino acid sequenceanalysisofVP-1 determinedthat the 5' end ofthe VP-1 gene waslocated atDNA sequence base 1972. There was a directcorrelation between the next 12 aminoacids of

thereading frame(Fig. 7) andthosefoundby theamino acid

sequencing (data not shown). To further confirm the

pro-posedopenreading frame,amino acid sequenceinformation

wasobtainedfrom the mixture of CNBr cleavage peptides of

purifiedVP-1 andofpurified VP-3 (C. D. Bennett and J. V.

Hughes, unpublisheddata). The amino acid sequences of the

mixtures offragmentswerecompared to the DNA sequence.

The correlations found with the projected amino acid

se-quenceof the open reading frame are shown in Fig. 7.

1

2

p-1.35. 1.08- 0.87--0.6

-0.31

- 0.27-

0.28-.0

j.

..."W

NW '.. . j.

"io

_

0.23- A

0.19- _

FIG. 8. Polyacrylamide gel analysis of oligonucleotide-primed

cDNA. An 18-baseoligonucleotide homologoustoHAVRNAnear

the5'end of thegenome waslabeled with32Pbyusing polynucleo-tide kinase and then was annealed to HAV RNA. First-strand cDNA synthesis was performed with reverse transcriptase, as

describedin thetextwith theaddition of42 ,ugofactinomycinD per ml. The resulting cDNA was heated 2 min at 90°C in 90% form-amide-1 mM EDTA and electrophoresed through an 8% poly-acrylamide-8 M ureagel with Tris-borate buffer (lane 2). Thegel

wasfixedin30% aceticacid-30%methanol, dried,andexposedto X-rayfilm. As molecular weight markers, HaeIII-digested iX174 DNA waslabeled with32Pbyusingkinase andwaselectrophoresed (lane1). Thesizesof the marker bandsareindicated in kb.

DISCUSSION

In this paperwe reportthe molecularcloning and partial

sequencing of cDNA prepared from purified Hepatitis A

virusRNA.The cloneswereobtained fromaseries ofcDNA

cloning experiments which yielded overlapping clones

rep-resenting atleast 97%oftheviralgenome.Thecloneswere

verified to be HAV-specific by labeling the inserts and

hybridizing them to various RNA preparations. These

la-beled inserts were shown to hybridize to RNA extracted from HAV grown in LLC-MK2 cells and to cellular RNA fromHAV-infected LLC-MK2cells,butnot tocellular RNA fromuninfected LLC-MK2cells. Inaddition, toensurethat

FIG. 7. Nucleotide sequenceofcloned HAV cDNAfrom the 5' structural generegion of the viral genome. Nucleotide sequence was

determined byusing clones T28-18, T28-71, T28-77, and T28-94 as shown in the schematic diagram in Fig. 6. In addition,thissequence was

verifiedby direct sequencing of the viral RNA in a few areas (see Fig. 6). An open reading frame of 854 consecutive coding triplets was

identified inthe sequence bycomputer analysis with the Intelligenetics software.Amino acids identified by stepwise Edman degradationof purified VP-1 and VP-3 products and matching with the above sequence are shown by underlining as follows (Bennett and Hughes,

unpublished data):VP-1, ;VP-3,---.The N terminus ofVP-1is indicated; however, the precise N terminusof VP-3 has not been

determined.Thelocationofpotentialinitiation codons for translation before this open reading frame areindicatedbybrackets, and those for

terminationcodons areindicatedby overlying dots. Asdescribed in the text, there are ca. 220 bases preceeding this sequence at the 5' end

ofthe viralgenome.

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the cloned sequences were HAV in origin, not from other sequences possibly found in infected LLC-MK2 cells, the labeled inserts were shown to hybridize to RNA extracted from HAV purified from the livers ofinfected marmosets. The labeled inserts also hybridized positively to cellular RNAextracted from thelivers ofinfectedmarmosets but not to cellular liver RNAfrom uninfected animals.

Arestriction enzyme map was prepared fromthe cloned inserts and was compared with amappublished recently by Ticehurst et al. (30) generated from clones of a different isolate ofHAV. There are many similarities in restriction enzyme sites between the two maps as well as many differences. Clearly, the degree of relatedness between the

two restriction maps is expected between two different

strains of the virus.

The clones we obtained do not extend to the extreme 5' endof theviral RNA andthereforedonot represent approx-imately the first 220 bases of the viral genome. The current

determination ofthe base sequence begins near the 5' end

andreaches a large open reading frame for translation after 493 bases. The first methionine in this large open reading frame is atthe third codon, and there is a second methionine at the fifth codon position. There is no data at present to indicate which methionine acts as the initiator methionine. Clearly, neither ATG codon isfollowed by a G; a G at this +4position has been suggested to be preferred for ribosome initiation sites in eucaryotic mRNAs by the findings of M. Kozak (13). However, both ATG codons have thepreferred A residue at the -3 position (13). The open reading frame continues for 2,562 bases to the end of theregionsequenced. Thelargest open reading frame in the first 493 bases contains only 20 amino acids after a methionine codon. If one examines the other two reading frames, there are only two open frames of significant size which follow methionine residues in the entire sequence presented, one of 56 amino acidsandasecond of55amino acids.It isunknownwhether anyof these or other open frames are used to encode protein products.

We have identified the locations ofthe sequences which encode the HAV structural proteins VP-1 and VP-3. This

was accomplished by comparing the predicted amino acid

sequence with amino acid sequence data generated from

sodium dodecyl sulfate-polyacrylamide gel-purified VP-1

and VP-3. Thiscomparison identified the amino terminus of VP-1 as the valine at DNA sequence position 1972. From CNBrcleavage products of VP-1, fragments beginningwith methionines at DNA sequence positions 2053, 2089, 2236, and 2437 were also identified as originating from VP-1. Amino acid sequencing of purified VP-3 showed theamino terminus to be blocked and unavailable for sequencing as purified; however, fragments beginning with methionines at DNA sequence positions 1237, 1312, 1495, 1552, and 1711

were identified from CNBr cleavage products of VP-3. It is

not possible at this time to determine the precise carboxy terminus of the VP-1 or the precise amino terminus of the VP-3. However, since VP-1 has been shown to have a molecular weight of 33,000, which would require a coding capacity of ca. 900 bases of DNA, the carboxy terminus mustbe near the histidine at DNA sequence position 2875. Since the molecular weight of VP-3 is 27,000, its amino terminus must be very close to the methionine at DNA sequence position 1237. These two structural proteins are in the same relative order on the genome as that found for poliovirus, but each one begins about 250 bases closer to the first methionine in the large open reading frame. In other words, the VP-3 and VP-1are shifted about 250 bases in the

5' direction of the HAV genome as compared with the

poliovirus genome.

Regardless of this apparent shift in the position of the structural gene sequences, the sequence ofthe clones indi-cates an overallgenomeorganization ofHAVwhichis very

similar to that of other picornaviruses. Also, as has been

describedfor poliovirus and FMDV (1), HAV has a stretch

ofpyrimidine residues

preceding

thefirst ATGcodonsin the

large open reading frame. This structure may be an

impor-tant site for the recognition ofribosome binding in

picorn-aviruses. However, there is no significant homology

be-tweenthe DNA sequence ofHAVand that ofpoliovirusor

foot and mouth disease virus. Also, there is no obvious

homology between these viruses at the level ofthe amino

acid sequence based on the translation of the nucleotide sequences. In addition, the amino acid pairs which are

cleaved during processing of the polyproteins are different

for these viruses. In poliovirus, proteolytic cleavage occurs

8outof10timesatGln-Gly pairs,theC-terminalcleavageof

VP-1 being at a Tyr-Gly pair and that between VP-4 and VP-2being at an Asn-Serpair (12). The sequence specificity

for the proteolytic processing is much broader in FMDV,

where different amino acid sequences are found at three

cleavage sites in the structural proteins (1, 2). Even in

poliovirusthere must beadditional determinants, other than

the Gln-Gly sequence, to specify the cleavage sites since proteolysis does not occur at all Gln-Gly pairs in the poliovirus proteins or in every protein in the infected cell. The only cleavage site we have located in HAV occurs at a Gln-Val pair atthejunctionbetween the VP-3 and VP-1; this assumes of course that there is no additional processing of the N terminus ofthe VP-1 to generate the matureprotein. Although this is not exactly the same as the Gln-Gly pair used in poliovirus or the Gln-Thrused in FMDV, there is a similarity in the VP-3-to-VP-1 cleavage sites for these picornaviruses.

It hasbeen shown recently that atleast one neutralization epitope for HAV is present on the VP-1 (10). This was demonstrated bycross-linking virus-neutralizing antibodyto virus particles and analyzing which viral proteins were cross-linked. This suggests the ratherobvioususe ofatleast

the VP-1 protein or a peptide of it for development of an

HAV vaccine. Now, with the cDNA clones and identifica-tion ofthestructural genesequences, itshould bepossibleto producevaccines forHAV by recombinant DNA or in vitro synthesis techniques. We are currently working toproduce HAV structural gene products by in vitro expression of the cDNA clones and by in vitrooligopeptidesynthesisbased on the translation of the HAV cDNA sequence presented here.

ACKNOWLEDGMENTS

We thank Carl D. Bennett for providing amino acid sequence analysis of thepurified VP-1andVP-3 proteins andJoanne Tomas-sini, Abner Schlabach, DarleneWilliams, Linda Stanton, and Paula

Giesa for the assistancein growth andpurificationofthevirus in cell

cultures. We also thank Joel Shapiro for synthesis ofthe

oligonu-cleotide primer.

A.M.G. is the recipient ofafellowship from the Ramon Areces

Foundation, Madrid, Spain.

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