Identification of a doubly spliced viral transcript joining the separated domains for putative protease and reverse transcriptase of hepatitis B virus.

Identification of

a

Doubly Spliced

Viral Transcript

Joining the

Separated Domains for Putative

Protease

and

Reverse

Transcriptase

of

Hepatitis B Virus

PEI-JER CHEN, CHIOU-RONG CHEN, JUEI-LOWSUNG, ANDDING-SHINNCHEN*

Graduate Institute of Clinical Medicine, HepatitisResearch Center, and Department of InternalMedicine, National

Taiwan

University

College

of

Medicine and the

University

Hospital, Taipei,

Taiwan

10016,

Republic of

China

Received24 March1989/Accepted13 June 1989

Hepatitis Bvirus(HBV), like retroviruses, replicates through reversetranscription. However, the identity

and mechanism for the synthesis of HBV reverse transcriptase remain unknown. The open reading frame

(ORF) forHBVputativereverse transcriptase(pol), as a consequenceof overlapping with the whole ORF of

envelope proteins (hepatitis B surface antigens), includes a hypervariable region at the N terminus. Thus,

comparedwithretroviruses, it isunlikely that HBVreversetranscriptase is translated fromcompletepol ORF inthe full-length pregenomicRNA. We havenowdetected ininfected human liversanoveldoublysplicedRNA in whichonesplicingeventremoved the hypervariable region of thepolgenebutretained the conserved region

homologoustoretroviral reversetranscriptase. The other splicingeventdeleted the central region of hepatitis B core antigen and thus brought the protease domain which is important for maturation of reverse transcriptase closetothat ofpol. For thissequenceorganization,the spliced RNAasthepossible template for thesynthesis ofHBVreversetranscriptase isdiscussed.

The hepatitis B virus (HBV) has been showntoreplicate throughreversetranscription (18, 27), and its overall genetic

organization is similar to that of retroviruses in containing

open reading frames (ORFs) for (i) the nucleocapsid protein (hepatitis Bcoreantigen [HBcAg]); (ii) the envelope proteins

(large, middle, and small hepatitis B surface antigens [HB-sAgs]); (iii)theputativereversetranscriptase (pol ORF),and

(iv) a protein of unknown function (hepatitis B X ORF),

respectively. However, the size of HBVgenomeis smaller, about 3.2 kilobasepairs (kbp)inlength, largelyas aresult of

the overlapping of the pol ORF with the whole ORF of envelope proteins (HBsAgs) (30).

The genetic organization of HBV creates a potential

problembecause of thepresenceofahypervariable regionin

HBsAgs. It has been noted that the N-terminal ORF of the envelope protein (pre-Sl and S2, about 500 bases) is the

most variable region in the genome of different HBV

sub-types (20). Thefrequency of nucleotide substitution in this

regionis about15%, comparedwith 5%for the totalgenome

(21). Many of the nucleotide sequence changes in this

hypervariable region affect not only the amino acid

se-quencesofpre-Sl and S2 but alsodramatically affect the N terminus of theputativepol ORF,whichoverlaps thepre-S

sequence in a differentreading frame (21). Incontrast, the

sequencesof the middleregionof thepol ORF,whichshares

some homology with retroviral reverse transcriptase, are

highlyconserved(5, 6). Thus, ithas beensuggestedthat the

part of the pol ORF that overlaps with the hypervariable region of HBsAgs (pre-Si and S2) is too variable to be

functional (21, 25). In a recent report, the putative viral

reverse transcriptase molecule identified by specific

anti-bodyis also noted to lack the N terminus of thepol ORF

(13). Nevertheless, how HBV overcomes this problem to

remove the hypervariable regionremains unknown. In this report, we present evidence for the presence of a novel

spliced mRNA species in HBV-infected human livers. This

* Correspondingauthor.

mRNA is coterminal with pregenomic RNA but lacks the hypervariable region ofpol, and we propose that it is a

candidate mRNA for the synthesis of HBV reverse

tran-scriptase.

MATERIALS ANDMETHODS

Primary hepatocellular carcinomas. Tissueswereobtained

fromtwochronicHBsAg carriers who developed

hepatocel-lularcarcinomas (HCCs)and underwentsurgical resection. Thetumortissuesweredissected fromnontumor partsofthe

liver and both were immediately cut into smallpieces and

thenputinto liquid nitrogenuntil use.

Southern blot analysis. The cellular DNAs were isolated

from tissuesby sodium dodecyl sulfate-protease Kdigestion

and phenol-chloroform extraction as described previously

(12). For blotting, 10 ,ug ofDNAswaseithernondigestedor

digested with EcoRI, HindIII, and BamHI (Bethesda Re-search Laboratories, Inc., Bethesda, Md.) in the

recom-mended buffers. The DNAswerefractionatedby

electropho-resis ina0.7% agarosegel, transferredtonylon membrane

(Nytran;Schleicher &Schuell, Inc., Dassel,Federal

Repub-lic ofGermany), and thenhybridizedwith an HBV-specific

probe (kindly provided by W. S. Robinson, Stanford

Uni-versity, Stanford, Calif.).

Northern (RNA)blotanalysis. RNAswereextracted from

tissues by the guanidium isothiocyanate-cesium chloride

centrifugationmethod(4). Poly(A)+ RNAswere purified by

passagethrough anoligo(dT) columntwice; theywerethen

preservedin 70% ethanol at -20°C. For Northernanalysis, poly(A)+ RNAswere glyoxalated, separatedinagelof 1%

agarose, transferred to nylon membrane, and then

hybrid-ized(3).

Construction and screening of cDNA library. For cDNA

library construction, 2 ,ug of poly(A)+ liver RNAs were

converted to double-stranded DNAs by the method of

Gubler and Hoffman (7) by using a kit supplied by

Boehr-inger Mannheim GmbH, Mannheim, Federal Republic of

Germany. The cDNAs were methylated with EcoRI

meth-4165

0022-538X/89/104165-07$02.00/0

Copyright C) 1989, American Society for Microbiology

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acids from liver or tumor tissues containing actively replicating

HBV. (A)Southern blotanalysis of totalDNAfromoneliver tissue sample. Lanes: M, HindIll fragments of lambda DNA; I, linear, 3.2-kbp HBV genome. Ten micrograms of tissue DNA was

undi-gested(lane 1)ordigested withHindIII(lane 2), EcoRI (lane 3),or

BamHI (lane 4) and then subjected to electrophoresis and

trans-ferred andhybridized with HBV-specific probe. (B) Northern blot analysis of poly(A)+ HBV RNA. Lane M, Hindlll fragments of lambdaDNA.Poly(A)+RNAsfrom the first HCC (lane 1), second HCC (lane 2), and nontumor liver (lane 3) were glyoxalated, fractionated by gelelectrophoresis, transferred, and hybridized with full-length HBV probe. Lane 4,Denatured 3.2-kbp HBVgenome.

(C) Hybridization of poly(A)+RNAfromasample ofnontumorliver tissue (lane 1) and HCC tissue (lane 2) with an HBcAg-specific probe. Lane M contains the size markers. Results offractionated poly(A)+ RNAs from two independent tissue samples hybridized withtheBglIl-BglII fragmentareshown (lanes1 and 2).

ylase, ligated with EcoRI linkers, and then inserted into a

Agtll vector by using a cloning kit purchased from

Amer-M 2 sham International plc, Amersham, United Kingdom. The library contained about 200,000 clones, and 90% were re-combinants. Forscreening, 10,000 plaqueswere platedin a

98-mm-diameter petri dish, transferredto nitrocellulose

pa-pers, and then hybridized with core-specific probes

(BglII-BgIII

fragment; see Fig. 2A).

Subcloning and sequencing. A positive clone (CRC12), which contained a 0.75-kbp insert and was found not to

hybridize with the BglII-EcoRI probe but with the

EcoRI-RsaIprobe, waspickedup for sequencing (see Fig. 2A).The

0.75-kbp

insert was subcloned into a

pGEM7

vector

(PromegaBiotec,Madison, Wis.),andsequencingwasdone

bythechain-terminationmethod with the supplied primers.

A

~3100_ tAn 3.6 kb RNA -CAtv An 2.6 kb RNA _1255 An 2.2 kb RNA

PbI

[INS,

13I

s ]J

Eceol_[cIRZ_.AsaI

-Ft:rI:

HBV

genome

1

__1x

L

(tandem)

FIG. 2. Mapping ofthe novel viraltranscript. (A)Organization ofthe HBV genome. Apartial dimer of theHBV genomeisshown, andthefour ORFsareindicated.C,ORFforHBcAg;Pol, ORF for theputativepolymerase of HBV; PreSl,S2, and S, HBsAgs. TheX

ORF is also shown. The first nucleotideof the initiation codon of HBcAg is designated 1. Three previously identified transcripts of

HBVareshown. The3.6-kbtranscript is thepregenomic RNA;the 2.6-kb species is the pre-Sl RNA; and the 2.2-kb species is the

pre-S2+S mRNA(11, 33). TerminalrepeatsofpregenomicRNAs areshown( )and arelabeledR. The 5'endsofthethree RNAs are shown (11). The probes used for subgenomic mapping are

indicated below the HBVgenome. (B)Mapping ofthe novel viral transcript with subgenomic probes. Lane M,HindIII fragments of lambdaDNA.Thepoly(A)+RNAsweresubjectedtoNorthernblot analysis,and thefilterwashybridized with wholeHBVprobe(lane

1).After stripping asdescribed previously (3), the samefilter was thenhybridizedwiththeBglII-BglIIfragment (lane 2). The results of hybridization with the BglII-EcoRIfragment (lane 3)and with the

EcoRI-RsaIfragment (lane 4)areshown. C

B

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B.

GCACTCAGACGAAGA--- ---TCTCAPTGGGOAACA cDNA

GCACTCAGACGACGA --TCTCAATGGGGAGCA HBV(adr)

CAAGC- TTGTTATrAGT----TTCTA

C.

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C f~~~~~~eSIS1S~~~~~~~ Xim~r HBV genome

(tandem)

I6 a4541 1564

An 2.2 kb RNA

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(vspliced) 46480

FIG. 3. Structure of thedoubly spliced HBV transcript deduced from cDNA clones. (A) Autoradiograms of sequences oftwosplicing junctions as revealed by the cDNA clone. The junction inside the HBcAg is shownattheleft; that between HBcAg and HBsAg isattheright. (B)Alignment of nucleotide sequences with that of HBV (subtype adr) (5). The intronsareindicated, and theconsensussequencesforsplicing

junctions are underlined. (C) The splicedRNAcontained theconserved andfunctional portions of the pol ORF.The twosplicing sitesare

marked. The conservedregionsof HBV ORFareindicatedbythe hatched boxes. Areas of 30% amino acidhomology( )andof 70to80%o

homology ( ) amongthehepadnaviruses (adapted from reference 25)areshown. Theregion with strong homologytoretroviralreverse

transcriptaseis marked withanasterix. Theoligonucleotide used forprimer extension analysis is also shown.

To complete the sequencing process, an oligonucleotide

complementary to bases 466to480ofthe HBV genome(5'

AGTTCTTCTTCTAGG 3') was synthesized (model 381A;

Applied Biosystem, Foster City, Calif.) and utilized as an

internalprimer.

Primer extension. The oligonucleotide described above

was labeled at its 5' end by using polynucleotide kinase

(Bethesda Research Laboratories) and

[32P]ATP

(5,000 Ci/

mmol). Labeled oligonucleotide was isolated by passage

throughaG-50column. About 100,000cpmof

oligonucleo-tide was mixed with 1 ,ug of poly(A)+ liver RNA in a

hybridization solution {40 mM PIPES

[piperazine-N,N'-bis(2-ethanesulfonic acid)] [pH 6.4], 1 mMEDTA, 400mM

NaCl, and 60% formamide}, denatured at

80°C

for 10 min,

and annealed at 42°Cfor 1 h (33). Total nucleic acidswere

ethanol precipitated. The pellet was dissolved in 10

,ll

of

reaction buffer (100 mM Tris hydrochloride [pH 8.3], 140

mM KCl, 8 mM MgCl2, 2.5 mM

dithiothreitol,

5 mM

deoxynucleoside triphosphates)

without and with dactino-mycin (1.5 ,ug/ml), and then 10 U ofreverse transcriptase

(Seikagaku America, Inc., St.

Petersberg, Fla.)

was added

and themixturewasincubatedat

42°C

for 2 h. The

elongated

products were then separatedin a7 M urea-5%

polyacryl-amidegel.

RESULTS

Identification of a novel viral transcript in liver tissues. Three

specimens,

twofroman HCC and onefrom

adjacent

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FIG. 4. Mappingof the 5' end ofsplicedRNAby primer exten-sion. An oligonucleotide complementary toexon2 waslabeled at the 5' end, annealed with poly(A)+ RNA from the liver, and

elongated byreverse transcriptase (33)withorwithout dactinomy-cin. Productswere analyzed by electrophoresisina7 M urea-5%

polyacrylamide gel. Lanes 1 and 2 show the products withoutor

with the presence ofdactinomycin, respectively.Lane M,HindlIl

fragmentsoflambda DNA. The positionsofHaeIII-digested

frag-ments of the PX174 replicative form are shown at the right.

Reactionproductsareindicatedbythearrowsat the left of thegel.

livertissues,were surgicallyremoved fromHBsAgcarriers. All three tissue samples were found to have only actively replicating HBV DNA (Fig. 1A), without integrated viral DNA. Northern blot analyses of poly(A)+ RNAs from the three samplesareshowninFig. 1. Usingwhole HBV DNA

as a probe, three discrete RNA species 3.6, 2.6, and 2.2 kilobases (kb) in length, respectively, were detected (Fig. 1B), corresponding to the pregenomic RNA,

pre-Sl

RNA,

and pre-S2+S RNA (Fig. 2A) previously characterized in

human liver (33) and HBV DNA-transfected hepatoma cell lines (11, 28).

The threespecimenswerefurtherexamined todetermine whetherthereisasplicedRNAwhich deletes the

hypervari-able region ofpre-S sequences, thus potentially servingas

themRNAforpolprotein. Since thisputativeRNAspecies

shouldtheoreticallycontainsequencesofnucleocapsid

pro-tein(34), anHBcAg gene-specific BglII-BglII fragment (Fig.

2A)wasused as aprobe for Northernanalysis.As shown in

Fig. 1C,two RNA speciesweredetected in twoindependent

tissue specimens, a 3.6-kb pregenomic RNA and an

addi-tional RNA approximately 2.2 kb in length. Although the

latterRNAhasapproximatelythesamesizeasthepre-S2+ S RNA, itisanovel RNAspecies sincepre-S2+S RNAdoes

notcontainHBcAgsequences(Fig. 2A).Thesamefilterwas

also hybridized with a glyceraldehyde phosphate

dehydro-genase cDNAprobe (32) and showed no RNAdegradation (data notshown).

determine the structure of this novel RNAspecies, hybrid-ization experiments were done with different subgenomic fragments of HBV (Fig. 2A). In Fig. 2B, lane 1, all three RNAs were detected with thefull-length HBVprobe. When the HBcAggene-specific BglII-BglII probe was used, only 3.6- and 2.2-kb species were detected (lane 2). The probe from the next region (BglII-EcoRI) detected two speciesas

expected, the 3.6-kb pregenomic and the 2.6-kb

pre-Sl

RNAs(lane 3). Another probe further downstream (EcoRI-RsaI), containing sequences of the HBsAg gene, hybridized with all three bands (lane 4). These results suggest that the 2.2-kb RNA contains sequences of the C and S regions but has an internal deletion in the pre-S region.

cDNAcloning and sequencing indicated that thetranscript is adoubly spliced RNA. Because the amounts of available RNAswere toolimitedtodoS1 nuclease mapping to further define the structure of this RNA, we performed cDNA cloning of poly(A)+ RNAs from these liver tissues and screened the clones withBgII-BgIII probes. Positive clones werefurtherhybridizedwithaBglII-EcoRI probe, and those which werenegative at thesecond hybridizationwere con-sidered candidate cDNA clones derived from this novel 2.2-kb RNA. By this approach, several cDNA clones were

picked up. The sequence of one representative clone

(CRC12) was obtained. After comparison with HBV

se-quences, this clone was found to start from the 5' end of pregenomic RNA and to skip the next 282 nucleotides

correspondingtothe middle ofHBcAg (Fig. 3A, left). There

isaseconddeletion of 1016nucleotidescorrespondingtothe

regionbetween the endofHBcAgand thebeginningof the S

region (Fig. 3A, right). The deleted sequences are bracketed

atthe 5' endby GCorGT andatthe 3' endby AG (Fig. 3B), theconsensusboundarysequencesof the introns(19, 24). In

aprevioustransfectionexperimentdone withCOS cells, the

second acceptor site definedbythiscDNAwasaccidentally

discovered (1). Thus, we concluded that the cDNA clone

represented adoubly spliced HBV transcript (Fig. 3C).

Spliced RNA is coterminal with pregenomic RNA. The

approximate 5' and 3' ends of this transcript were

subse-quentlydetermined. Since there isonlyonepolyadenylation

signal in the HBV genome (33), all poly(A)+ transcripts of

HBV were 3'-end coterminal. Subtraction of two introns from the pregenomicRNA wouldgive a size equivalent to that of the spliced2.2-kb RNA (Fig. 3C). Thus, it is likely that the spliced transcript is coterminal with pregenomic RNA. To test this possibility, an oligonucleotide comple-mentaryto nucleotides466 to480ofthe HBV genome was used forprimer extension. Usingpoly(A)+ RNA from these humantissuesasthe template, three primer-extended

prod-uctswereobtained(Fig.4).The product, about 600 bases in

length, should represent the extended primer on unspliced

pregenomicRNA. The other two products, about 300 bases

inlength, probablyrepresented the extended primer on the

spliced2.2-kb RNA. The presenceof two bands may be due

tomicroheterogeneityatthe 5' ends of the HBV transcripts.

This result suggests that the 2.2-kb spliced RNA is cotermi-nal with the 3.6-kb pregenomic RNA.

Analyses oftheORFs of the spliced RNA and the

conserva-tion of sequences around splicing junctions. The coding

ca-pacity of this splicedRNAwas analyzed. The first splicing

eventtookplace in the ORF of the HBcAg gene. Since 282 bases were removed, the reading frame of HBcAg was

preserved.The secondsplicing event removed the

termina-tion codon ofHBcAg and the whole hypervariable regions of

I

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TCATCTCATAGCATGTCCTACTOTTCAAGCCTCCAAGC

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70 80 90 100 110 120

TGTGCCTTGGCTGGCTTTOGOGCATGGACATTGACCCOTATMAGAATTTOGAGCTTCTO

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130 140 150

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adyw actcag/GCAAGC--TGTTAG/acgaga.--ctoaat/GTTAGT--TTCTAG/ggg9aa

FIG. 5. Analyses of coding capacities of the spliced RNAs andsequenceconservation around the splicing sites. (A) The mapped5'end ofpregenomic RNAwasusedasthe first nucleotide (11). S1, The first splicing junction;S2,the second splicingjunction. The overlapping ORFs between shortened HBcAg andpol are shown. (B) Conservation of nucleotide sequences around the splicing sites among HBV

subtypes. Sequences from HBV subtypes adr (5), adr4 (20), adw (20), ayw(6),and adyw (21)arealigned with respecttothetwosplicing junctions. S.D., Splice donor site; S.A., spliceacceptorsites.

pre-Sl and pre-S2.Asaresult, the shortened ORF ofHBcAg

is followed by a Trp and then is contiguous with that of

HBsAg. The N terminus of the putative polymerase gene

was removed, and a new initiation codon was generated,

which is in phase with the truncated ORFs ofthepolymerase

gene (Fig. 5A). The truncated pol includes the most

con-servedregionand theregionhomologoustothat ofretroviral

reversetranscriptase (Fig. 3C).

The sequences around the two splicing junctions were

found to be remarkably conserved amongthe various

sub-types of HBV (Fig. 5B) (except for one nucleotide). The

homology could be extended somewhat to that of

wood-1o8

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second

splicing

acceptor site

(data

not

shown).

It will be

interesting

todeterminewhetheranimal

hepadnaviruses

also express similar

spliced transcripts.

DISCUSSION

The

transcription

ofHBVboth invivo and in transfected cell lines has been studied

extensively,

and mRNAs for

major

viral

protein

products

havebeenidentified

(11,

28, 33).

A2.2-kbviralRNA is showntobe thetranslation

template

for

hepatitis

B surface

proteins,

and a 3.6-kb RNA is the

template

for core

protein (11, 33). However,

whether the

3.6-kb RNA also serves as the mRNA for viral reverse

transcriptase

is not yet settled.

Considering

the

special

genetic organization

of

HBV,

welooked forthe presence of

spliced

RNAas analternative RNA

template.

Intwo

HBV-infected human liver

specimens,

evidence ofa2.2-kb

doubly

spliced

viral

transcript

was obtained. In

fact,

similar RNA

species

were

recently

also detected in infected human

tis-sues

(26).

The results

clearly

indicate theexistence ofviral

transcripts

of HBV not yet discovered and the need to

explore

the

complex

viral gene

expression

during persistent

infections.

The

biological

significance

ofthis

spliced

RNA

currently

is

unknown.Itshould be

pointed

out

that,

exceptin the report

ofSu et al.

(26),

the RNA has not been demonstrated in

previous

studies. This factmayreflectthe lack ofan

essen-tial role for this RNA in the viral life

cycle.

However,

the

failure could be due to

(i)

an

obscuring

effect

by

the

pre-S2+S

mRNA that

comigrates

with the

spliced

mRNA

species

or

(ii)

adifferent levelof

splicing efficiency,

since it

has been well documented that retroviruses can

regulate

viralgene

products by

controlling

the extent of

splicing (8,

14).

In

addition,

the conservation ofsequences around the

splicing

donor andacceptorsitesamongHBV

subtypes

(Fig.

5B)

suggeststhat RNA

splicing

may havefunctional role. For all the retroviruses

characterized,

the

unspliced

ge-nomicRNA is considered tobethe

template

for translation

ofreverse

transcriptase

(10, 29). However,

in

retroviruses,

the ORF for reverse

transcriptase

is

separated

from that of

the

envelope

proteins

which contain

highly

variable

regions

(29).

In contrast, the two ORFs in HBV are

completely

overlapping.

The

doubly spliced

form of

pregenomic

RNA

identified inour

study

may

provide

amechanismtoremove

this

hypervariable region

so that the

polymerase

remains

functional.In

addition,

the

region

within the HBVpolORF

which is

homologous

to theretroviral reverse transcriptase

is retained in this

spliced

RNA

(Fig.

3C).

Therefore,

it is

possible

thatthis

spliced

RNAisthetranslation

template

for

reverse

transcriptase

ofHBV. Ina

plant

virus,

cauliflower

mosaic

virus,

thatalso

replicates through

reverse

transcrip-tion

(9),

the

template

for translation ofreversetranscriptase has been shown to bea

subgenomic

RNAof about 2.5 kb,

whichcouldbea

spliced

RNA(22). This result is consistent

with

findings

presented

inourreport. Tofurthersupportit,

a

putative

reverse

transcriptase

identified in virions by

specific

antibody

has been shown not to contain the N

terminus ofpol

ORF,

the

hypervariable

region (13). The

primary

translation product of retroviral reverse

transcriptase

isagag-pol fusion

protein

which isfunctionally

inactive

(10, 29).

After

being

packaged into virionparticles,

the fusion

protein

is cleaved byaproteaselocated eitherat

the C terminusof gagor atthe N terminusofpoltorelease

theactivereverse

transcriptase

(16, 29). Asimilar

protease-cleavage

mechanismmayalso be

important

for activation of

protease has beenproposedtobe locatedatthe Nterminus of thenucleocapsid protein (HBcAg)

(17).

The removalofa

portionof

HBcAg by

RNA splicingcould

bring

the domain

of theputativeprotease closerto thehypothetical

cleavage

site ofreversetranscriptase. Thus,thismaybeacrucialstep

inactivatingthe distantly spaced protease.

Inthisspliced RNA,thetwo shortened ORFs of

HBcAg

and pol are contiguous but out of phase (Fig. 5A). This

organization is reminiscent of that of theretroviral gene for

the

gag-pol

fusion protein (10, 29). The genetic structure

allows the translation of this spliced RNA viaa ribosomal frame-shifting mechanism (34) to generate a

protease-pol

fusionprotein. However,itis noted that the second

splicing

eventcreatesa new initiation codon in shortened

pol

ORF. Therefore, the synthesis of viral reverse transcriptase

by

internal initiationfrom thesplicing site isequally possible,as

isdocumentedbythemechanism for translation of the duck

HBVpolgene (2, 23). Further studiesare needed to show

which modelactually works intranslating thisdoublyspliced transcript of HBV.

Although arguments that thissplicedRNA is the

template

for HBV reverse transcriptase have beenpresented,

previ-ous genetic studies on the duck HBVpol ORF identified severalfeatures whicharerelevanttopolexpression butare

not present in the spliced RNA. For example, the spliced RNAdoesnotcontain the first AUG in thepolORF that is shown to be important for polymerase expression (2,

23).

Certainly it is possible to evoke a mechanismdefending such

adefect(e.g., that the sequence is required for removal of the intron), but until further experimental data become available, the functional assignment of this spliced RNA shouldbe considered hypothetical.

Another consequence of this RNA splicing event is the possible generation of an HBcAg-HBsAg fusion protein, since the two ORFs are fused in this spliced RNA. Such a

protein remains tobe identified.

Finally, since the spliced RNA is coterminal with the pregenome, all thecis-acting sequences required for replica-tion are probably present. However, this RNA species is unlikely to be packaged into virus particles; otherwise, its reverse transcription might interfere with viral replication. Thus, the sequences missing in this spliced mRNA, analo-gous tothose ofretroviruses, may be important for

packag-ing (15).

ACKNOWLEDGMENTS

Wethank Michael M. C. Lai for critical reading of the manuscript and helpful comments, W. S. Robinson for providing the HBV

plasmid, S. J. Tu for invaluable technical assistance, and M. S. Chuang for secretarial assistance.

The workwassupported bygrantsfrom theInstitute of Biomed-icalSciences,AcademiaSinica,the National Science Council, and

theDepartment ofHealth, Taiwan, Republic ofChina.

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