Mutational analysis of delta antigen: effect on assembly and replication of hepatitis delta virus.

0022-538X/94/$04.00+0

Copyright©D 1994, American Society for

Microbiology

Mutational

Analysis

of

Delta

Antigen:

Effect

on

Assembly

and

Replication

of

Hepatitis

Delta Virus

MING-FU

CHANG,'*

CHUN-JUNG

CHEN,'

ANDSHIN C.

CHANG2

Institutesof

Biochemistry'

andMicrobiology,2 College of Medicine, National Taiwan University, Taipei, Taiwan, Republic of China

Received 27 August 1993/Accepted 2 November1993

Hepatitis delta virus requires a helperfunction fromhepatitisBvirus forpackaging, release, andinfection ofhepatocytes. The assemblyof large deltaantigen(HDAg) ismediated bycopackagingwith the small surface antigenofhepatitisBvirus (HBsAg),andtheassemblyof smallHDAg requiresinteractions withlarge HDAg. Toexamine the molecular mechanisms by which smallHBsAg, largeHDAg,and smallHDAg interact,wehave established a virionassemblysystem inCOS7cellsbycotransfecting plasmids encodingthe smallHBsAg,the smallHDAg, and largeHDAgmutants.Resultsindicate that sequences within theC-terminal 19-amino-acid domainflankingtheCxxxisoprenylation motif areimportantfor theassemblyoflarge HDAg.Inaddition, a large HDAg mutant bearing extra sequences separating the C-terminal 19-amino-acid domain from the commonregionsof the small andlargeHDAgsiscapable, likethewild-type largeHDAg,of

copackaging

with small HBsAg. The ability ofassembly is also demonstrated for a large HDAg mutant from which nuclear localization signals have beenremoved. Furthermore, acryptic signal within the N-terminal50 amino acid residues other than the putative N-terminal coiled-coil structure and a subdomain betweenamino acid residues 50 and 65 of the largeHDAg are important for the assembly of small HDAgas well as the trans-dominant negative regulation of largeHDAg inhepatitisdelta virusreplication.

Hepatitis delta virus(HDV) isasatellite virus ofhepatitisB virus (HBV). HDV particles contain a negative single-stranded, covalently closed circular RNA genome and virus-specific delta antigens (HDAgs)(1, 2,6, 18, 24, 27, 34,35). The RNA genome possessesextensive intramolecular complemen-tary sequencesthat formanunbranched rod-likestructure(18) and isaribozyme similartoplant viroidRNA(3, 20, 26, 31, 36, 37). The envelope ofHDVis composed of three forms ofHBV surface antigen (HBsAg), L, M, and S, thatare presentinthe ratio of 1:5:95 (2). The S protein (small HBsAg) contains information sufficient for producingemptyparticles ofHDVin thepresenceof HDAgs(28, 32). TheLprotein is essential for infectivity (30), whereas the roles of M protein in HDV multiplication are unknown. In addition, encapsidation of HDVgenomic RNAis mediatedby interactionsbetween the RNA and HDAgs and requires protein-protein interactions betweenHDAgsand small HBsAg (9a).

HDAgsarenuclearphosphoproteins(6, 17) that exist intwo forms. The onlysequence difference between thetwoforms is thepresenceinthelarge HDAg ofanadditional 19-amino-acid domain at the C terminus resulting from a specific point mutation (uridineto cytidine) atthe stop codon of the small HDAg (4, 39, 42). However, they have very distinctive func-tions. ThesmallHDAg iscapable ofsupporting replication of HDV (19), while the large HDAg inhibits the replication of HDVand is essential for virion assembly (5, 9, 28). Recent studiesonthe functional motifs ofHDAgsrevealed that there exist multiple functions within the N-terminal regions of HDAgs. Twoindependent nuclear localization signals (NLS1 andNLS2) importantfor nuclear transport of the HDAgs are located between amino acid residues 35 and 88 (7). The N-terminal 65-amino-acid domain is important for the

inter-*Corresponding author. Mailing address: Institute of Biochemistry,

National Taiwan University College of Medicine, No. 1, Jen-Ai Rd., 1stSection,Taipei, Taiwan, Republic of China. Phone: (02) 397-0800,

ext. 8217. Fax:(02)341-1695.

actions betweenHDAgsboth in vivo and in vitro(7, 8, 33, 40). Furthermore, aputative coiled-coil structure oran unidenti-fiedsignal within the N-terminal 50 amino acid residuesof the large HDAg has been proven to be involved in

negative

regulation of viral replication and in

copackaging

with the small HDAg

(8,

10, 21,

40).

In

addition,

the Leu-115

(8)

and two

arginine-rich

motifs in the middle domain

(22)

of the small HDAg arecrucial forRNAbinding andHDVreplication. The isoprenylation motif at the C terminus of large HDAg is involved inHDVassembly

(14).

Eventhough themanystudies examining the functional motifs of HDAgs have

greatly

con-tributed to ourunderstanding of the replication and assembly of HDV, the HDV multiplication process has not been

fully

elucidated.

Previous studies have demonstrated that the large HDAg could be copackaged with small HBsAg to form virus-like particles in Huh-7 cells. The virus-likeparticles secreted into culture media could be isolated through sucrose

gradient

centrifugation

and observedunderanelectron

microscope

(5,

28, 32,34).Inthecurrentstudy,wehavefurther established a packagingsystemforHDV inCOS7 cells. Detailed mutational analyses have revealed that

flanking

sequences of the Cxxx isoprenylation motif within the C-terminal 19-amino-acid do-main ofthe large HDAg are important for the assembly of HDV.Moreover,alarge HDAg lacking amino acid residues 50 to 75 failsto inhibit viral

replication

andto

copackage

small HDAg.

MATERIALSAND METHODS

Cell lines and DNA transfection. Huh-7 cells

(a

human hepatoma cell

line)

and COS7 cells

(a

monkeykidney cell

line)

were cultured at 37°C in Dulbecco's modified Eagle medium supplemented with 10% fetal calf serum

plus

100 U of penicillin and 100

pug

ofstreptomycin perml. DNA transfec-tion was performed with cationic liposomes as previously described

(7).

646

on November 9, 2019 by guest

http://jvi.asm.org/

Plasmid construction. (i) Plasmids pECE-d-BE, pECE-d-SM, and pSVD2. Plasmid pECE-d-BE encodes the wild-type large HDAg consisting of 214 amino acid residues, and plas-mid pECE-d-SMencodesthe wild-type small HDAg consisting of 195 amino acid residues. Both plasmids have beendescribed previously (6, 41). For construction of plasmid pSVD2, the 3,397-bpSacl-HindIll DNA fragment of plasmid pD2 (8)was

subcloned into modified pECE vector (13) from which the polylinkersequencesfrom theSacl sitetotheHindlIl site had been deleted. The resultant plasmid, pSVD2, contains a

dimeric HDV cDNA under the control of the simian virus 40 early promoter.

(ii) Plasmid pECE-C-ES. For construction of plasmid pECE-C-ES, plasmid pECE-C (a gift from James Ou, Univer-sity ofSouthern California), which contains monomeric HBV

cDNA,wasdigested with HindlIl and EcoRI and treated with the Klenow fragment of DNA polymerase I before self-ligation. The resultant plasmid, pECE-C-ES,encodesthe small form of HBsAg (Fig. IA).

(iii) PlasmidspECEL-d35/75andpECEL-d89/163. For

con-struction of plasmids pECEL-d35/75 and pECEL-d89/163, plasmid pECE-d-BE containing the BamHI-EcoRI fragment of HDVcDNA was digested withMroI and StuIl plus SmaI, respectively, before self-ligation. The resultant plasmids, pECEL-d35/75 andpECEL-d89/163, encode largeHDAg

mu-tantswith deletions from amino acid residues 35 to 75 and 89

to 163, respectively. Specific mutations in these plasmids and

the following constructs used in this studywere confirmed by

DNAsequencing, using the dideoxychain termination method (29). Positions ofmutations for each correspondingHDAgare

summarized in Fig. 2 and 5A.

(iv)Plasmids pECEL-d35/88and pECEL-CRLDK. For

con-struction of plasmids pECEL-d35/88 and pECEL-CRLDK, plasmid pECE-d-BEwasdigested withMroIplus StuI andSall

plus XbaI,respectively, and treated with the Klenow fragment of DNApolymerase I before self-ligation. The resultant plas-mid, pECEL-d35/88, encodes a large HDAg mutant missing the domain from amino acid residues 35 to 88, and plasmid pECEL-CRLDKencodes alarge HDAgwiththe Cxxx

isopre-nylation motif (14, 25) (211-Cys-Arg-Pro-Gln-214; numbers indicate thepositions of amino acid residues) attheextreme C

terminuschangedto211-Cys-Arg-Leu-Asp-Lys-215 (CRLDK;

Cxxxx).

(v) Plasmid pECEL-aCAT. Plasmid pT7L-aCAT was

cre-ated and used forconstructionof pECEL-aCAT.For

construc-tion ofpT7L-aCAT, plasmidpSV2CAT (15)wasdigested with

StuI plus RsaI. The StuI-RsaI fragment which contains the chloramphenicol acetyltransferase gene was then subcloned

into a modified pT7-d-BP (6) from which the 143-bp

SaclI-Sacl DNA fragment had been deleted and the resultant ends blunted with T4 DNA polymerase. For construction of pECEL-aCAT,plasmid pT7L-aCATwas furtherdigested with

HindIII for subsequent cloning of the HDAg-containing HindIII fragment into the unique HindlIl site of the pECE

vector (13). This resultant plasmid, pECEL-aCAT, encodes a

fusionprotein with the N-terminal 31 amino acid residues of chloramphenicol acetyltransferase linked to amino acid resi-dues 10to 214of the large HDAg.

(vi) Plasmid pECEL-dl96/210.Forconstruction of pECEL-d196/210, plasmid pT7-d-BP (6)wastreated withNcoIand the

Klenowfragmentof DNA polymerase I before SacII digestion. The 559-bp SacII-NcoI DNA fragment was then subcloned

into modified pECE-d-BE from which the 608-bp SacII-SalI DNA fragment had been deleted and the remaining DNA blunted at the Sall site with the Klenow fragment of DNA polymerase I. The resultant plasmid, pECEL-dI96/210,

en-codes a large HDAg mutant in which amino acid residues 196 to 210 have been deleted but the Cxxx isoprenylation motif (211-CRPQ-214) is unchanged.

(vii) Plasmid pECEL-dlO/55. A 495-bp DNA fragment that represents the HDV sequence from nucleotides 720 to 1214 was synthesized by PCR as previously described (8). After further digestion of the PCR productwithSall, a341-bp DNA fragment was generated and subcloned into modified pECE-d-BE from which the 608-bp

SacII-SalI

fragment had been deleted and the remaining DNA blunted at the

SacII

site with T4 DNA polymerase. The resultant plasmid, designated pECEL-dlO/55, encodes the large HDAg with an internal deletion from amino acid residues 10 to 55.

(viii) PlasmidpECEL-d164/195. For construction of plasmid pECEL-dl64/195, the 365-bp NcoI-HindIII DNA fragment of plasmid pT7-d-BP (6) was blunted at theNcoI site and inserted into a modified pBluescript KS(+) vector (Stratagene) that had been treated withEcoRI plus

HindIll

and blunted at the EcoRI site with the Klenow fragment of DNA polymerase I. The recombinant plasmid, designated pKSL(NH), was further treated withPstI to generate a 374-bp DNA fragment. Follow-ing a blunt-end reaction with T4 DNA polymerase, the 374-bp PstI fragment was further subcloned into modified pECE-d-BE from which the 519-bp SmaI-EcoRI fragment had been de-leted and the remaining DNA blunted at theEcoRI site with the Klenow fragment of DNA polymerase I. The resultant plasmid, pECEL-d164/195, encodes the large HDAg with an internal deletion from amino acid residues 164 to 195.

(ix) Plasmids pECEL-d5O/75 and pECEL-d65/75. Recombi-nant plasmids pKSN50Z(SB) and pKSN65Z(SB) were first obtained for further construction of pECEL-d50/75 and pECEL-d65/75, respectively. To construct pKSN50Z(SB), a 125-bpSaclI-BamHIfragment generated from plasmid pN50Z (7) was subcloned into a modified pBluescript KS(+) vector that had been treated with SacII plus BamHI. A similar approach was taken to construct plasmid pKSN65Z(SB) except that the starting plasmid was pN65Z (7) instead of

pN5OZ,

and a 170-bp SacII-BamHI DNA fragment was generated from plasmid pN65Z. Both

pKSN5OZ(SB)

and pKSN65Z(SB) were digested with SacII plus SmaI. The SacII-SmaI DNA frag-ments were then individually subcloned into modified pECE-d-BE from which the 317-bp SacII-MroI fragment had been deleted and the remaining DNA blunted at the MroI site with the Klenow fragment of DNA polymerase I to generate plasmids pECEL-d50/75 and pECEL-d65/75, respectively. Plasmid pECEL-d5O/75 encodes the large HDAg with an internal deletion from amino acid residues 50 to 75 whereas plasmid pECEL-d65/75 encodes the large HDAg deleted from amino acid residues 65 to 75. Both of the mutant proteins possess the extra amino acids GDPPR (Gly-Asp-Pro-Pro-Arg) located at the site of deletion, resulting from the processes of plasmid construction.

(x) Plasmid pECES-aSR. For construction of plasmid pECES-aSR, plasmid pECE-d-BE was digested with

SalIl,

deleted with exonuclease III

(Exolll)

and mung bean exonu-clease (16), and further digested with XbaI and blunt ended with the Klenow fragment of DNA polymerase I before self-ligation. The resultant plasmid, pECES-aSR, encodes a small HDAg with two extra amino acid residues, Ser-Arg, at the Cterminus.

(xi) PlasmidspECEL8-al95/196andpECEL7-al95/198.For construction of plasmids pECEL8-al95/196 and pECEL7-a195/198, intermediate recombinant plasmids were first gener-ated asfollows.Plasmid pT7-d-BP (6) was digested withNcoI and treated with

Exolll

and mung bean exonuclease (16) and then withHindIll. DNA fragments with sizes around 300 bp

on November 9, 2019 by guest

http://jvi.asm.org/

wereinserted into modified

pBluescript KS(+)

fromwhich the

polylinker

sequences from the Hindlll site totheEcoRI site had been deleted and blunted at the EcoRI site with the Klenow

fragment

of DNA

polymerase

I. Recombinant

plas-mids

pKSL8

and

pKSL7

containing

the HDV sequencesfrom nucleotides 654 to 1012 and 654 to 1003

(24),

respectively,

were

generated.

Plasmids

pKSL8

and

pKSL7

were further treated withBamHI andthe Klenow

fragment

ofDNA

poly-merase I before HindlIl

digestion.

The resultant

HDV-con-taining

BamHI-HindIIIDNA

fragments

werethen

individually

subcloned intomodified

pT7-d-BP

(6)

from which the

365-bp

HindIII-NcoI

fragment

had been deleted and the

remaining

DNA blunted at the NcoI site with the Klenow

fragment

of DNA

polymerase

I to generate recombinant

plasmids

pT7L8-a195/196

and

pT7L7-a195/198, respectively.

Plasmids

pECEL8-al95/196

and

pECEL7-al95/198

were then obtained

by replacing

the SacII-SalI

HDV-containing

DNA

fragment

in the

plasmid

pECE-d-SM

with their cognate

fragments

from

pT7L8-a195/196

and

pT7L7-a195/198,

respectively.

Plas-mid

pECEL8-al95/196

encodes a

large HDAg

mutant with

anextraamino acid

domain, Trp-Ile-Pro-Arg-Ala-Ala-Gly-Ile

(WIPRAAGI),

inserted between residues 195 and 196. Plas-mid

pECEL7-al95/198

encodesa

large HDAg

mutant

lacking

amino acid residues 196 and 197 and

containing

an extra

domain, WIPRAAG,

inserted between amino acid residues 195 and 198.

(xii)

Plasmids

pECE-d-BE(pro)

and

pECE-d-SM(pro).

Plasmid

pECE-d-BE(pro)

has been described

previously (8).

It encodesa

large HDAg

with

proline

substitutionsatLeu-37 and Ile-41. The strategy used for

constructing pECE-d-SM(pro)

wassimilartothat used for

constructing pECE-d-BE(pro) (8)

except that the mutated

234-bp

Sacll-Stul

fragment

was

subcloned into modified

pECE-d-SM

from which the

234-bp

SaclI-Stul

fragment

had been deleted.

Harvest of HBV- and HDV-like

particles.

Virus-like

parti-cleswerecollectedasdescribed

previously (38)

with modifica-tions. In

brief,

culture mediawereharvested 3

days

posttrans-fection and clarified

by spinning

at

9,000

rpm in an RA-4F rotor

(Kubota)

for 10 min. Thesupernatantwas

layered

over a

20%

sucrose cushion

(20%

sucrose, 20 mM HEPES

[N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid] [pH 7.4],

0.1% bovine serum albumin

[BSA])

and then

centrifuged

at

40,000

rpm in an

SW41

rotor

(Beckman)

for 5 h. Virus-like

particles

secreted into the culture media were spun to the bottom. The

pellet

was

resuspended

in

phosphate-buffered

saline

(PBS)

and

analyzed

for the presence of

HBsAg

and

HDAgs.

Immunostaining

assay.Immunoblot

analysis

was

performed

as

previously

described

(8).

In

brief,

whole-cell extracts and

protein lysates

from virus-like

particles

were

prepared

3

days

posttransfection, separated by

sodium

dodecyl

sulfate-polyac-rylamide gel electrophoresis

and electrotransferred onto Im-mobilon-P membrane

(Millipore).

Themembranewas immu-nostained withantibodies

specific

to

HDAg

(23)

orto

HBsAg

(Dako).

Indirect immunofluorescence

staining.

Transfected COS7 cells were fixed on

coverslips by

treatment with

precooled

acetone-methanol

(4:1)

at -

20°C

for2h.Thefixed cellswere

incubatedwith 1% BSAat

37°C

for15 minandthen ina37°C moistchamber for30 minwithan

antibody specific

toHDAg

(23)

which had been

purified

on immobilized

protein

G

gel

(Pierce).

After

incubation,

the cells were washed with PBS. Fluorescein

isothiocyanate-conjugated

goat anti-rabbit

immu-noglobulin

G

(Jackson

ImmunoResearch

Laboratories, Inc.)

wasadded at a1:40

dilution,

and incubationwascontinuedat

37°C

for another 30 min. After a

subsequent

treatmentwith

Evansblue counterstain solution (Sigma) for 1 min, the cells were washed with PBS, covered with mounting fluid (no. 9; Organon TeknikaCorp.), and photographedunder a fluores-cencemicroscope.

Northern (RNA) blot analysis. Isolation of total cellular RNAand Northern blot analysiswereperformedaspreviously described (8, 11). The probe usedwasthe antigenomicstrand of HDV RNA that was transcribed in vitro from the Sall dimeric HDV cDNA plasmid, pD2 (8), with T7 RNA poly-merase (Promega) in the presence of [cx-32P]UTP. The 28S rRNA detected by hybridization with a 32P-labeled cDNA fragment of 28SrRNA wasusedas an internal control

(8).

RESULTS

Large HDAg could be detected in culture medium when coexpressed with small HBsAg in both nonhepatic and hepatic cells. To elucidate the mechanisms by which HDAgs are involved in the assembly of HDV, we first established a cotransfectionsystemin COS7 cells withplasmid pECE-C-ES (Fig. 1A) that encodes the small HBsAg and plasmid pECE-d-BE that encodes the large HDAg. Two formsof the small HBsAg, p24 and gp27, were detected in culture medium by Western blot

(immunoblot)

analysis with anantibody specific to HBsAg when plasmid pECE-C-ES alone was transfected intoCOS7 cells(Fig. 1B, lane 2).Asimilarresultwasobserved for protein lysate isolated from the transfected COS7 cells (data not shown). Therefore, the small HBsAg could be expressed in COS7 cells and present in both cell lysates and culturemedia. Thisprovides amodel systemforstudyingthe assembly ofHDV.Cotransfection of plasmid pECE-C-ES with pECE-d-BE further demonstrated that the large HDAg ex-pressed in COS7 cells is capable of copackaging with the small HBsAg (Fig. 1C, lane 1). Nevertheless, small HDAg was detected in culture medium only in the presence of large HDAg (Fig. 1C, lanes 2 and 3). Similarpatterns were repro-duced in Huh-7 cells

(Fig.

1C,lanes 7 to

9).

Functional motifs of the

large

HDAg essential for virion assemblywere further examined.

The Cxxxisoprenylation motif is not the only prerequisite for virion assembly of the large HDAg. A series of mutant constructs for large HDAg were generated (summarized in Fig.

2)

and used in cotransfection experiments with small HBsAg to determine the functional motifs of large HDAg required for virion assembly. Neither the deletionnearthe N terminus encoded by pECEL-d35/88, the deletion nearthe C terminusencodedby pECEL-dl64/195,northe deletion in the middle domain encoded by pECEL-d89/163 affected the ability oflarge HDAgto form virus-likeparticleswith smallHBsAg (Fig. 3A, lanes3 to5), suggesting that the amino acid residues between35 and 195 aredispensable for the assembly of large HDAg. Todetermine whether the N-terminal 35 amino acid residues contribute to the assembly of HDV, we further generatedtwo mutantconstructs,dlO/55 and pECEL-aCAT, that encode large HDAgs with deletions from amino acid residues 10 to55 andof the first10amino acid residues, respectively. In plasmid pECEL-aCAT, the sequences for translation initiationwere derived from the chloramphenicol acetyltransferasegene.Results indicatethat theN-terminal 55 amino acid residues may only contribute to the efficiency of virion assembly of the large HDAg as both HDAg mutants were detected in theculture media (Fig. 3B, lanes 3 and 4). Takentogether, results from mutational analysis suggestthat the 19 amino acid residues at the C terminus of the large HDAgplay amajor role in theassembly ofHDV.

Isoprenylation of large HDAgat the 211-CRPQ-214 motif

on November 9, 2019 by guest

http://jvi.asm.org/

A _{B assembly}

(kD)

HBsAg

1 2

29-226 gp27,

p24,

14-Sac I

c

1 2 3 4 5 6 7 8 9

3 45

10 11 12

-L

FIG. 1. Westernblot analysis ofHDAgsfollowing cotransfection of plasmids encoding the small HBsAg and wild-type HDAgs. (A) Structure ofthe recombinant plasmid pECE-C-ES. Plasmid pECE-C-ES encodes the small HBsAg (p24) consistingof 226 amino acid residues. The p24 smallHBsAg canbeglycosylated to form the gp27 product.The solid circlerepresentsthe simianvirus 40 early promoter. The stippled domain represents the complete coding region of the small HBsAg, and the solid boxes represent sequences flankingthe coding region of the small HBsAg. (B) Western blot analysis of HBsAg.Plasmidsused in the transfection studieswere pECE vector (13) (lane1) and pECE-C-ES (lane 2), respectively.HBV-likeparticles were harvested from medium 3 days posttransfection, and protein lysateswereanalyzedwithanantibodyspecifictoHBsAg(Dako).Two formsof the smallHBsAg,p24andgp27,wereidentified.(C)Western blot analysisofHDAgs.Cotransfectionwasperformedwithplasmids pECE-C-ES andpECE-d-BE (lanes1, 4, 7, and10), plasmids pECE-C-ES andpECE-d-SM(lanes 2, 5, 8,and11),orplasmids pECE-C-ES, pECE-d-BE, andpECE-d-SM(lanes 3, 6, 9,and12). Plasmid pECE-d-BEencodes thelarge HDAgwhereasplasmid pECE-d-SMencodes the small HDAg. Protein lysates were prepared either from the virus-likeparticlessecretedinto culture media(lanes 1to3 and 7 to9)

orfrom transfected cells(lanes4to6 and 10to12)andimmunoblotted with an antibodyspecifictoHDAgs. Cell lines usedforthe cotrans-fection studieswereCOS7cells(lanes1to6)and Huh-7 cells(lanes7

to 12). Large (L)andsmall (S) HDAgsareindicated.

has been showntobe

important

fortheassembly ofHDV

(14).

However, whether the Cxxx motif issufficient for thiseventhas notbeenstudied. Here,wefurther examined the

participation

of the

surrounding

sequence in the

assembly

ofHDV. Muta-tions that have either the Cxxx

(21

1-CRPQ-214)

motif re-placed by Cxxxx (21 1-CRLDK-215, encoded by

pECEL-CRLDK)

or a short extension of two unrelated amino acid

residues,

Ser-Arg, at the C terminus of the small HDAg

(encoded

by

pECES-aSR)

abolished the

ability

of HDAgto form virus-like

particles

with small HBsAg as both of the HDAg mutants were notdetected in the culture media (Fig. 3A, lanes7and

8).

Itis

noteworthy

that the

Cys-211

residue in the former mutant isnot located in the fourth

position

from the C

terminus,

whereas the latter mutant lacks both the 211-CRPQ-214

isoprenylation

motif and the

flanking

sequence

1 214

1 195

35 88

pECEL-d35/88 _ ,__

89 163

164 195 10 55

10

210 CRLDK pECELd89/163

pECEL-d164/195 pECEL-dlO/55 pECEL-aCAT

=

pECEL-CRLDK pECELd196/210 pECES-&SR pECEL8-a195/196 pECE-d-BE pECE-d-SM

+

+

+

196 210 195 MSR

195/196

WIPRAAGI

+

195/198

pECEL7-a195/198 +

WIPRAAG

FIG. 2. Structure of large HDAg mutants and their characteristics in the assembly of virus-like particles. Plasmids pECE-d-BE and pECE-d-SM encode thewild-typelarge HDAg consistingof214amino acid residues and smallHDAgconsisting of195 amino acid residues, respectively. Structural representations of individual mutants are shown. Numbers denote thepositionsofaminoacid residuesflanking eachdeletionandinsertion.Specific amino acid residuesthatreplaced the cognateresiduesin thewild-typelargeHDAgor extraamino acid residues that were insertedintowild-typeHDAg are indicated. The abilityof each large HDAgmutant to form virus-like particleswith smallHBsAgis indicatedbyaplusor aminussign.

from amino acid residues 196 to 210. Interestingly, in the presence of small HBsAg, an HDAg mutant (encoded by

pECEL-d196/210)

that possesses the211-CRPQ-214 motif but lacks the 15 amino acid residues from 196 to 210 was not detected inthe culture medium either

(Fig.

3A, lane

6).

These results strongly suggest that sequences within the C-terminal 19amino acid residues

flanking

the Cxxxmotif of

large

HDAg are

important

forvirion

assembly.

Toexamine whether conformation of theextreme C-termi-nal

domain

of

large

HDAg would affect virion

assembly,

we constructed two insertion mutants, pECEL8-al95/196 and pECEL7-al95/198. The insertions resulted in

separation

of the C-terminal 19-amino-acid domain of the

large HDAg

from the common 195

amino

acid residues present in both small and large HDAgs. It was found that insertion of

eight

or seven unrelated amino acids between amino acid residues 195 and 196orbetween aminoacid residues 195and

198,

respectively,

did notaffect

large

HDAg in

copackaging

withsmall

HBsAg

(Fig.

3B, lanes 5 and

6).

Taken

together,

theseresults indicate that the Cxxx

isoprenylation

motif

coupled

with the

on November 9, 2019 by guest

http://jvi.asm.org/

[image:4.612.58.295.75.329.2] [image:4.612.315.544.77.411.2]

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

a

1 2 3 4 5 6 7 8 9 10 11 12

FIG. 3. Western blot analysis of large HDAg mutants following cotransfection ofplasmids encoding wild-type small HBsAg and large HDAgmutants.Cotransfection studieswerecarriedoutinCOS7 cells. HDAgs expressedwere thewild-type large HDAg (panel A, lanes1

and9; panel B, lanes 2 and 8), the wild-type small HDAg (panel A, lanes 2 and 10; panel B, lanes 1 and 7), and large HDAgmutants.

Plasmids encoding large HDAg mutants used in this study were

pECEL-d35/88 (panel A, lanes 3 and 11), pECEL-d89/163 (panel A, lanes 4 and12),d164/195 (panel A, lanes 5 and 13), pECEL-d196/210 (panel A, lanes 6 and14), pECEL-CRLDK (panel A, lanes 7 and 15), pECES-aSR (panel A, lanes 8 and 16), pECEL-aCAT (panel B, lanes 3 and 9), pECEL-d10/55 (panel B, lanes 4 and 10),

pECEL8-al95/196 (panel B, lanes 5 and 11), andpECEL7-al95/198

(panelB, lanes 6 and 12). Western blot analysiswasperformedwithan

antibody specifictoHDAgs asdescribed in Materials andMethods. Protein lysates were prepared either from the virus-like particles

secreted into culture media(panel A, lanes 1to8; panel B, lanes1 to

6)orfrom transfected COS7 cells (panel A, lanes9 to 16; panel B, lanes 7to12).Wild-type HDAgs and large HDAgmutantsdetected in the culture mediaareindicatedby asterisks.

ing sequence located within the C-terminal 19 amino acid residues isimportant for the assembly of largeHDAg.

More-over, the 19-amino-acid domain doesnot havetobe immedi-ately adjacent to the sequences common to both small and largeHDAgs for virion assembly.

Alarge HDAgmutantlacking nuclear localization signals is capableofcopackaging withsmallHBsAg. We have previously shown that the large HDAg isa nuclear phosphoprotein (6).

Two nuclearlocalizationsignalsarepresentwithinthe domain from amino acid residue 35 to 88 (7). However, HBsAg is localized in the cytoplasm (12). Therefore, where and how nuclear HDAg and cytoplasmic HBsAg interact have been puzzles. Our previous results showed thatan

HDAg-,B-galac-tosidase fusion protein, N163Z(MS), from which amino acid residues 35 to 88 ofthe HDAg had been deleted, localized exclusively in the cytoplasm of transfected cells (7). Current studiesdemonstrate thatdeletion ofthis domain(pECEL-d35/ 88) has little effect on the assembly oflarge HDAg to form virus-like particles (Fig. 3A, lane 3). Indirect

immunofluores-cence staining shows that the plasmid pECEL-d35/88 does

encode a cytoplasm-localized large HDAg (Fig. 4c). These

findings suggest that the nuclear localization signals of large HDAg are not prerequisites for assembly of virus-like parti-cles.

b

_

FIG. 4. Indirect immunofluorescence

staining

of the

large

HDAg in transfected COS7 cells. COS7 cells were transfected with

pECE

vector

(a), pECE-d-BE (b),

or

pECEL-d35/88

(c).

At48h posttrans-fection, cells were fixed and

processed

for detection of

HDAg

by

indirect immunofluorescence

staining

as described in Materials and Methods. Nuclear

staining

wasobserved for the

wild-type large

HDAg encoded

by

pECE-d-BE

whereas the

pECEL-d35/88-transfected

COS7 cellswerestained in the

cytoplasm.

Plasmid

pECE

wasusedas a

negative

control.

A

signal

within the N-terminal50 amino acid residues and

a subdomain between amino acid residues 50 and 65 of the

large HDAg

areinvolved in the

assembly

of small

HDAg.

Ithas been shown that the

large HDAg

isessential for the

assembly

of small

HDAg

(10, 28).

A

signal

within thefirst50amino acid residues of the

large HDAg plays

an

important

role for the event

(10, 21). Interestingly, proline

substitutions at Leu-37 and Ile-41 of

large HDAg

affect the interactions between

HDAgs

but retain the

ability

of

large HDAg

to act as a

negative regulator

in HDV

replication

(8).

Here,

we further demonstrate that the

large HDAg

with

proline

substitutions is still

capable

of

copackaging

the

wild-type

small

HDAg

(Fig.

5C,

lane

5).

Moreover,

copackaging

of small

HDAg

could

proceed

whether the

proline

substitutions were

generated

in the

large HDAg

(Fig.

SC,

lane

5),

inthe small

HDAg

(Fig.

SC,

lane

4),

orinboth

(Fig. SC,

lane

6),

even

though

the

efficiency

may differ between

wild-type

andmutant

HDAgs.

In

addition,

wefound thatthe N-terminal 50-amino-acid domainisnotthe

only

determinant for the

assembly

ofsmall

HDAg.

Plasmids

on November 9, 2019 by guest

http://jvi.asm.org/

[image:5.612.65.304.72.278.2] [image:5.612.353.530.78.433.2]

M

1

2

3

4

5

A

pECE-d-BE I

35 75

pECEL-d35/75 _

50 75

pECEL-dSO/75 _ _ 6575

pECEL-d65/75

1 2 3 4 5 6 7 8

c

1 2 3 4 5 6

[image:6.612.323.541.72.277.2]

_L*

FIG. 5. WesternblotanalysisofHDAgs following cotransfection of plasmids encodingthe smallHBsAgandHDAgmutants.(A) Diagram showingthestructureofwild-typeandmutantlarge HDAgsand their characteristics in virionassembly.Numbers shownon individual

mu-tants denote the positions of amino acid residues flanking each deletion. Theabilityof each large HDAgmutant tocopackage small

HDAgisindicated byaplusorminussign. (B)Western blotanalysis

ofHDAgsfollowing cotransfection withplasmids C-ES, pECE-d-SM, and either the wild type (pECE-d-BE) (lanes 1 and 5) or a mutant construct ofthe large HDAg (lanes 2 to4 and 6 to8).The mutantconstructsoflarge HDAg usedwerepECEL-d35/75 (lanes2 and 6),pECEL-d65/75 (lanes 3 and 7),and pECEL-d50/75 (lanes4 and8).Western blotanalysiswasperformedwith theantibody specific toHDAgsasdescribed in Materials and Methods. Proteinlysateswere

prepared either from the virus-like particles secreted into culture media (lanes I to4)orfrom transfected COS7 cells (lanes5 to8).

Wild-type large HDAg and its mutated forms are indicated by

asterisks. S denotes thewild-type smallHDAg. Twolighter signalsin lane 2 representing wild-type small and large HDAgsresulted from

overflowingofasamplefrom thenextlane. (C)Western blotanalysis

ofHDAgswithprolinesubstitutionsatLeu-37 and Ile-41. Plasmidsof

HDAgsusedwerepECE-d-BEandpECE-d-SM(pro) (lanes1 and4),

pECE-d-BE(pro) and pECE-d-SM (lanes 2 and 5), and pECE-d-BE(pro) and pECE-d-SM(pro) (lanes 3 and 6). Plasmids pECE-d-BE(pro) and pECE-d-SM(pro) encode large and small HDAgs,

re-spectively, with proline substitutions at Leu-37 and Ile-41. Protein

lysateswerepreparedfrom either transfectedCOS7 cells(lanes1to3)

orvirus-likeparticles secreted into culture media (lanes4 to 6).L*

denotes thewild-type and proline-substituted large HDAgs. S* de-notesthewild-type andproline-substitutedsmallHDAgs.

FIG. 6. Northern blot analysis of HDV RNA following cotransfec-tion of Huh-7 cells with dimericHDV cDNAandaplasmid encoding

large HDAg.Plasmids usedinthe cotransfection studiesrepresentthe dimericHDVcDNAonly (lane 1) and thedimeric HDV cDNAplus

either thewild-type large HDAg (lane 2)orlarge HDAgmutantsthat

weredeleted between amino acid residues65 and 75 (lane3), 50 and

75(lane 4),or35and75 (lane 5).Theprobe usedwastheantigenomic

strand of HDV RNAandwaspreparedasdescribedinMaterialsand

Methods. Invitrotranscripts representing the 1.7-kb monomeric and

3.4-kb dimeric HDV genomic RNAs hybridized to the antigenomic RNAprobewereusedasmarkers (M). 28Srepresentsthe 28S rRNA

as an internal control.

pECEL-d35/75, pECEL-d5O/75, and pECEL-d65/75, which encode mutant large HDAgs with deletions from amino acid residues 35to75,50to75, and 65to75,respectively,wereused

incotransfectionexperiments. Data show that the small HDAg could be copackaged and secreted into culture media only whencotransfectionwascarriedoutwith the plasmid pECEL-d65/75 (Fig. 5B, lane 3), butnot theothertwo plasmids (Fig. SB, lanes2 and4). Takentogether, these results indicate that

asubdomain from amino acid residues 50 to 65 aswell as a

signal within the N-terminal 50 amino acid residues of large HDAg may be important for the interactions between small and large HDAgs and is involved in virion assembly of the smallHDAg.

Alarge HDAgmutantlackingaminoacid residues 50to75 fails torepress thereplication of HDV.To learn whether the subdomain from amino acid residues 50 to 65, which is important for the assembly of HDV, also contributes to the negative effect of large HDAg on HDV replication, we

co-transfected Huh-7 cellswith the plasmid pSVD2, which

con-tains dimericHDVcDNA, andaplasmid that encodeseither thewild-typeoramutatedlarge HDAg.Aswehavepreviously shown(8),thedimeric cDNA alone couldundergo replication (Fig. 6, lane 1). Interestingly, wefound thatlarge HDAgwith adeletion of amino acidresidues 50to75 failedtorepressthe replication of HDV (Fig. 6, lane 4) whereas a deletion from

amino acid residues65to75 retainedinhibitory activity (Fig. 6, lane 3). These findings indicate that the subdomain between

amino acid residues50 and 65 of the large HDAgisinvolved

in the negative regulation of HDV replication. copackage

214

small

HDAg

U

+

3.4-

1.7-B

+

28S-0

#Olb* --m^Aw* qmlp*

4m ANIML4s

44%%bb.* -VOWOO#*

on November 9, 2019 by guest

http://jvi.asm.org/

[image:6.612.54.291.96.422.2]

DISCUSSION

In this study, we have examined subdomains of the large HDAg

involved

in the

assembly of

HDV-like

particles.

De-tailed mutational

analyses

demonstrate that

the

C-terminal 19-amino-acid domain

containing

the

isoprenylation

motif is

important

for the

assembly

of

large HDAg. Furthermore,

a subdomain from amino acid residues 50 to 65 and a

signal

within the N-terminal 50 amino

acid residues

of the

large

HDAgareinvolved in the

assembly

of smallHDAgaswellas the trans-dominant

negative regulation

ofHDV

replication.

The process by which nuclear HDAg

(6)

and

cytoplasmic

HBsAg

(12)

meet toform HDV

particles

is unclear. An earlier

study

correlated the

assembly

ofHDVwith

isoprenylation

of large HDAg at the 211-Cxxx-214 motif

(14).

A serine substi-tutionat

Cys-211 abolished

the

assembly

of

large HDAg

(14).

Therefore,

it

is possible

that

isoprenylation of large

HDAg results in membrane

anchoring

which,

in turn,

facilitates

the interactions between

large HDAg

and small

HBsAg

on the membrane of the

endoplasmic

reticulum.

Interestingly,

we found that a

large HDAg

from which the nuclear localization

signals

have beendeleted is

capable

of

copackaging

with small

HBsAg

(Fig. 3A,

lane

3).

HDAgsarenuclear

phosphoproteins

(6,

17).

The role of nuclear localization

signals

in virion

assembly

is unclear.However, nuclear localization of

wild-type

large HDAg is likely to be involved in the assembly of both smallHDAg and HDV

genomic

RNAlocatedin the nucleus in order to form infectious HDVparticles.

The C-terminal 15 amino acid residues

adjacent

to the 211-Cxxx-214

isoprenylation

motifareinvolved in the

assembly

of

large HDAg.

A

large HDAg

mutant, encoded

by

pECEL-d196/210,

that retains the Cxxx

isoprenylation

motif but lacks its

flanking

amino acid residues from 196 to 210 failed to

undergo assembly (Fig.

3A, lane

6).

Theeffect of the 15-amino-acid deletiononassembly may be dueto a

disruption

of direct

interactions

between thelarge HDAg and small HBsAgordue to aninfluenceonisoprenylation. Alternatively, the conforma-tion of the

flanking

sequencestogether with the

isoprenylation

motif may becritical for

assembly.

However, the latter

hypoth-esis isnot

supported by

ourresults

showing

thata

large HDAg

mutant in which the extreme C-terminal 19-amino-acid do-mainwas

separated

from thecommonN-terminal 195-amino-aciddomain

by

an

8-amino-acid

peptide

retained the

ability

to form virus-like

particles

(Fig.

3B,

lane

5).

In

addition,

a

large

HDAg

mutant in which a seven-amino-acid

peptide

was in-sertedat thesame

position

and the

wild-type

amino acids 196 and 197weredeleted could bedetected in the culture medium

(Fig.

3B,

lane

6). Furthermore,

a

large

HDAgmutantin which the C-terminal

Pro/Gly-rich region

between

amino

acid resi-dues 164 and 195wasdeleted stillretained the

ability

toform virus-like

particles

(Fig.

3A, lane

5). Therefore,

we propose that the

isoprenylation

motif

together

with the

flanking

amino acid residues from 198to210 is

important

for the

assembly

of

large

HDAg and may be involved in the interactions between

large

HDAg and small HBsAg. The question of whether cellular

proteins

areinvolved in the interactions is unanswered.

Protein-protein

interactionsbetween the HDAgs have been

hypothesized

toplayakey role in virion assembly as well as the

negative

regulation ofHDVreplication (8, 10, 21, 40). These studies concluded that a putative coiled-coil structure or an unidentified

signal

located betweenamino acid residues 13 and 48 is

important

for both events. Our present study on the

assembly

of smallHDAg further suggests that a cryptic signal within the N-terminal 50 amino acid residues other than the N-terminal coiled-coilstructureisresponsible for the interac-tions betweenHDAgs; small HDAgwascapable of

copackag-ing with a large HDAg containing proline substitutions at Leu-37 andIle-41,eventhoughtheefficiency maybedifferent

(Fig.

SC).

As

predicted by

computer

analysis (PepPlot

of

Genetics

Computer Group programs), proline

substitutions break the a.-helix and are likely to destroy the N-terminal coiled-coil structure. Results from the present

study

also suggest that a subdomain located between amino acid residues 50 and 65 ofthe

large

HDAg

is involved in the virion

assembly

of small

HDAg

and the

negative

effect of

large HDAg

onthe

replication

of HDV

(Fig.

5 and

6). However,

the exact conformation of the N-terminal 65 amino acid residues of HDAgs involved in the HDV multiplication remains to be elucidated.

ACKNOWLEDGMENTS

Wethank J.-C.Chengand C.-W. Tsai fortechnical assistance.We

aregratefultoJames Ouforproviding plasmid pECE-CandtoJeffrey Yenand SteveRoffierforhelpfulcommentsand criticalreadingofthe manuscript.

This workwassupported byaresearchgrant (NSC82-0419-B-002-438-MB) from the National Science Council of theRepublicof China.

REFERENCES

1. Bergmann, K.F.,andJ.L.Gerin. 1986.Antigensofhepatitisdelta virus in the liver andserumof humans andanimals.J.Infect. Dis. 154:702-705.

2. Bonino, F.,K. H.Heermann,M.Rizzetto,and W. H. Gerlich.1986. Hepatitisdelta virus:protein compositionof deltaantigenandits hepatitisBvirus-derivedenvelope.J.Virol.58:945-950. 3. Branch,A.D.,and H. D. Robertson.1991.Efficienttranscleavage

and a common structural motif for theribozymes of thehuman hepatitis 8agent. Proc.Natl. Acad. Sci. USA 88:10163-10167. 4. Casey, J. L.,K. F.Bergmann,T. L.Brown,andJ.L.Gerin.1992.

Structural requirements for RNA editing in hepatitis 8 virus: evidence forauridine-to-cytidineeditingmechanism.Proc. Natl. Acad. Sci.USA 89:7149-7153.

5. Chang, F.-L., P.-J. Chen,S.-J. Tu, C.-J. Wang,andD.-S. Chen. 1991. The large form of hepatitis delta antigen is crucial for assembly of hepatitis delta virus. Proc. Natl. Acad. Sci. USA 88:8490-8494.

6. Chang, M.-F.,S. C.Baker,L. H.Soe,T.Kamahora, J.G.Keck,S. Makino, S. Govindarajan, and M. M. C. Lai. 1988. Human hepatitis delta antigen is a nuclearphosphoprotein with RNA-binding activity.J.Virol.62:2403-2410.

7. Chang, M.-F.,S. C.Chang,C.-I.Chang,K.Wu,and H.-Y.Kang. 1992. Nuclearlocalizationsignals,butnotputativeleucinezipper motifs, are essential for nuclear transport of hepatitis delta antigen.J.Virol.66:6019-6027.

8. Chang, M.-F., C.-Y. Sun, C.-J. Chen, and S. C. Chang. 1993. Functional motifsof deltaantigen essentialfor RNAbindingand replicationofhepatitisdeltavirus.J.Virol. 67:2529-2536. 9. Chao, M.,S.-Y.Hsieh,andJ.Taylor. 1990. Role oftwoforms of

hepatitis delta virus antigen: evidence for a mechanism of self-limitinggenomereplication.J.Virol. 64:5066-5069.

9a.Chen, C.-H.,and M.-F.Chang.Unpublishedobservation. 10. Chen, P.-J.,F.-L.Chang, C.-J. Wang, C.-J. Lin,S.-Y.Sung,and

D.-S. Chen. 1992. Functional study ofhepatitisdeltaviruslarge antigeninpackagingandreplicationinhibition:role of the amino-terminal leucinezipper.J.Virol. 66:2853-2859.

11. Chomczynski, P.,and N.Sacchi. 1987.Single-step method ofRNA isolation by acid guanidium thiocyanate-phenol-chloroform

ex-traction. Anal.Biochem. 162:152-159.

12. Eble,B.E.,D. R.Macrae,V. R.Lingappa,and D.Ganem.1987. Multiple topogenicsequencesdetermine the transmembrane ori-entation ofhepatitisBsurfaceantigen. Mol. Cell.Biol.7:3591-3601. 13. Ellis, L., E. Clauser, D.0. Morgan, M. Edery, R. A. Roth, and W. J. Rutter. 1986. Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activityanduptakeof2-deoxyglucose. Cell 45:721-732.

14. Glenn, J. S., J.A.Watson,C. M.Havel, andJ.M.White. 1992.

on November 9, 2019 by guest

http://jvi.asm.org/

Identification of a prenylation site in delta virus large antigen. Science 256:1331-1333.

15. Gorman, C. M., L. F. Moffat, and B. H. Howard. 1982. Recombi-nantgenomes which expresschloramphenicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2:1044-1051.

16. Henikoff, S. 1984. Unidirectional digestion with exonuclease III createstargeted breakpoints for DNA sequencing. Gene 28:351-359.

17. Hwang, S., C. Z. Lee, and M. M. C. Lai. 1992. Hepatitis delta antigen expressed by recombinant baculoviruses: comparison of biochemical properties and post-translational modifications be-tweenthelarge and small forms. Virology 190:413-422. 18. Kos, A., R. Dijkema, A. C. Arnberg, P. H. van der Merde,and H.

Schelekens. 1986. The HDV possesses a circular RNA. Nature (London)323:558-560.

19. Kuo, M. Y.-P., M. Chao, and J. Taylor. 1989. Initiation of replication of the human hepatitis delta virus genome from cloned DNA:role of deltaantigen. J. Virol. 63:1945-1950.

20. Kuo, M. Y.-P., L. Sharmeen, G. Dinter-Gottlieb, and J. Taylor. 1988. Characterization of self-cleaving RNA sequences on the genome andantigenomeof humanhepatitisdelta virus. J.Virol. 62:4439-4444.

21. Lazinski, D. W., and J. M. Taylor. 1993. Relating structure to function in thehepatitisdeltavirusantigen.J.Virol. 67:2672-2680. 22. Lee, C.-Z., J.-H. Lin, M. Chao, K.McKnight, and M. M. C. Lai. 1993.RNA-binding activity of hepatitis deltaantigeninvolves two arginine-richmotifs and isrequired for hepatitisdeltavirusRNA replication.J.Virol. 67:2221-2227.

23. Lin, J.-H., M.-F. Chang, S. C. Baker, S. Govindarajan, and M. M. C. Lai. 1990. Characterization ofhepatitis delta antigen: specificbinding to hepatitis delta virus RNA. J.Virol. 64:4051-4058.

24. Makino, S., M.-F. Chang, C.-K. Shieh, T. Kamahora, D. M. Vannier, S. Govindarajan, and M. M. C. Lai. 1987. Molecular cloning and sequencing of human hepatitis delta virus RNA. Nature (London)329:343-346.

25. Maltese, W.A.1990.Posttranslational modificationofproteins by isoprenoids inmammalian cells. FASEB J. 4:3319-3328. 26. Perrotta, A. T., and M. D.Been.1991. Apseudoknot-likestructure

required for efficient self-cleavage ofhepatitis delta virus RNA. Nature (London)350:434-436.

27. Rizzetto, M.,B.Hoyer,M.G.Canese, J.W.-K.Shih,R. H.Purcell, and J.L.Gerin. 1980. Delta agent: association of delta antigen with hepatitis B surface antigen and RNA in serum of delta-infectedchimpanzees. Proc. Natl. Acad. Sci.USA77:6124-6128. 28. Ryu, W.-S., M. Bayer, andJ. Taylor. 1992.Assemblyofhepatitis

delta virusparticles.J.Virol.66:2310-2315.

29. Sanger, F., S.Nicklen, andA. R.Coulson.1977. DNAsequencing

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

30. Sureau, C., B. Guerra, and R. E. Lanford. 1993. Role of the large hepatitis B virus envelope protein in infectivity of the hepatitis delta virion. J. Virol. 67:366-372.

31. Uhlenbeck, 0. C. 1987. A small catalytic oligoribonucleotide. Nature(London)328:596-600.

32. Wang, C.-J., P.-J. Chen, J.-C. Wu, D. Patel, and D.-S. Chen. 1991. Small-formhepatitisBsurface antigen issufficienttohelp in the assemblyof hepatitis delta virus-like particles. J. Virol. 65:6630-6636.

33. Wang, J.-G., and S. M. Lemon. 1993.Hepatitisdelta virusantigen forms dimers and multimeric complexes in vivo. J. Virol. 67:446-454.

34. Wang, K.-S., Q.-L. Choo, A.-J. Weiner, J.-H. Ou, R. C. Najarian, R.M. Thayer,G. T. Mullenbach, K. J. Denniston, J. L. Gerin, and M. Houghton. 1986. Structure, sequence and expression of the hepatitisdelta virus genome. Nature (London)323:508-513. 35. Weiner, A. J., Q.-L. Choo, K.-S. Wang, S. Govindarajan, A. G.

Redeker, J. L. Gerin, and M. Houghton. 1988. A single anti-genomicopenreading frame ofthehepatitis delta virusencodes the epitope(s) of both hepatitis delta antigen polypeptides p248 andp278.J.Virol. 62:594-599.

36. Wu, H.-N., and Z.-S. Huang. 1992. Mutagenesis analysis of the self-cleavage domain ofhepatitis deltavirus antigenomic RNA. NucleicAcids Res. 20:5937-5941.

37. Wu, H.-N., Y.-J. Lin, F.-P. Lin, S. Makino, M.-F. Chang, and M. M. C. Lai. 1989. Humanhepatitis delta virus RNA subfrag-mentscontain an autocleavageactivity.Proc.Natl. Acad.Sci.USA 86:1831-1835.

38. Wu, J.-C.,P.-J. Chen,M. Y.-P.Kuo, S.-D.Lee,D.-S. Chen,and L.-P. Ting. 1991.Productionofhepatitisdeltavirusand suppres-sion ofhelper B virus in a human hepatoma cell line. J. Virol. 65:1099-1104.

39. Xia, Y.-P., M.-F. Chang, D.Wei, S. Govindarajan,and M. M.C. Lai. 1990. Heterogeneity of hepatitis delta antigen. Virology 178:331-336.

40. Xia, Y.-P., andM. M.C. Lai. 1992. Oligomerizationofhepatitis delta antigen is required for both thetrans-activating and

trans-dominant inhibitory activities of the delta antigen. J. Virol. 66:6641-6648.

41. Xia, Y.-P., C.-T. Yeh, J.-H.Ou,and M.M. C.Lai. 1992. Charac-terization of nuclear targeting signal of hepatitis delta antigen: nuclear transportas aprotein complex.J.Virol. 66:914-921. 42. Zheng, H.,T.-B.Fu, D.Lazinski, and J. Taylor. 1992.Editingon

the genomic RNA of human hepatitis delta virus. J. Virol. 66:4693-4697.

on November 9, 2019 by guest

http://jvi.asm.org/