JOURNAL OF VIROLOGY, May 1991, p. 2357-23610022-538X/91/052357-05$02.00/0Copyright C) 1991, American Society for Microbiology

trans-Dominant Inhibition of Human Hepatitis DeltaVirus Genome Replication

JEFFREY S. GLENN' AND JUDITH M. WHITE' 2*

Departments of Biochemistry and Biophysics' and Pharmacology,2 University of California,San Francisco, California 94143-0450

Received 18 October 1990/Accepted 5 February 1991

Infection with hepatitis delta virus (HDV) is an important cause of acute and chronic liver disease and can

be rapidly fatal. Sequencing of the HDV RNA genome has revealed variability at the C-terminal end of the deltaantigen reading frame. One genome type (termed the S genome) synthesizes a 24-kDa protein thought to berequired for genome replication. Another genome type (termed the L genome) extends the reading frame by 19amino acids as a result of a single base change. Replication of the S and L genomes was studied in culturedfibroblasts. While the S genome efficiently initiated genome replication, the L genome did not. Moreover, in acodelivery experiment, L genome RNA inhibited replication of the S genome. Potent trans inhibition was alsoobserved following cotransfection of the S genome and a plasmid encoding the larger delta antigen. Mutationalanalysis indicated that the inhibitory activity was not a simple function of the large delta antigen reading frame'sextra length. Implications for the viral life cycle, clinical infection, and potential treatment are discussed.

The outcome of infection with human hepatitis delta virus(HDV) is varied and includes chronic liver disease or fulmi-nant hepatitis with rapid progression to death (12, 19). HDVvirions contain a 1.7-kb single-stranded circular RNA ge-

nome, delta antigen, and a surrounding lipid envelope em-

bedded with hepatitis B virus (HBV) surface antigen pro-

teins. HBV's helper role appears to be limited to providing a

means of cellular entry for the HDV genome, as deltagenome replication proceeds in the absence of any HBVgene products. Moreover, the replication strategies of HBVand HDV are quite different. Specifically, HDV replicationis thought to proceed entirely through RNA intermediatesvia a rolling-circle mechanism, such as that proposed forviroids (4). RNA of genomic polarity found in incomingvirions serves as the template for the production of antige-nomic RNA of opposite polarity. Antigenomic RNA canserve as a template for new genomic RNA (7a) and is thecoding strand for delta antigen, the only known protein to beexpressed from the viral genome.

Examination of independently derived nucleotide se-quences of the HDV genome (14, 16, 23) reveals microhet-erogeneity at codon 196 of the delta antigen reading frame.One genome type (here termed the S genome, for small deltaantigen) has an amber stop codon at this position. In theother genome type (termed the L genome, for large deltaantigen), the delta antigen reading frame is extended by 19amino acids. Both small and large forms of delta antigen canbe found in infected livers and serum (2, 3).

In this study, we compared the effects of the small andlarge delta antigen isoforms on HDV genome replication.Our major findings are that not only do the two closelyrelated delta antigen forms have very different effects, butone acts in a trans-dominant fashion over the other to inhibitgenome replication. Mutational analysis indicated that thispotent inhibition exerted by the large delta antigen was not asimple function of the extra 19 amino acids at its C-terminalend.

* Corresponding author.

MATERIALS AND METHODS

Plasmids and transfections. Plasmid pT7GM directs thesynthesis ofHDV monomeric genomic RNA upon transcrip-tion with T7 RNA polymerase (9). Plasmid pT7GMC wasmade from plasmid pT7GM by in vitro mutagenesis (22) witholigonucleotide C (5'-AAGAGTATATCCCATGGAAATCCC-3'). In vitro transcription of plasmids pT7GM andpT7GMC yields the S and L genomes, respectively (Fig. 1).

Plasmid pSVL(D3) contains a head-to-tail tandem trimerof the delta genome. Transcription initiated at the simianvirus 40 (SV40) late promoter yields a trimeric HDV RNA ofgenomic polarity (13). Plasmid pSVLlarge (encoding thelarge delta antigen) was made from plasmid pSVL(Ag) (9)(encoding the small delta antigen) by replacing the SacII(position 1567)-BstXI (826) fragment of pSVL(Ag) with theanalogous fragment of T7GMC. DNA transfections weredone as described before (7).

Cells. GAG cells were used for all experiments exceptthose in Fig. 3, for which GP4f cells were used. GP4f cellsare an NIH 3T3 fibroblast cell line which stably expressesthe influenza virus hemagglutinin (HA) (6). GAG cells areGP4f cells which stably express the small delta antigen (9).

Production and delivery of in vitro-synthesized RNA. Forthe experiments described in this article, in vitro-transcribedRNAs of genomic polarity were made essentially by themethod of Yisraeli and Melton (25) and delivered to GAGcells by previously described methods (8, 9, 18). Briefly, forliposome deliveries, in vitro-transcribed RNAs were encap-sulated in glycophorin-containing liposomes, which werethen bound to cells expressing the influenza virus HA ontheir surface. A transient lowering of medium pH activatesthe well-characterized acid-induced conformational changein HA (24), which promotes fusion between the boundliposomes and the target cells, and hence delivery of theencapsulated RNAs.RNA extraction and Northern (RNA) blot analysis. Total

cellular RNA was isolated by a guanidinium-CsCl procedure(9) at 7 days after delivery of nucleic acids unless otherwiseindicated. Glyoxalated RNAs (13) were fractionated on 1.5%agarose gels, transferred to nylon membranes, and probedfor antigenomic HDV RNA as described previously (9).

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2358 GLENN AND WHITE

3 5

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would not be related to the size of the delta antigen species.Oligonucleotides containing the desired mutation served ashalf of a primer pair. Short DNA fragments were amplifiedby using plasmids pT7GM or pT7GMC as the template,restricted with appropriate enzymes and cloned into simi-larly restricted vector plasmids (pT7GM or pT7GMC). TheS+8 genome was constructed by using oligonucleotides S8(5'-CTGGGGTCGACAACTCTGGGGAGAAAAGGACIAGTCGGCTGG-3') and StuP (5'-CCGAACGGACCAGATGGAGGTAGACTCCGG-3') and extends the small deltaantigen reading frame by 8 amino acids. Similarly, the S+13(made with oligonucleotides S13 [5'-CTGGGGTCGACAACTCTAGGGAGAAAAGGG-3'] and StuP), S+16 (made witholigonucleotides S16 [5'-CTGGGGTCAACAACTCTGGGGAGAAAAGGG-3'] and StuP), and S+18 (made with oli-gonucleotides S18 [5'-CCAGAGTTGTCGACCCIAGTGAATAAAGCG-3'] and BSP [5'-CCCACAAATCTCTCTAGATTCCG-3']) genomes extend the small delta antigenreading frame by 13, 16, and 18 amino acids, respectively.Western immunoblot analysis. Seven days after delivery of

in vitro-transcribed HDV RNA to GAG cells, total cellularprotein was isolated, fractionated on 12% polyacrylamidegels, and probed with rabbit anti-delta antigen antibody (giftfrom John Taylor) and alkaline phosphatase-linked goatanti-rabbit antibody (Promega Biotec, Madison, Wis.) essen-tially as described before (9).

FIG. 1. Replication of the S and L genomes. (A) Schematicdiagram of the in vitro-transcribed HDV S and L RNAs. Transcrip-tion of plasmids pT7GM and pT7GMC (see Materials and Methods)yielded HDV monomeric RNAs of genomic polarity termed S andL, respectively. S RNA differs from L RNA only in that the formerhas a uridine instead of a cytosine at nucleotide position 1012 on thegenomic strand of the delta genome. (B) Schematic diagram of thedelta antigen reading frame encoded on the antigenomic (comple-mentary) strand of the S and L genomes. The delta antigen readingframe of the S antigenome is terminated by the UAG stop codon atcodon number 196 and encodes a predicted protein of 195 aminoacids (a.a.). The corresponding codon on the L antigenome is UGG.As a result, the delta antigen reading frame is extended an additional19 amino acids to the next stop codon at codon number 215. (C)Northern blot to detect de novo-synthesized antigenomic HDVRNA following initiation of genome replication with in vitro-tran-scribed S (left panel) and L (right panel) genomes. Equal amounts ofcellular RNA were loaded in each panel and confirmed by cohybrid-ization with a probe for SRPR mRNA. (Note that in this exposurethe SRPR mRNA signal in the left panel is overshadowed by thedelta dimer signal.)

The probe used was designed not to detect either the inputgenomic HDV RNA or the endogenous delta antigen mRNApresent in GAG cells (9). A probe for the signal recognitionparticle receptor (SRPR) mRNA was included in the hybrid-izations to allow comparison of gel sample loading.

Engineering of mutant genomes. A mutagenesis strategywas designed to generate a series of delta genomes whichdiffer at the C-terminal end of the delta antigen reading frameand thus encode delta antigen species of various specifiedsizes between those of the small and large delta antigens. Todo this, stop codons were inserted at various positionsupstream of the large delta antigen termination codon bypolymerase chain reaction mutagenesis (20), with slightmodifications, of plasmid pT7GMC. Identical mutationswere introduced into plasmid pT7GM to serve as controls forany cis-acting effects resulting from these mutations which

RESULTS AND DISCUSSION

Replication of the S and L genomes. We previously dem-onstrated that an in vitro-transcribed HDV RNA (S genome)encoding the small form of delta antigen can initiate genomereplication (9). Briefly, an HDV monomer of genomic polar-ity was delivered to NIH 3T3 fibroblasts which stablyexpress the small form of delta antigen (GAG cells). Oneweek later, total cellular RNA was analyzed with strand-specific probes for the appearance of newly synthesizedHDV RNA. Amplified input genomic RNA as well as bothmonomers and dimers of antigenomic (complementary) po-larity were detected, indicating genome replication. Priorexpression of the small delta antigen in the target cells wasrequired.To our surprise, an RNA (L genome) which differs only at

nucleotide 1012 (numbering scheme of Wang et al. [23]), sothat it would direct expression of the large delta antigenisoform, was defective for replication in this assay (Fig. 1).Levels of both antigenomic (Fig. 1) and genomic (data notshown) delta RNA were dramatically reduced comparedwith those observed for the S genome. This impaired ge-nome replication could be due to the predicted addition of 19amino acids at the C-terminal end of the delta antigen.Alternatively, the presence of a guanine rather than anadenosine at position 1012 might impose a cis-acting defecton the RNA genome.

Effect of L genome on S genome replication. To investigatethe effect of the defective L genome on replication of thefunctional S genome, we codelivered both genomes to GAGcells and assayed for the appearance of antigenomic HDVRNA. As shown in Fig. 2, the L genome was not onlydefective for its own replication; it also inhibited the repli-cation of S genome RNA in trans. At day 7, markedinhibition was observed. By day 14, genome replication wasreduced still further and approached the low level detectedin cells harboring the L genome alone. Inhibition of genomereplication may be even greater than that inferred by simplycomparing the band intensities in lanes D and E, since the

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J. VIROL.

INHIBITION OF HUMAN HDV GENOME REPLICATION

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FIG. 2. Northern blot to detect antigenomic HDV RNA follow-ing codelivery of S and L RNAs. Cells were analyzed at days 7 and14 after delivery of genomic RNAs: 3 ,ug of S RNA plus 3 pug of yeasttRNA (lanes A and D), 3 Fg of S RNA plus 3 p.g of L RNA (lanes Band E), or 3 ,ug of L RNA plus 3 ,ug of yeast tRNA (lanes C and F).Day 14 lanes were exposed approximately 10 times longer to allowvisualization of HDV monomeric RNA in lanes E and F.

antigenomic RNA in lane E represents the sum of residualHDV RNA accumulated prior to inhibition and that due toon-going replication, if any.

Effect of delta antigen isoforms on HDV genome replication.We next addressed whether inhibition of genome replicationcould be exerted via large delta antigen protein suppliedseparately in trans. Delta genome replication was initiatedby transfecting a derivative of NIH 3T3 fibroblasts (GP4fcells) with a plasmid bearing a tandem head-to-tail trimer ofthe S genome under the transcriptional control of the SV40 .

late promoter, as described previously (13) (Fig. 3, lane B).When cotransfected with a plasmid encoding the small deltaantigen, replication was enhanced (Fig. 3, lane A). In sharpcontrast, cotransfection with a plasmid encoding the largedelta antigen inhibited genome replication to below our limitof detection (Fig. 3, lane C). Similar results were alsorecently obtained by Chao et al. (5).

Mutational analysis of C-terminal end of delta antigen

A B C

SRPR mRNA of _ql

delta monomer _

FIG. 3. Northern blot to detect antigenomic HDV RNA follow-ing transfection of GP4f cells with pSVLD3, a plasmid that is able toinitiate HDV genome replication (13) (lanes A, B, and C). Cellsanalyzed in lanes A and C were cotransfected with a 10-fold molarexcess of a plasmid encoding the small [pSVL(Ag)] or large (pSVL-large) delta antigen, respectively.

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FIG. 4. Characterization of delta antigen reading frame mutants.(A) Schematic diagram of the C-terminal end of the delta antigenreading frame in the S, L, and genetically engineered mutantgenomes. The mutant S+8 genome extends the short delta antigenreading frame by 8 amino acids. Similarly, the S+13, S+16, andS+18 genomes extend the small delta antigen reading frame by 13,16, and 18 amino acids, respectively. The terminal amino acid of thedelta antigen reading frame in each case is indicated. (B) Northernblot to detect de novo-synthesized antigenomic HDV RNA follow-ing initiation of genome replication with indicated in vitro-tran-scribed RNAs. Lane 1, S genome; lane 2, S+8 genome; lane 3, S+ 13genome; lane 4, S+16 genome; lane 5, S+18 genome; lane 6, Lgenome. (C) Western blot to detect the delta antigen species made 7days after delivery of the indicated genomes. Lanes 1 to 6 corre-

spond to lanes 1 to 6 in panel B. Protein from uninfected GAG cellswas analyzed in lane 7. kd, Kilodaltons.

reading frame. The trans-dominant inhibition of replicationis thus most likely exerted by the difference between thelarge and small forms of delta antigen, which differ by 19C-terminal amino acids. In an initial attempt to further definethe molecular details of large-antigen-mediated inhibition,we undertook a mutational analysis of the extreme C-termi-nal region of the protein. These mutants and their effects on

genome replication are shown in Fig. 4. Mutations designedto prematurely terminate translation of the large delta anti-gen were placed into both the L and S genomes. The latterconstructs were designed to serve as internal controls forpossible cis-acting defects resulting from these mutations.As demonstrated before (Fig. 1C), the amount of genomereplication observed was extensive with the S genome (Fig.4B, lane 1), which encodes the small delta antigen, but was

genorre ,yvedelivered S S+L4 S S4-L L

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greatly reduced when the delta antigen reading frame is 19amino acids longer, in the L genome (Fig. 4B, lane 6).Significant genome replication still proceeded when thesmall form was extended by 13 (Fig. 4B, lane 3) or remark-ably even 18 (Fig. 4B, lane 5) amino acids at the C terminus.In contrast, extending the small delta antigen reading frameby 8 (Fig. 4B, lane 2) or 16 (Fig. 4B, lane 4) amino acidsinhibited genome replication. Analogous mutations placedinto the S genome did not affect genome replication (data notshown).The absence of inhibition by the S+13 and S+18 mutant

forms of delta antigen cannot be explained by a lack of theseproteins. In fact, they are readily detected by Western blotanalysis (Fig. 4C, lanes 3 and 5, respectively). This is anexpected consequence of successful genome replication.Similarly, the strong inhibition of genome replication ob-served, for example, in the L (Fig. 4B, lane 6) and mutantS+8 (Fig. 4B, lane 2) genomes is most likely responsible forthe resulting low levels of their corresponding delta antigenspecies present at day 7 postdelivery (Fig. 4C, lanes 2 and 6,respectively). Curiously, the forms of delta antigen whichallow genome replication to proceed the most share the sameC-terminal amino acid, proline. These results suggest thatinhibition of genome replication by large delta antigen is notdetermined simply by its increase in length. Rather, morecomplex molecular interactions, perhaps influenced by thenature of the C-terminal amino acid, mediate the disruptionof genome replication.

Implications of large delta antigen inhibition of genomereplication. The results shown here demonstrate trans-dom-inant inhibition of a human pathogen in vitro. Moreover, thenow apparent opposing functions of the small and largeforms of delta antigen leads to the proposal that a molecularswitch from the small to the large form might play a role inthe HDV life cycle. Such a switch could explain previousreports in the literature. Sequencing of various delta RNAisolates reveals the presence of both S and L genomes (14,16, 23). Both large and small forms of delta antigen are foundin liver- and serum-derived virus particles from HDV-in-fected humans and chimpanzees (2, 3). Thus, the heteroge-neity at nucleotide 1012 that is responsible for the two deltaantigen isoforms is a naturally occurring phenomenon. Spon-taneous mutation to the L genome has been observedfollowing replication initiated exclusively with the S genome(7a, 15). It has been reported that this mutation may occur ata 500-fold greater frequency than the rate observed for othernucleotide positions on the delta genome (15). One role forsuch a mutation, whether programmed by specific hypermu-tation or the result of random mutation by error-proneRNA-dependent RNA transcription (21), could be to serveas a switch to promote attenuation of viral replication andthe establishment of a form of latency.

If indeed the propensity to mutate to the L genome is adeterminant of replication inhibition in vivo, this may helpexplain the various clinical outcomes of HDV infectionobserved in patients, ranging from rapid fulminant hepatitisto long-term chronic infection (12, 19). Higher initial ratios ofsmall to large delta antigen might be expected early ininfection or associated with a more severe clinical outcome.

Several mechanisms have previously been outlined forhow trans-dominant inhibitors might function (11). One ormore of these may explain the effects of large delta antigen.For example, one of small delta antigen's normal functionsmight require oligomerization. Alternatively, a regulatoryrole, such as promoting RNA-directed RNA synthesis, mayrequire precise molecular interactions. The presence of extra

amino acids, as encoded in large delta antigen, could disruptthe functions of small delta antigen in either or both of thesemodels. Whatever the mechanism, the effect of the largeantigen appears to be quite potent, as inhibition was ob-served in GAG cells, which themselves constitutively ex-press relatively large amounts of the small delta antigen.

Others have proposed the use of dominant negative mu-tants to combat viral infections (1, 10, 17). The HDV lifecycle may represent a situation in which such an activity isactually used in nature to repress viral genome replication.Furthermore, one might be able to exploit this activity andturn one of the viral gene products into a therapeutic weaponagainst its own genome replication. We have shown thatlarge delta antigen supplied separately, as we have done herefrom an expression plasmid, inhibits genome replication. Wehave also demonstrated inhibition when the large deltaantigen is supplied from an HDV vector. The L genomevariant used for this purpose is particularly appealing be-cause of its theoretical specificity; since uninfected cells lacka source of small antigen, which is required to initiateefficient delta genome replication, L genome RNA will onlyreplicate to yield inhibitory levels of the large delta antigen incells which harbor the target S genomes. Until the precisemechanism of HDV pathogenesis is elucidated, however, itremains speculative whether inhibiting genome replicationby either of these approaches could alter the clinical out-come of HDV infection in vivo. Nevertheless, further un-derstanding the mechanism of large delta antigen-mediatedinhibition and what factors promote the switch from small tolarge antigen may yield novel insights into how HDV repli-cates and lead to strategies to combat this and other viralagents.

ACKNOWLEDGMENTS

We thank John Taylor, Don Ganem, George Kemble, and CarlBlobel for critical reading of the manuscript and stimulating discus-sions.

This work was supported by a grant from the NIH to J.M.W. J.Glenn is supported by the Medical Scientist Training Program at theUniversity of California, San Francisco.

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