Institlite for Cancer Research, Fox Chase Cancer Center, 7701 Bulrholme Avenuie, Philadelphia, Pennisylvanlia 19111. Received 25 June 1993/Accepted ...
JOUIRNAL Ol VIRoLOGNY, Nov. 1993, p. 6507-6512 0022-538X/93/1 16507-()6$02.00/0 Copyright OK 1993, Amcrican Society for Microbiology
Vol. 67, No. 11
Novel Mechanism for Reverse Transcription in Hepatitis B Viruses GUANG-HUA WANG AND CHRISTOPH SEEGER* Institlite for Cancer Research, Fox Chase Cancer Center, 7701 Bulrholme A venuie, Philadelphia, Pennisylvanlia 19111 Received 25 June 1993/Accepted 13 August 1993
Reverse transcription of all retroviruses and most retroid elements requires tRNA as a primer for DNA synthesis. However, in hepatitis B viruses the viral polymerase itself acts as a primer for reverse transcription (G.-H. Wang and C. Seeger, Cell 71:663-670, 1992). We have now demonstrated that in order to prime DNA synthesis, the polymerase binds to an RNA hairpin, which then serves as a template for the formation of a short DNA primer that is covalently linked to protein. Following its synthesis, the nascent DNA strand apparently dissociates from its template and reanneals with complementary sequences at the 3' end of the RNA genome, where DNA synthesis continues. Since this RNA hairpin also functions as a packaging signal for viral RNA, hepadnaviruses have adopted a replication strategy that relies on the same signal for two biochemically distinct events, RNA packaging and reverse transcription. This mechanism is without precedent among all known retroid elements and among other viruses and bacteriophages that use protein as a primer for RNA or DNA synthesis. It could provide an effective target for antiviral therapy, which is required for the treatment of more than 300 million carriers of hepatitis B virus.
previously been recognized a cryptic initiation site used for replication of DHBV genomes in tissue culturc cells that lacked a natural initiation site for minus-strand DNA synthesis (2). We now report that initiation of reverse transcription in hepadnaviruses occurs at sequences within the bulgc of and not, as previously assumed, at DRI. Following the synthesis of the first four nucleotides of minus-strand DNA, the polymerase-deoxynucleoside monophosphate (dNMP) complcx dissociates from its template and rcanneals with complementary sequences at DRI near the 3' cnd of pregenomic RNA, where DNA synthesis continues. Thus, our results indicate that a signal for RNA packaging but also serves e not only acts as a templatc for the initiation of viral DNA synthesis.
The reverse transcriptases of retroviruses and most known retroid elements require tRNAs as primers for RNA-directed DNA synthesis (10). In hepadnaviruses, however, the reverse transcriptase itself acts as a primer for this reaction (26). As a consequence of the priming reaction, the polymerase is covalently linked to the 5' end of the reverse transcribed (minus) DNA strand (5, 14). The 5' end of minus-strand DNA maps to a specific base localized within an 11- to 12-bp region near the 3' end of viral RNA, known as DR] (12, 20). Genetic experiments with mammalian and avian hepadnaviruses revealed that three or four nucleotides near the 3' end of viral RNA, which correspond to the first nucleotides of minusstrand DNA, are required to specify the position of the 5' ends of the reverse transcribed DNA strand (2, 22). This signal is a unique recognition site for the reverse too short to serve transcriptase; however, a specific binding site, epsilon (E), for the reverse transcriptase has been identified near the 5' end of pregenomic RNA. The sequence of contains a palindrome, which has the potential to form a stem-loop structure with a bulge and a loop (see Fig. 1) (8). Since interaction between the reverse transcriptase and E is required for the assembly of RNA containing subviral core particles, e is referred to as the RNA-packaging signal (7, 8). We have recently demonstrated that the reverse transcriptase of duck hepatitis B virus (DHBV) can be expressed in enzymatically active form in a ccll-free system (26). The ability of the in vitro-expressed polymerase to initiate DNA synthesis depended on RNA sequenccs that contained DRI and Surprisingly, DNA synthesis was often arrested following the synthesis of a 4-nucleotide-long DNA oligomer with the sequence 5'-GTAA-3'. This motif is identical to complementary sequences on pregenomic RNA that corrcspond to the 5' end of minus-strand DNA and are critical for correct initiation of DNA synthesis. Moreover, identification of the 5' ends of in vitro-synthesized minus-strand DNA revealed that in addition located in to the normal initiation site, a second site, which used for DNA synthesis. This sitc has the bulge region of 6?,
as
as
as
MATERIALS AND METHODS Molecular clones. Plasmid p5.lGal contains
completc
generated by oligomer-directed site-specific described by Taylor et al. (23). Plasmid pCMDHBV directs the transcription of prcgcnomic RNA from the cytomcgalovirus immcdiate-early promoter (27). Variants of pCMDHBV were constructed by substitution of the relevant DNA restriction fragments with the corrcsponding fragments released from the respective variants of p5.lGal. Polymerase assay. The RNA template for the translation of the DHBV polymerasc genc synthesized from linearized plasmid DNA with the help of the MEGAscript kit (AMBION Inc., Austin, Tex.). The in vitro translation reaction was carried recommended by the supplier (Promega Biotec, Madiout son, Wis.). Reaction mixtures were incubatcd for 60 min at 30(C. The DNA polymerization reactions were carried out essentially as described previously (26). To S p.l of the reticulocyte lysate with the polymerase, 4 .1I of a reaction mixturc containing buffer (100 mM Tris-HCI [pH 7.5]), salts (30 mM NaCI, 20 mM MgCL,), deoxynucleoside triphosphatc (dNTP) (28 IiM), and [32 P]dNTP (2.4 F.M, 400 Ci/mmol) was added. Reaction mixturcs were incubated at 30(C for 30 min and
p5.11BC,
were
mutagenesis,
as
was
as
was
was
*
a
gcnomc of an infectious clonc of DHBV fused to the SP6 promoter (13, 16). The derivatives of p5.lGal, p5.IBG and
Corresponding cauthor. 6507
6508
J. VIROL.
WANG AND SEEGER
Ca u ua
a UU
u c
c GUUu CU-
a u a-c
U-u acn
u-G u U-G
A-U
A-U
2576 U A-U
2576 u A-U
U-A \,pa-c
U-A
c-a
c-a
.u A-U U-A
cU-a A-U U-A
a-C
a-C
A-U
2537
A-U
A-U
(-)DNA 5'-aAAUUACACCCCUCUCCUUCGGAGCUaCU C-aU-A //IPOLYMERASE ORF AccccucUc cuUcG'~AGcuGCU
a-c
A-U U
A-U c-a c-a a-u
U-A..3
I~~~~~~~~~:DR1I--
(+)DNA
I 2488
FIG. 1. Structure of pregenomic RNA of DHBV (13). The RNA bears terminal repetitions that contain copies of the packaging signal (s). The predicted stem-loop structure of £ is depicted as described by Junker-Niepmann et al. (8). The positions of the polymerase open reading frame (ORF) and the 5' ends of minus- and plus-strand DNAs are indicated. The UUAC motifs important for minus-strand DNA synthesis at positions 2576 in £ and 2537 in DRI (bracketed) are shaded. The position of the 5' end of DHBV RNA used for expression of the polymerase in vitro is indicated (position 1). Note that the RNA template used for expression of the polymerase in vitro contains only one copy of s at the 3' end.
stopped by the addition of protein-loading buffer (17). Samples were electrophoresed through 0.1% sodium dodecyl sulfate (SDS)-10% polyacrylamide gels, dried, and autoradiographed. Isolation of viral DNA. Isolation of viral DNA synthesized in the in vitro reaction and primer extension analysis were carried out as described previously (26). Isolation of viral DNA from transfected tissue culture cells and Southern blot analysis were done as described previously (22). Tissue culture. The chicken hepatoma cell line LMH (9) was maintained as described by Condreay et al. (1). DNA transfections were carried out with the help of a calcium phosphate cell transfection kit (5 Prime-3 Prime Inc., Boulder, Colo.). PCR. Virion DNA was amplified directly from culture medium of transfected cells by polymerase chain reaction (PCR) (24). The supernatants were cleared from cellular debris by centrifugation and incubated with DNase I to digest residual plasmid DNA used for the transfection. Following an incubation with SDS (0.13%) and proteinase K (0.13 mg/ml) at 45°C for 2 h, an aliquot of the reaction mixture was used for the amplification of a DNA segment spanning positions 2472 and 3000 on the DHBV genome. For sequencing, the amplified DNA was purified with a kit purchased from Promega Biotec. RESULTS The packaging signal provides the template for the incorporation of the first four nucleotides of minus-strand DNA. The in vitro polymerase reaction yields at least two species of elongated minus-strand DNA with the same four nucleotides at their 5' ends (26). They are localized at position 2537 in DR1 and at position 2576 in s (Fig. 1). To determine which of the two sites provided the template for the initiation reaction, we introduced mutations into both sequences and examined their effects on the order of nucleotides incorporated into the reverse transcriptase. Following the in vitro expression of polymerase, aliquots of the reaction mixtures were incubated with solutions that contained all four dNTPs, one of which was labeled with 32p. As expected from the predicted nucleotide sequence at the 5' end of minus-strand DNA, incubation of
reverse transcriptase in the presence of wild-type RNA with 32P-labeled dGTP, dTTP, or dATP, but not with [32P]dCTP, yielded polymerase polypeptides that had acquired radioactivity (Fig. 2A and B, lanes 1, 3, 5, and 7). However, when the polymerase was expressed from pS.1BG, in which the UUAC motif located at position 2576 in the bulge of s had been changed to GGAC, a different incorporation pattern was observed. The polymerase now incorporated [32P]dCMP instead of [32P]dAMP (Fig. 2B, lanes 6 and 8). This result suggested that the sequence of the first four nucleotides attached to polymerase expressed with p5.1BG was changed from GTAA to GTCC. Confirmation of this result was obtained with a second construct, p5.1BC, in which the C residue at position 2576 was changed to G. This base change led to the incorporation of [32P]dCMP into the polymerase (Fig. 2C, lane 4), while the reaction with [32P]dGTP did not yield a labeled polymerase polypeptide (lane 3). These results suggested that the template for the synthesis of the first four nucleotides attached to the viral polymerase was located in the bulge of s and that the sequences on pregenomic RNA in DR1 that corresponded to the position of the natural 5' end of minus-strand DNA were not actually copied into minus-strand DNA. This observation was confirmed with the help of two independent variants of p5.1Gal, which carried mutations in the UUAC motif at position 2537 in DR1 that changed the sequence to UUAA and GGiAC, respectively. None of the mutations tested altered the order of the first four nucleotides of minus-strand DNAs that were covalently linked to the polymerase (18). Mutations in e interfere with the formation of minus-strand DNA. On the basis of these observations, we surmised that the UUAC motif located in the bulge of s (referred to as the donor site) provided the template for the synthesis of the first four nucleotides of minus-strand DNA and that subsequently, the polymerase-dNMP complex translocated to the site in DR1 (referred to as the acceptor site) to continue DNA synthesis. This model predicts that mutations like the one in p5.1BG that alter the UUAC motif in s would interfere with the synthesis of minus-strand DNAs with 5' ends at position 2537. To test
VOL. 67, 1993
REVERSE TRANSCRIPTION IN HEPATITIS B VIRUSES
A
2537
1
2576
2
3
6509
4
pos. 5'-- UUAC 5'
UUAC
---
II--- UUAC GGAC
3'
pS.1GAL
3'
p5.1 BG
5'-- UUAC ---11--UUAG-3'
p5.1 BC
--
--- //
--
--
a
B
2
1
to.
2576 --o-I4
_O
_
NV
3
4
5
6
7
8
POL-'2537--_
..:i
FIG. 3. Initiation of minus-strand DNA depends on nucleotide in Primer extension analysis was carried out on DNA purified from the in vitro polymerase reaction products obtained with wild-type plasmid p5.lGal (lane 1) and mutants p5.1BG, p5.1AG, and p5.1ABG (lanes 2 through 4, respectively). In p5.1AG. the UUAC motif at position 2537 in DRI was changed to GGAC, and in p5.IABG, the UUAC motifs at positions 2537 and 2576 were changed to GGAC. A DNA oligomer spanning positions 2472 to 2498 on the DHBV genome served as the primer for DNA extension reactions. sequences
G
T
1 2
3 4
A
C
POL -"
G C G C FIG. 2. Identification of the template for the synthesis of the first four nucleotides of minus-strand DNA. (A) The UUAC motif at position 2576 in E in p5.1Gal was changed to GGAC in p5.IBG and UUAG in p5.1BC. (B) In vitro reaction products obtained with reverse transcriptase expressed with wild-type plasmid p5.lGal (GAL) (lanes 1, 3, 5, and 7) and with mutant p5.1BG (BG) (lanes 2, 4, 6, and 8). (C) Same as panel B but with mutant p5.1BC (BC) in lanes 3 and 4. For panels B and C, the nature of the alpha-labeled [32P]dNTP present in the reaction mixtures is indicated below the lanes (G, T, A, or C). Plasmid p5.1Gal contains a complete genome of an infectious clone of DHBV fused to the SP6 promoter (13, 16). POL, polymerase.
this prediction, we analyzed the 5' ends of minus-strand DNA synthesized in vitro from RNA templates transcribed from plasmids with mutations at either position 2576 (p5.1BG) or 2537 (p5.lAG) and at both sites (p5.1ABG). As expected from previous results, the polymerase expressed with wild-type plasmid p5.1Gal produced minus-strand DNAs with 5' ends mapping to positions 2537 and 2576, corresponding to the UUAC motifs in DRI and respectively (Fig. 3, lane 1). A third extension product that mapped to a CUAC motif at position 2582 was obscrved. When the polymerase was expressed from pS.lBG, which contained the GGAC motif at position 2576, only one cxtension product was detected that corresponded to minus-strand DNAs with 5' ends at position 2576 (Fig. 3, lane 2). However, initiation from position 2537 in DRI could partially be restored when the compensatory base changes were introduced at the putative acceptor site 8,
*
E.
(p5. IABG; Fig. 3, lane 4), as expected if initiation from this site depended upon base pairing with DNA derived from the donor site. Finally, the GGAC mutation at position 2537 in DRI (pS.AG) prevented the initiation of minus-strand DNA synthesis from the acceptor site but did not affect the synthesis of minus strands from the donor site in £ at position 2576 or from position 2582 (Fig. 3, lane 3). These results established that the synthesis of minus-strand DNA from position 2576 in the bulge of E can occur independently of the reaction that leads to the synthesis of minus strands beginning at position 2537. In contrast, the formation of minus strands beginning at position 2537 in DRI, the position of the 5' ends of minus-strand DNA in virions, and appears to be regulated by sequences in the bulge of depends on sequence homology between the two sites. Hence, our results are consistent with the hypothesis that the template for the synthesis of the first four nucleotides of minus-strand DNA is located in the bulge of E and that a short DNA strand is transferred to the acceptor site in DRI, where DNA synthesis can continue. Transfer of nascent minus-strand DNA from the 5' to the 3' end of pregenomic RNA. We next tested whether the results obtained under the selected conditions in vitro were relevant for the mechanism of viral replication in vivo. For this purpose, we constructed variants of pCMDHBV, in which the UUAC motifs at the donor and acceptor sites were mutated to GGAC. pCMDHBV contains an infectious DHBV clone fused to the cytomegalovirus sequences so that pregenomic RNA is expressed in transfected cells from the cytomegalovirus immediate-early promoter (Fig. 1) (27). While pregenomic RNA contains two copies of prcvious experiments have established that the 3' copy is dispensable for RNA packaging and reverse transcription (21). Mutation of the UUAC motif in the 3' copy of e also had no apparent effect on the synthesis of DHBV DNA in transfected tissue culture cells (Fig. 4A). 8,
J. VI ROL.
WANG AND SEEGER
6510
-4:
12
3
12
I RC DSL
__
2537
2576
A
5' -- UUAC --- 1--- UUAC -- 31
CMDHBV
5' -- UCAC ---11--- UUAC -- 3'
DHAC
5'--UUAC---//---UUAA-.3'
DHAA
-
Ss
B 3'
FIG. 4. Southern blot analysis of DNA extracted from subviral core particles in transfected LMH cells. (A) Cells were transfected with pCMB3G, in which the UUAC motif in the 3' copy of E at position 2576 was changed to GGAC (lane 2), and with wild-type plasmid pCMDHBV (lane 1). (B) Same as panel A but with pCMBG, in which the UUAC motif in the 5' copy of E(at position 2576 was changed to GGAC (lane 2), and pCMABG, which is a derivative of pCMBG, in which the UUAC motif at position 2537 in DRI was also changed to GGAC (lane 1). Lane 3 shows the result of the transfection with pCMDHBV. RC, relaxed circular DNA; DSL, double-stranded linear DNA; SS, single-stranded DNA.
C C C C
p
p
V
V
1 2
3 4
5 6
7 8
A C A 2535--_ T-C
* .
T A A
CT
8,
,,,
CT
5,
C T When the same mutation was introduced into the 5' copy of £ (the donor site), expression of viral DNA was almost completely abolished (Fig. 4B, lane 2). This mutation could be partially rescued when the UUAC at position 2537 in DRI (the acceptor site) was also changed to GGAC (Fig. 4B, lane 1). This result was consistent with the observation made with the in vitro assay, which showed that the GGAC mutation in E led to an overall reduction of viral DNA synthesis (Fig. 3). Since our model predicts that the 5' end of minus-strand DNA is not collinear with the corresponding sequences on pregenomic RNA (position 2537), we introduced a mutation into the UUAC motif in e and asked whether a base change would be observed at the 5' end of newly synthesized minusstrand DNA. For this purpose, we changed the UUAC motif to UCAC in the 5' copy of in plasmid pCMDHBV (Fig. SA). This mutation was selected because it did not appear to inhibit viral DNA replication, even without compensatory changes in the UUAC motif at position 2537 (18). The resulting plasmid, termed pDHAC, was transfected into tissue culture cells, and after 5 days, supernatants containing progeny virus were collected from the transfected cells. A segment of the viral DNA, which spanned the position of the 5' end of viral DNA and was amplified by PCR, and the amplified DNA was sequenced. The result indicated that the T residue at position 2535 in pDHAC was changed to C in progeny virion DNA (Fig. 5B; compare lanes 1 through 4 with lanes 5 through 8), and thus the nucleotide sequence at the 5' end of minus-strand DNA was changed from 5'-GTAA-3' to 5'-GTGiA. To confirm this result, we molecularly cloned the PCR-amplified DNA fragments that were synthesized from virion DNA obtained from three independent transfection experiments. From a total of 12 plasmids that were sequenced, 10 contained the C residue at position 2535 and 2 had the wild-type sequence (18). Since the 3' end of the terminally redundant minus-strand DNA bears a second copy of the UUAC motif (Fig. 1) (12), it
W
C T
C
p 1
V
2 3 4
T T A
2537--. _ A-C A C
c~~~~~~ -----
3' G
T G T
FIG. 5. Mutations in change the nucleotide sequence at the 5' end of minus-strand DNA. (A) For the construction of variant pDHAC, the UUAC motif at position 2576 in the 5' copy of in pCMDHBV was changed to UCAC, and for pDHAA, the UUAC motif at position 2537 in DRI was changed to UUAA. (B) The C and T tracks of the nucleotide sequence ladders obtained from reactions with pDHAC (P) (lanes 1 through 4) and DNA obtained after amplification of virion DNA (V) expressed with pDHAC (lanes 5 through 8). The primer used for the sequencing reactions corresponded to positions 2472 to 2499 on the DHBV genome. The sequence ladder shows the nucleotide sequence of the plus strand of DHBV. (C) The G and T tracks of the nucleotide sequence ladders obtained from reactions with pDHAA (P) (lanes 1 and 2) and virion DNA (V) expressed from pDHAA (lanes 3 and 4). The sequencing reactions were primed with a DNA oligomer spanning positions 2655 to 2632. The sequence ladder corresponds to the minus strand of DHBV DNA.
VC)L. 67, 1993
expected that some clones with the wild-type genotype would be obtained under the conditions selected for viral DNA
was
REVERSE TRANSCRIPTION IN HEPATITIS B VIRUSES
6511
A
amplification.
I
A similar result was obtained with a second derivative of
pCMDHBV, pDHAA. In this plasmid, the UUAC motif at the acceptor site in DRI (position 2537) was changed to UUAA (Fig. 5A) while the motif in £ remained unchanged. As predicted by our model, the nucleotide sequence of the amplified DNA from the progeny virus revealed that as a consequence of DNA replication, the A residue at position 2537 was reverted to C (Fig. 5C). In summary, these results directly demonstrated that a mutation introduced into the UUAC motif in E is genetically transferred to the 5' end of minus-strand DNA during DNA replication. This observation, therefore, provides compelling evidence for a mechanism in which £ provides the template for a short DNA strand that becomes the 5' end of minus-strand DNA. DISCUSSION Previous experiments led to the conclusion that the initiation site for the reverse transcription of viral DNA coincided with sequences on pregenomic RNA that correspond to the position of the 5' end of minus-strand DNA (21, 22). On the basis of new evidence described in this report, we propose a revised model for the replication of the hepadnavirus genome. This model predicts that the reaction which leads to the priming for reverse transcription of minus-strand DNA synthesis employs RNA sequences present in the RNA-packaging signal, s, as a template for the polymerization of a short 4-nucleotide-long DNA strand. Following the synthesis of this strand, the nascent DNA strand switches templates to reanneal with the sequences on pregenomic RNA that correspond to the previously mapped 5' end of minus-strand DNA. DNA synthesis then continues toward the 5' end of pregenomic RNA (Fig. 6A). Thus, the priming reaction occurs in two biochemically distinct steps. In the first, a tyrosine residue at position 96 of the polymerase acts as the primer for the formation of a covalent bond between protein and dGMP (28). In the second step, the G residue is extended through the addition of three nucleotides to create the actual primer for minus-strand DNA synthesis. Our model thus links the reaction that leads to the binding of the polymerase to the 5' end of pregenomic RNA, which was previously considered a requirement for RNA packaging, with the reaction that controls viral DNA synthesis. Pollak and Ganem (15) recently demonstrated that RNA packaging, and hence the binding of the polymerase to £, can occur independently of the primary sequences in the bulge of s. In agreement with these results, we observed that mutations in the bulge of E did not interfere with the ability of the reverse transcriptase to initiate DNA synthesis in vitro (Fig. 2B). However, these mutations apparently can influence the levels by which minus strands are extended beyond the first four nucleotides of minus-strand DNA (Fig. 3 and 4). Moreover, our results showed that sequence identity between the donor and acceptor sites on pregenomic RNA is not an absolute requirement for viral DNA synthesis, since the mutation in pDHAC did not interfere with DNA replication in vivo (Fig. 5). Synthesis of minus strands expressed with this variant required base pairing between a dG residue corresponding to the third nucleotide of minus-strand DNA and U on viral RNA. While it is well-known that GU residues form base pairs stabilized by two hydrogen bonds in RNA, our results indicate that base pairing can also occur between dG and U residues in RNA-DNA hybrids.
AAAAAAAA
II
r
AAAAAAAA
(-) DNA
B 2576
2537
DHBV 5'- CUUUUACGT -3'
5'- AAUUACACC-3'
HBV WHV 5'- CUGUUCAAG -3' GSHV
5'- UUUUUCACC-3'
FIG. 6. Model for the priming and initiation of minus-strand DNA synthesis. (A) Priming of reverse transcription of the hepadnavirus genome occurs at the 5' end of pregenomic RNA. (I) The polymerase (shaded oval) uses RNA sequences located in the bulge region of £ as a template for the synthesis of a short DNA oligomer. (II) To initiate minus-strand DNA synthesis, the nascent DNA strand is transferred to DRI at the 3' end of pregenomic RNA, where it can reanneal with complementary sequences (T) and prime minus-strand DNA synthesis. (B) Comparison of the nucleotide sequence of DHBV with those of the mammalian hepadnaviruses hepatitis B virus (HBV) (25), woodchuck hepatitis virus (WHV) (4), and ground squirrel hepatitis virus (GSHV) (19) in the bulge of E and at DRI.
While the results presented in this report were obtained with an avian hepadnavirus, they are with all likelihood applicable to the mammalian counterparts of these avian hepadnaviruses. The most compelling argument supporting this view comes from genetic analyses of the initiation site for reverse transcription of woodchuck hepatitis virus, which had shown that a UUC sequence motif at DRI is sufficient to specify the 5' end of minus-strand DNA (22). As expected from our model, the same sequence motif is indeed located in the bulge of e of the mammalian genomes, exactly at the position corresponding to the UUAC motif found in DHBV (Fig. 6B) (8). The ability of the reverse transcriptase to arrest DNA synthesis following the incorporation of four nucleotides is reminiscent of a feature attributed to telomerase, which likewise copies only a limited portion of its internal RNA template that corresponds to the telomeric DNA repeats (6). The
6512
J. VIROL.
WANG AND SEEGER
mechanism by which the polymerase accomplishes the transfer of the primer from one end of the RNA pregenome to the other still remains elusive. It is likely that the RNA-polymerase complex is arranged in a manner such that E and DRI are held in close proximity. Such an arrangement would facilitate the transfer of the nascent DNA strand and, most importantly, specify the annealing site for the short DNA primer on pregenomic RNA. It is noteworthy that the reaction leading to priming of plus-strand DNA also depends upon the transfer of nucleic acid, in this case, an RNA primer, across the viral genome (11, 20). Our results demonstrate that in hepadnaviruses, the reactions leading to RNA packaging and reverse transcription are controlled by the same signal on pregenomic RNA. Since pregenomic RNA also acts as an mRNA template for the translation of the polymerase polypeptide, the reverse transcriptase could bind to 6 during or immediately following translation. This model explains why in cotransfection experiments, pregenomic RNAs that express active polymerase polypeptides are preferred over RNAs with defective polymerase genes as substrates for reverse transcription (7, 27). An interesting paradigm for the mechanism in hepadnaviruses described here is the strategy adopted by the yeast L-A viruses, in which RNA packaging also requires the binding of the viral polymerase to a stem-loop structure on viral RNA (3). However, it is not yet known whether this interaction plays a role in RNA-directed RNA synthesis. In contrast, in retroviruses and most other known retroid elements, where the reverse transcriptase is synthesized as a Gag-Pol fusion protein, the Gag domain is believed to direct incorporation of the polymerase into the viral tegument. Since the reaction that controls the binding of the hepadnaviral reverse transcriptase to 6 is critical for genome replication, it could provide an important target for the development of effective antiviral agents required for the treatment of chronic hepatitis infection in more than 300 million individuals.
8.
9.
10.
11. 12.
13.
14.
15. 16.
17. 18. 19.
20. ACKNOWLEDGMENTS We thank Rich Katz, David Lazinsky, Bill Mason, James Sherley, Ann Skalka, and John Taylor for critical review of the manuscript. We thank Anthony Yeung for the synthesis of DNA oligomers. This work was supported by Public Health Service grants from the National Institutes of Health and an appropriation from the Commonwealth of Pennsylvania. 1.
2.
3. 4. 5. 6.
7.
REFERENCES Condreay, L. D., C. E. Aldrich, L. Coates, and W. Mason. 1990. Efficient duck hepatitis B virus production by an avian liver tumor cell line. J. Virol. 64:3249-3258. Condreay, L. D., T.-T. Wu, C. E. Aldrich, M. A. Delaney, J. Summers, C. Seeger, and W. S. Mason. 1992. Replication of DHBV genomes with mutations at the sites of initiation of minusand plus-strand DNA synthesis. Virology 188:208-216. Fujimura, T., J. C. Ribas, A. M. Makhov, and R. B. Wickner. 1992. Pol of gag-pol fusion protein required for encapsidation of viral RNA of yeast L-A virus. Nature (London) 359:746-749. Galibert, F., T. N. Chen, and E. Mandart. 1982. Nucleotide sequence of a cloned woodchuck hepatitis virus genome: comparison with the hepatitis B virus sequence. J. Virol. 41:51-65. Gerlich, W. H., and W. S. Robinson. 1980. Hepatitis B virus contains protein attached to the 5' terminus of its complete DNA strand. Cell 21:801-809. Greider, C. W., and E. H. Blackburn. 1989. A telomeric sequencc in the RNA of Tetrahymena telormerase required for telomere repeat sequence. Nature (London) 337:331-337. Hirsch, R. C., J. E. Lavine, L.-J. Chang, H. E. Varmus, and D. Ganem. 1990. Polymerase gene products of hepatitis B viruses are
21.
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