JOURNAL OF VIROLOGY,0022-538X/00/$04.0010

Oct. 2000, p. 9010–9018 Vol. 74, No. 19

Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Effects of Genomic Length on Translocation of Hepatitis BVirus Polymerase-Linked Oligomer

TSUNG-CHUAN HO,1 KING-SONG JENG,2 CHENG-PO HU,1,3 AND CHUNGMING CHANG1,2*

Institute of Microbiology and Immunology, School of Life Science, National Yang-Ming University,1 and Department ofMedical Research, Veterans General Hospital,3 Shih-Pai, Taipei 112, and Division of Molecular and Genomic

Medicine Research, National Health Research Institutes,2 Taipei 115, Taiwan, Republic of China

Received 12 April 2000/Accepted 9 July 2000

Accurate translocation of the polymerase-linked oligomer to the acceptor site (DR1*) in reverse transcrip-tion is crucial for maintaining the correct size of the hepatitis B virus (HBV) genome. Various sizes of foreignsequences were inserted at different sites of the HBV genome, and their effects on accurate translocation ofpolymerase-linked oligomer to DR1* were tested. Three types of replicate DNA products were observed in theseinsertion mutants: RC (relaxed circle) and type I and type II DL (duplex linear) DNA. Our results indicatedthat the minus strand of RC and type I DL form was elongated from DR1*, while the minus strand of the typeII DL form was elongated from multiple internal acceptor sites (IAS), such as IAS2. These IASs were alsofound to be used by wild-type HBV but with a very low frequency. Mutation of IAS2 by base substitutionabrogated polymerase-linked oligomer transferring to IAS2, demonstrating that base pairing also plays animportant role in the function of IAS2 as a polymerase-linked oligomer acceptor site. Data obtained from ourinsertion mutants also demonstrate that the distance between the polymerase-linked oligomer priming site andthe acceptor is important. The polymerase-linked oligomer prefers to translocate to an acceptor, DR1* or IAS2,which are ca. 3.2 kb apart. However, it will translocate to both DR1* and IAS2 if they are not located 3.2 kbapart. These results suggest that the polymerase-linked oligomer may be able to scan bidirectionally forappropriate acceptor sites at a distance of 3.2 kb. A model is proposed to discuss the possible mechanism ofpolymerase-linked oligomer translocation.

Hepadnaviruses are a group of small, enveloped DNA vi-ruses of which hepatitis B virus (HBV) is the prototype. Al-though the mature virus contains a circular partially double-stranded 3.2-kb DNA genome, hepadnaviruses replicate solelythrough reverse transcription from a pregenomic RNA (pgRNA)intermediate within cytoplasmic nucleocapsids (2, 26).

A stem-loop structure that resides near the 59 end of pgRNAis the primary element of the hepadnavirus RNA packagingsignal (ε) (4, 7, 11, 13, 21) and serves as the origin for reversetranscription (19, 23, 27, 29). The viral polymerase initiatesreverse transcription from the bulge of the stem-loop andsynthesizes a 3- to 4-nucleotide (nt) oligomer, which is co-valently linked to the polymerase (1, 6, 19). This polymerase-linked oligomer functions as minus-strand DNA primer for thesynthesis of minus-strand DNA (5, 22, 28). The polymerase-linked oligomer is then transferred to a complementary UUCmotif within direct repeat 1 (DR1*) near the 39 end of thepgRNA, where minus-strand DNA is elongated (19, 22, 23, 27,29).

Elongation of minus-strand DNA is accompanied by degra-dation of the pgRNA (17). The terminal 15- to 18-oligonucle-otide stretch of the pgRNA is resistant to the RNase H activityof HBV polymerase when the reverse transcription proceeds tothe 59 end of the RNA template (14). This short RNA istranslocated to a complementary sequence in DR2 near the 59end of the minus strand, where the plus-strand DNA is initi-ated. This process leads to the formation of the relaxed circular(RC) DNA genome (25). Part of the plus-strand RNA primer

does not transfer to DR2 but initiates the synthesis of plus-strand DNA in situ. This process results in the formation ofduplex linear (DL) DNA genome (25).

The mechanism of the translocation of the polymerase-linked oligomer to the primer acceptor site is poorly under-stood. Previous results showed that complementarity betweenthe polymerase-linked oligomer and the DR1* is required forthe transfer to occur (19). Sequence analyses revealed that theadjacent sequence of DR1* contains several copies of theUUC motif as well as DR2. However, the polymerase-linkedoligomer does not transfer to such a UUC motif, indicatingthat the UUC motif alone is not sufficient for polymerase-linked oligomer translocation. In mutants in which the comple-mentarity between the polymerase-linked oligomer and DR1*have been destroyed, the polymerase-linked oligomer still canbe transferred to the location of the altered DR1* but not theother UUC motifs (16, 19). Additionally, deletion of DR1* inwoodchuck hepatitis virus can lead to the initiation of minus-strand DNA synthesis at an internal site (24). These resultsstrongly suggested that a well-controlled mechanism beyondcomplementarity may exist to control the specificity of poly-merase-linked oligomer transfer. These results also raise theinteresting question whether the distance between the primingsite (bulge site on ε) and the DR1* in the pgRNA is importantfor polymerase-linked oligomer translocation. To address thisissue, a series of mutants with insertions of various lengths offoreign sequence into different sites on the HBV genome wereconstructed to explore the effects of the altered distance be-tween the priming site and DR1*. Our data show that thepolymerase-linked oligomer was transferred to multiple inter-nal acceptor sites in various insertion mutants. Among them, nt2091 to 2093 (IAS2) is the major one used by the polymerase-linked oligomer. Remarkably, this event resulted in the pro-duction of restricted sizes of genome DNA, i.e., approximately

* Corresponding author. Mailing address: Department of Intramu-ral Research Affairs, Division of Molecular and Genomic MedicineResearch, National Health Research Institutes, 128, Yen-Chiu-YuanRd., Sec. 2, Taipei 115, Taiwan. Phone: 886-2-2653-4401 ext. 8300. Fax:886-2-2651-3723. E-mail: tonychang@nhri.org.tw.

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3.2 kb; thus, almost a unit length of HBV genome is main-tained. The significance of this finding is discussed.

MATERIALS AND METHODS

Plasmids. HBV mutants used in this report were derived from plasmid pMH-9/3091 subtype ayw (10). Plasmid pSHH2.1 and helper plasmids pMTP andpMT1883 were described previously (3, 4, 30). The HBV sequence was num-bered according to the system of Pasek et al. (20), beginning with the A residueof the C gene initiation codon. For construction of plasmids X200, X400, X600,X825, and X1021, the SauI-HpaI (nt 236 to 438), AluI (nt 2250 to 2661), HpaI (nt438 to 1062), EcoRV-AluI (nt 1125 to 1950), or PvuII-EcoRV (nt 104 to 1125)restriction fragments of the lacZ DNA of pCH110 (Pharmacia, Inc., Piscataway,N.J.) were cloned into the Klenow-filled-in XhoI site of pMH9/3091-m8. pMH-9/3091-m8 was a derivative of pMH-9/3091, which contained a created XhoI siteat nt 37 on the HBV genome (3). To generate X1215, the 194-bp restrictionfragment of HaeIII of the fX174 DNA sequence was inserted into the Klenow-filled-in ClaI site located at the lacZ sequence of X1021. The EcoRV-AluIfragment of the lacZ DNA was inserted into three restriction sites on pMH-9/3091 (BspEI [nt 433], AvrII [nt 1461], and SpeI [nt 1962]) to generate B825, A825,and S825 insertion mutants with a positive orientation of the lacZ sequence. Togenerate plasmid X-GFP, a 745-bp BamHI-NotI (nt 661 to 1406) restrictionfragment of plasmid pEGFP-N2 (Clontech, Palo Alto, Calif.) containing thegreen fluorescent protein gene sequence was inserted into the XhoI-restricted,Klenow-blunted vector pMH-9/3091-m8. The C2093G mutant carried a C-to-Gmutation at nt 2093 (see Fig. 4A) and was generated by jumping PCR usingplasmid X1021 as the template. The mutagenic primer was mHBV2498 (59-CGCTGTTACCAATTTTGTTTTG [nt 2077 to 2098]). To construct εm mutants,PCR fragments were amplified by a primer-carried C-to-G mutation in thepriming site (59-GTCCTACTGTTGAAGCCTCC [nt 3136 to 3155]; mutatedbase in bold). Then the amplified products were used to replace the correspond-ing region of plasmid X1021. εm is similar to plasmid 1 published by Nassal andRieger (19). Plasmid EV825 was constructed by the following procedure. AnEcoRV site was first created by jumping PCR at a position just behind DR1*.The mutagenic primer used in the jumping PCR was mHBV3511 (59-CTCTGCCTAATGATATCTTGTTC [nt 3111 to 3133]; EcoRV site in bold). The EcoRV-AluI restriction fragment (nt 1125 to 1950) of the lacZ DNA sequence was theninserted into this created EcoRV site on pMH-9/3091. Constructs harboringPCR products were confirmed by DNA sequencing.

Transient transfection. HuH-7 human hepatoma cells (18) were transfected bythe calcium phosphate coprecipitation method as described previously (4). Forcotransfection, 15 mg of HBV mutant plasmid was cotransfected with 15 mg eachof plasmids pMTP and pMT1883 per 15-cm plate.

Isolation of viral core particles. The intracellular core particles and nucleicacid were purified as described previously (4). Core particles were immunopre-cipitated with 10 ml of human anti-HBV core protein antiserum which wascoated on protein A-agarose beads.

RNA preparation. Total cellular RNA was extracted with RNAzol B (Biotecx,Houston, Tex.) from day 2 posttransfection HuH-7 cells. For detection of en-capsidated pgRNA, immunoprecipitated cores from the cytoplasm were treatedwith micrococcal nuclease for 30 min at 37°C as described previously (11). ThenRNA was prepared by digestion with proteinase K (200 mg/ml in 1% sodiumdodecyl sulfate) at 37°C for 1 h followed by phenol-chloroform extraction. Afterethanol precipitation, nucleic acids were treated with DNase I for 20 min (11).

Detection of HBV nucleic acids. The endogenous polymerase assay (12) wasperformed as described previously (3) to detect HBV DNA of core particles.Southern and Northern blot analyses were performed as described previously(4). The SalI-SmaI HBV fragment containing the full-length HBV sequencefrom pMH-9/3091 was labeled by random priming (Promega Corp., Madison,Wis.) to serve as a probe.

Primer extension analysis. Primer extension analysis was carried out to detectthe 59 end of the minus-strand DNA as described by Nassal and Rieger (19). Thethermocycling parameters were 95°C for 1 min, 56°C for 1 min for primer KN-23or 52°C for 1 min for primers HBV2414, HBV1771, and HBV3341, and 72°C for1 min for 15 to 30 cycles. The extension products were mixed with loading bufferand subjected to electrophoresis in a 6% polyacrylamide sequencing gel. Afterbeing dried, the gel was autoradiographed at 270°C. Autoradiograms fromSouthern and Northern analyses were transformed to computer images usingAdobe Photoshop version 5.0. Oligonucleotides for primer extension analysescorrespond to nt 1933 to 1952 (KN-23), nt 2014 to 2035 (HBV2414), nt 1390 to1371 (HBV1771), and nt 3041 to 3060 (HBV3341) on the HBV genome. Oligo-nucleotide sequence are sense strand (plus-strand polarity) except for HBV1771(minus-strand polarity).

RESULTS

The HBV genome with foreign DNA inserts affects the for-mation of RC form DNA. To explore if the insertion of foreignDNA into its genome affects HBV DNA replication, HBVmutants containing lacZ gene sequences, X825 and X1021,

were generated. These insertion mutants produced pgRNAs of4.1 and 4.3 kb, respectively (Fig. 1D, lanes 2 and 3), comparedwith the wild-type 3.3-kb pgRNA [not including the poly(A)sequence] (lane 1). Southern blot analysis was employed tomonitor the viral genome by using cytoplasmic core particlesproduced from the HBV insertion mutants along with helperHBV genomes, pMT1883 and pMTP, which provided in transall the viral proteins required for encapsidation. As shown inFig. 1B, the wild-type HBV genome (pMH-9/3091) producedtypical RC and DL DNAs (lane 1). However, mutants X825and X1021 exhibited only a major band with a molecular sizeof approximately 3.0 to 3.2 kb (lanes 2 and 3), similar to that ofthe wild-type DL DNA product. No replication signals weredetected when mutant genomes were transfected alone (datanot shown). Furthermore, mutants that contain the green flu-orescent protein-encoding gene at the same site also gave riseto the same replication pattern as mutants X825 and X1021(data not shown), suggesting that the size of the insertionsequence, not the context of foreign sequences, contributes tothis change.

To investigate the nature of DNA genomes produced bymutants X825 and X1021, EcoRI, which has a single restrictionsite on wild-type and mutant genomes, was employed. Afterdigestion, the DL DNA of the wild-type genome would pro-duce 1.4- and 1.8-kb fragments whereas the RC DNA wasshifted downward to the position of the DL DNA (Fig. 1B,lane 4). In mutants X825 and X1021, the 3.2-kb DNAs werecleaved into 2.2- or 2.4-kb and 0.8-kb fragments, respectively(Fig. 1B, lanes 5 and 6). Based on the insertion position andlength as illustrated in Fig. 1A, the 2.2- and 2.4-kb fragmentscorresponded to the 59 end of mutant genomes. In contrast, the0.8-kb fragment was derived from the 39 end of replicate prod-uct. This 0.8-kb DNA fragment indeed did not hybridize withlacZ DNA (Fig. 1C, lane 5 and 6). The result is consistent withthe assumption that the 0.8-kb DNA fragment was derivedfrom the 39 end of X825 or X1021 replicate products. Theundigested DNA species may represent replicate intermedi-ates (compare lanes 5 and 6 to lanes 2 and 3); thus, they maybe resistant to EcoRI digestion. Taken together, these resultsstrongly suggest that the DNA genomes produced by mutantsX825 and X1021 were linear DNA with a size similar to wild-type DL DNA.

Polymerase-linked oligomer transfers to internal novel ac-ceptor sites in insertion mutants. The above-described datasuggest that the major replication products of X825 and X1021were DL DNA of approximately 3.0 to 3.2 kb even though thesizes of the pgRNAs are 4.1 or 4.3 kb. This result could beexplained if the polymerase-linked oligomer is transferred notto DR1* but to a new internal sequence on the HBV genome.Analysis of the terminus of minus-strand DNA by primer ex-tension, as shown in Fig. 2, revealed that the most 59 end of theminus-strand DNA of X825 and X1021 was mapped to nt 2093and minor amounts of extended products ended at nt 2074 andnt 2158 (lanes 1 and 2). A trace amount of extended productthat terminated at nt 2093 was also observed in wild-typegenomes pMH-9/3091 and pSHH2.1 (lanes 3 and 4). The sitesat 2074, 2093, and 2158 were named the internal acceptor sitesIAS1, IAS2, and IAS3, respectively. Taken together, the resultsindicated that the linear DNAs produced by X825 and X1021were elongated from IASs rather than DR1*. Furthermore,the length from the IAS to the EcoRI site is 0.8 kb, consistentwith the EcoRI digestion of mutant genomes.

Translocation of the polymerase-linked oligomer to IAS2 inthe insertion mutant is not dependent on insertion sites on theHBV genome. To examine whether other sites on the HBVgenome of the insertion mutant lead to change in the primer

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acceptor site from DR1* to IAS, a fragment containing 825 bpof the lacZ gene sequence was inserted into BspEI, AvrII, andSpeI sites on the HBV genome to generate HBV mutantsB825, A825, and S825, respectively (Fig. 3A). Northern blot-ting detected a 4-kb pgRNA in all mutants, as predicted (datanot shown). The replication products of these mutants dis-played DNA arrays similar to X825 as demonstrated by theendogenous polymerase assay (Fig. 3B). Primer extensionanalysis of the replicate products revealed that the 59 end ofminus-strand DNA mapped primarily to nt 2093, which wassimilar to that obtained with mutants X825 and X1021 (Fig.3C). The results indicate that the polymerase-linked oligomertransferring to IAS is not dependent on the insertion site onHBV genome.

Based on data described above, we may predict that if asimilar insertion was introduced into a site behind DR1*, poly-merase-linked oligomer translocation should not be affected.To address this issue, the fragment containing 825 bp of lacZgene sequence was inserted into a site behind DR1* to gener-

ate HBV mutant EV825 (Fig. 3A). As predicted, the pattern ofthe replication products (i.e., RC and DL DNA) of such amutant was the same as that of the products of the wild-typegenome (Fig. 3D, compare lane 2 with lane 1). Taken together,our results suggest that the production of a unit-length genomefrom a longer-than-unit-length pgRNA may be controlled by amechanism involving a fixed distance between the priming siteand the primer acceptor site.

Mutational analysis of the role of IAS2 in DNA replication.To confirm that IASs function as HBV minus-strand primeracceptor sites, a mutant (C2093G) of IAS2 in which C waschanged to G at position 2093 in X1021 was constructed. ThisX1021 mutant resulted in a dramatic decrease (up to 90%) ofreplicate DNA content, as demonstrated by the endogenouspolymerase assay (Fig. 4B, compare lane 2 with lane 1 [paren-tal type]). Analysis of the 59 end of minus-strand DNA byprimer extension revealed that the usage of IAS2 by the poly-merase-linked oligomer was indeed abolished whereas the us-age of IAS1 and IAS3 by the polymerase-linked oligomer was,

FIG. 1. Southern and Northern blot analyses of replicate products of HBV insertion mutants. (A) Schematic representation of restriction sites and genomeorganization of HBV. Four open reading frames of HBV are shown at the top, i.e. genes of core protein (C), surface protein (S), DNA polymerase (P), and X protein(X). The XhoI site was used to insert either 825 or 1,021 bp of the lacZ DNA as indicated. The cis elements required for HBV replication are located on the genome,including direct-repeat elements (DR1, DR1*, and DR2) and the RNA encapsidation signal sequence (ε) located at the 59 end of pgRNA. The polymerase-linkedoligomer priming site is located at the bulge region of ε. (B and C) Southern blot analysis of HBV nucleic acids. Cytoplasmic cores were isolated from HuH-7-transfected cell 5 days posttransfection. HBV DNAs which had been repaired using endogenous polymerase reaction with cold deoxynucleoside triphosphates (12) wereisolated from core particles produced by HuH-7 cells transfected with various plasmids as indicated at the top of the figures. Undigested (lanes 1 to 3) or EcoRI-digested(lanes 4 to 6) DNA samples were separated by agarose gel electrophoresis (1.3% agarose), and transferred to a filter, hybridized with 32P-labeled full-length HBV DNA(B) or the whole gene of lacZ DNA (C). Panel C shows the recombinant HBV hybridization with lacZ probe after stripping off the HBV DNA probe. DNA size markersare indicated at the right. (D) Northern blot analysis of HBV transcripts. Total RNAs isolated from HuH-7 cells that were transfected with various plasmids as in panelA were separated through a formaldehyde denaturing gel, transferred to a filter, and hybridized with 32P-labeled full-length HBV DNA. pgRNA and surface (preS1/S2)transcripts are indicated.

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at most, slightly affected by the IAS2 mutation (Fig. 4C, lane2). This result indicates that base pairing between the poly-merase-linked oligomer and IAS2 plays an important role inpolymerase-linked oligomer translocation. Since the C2093Gmutant did not demonstrate synthesis of other DNA species(Fig. 4B, lane 2), the results also clearly show that the IAS2 isthe major polymerase-linked oligomer acceptor site for X1021.Northern blotting revealed that the quantity and quality ofRNAs isolated from core particles produced by each mutantwere similar, suggesting that the abolition of DL DNA in theC2093G mutant is not due to the failure of pgRNA encapsi-dation (Fig. 4D).

In a reverse mutant, the C at nt 3147 of X1021 mutant withinthe bulge region of ε was changed to G (mutant εm). Thisresults in changing the nucleotide sequence on the polymerase-linked oligomer from GAA to CAA. The endogenous poly-merase assay revealed that the DNA array produced was sim-ilar to that of X1021 mutant (Fig. 4B, lane 3). However, primerextension analysis indicated that the 59 end of the minus-strandDNA mapped to nt 2098 and a minor part mapped to nt 2104(Fig. 4C, lane 3), suggesting that the altered polymerase-linkedoligomer may have the ability to scan the appropriate acceptorsite near IAS2. This altered mobility of the primer extensionproducts was also seen in IAS3 but not in IAS1 (Fig. 4C,compare lane 3 with lane 2). Previous reports also indicated

that mutant primers can translocate to better-fitting aberrantsites closed to DR1* (16, 19). Taken together, these resultsindicate that IAS2 functions as a polymerase-linked oligomeracceptor site and also suggest that polymerase-linked oligomermay possess scanning ability in order to find a matching ac-ceptor site.

Effects of insertion sizes on polymerase-linked oligomertranslocation. To further understand the relevance of thelength of the insertion sequence and the acceptor site selectionof the polymerase-linked oligomer, various lengths of lacZgene sequence were inserted into the XhoI site at nt 37 on theHBV genome to generate mutants X200, X400, X600, X825,X1021, and X1215. Several interesting results were obtainedfrom this panel of insertion mutants. (i) Production of RCDNA in each mutant was seriously affected (Fig. 5A, lanes 2 to7). Analysis of plus-strand DNA by primer extension indicatedthat the usage of DR2 by the plus-strand primer was very lowin mutant X400 and undetectable in mutants with insertionslarger than 625 bp (data not shown), a finding consistent withthe disappearance of RC DNA in insertion mutants. (ii) Thesize of the upper DL (largest) DNA produced by insertionmutants increased in parallel with the length of the insertion(Fig. 5A) and the amount was dramatically reduced in mutantscontaining insertions larger than 825 bp. However, this DLDNA was gradually replaced by novel bands of approximately2.6 to 3.4 kb as the insertion length increased (Fig. 5A). Primerextension analysis of the 59 end of minus-strand DNA indi-cated that the usage of DR1* by the polymerase-linked oli-gomer gradually decreased when the insertion size was in-creased (Fig. 5B). In contrast, the usage of IAS2 was increasedin a parallel manner (Fig. 5B). In control experiments withhelper plasmids (pMT1883 plus pMTP) alone, no primer ex-tension products were detected (Fig. 5B, lane 8). The intensi-ties of the primer extension products in Fig. 5B (lanes 1 to 8)were determined by amplification of templates with 15 cyclesof one-way PCR; if the reaction was further subjected to an-other 15 cycles, the intensity (lanes 10 and 12) was twice that ofthe value obtained after the first 15 cycles (lanes 9 and 11),indicating that the amounts of input primer still can quantifythe amounts of template within 15 to 30 cycles under ourexperimental conditions. (iii) The RC and DL DNA weredemonstrated by digesting repaired DNA with EcoRI. Asshown in Fig. 5A (lanes 9 to 16), RC DNA migrated down tothe position of the upper DL DNA (lanes 9 to 11) and thelower DL DNA produced two fragments smaller than those ofundigested DL DNA. One of these fragments, the 0.8-kb DNAfragment, was excised from the EcoRI site to IAS2 (lanes 11 to15); therefore, all mutants generated this DNA fragment. Thisresult, together with data obtained from primer extension anal-ysis (Fig. 5B), strongly suggested that the upper DL DNAswere elongated from DR1* and the lower DL DNAs wereelongated from IAS2. We also noticed that the additional bandincreased its size in parallel with the insertion length (Fig. 5A).These bands were resistant to EcoRI digestion and thereforemight represent a replication intermediate or DNA speciesthat lacks EcoRI site.

To gain more insight into the relationship between theprimer acceptor site used by the polymerase-linked oligomerand distance, we plotted the frequency (intensity) of primeracceptor sites used by the polymerase-linked oligomer againstthe distance from the priming site to IAS2 or DR1*, as shownin Fig. 5C. It is interesting that the polymerase-linked oligomerprefers to translocate to a site where the distance from primingsite to the primer acceptor site is approximately 3.2 kb, as forDR1* in the wild type or IAS2 in the X1021 mutant. Interest-ingly, such a translocation in this particular insertion mutant

FIG. 2. Determination of the 59 termini of minus-strand DNA of replicateproducts by primer extension. Core particle DNAs were prepared as described inthe legend to Fig. 1A and hybridized with KN-23 oligonucleotide as indicated inFig. 1A. A sequencing ladder primed with the same oligonucleotide using clonedHBV DNA as template was loaded in parallel to serve as a DNA marker. Arrowswith nucleotide number at the right side indicate the 59 end of minus-strand DNA.

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leads to maintaining one unit length of genome. In other in-sertion mutants, both primer acceptor sites (DR1* and IAS2)could be selected by the polymerase-linked oligomer, althoughthere is a tendency for DR1* to be more frequently selected.This observation also suggests that the polymerase-linked oli-gomer may be able to scan appropriate primer acceptor sitebidirectionally on pgRNA within the core particle.

DISCUSSION

Our results show that the polymerase-linked oligomer wastransferred to multiple novel acceptor sites when foreign se-quences were inserted into sites between ε and DR1* in theintact HBV genome. These identified IASs, which are located

approximately 2 kb from the priming site (bulge region of ε) onthe 59 end of wild-type pgRNA, contain a stretch of poly(U)sequence at the polymerase-linked oligomer binding sequence.They function as the acceptor site for polymerase-linked oli-gomer similar to that of DR1*. These IASs were also utilizedby the wild-type genome although at a very low frequency.However, the utility of these IASs become a major one in ourinsertion mutants.

The mechanisms of polymerase-linked oligomer transloca-tion to the DR1* acceptor site are largely unknown. It has beenproposed that the 59 and 39 ends of the RNA template arejuxtaposed within the capsid in a situation that allows efficientstrand transfer to proceed (16, 19, 23). Our data presentedhere may provide a clue to whether this does occur. The results

FIG. 3. Translocation of the polymerase-linked oligomer to IAS2 is independent of insertion sites on the HBV genome. (A) Schematic representations of theinsertion constructs. Symbols are identical to those in Fig. 1, except that X825, B825, A825, S825, and EV825 stand for mutants with the 825-bp insertion at XhoI (X),BstEII (B), AvrII (A), SpeI (S), and EcoRV (EV), respectively. The triple vertical line indicates the position of IASs. (B and D) The performance of the endogenouspolymerase assay is described in Materials and Methods. (C) Primer extension was done as described in the legend to Fig. 2, except that oligonucleotide HBV2414 wasused as a primer for extension and sequencing. WT, wild type; NC, negative control.

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obtained from the experiment in Fig. 5 indicate that the poly-merase-linked oligomer could translocate to either DR1* orIAS2 or both depending on the insertion size. In smaller orlarger insertion mutants, only one site is preferentially selectedby the polymerase-linked oligomer. For example, DR1* is usedprimarily in X200 and IAS2 is used in insertion mutants withinsertions larger than 825 bp, which makes the distance fromthe priming site to the acceptor site in those mutants approx-imately 3.1 6 0.2 kb. Interestingly, in the intermediate-inser-tion mutants (X400 and X600), both sites are utilized by poly-merase-linked oligomer, which makes the distance from IAS2less than 3.1 kb and that from DR1* greater than 3.1 kb. Howcould this happen? One explanation is that the priming sitemay juxtapose with a region on pgRNA within the nucleocap-

sid and the polymerase-linked oligomer may have the ability toscan the appropriate acceptor site bidirectionally. In otherexperiments, we showed that the polymerase-linked oligomerproduced from the εm mutant does not match IAS2. Interest-ingly, our results also show that this mutated polymerase-linked oligomer was translocated to a region near IAS2 (at nt2098) with a perfect sequence complementary to it (Fig. 4C).Therefore, this result supports the idea that the polymerase-linked oligomer may have the ability to scan appropriate ac-ceptor sites. These results, together with the fact that the IAS2mutation (C2093G) resulted in a loss of the function of thepolymerase-linked oligomer acceptor site, are consistent withthe hypothesis that (i) complementarity between the polymer-ase-linked oligomer and the primer acceptor site is required

FIG. 4. (A) Schematic representation of priming and polymerase-linked oligomer acceptor sites on pgRNA. The shaded oval tailing with the GAA trinucleotiderepresents the polymerase-linked oligomer; GAA is copied from UUC within the bulge of ε as indicated. Nucleotide sequences within the rectangle indicate thesequence around the polymerase-linked oligomer acceptor site, and arrows with nucleotide number indicate the 59 end of minus-strand DNA. The distance from thepriming site to the polymerase-linked oligomer acceptor is shown at the top in base pairs. (B and C) Mutation analysis of polymerase-linked oligomer translocation.Replicate DNAs were isolated from intracellular viral core particles produced by HuH-7 cells cotransfected with various constructs as indicated along with pMTP andpMH1883. The endogenous polymerase assay (B) and primer extension (C) were carried out as described in the legends to Fig. 1 and Fig. 2. (D) A portion of the coreparticles from panel B was prepared for the analysis of encapsidated pgRNA. The HBV sequence between nt 2089 and 2104 is shown at the left side of panel C.

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but not sufficient for primer translocation and (ii) the primingsite and IASs of pgRNAs may be juxtaposed with each otherwithin core particles of insertion mutants in order to facilitateprimer translocation. The latter hypothesis is further sup-ported by the result that sequence inserted downstream ofDR1* did not affect the transfer of the polymerase-linkedoligomer to DR1* (Fig. 3D).

Loss of RC DNA in our insertion mutants with insertionsizes greater than 400 bp could be caused by one of two pos-sibilities: (i) minus- or plus-strand primer transfers to an in-correct accepter site or (ii) lack of a terminally redundantsequence on pgRNA. Two types of DL DNA were detected inthese mutants. Our results clearly demonstrated that type IIDNA were generated by transferring the polymerase-linkedoligomer to IASs. This kind of aberrant translocation leads toboth a lack of the DR2 sequence in minus-strand DNA and aloss of terminal redundancy, both of which are required for RCDNA formation (15). Our results also showed that type Iminus-strand DNA (elongated from DR1*) genomes retainthe sequence determinants required for RC DNA formation,i.e., DR2 and terminal redundancy. However, the productionof RC DNA in these insertion mutants was hardly detected. Atpresent, we do not know why minus-strand DNA initiated fromDR1* could not generate RC DNA in our insertion mutant(Fig. 5A).

On the basis of these discussions, a model is proposed, asshown in Fig. 6, to account for each synthesized DNA productin various insertion mutants as well as in the wild-type genome.Mutants are classified into four classes according to whichregion or cis element juxtaposes with the priming site withincore particles. In class I mutants, the priming site and DR1*are juxtaposed with each other as depicted in Fig. 6a. Minus-strand DNA was elongated primarily from DR1*; thus, theproduction of RC and DL DNA resembled that in the wild-type genome. EV825 belongs to this phenotype. Class II mu-tants are those in which the region to be juxtaposed with thepriming site is located between DR1* and IAS2, as shown inFig. 6b. To elongate their minus-strand DNA, the polymerase-linked oligomer may have the ability to scan acceptor sitesbidirectionally in these mutants. If the polymerase-linked oli-gomer is elongated from DR1*, type I DL DNA or/and RCDNA was produced, as in the X200, X400, and X600 mutants.If the polymerase-linked oligomer is elongated from IAS2,type II DL DNA were formed, as in the X400, X600, and X825mutants. As shown in Fig. 6f, following translocation of thenascent polymerase-linked oligomer from the priming site tothe IAS on pgRNA, minus-strand DNA is elongated from the39 end to the 59 end along pgRNA that started at IAS. Elon-gation is concomitant with degrading pgRNA by the viralRNase H (17); thus, the 39-end RNA of IAS is removed frompgRNA [indicated by the RNA fragment with the poly(A)sequence in Fig. 6f]. The polymerase-linked oligomer was nottransferred to DR1* of pgRNA, which results in the loss ofDR2 on minus-strand DNA. In class III mutants, the primingsite is juxtaposed with IAS2, as depicted in Fig. 6c. An exampleis mutant X1021, in which only type II DL DNA was produced.Class IV mutants resemble class III mutants in terms of pro-duction of their DNA phenotype, except that IAS2 is notjuxtaposed with priming site. As depicted in Fig. 6d, the poly-merase-linked oligomer must back-scan to IAS2 in order forthe minus-strand DNA elongation to occur. This model isconsistent with our data presented. Therefore, our data alsoprovide indirect evidence that the priming site and DR1* maybe juxtaposed with each other within core particles in thewild-type genome. It has been reported that several cellularproteins such as the chaperone complex of heat shock protein

FIG. 5. Size effects of polymerase-linked oligomer translocation. (A) Thepreparation of replicate DNAs of intracellular viral core particles and the per-formance of Southern blot analysis are the same as in Fig. 1B. p, largest DL DNAproduced by insertion mutants; Š, novel bands produced as the insertion lengthincreased; p (lanes 9 to 11), RC DNA migrating at the DL DNA position; E,additional band produced by lower DL DNA. NC, negative control. (B) Primerextension analysis of the 59 end of minus-strand DNA. Viral DNA was digestedwith EcoRI prior to primer extension. The primers used for primer extension areHBV3341 (for mapping DR1*), HBV2414 (for mapping IAS2), and HBV1771(for detecting the viral template containing the EcoRI site). A sequencing ladderwith cloned HBV DNA as the template primed by the DNA oligomer HBV3341(left) or HBV2414 (right) was loaded in parallel to serve as the DNA marker. Apilot test was done to determine suitable amounts of DNA template to obtain asimilar signal primed by the HBV1771 primer. (C) Frequency of DR1* and IAS2minus-strand primer acceptor site used by the polymerase-linked oligomer ineach individual insertion mutant. The intensities of primer extension productswere normalized based on equal amounts of minus-strand DNA derived from theHBV1771 primer. The distances from priming site (ε) to IAS2 and to DR1* are2.13 and 3.15 kb, respectively, in wild-type pgRNA.

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90 and p23 were copackaged and interacted with pgRNA in-side core particles (8, 9). These cellular proteins may be in-volved in maintaining pgRNA in such a particular structure tofacilitate polymerase-linked oligomer translocation. Alterna-tively, pgRNA may organize into a particular structure withincore particles, which brings the priming site and DR1* or IAS(in insertion mutants) together to facilitate translocation.These two possibilities may not be mutually exclusive. Finally,our result also shows that IAS2 can be utilized in wild-typepgRNA during polymerase-linked oligomer translocation.Consequently, this process will also contribute to genome het-erogeneity or production of defective virus.

ACKNOWLEDGMENTS

We thank S.-J. Lo, L.-P. Ting, T.-S. Su, T. Y. Shih, and T. J. Liangfor helpful discussions and C.-M. Tseng for experimental assistance.

This work was supported by an intramural research grant of theNational Health Research Institutes and by grants NSC 86-2314-B-010-040 and NSC 87-2314-B-010-075 from the National Science Coun-cil, Republic of China.

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