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Originally published In Press as doi:10.1074/jbc.M608031200 on September 27, 2006

J. Biol. Chem., Vol. 281, Issue 47, 35794-35801, November 24, 2006
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The Duck Hepatitis B Virus Reverse Transcriptase Functions as a Full-length Monomer*

Zhian Zhang{ddagger} and John E. Tavis{ddagger}§1

From the {ddagger}Department of Molecular Microbiology and Immunology and §Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, Missouri 63104

Received for publication, August 22, 2006 , and in revised form, September 26, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepadnaviral reverse transcription occurs within cytoplasmic capsid particles and is catalyzed by a virally encoded reverse transcriptase, but the primary structure and multimeric state of the polymerase during reverse transcription are poorly understood. We measured these parameters for the duck hepatitis B virus polymerase employing active enzyme translated in vitro and derived from intracellular core particles and mature virions. In vitro-translated polymerase immunoprecipitated as a monomer, and polymerase molecules with complementary defects in the enzymatic active site and tyrosine 96, which primes DNA synthesis, could not complement or inhibit each other in priming assays. Western analysis using antibodies recognizing epitopes throughout the polymerase combined with nuclease digestion of permeabilized virion-derived capsid particles revealed that only full-length polymerase molecules were in virions and that they were all covalently attached to large DNA molecules. Because DNA synthesis is primed by the polymerase itself and only one copy of the viral DNA is in each capsid, the polymerase must function as an uncleaved monomer. Therefore, a single polymerase monomer is encapsidated, primes DNA synthesis, synthesizes both DNA strands, and participates in the three-strand transfers of DNA synthesis, with all steps after DNA priming performed while the polymerase is covalently coupled to the product DNA. Because the N-terminal domain of the polymerase is displaced from the active site on the same molecule by the viral DNA during reverse transcription, P must be structurally dynamic during DNA synthesis. Therefore, non-nucleoside compounds that interfere with this change may be novel antiviral agents.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Hepadnaviruses are small DNA-containing viruses that replicate by reverse transcription and infect primates, rodents, and birds (reviewed in Ref. 1). Significant differences exist in the biology of these viruses, but they all share a very similar genetic organization, hepatic tropism, and nearly identical replication mechanisms. The human hepadnavirus, hepatitis B virus (HBV),2 is a major cause of liver disease and liver cancer worldwide (2). Duck hepatitis B virus (DHBV) is a common model for HBV.

Hepadnaviral reverse transcription (Ref. 3, and reviewed in Ref. 1) begins with the binding of P to an RNA stem-loop ({epsilon}) on the pregenomic RNA (pgRNA), and this ribonucleoprotein complex is then encapsidated (4, 5). Reverse transcription occurs within cytoplasmic capsid particles and is catalyzed by a virally encoded reverse transcriptase (abbreviated as "P" for "polymerase"). DNA synthesis is primed by a tyrosine in the N-terminal domain of P employing {epsilon} as a template, and thus, the viral DNA is covalently linked to P (6–8). P synthesizes negative polarity DNA by RNA-dependent DNA synthesis and then positive polarity DNA by DNA-dependent synthesis. This process employs three-strand transfer reactions that presumably are promoted by P, possibly with contributions from the viral capsid structure.

P is synthesized by de novo translational initiation (9, 10) rather than as a fusion protein such as the orthoretroviral reverse transcriptases (11, 12). P has four domains (Fig. 1) (13–15). The terminal protein domain contains a tyrosine residue that primes DNA synthesis and covalently links P to the viral DNA (Tyr-96 in DHBV (6, 7)). The spacer domain has no known function and can tolerate significant insertions and deletions. The reverse transcriptase domain contains the DNA polymerase activity. Mutation of an essential YMDD motif in the reverse transcriptase domain leads to nucleoside analog resistance or ablation of DNA synthesis activity (2, 16). The RNaseH domain contains the RNaseH activity, which degrades the viral RNA following its conversion to DNA.

Despite the importance of P as a target for three approved nucleoside analog drugs, the size of P in viral particles has been hard to discern. Various activity gel, Western, and immunoprecipitation analyses have revealed P sizes of 155, 109, 98, 85, 80, 78, 70, 68, and 63 kDa associated with DHBV cores (17–19) and 100, 90, 70, and 65 kDa associated with HBV cores (20–25). We have detected DHBV P in cores at its predicted 90-kDa mass by Western analysis (26), but the antibodies recognized epitopes only in the terminal protein domain and would not have detected P fragments lacking these sequences.

P is widely assumed to function as a monomer based on indirect evidence. The strongest circumstantial argument comes from the cis-preference for encapsidation of P as a ribonucleoprotein complex with the pgRNA (4, 5) (the pgRNA is also the mRNA for P). Although the simplest mechanism would be an interaction between monomeric P and the pgRNA, the complex could contain one, two, or more copies of P bound to the pgRNA. Bartenschlager and Schaller (21) performed the most direct measurement of the multimeric state of P and concluded that P probably acts as a monomer, but as discussed below, their experiment could not measure this parameter because it did not account for the effects of the covalent linkage of DNA on the detection of P. Retroviral reverse transcriptases can be monomers, homodimers, or heterodimers (reviewed in Refs. 27 and 28), and thus, it is mechanistically possible for P to function as a monomer, dimer, or higher-order multimer.


Figure 1
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  		FIGURE 1.
Structure of P derivatives employed. The four domains of P and the amino acid numbers of the ends of the protein are indicated. Tyr-96, which primes DNA synthesis, and the YMDD motif of the DNA polymerase active site are indicated. HA indicates the influenza virus hemagglutinin epitope tag. TP, terminal protein; RT, reverse transcriptase.

 
Therefore, it is still unclear whether P functions as a full-length molecule and whether it works as a monomer or a multimer, despite the fundamental implications that these physical properties of P have on the mechanism of reverse transcription. We wish to understand hepadnaviral reverse transcription to promote development of novel antiviral drugs, and thus, we used a combination of immunologic and mutagenesis assays to determine whether active P is proteolytically processed and whether it functions as a monomer or a multimer.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Viruses and DNA Clones—LMH cells are a chicken hepatoma line that produces DHBV upon transfection with DHBV genomic expression constructs (29). LMH cells were stably transfected with a DHBV16 dimer to create LMH-D2 cells that constitutively secrete DHBV16 virions (30). pT7DPol contains DHBV3 (31) nucleotides (nt) 170-3021 encoding P in pBluescript (Promega) with a 33-nt insertion at nt 792 encoding the influenza virus hemagglutinin epitope (HA tag) (32) in the spacer domain. The Brome Mosaic virus internal ribosomal entry site was introduced upstream of the P open reading frame in pT7DPol to produce pT7BDPol to increase the fidelity of initiation at the first AUG of the P open reading frame. pT7BDPol-3'-HA is pT7BDPol with an insertion of the HA tag at the 3' end of the P gene instead of at nt 792. pT7DPol(Y96F) contains a point mutation in the P gene altering Tyr-96 to Phe to prevent covalent attachment of DNA to P. pT7DPol(YMHA) contains D513H and D514A active site missense mutations that ablate DNA synthesis (14). D1.5G contains 1.5 copies of DHBV3 (duplication of nt 1658–3021) in pBluescript (Stratagene) and produces wild-type DHBV following transfection into cells. D1.5G-3'-HA contains the HA tag in the 3' end of the P gene. D1.5G-SH has the insertion of the HA tag at nt 792 encoding the spacer domain. Fig. 1 shows the structure of all P derivatives employed in this study.

PCR—Template for T7 RNA polymerase-mediated transcription of 3'-HA-tagged P (P(3'-HA); 806 amino acids long) was generated by amplification of pT7BDPol-3'-HA with primers T7 (5'-TAA TAC GAC TCA CTA TAG GG-3') and HA-R1 (5'-CCA TCG ATT TAA GCG TAG TCT GGG ACG TCG TAT GGG TAA GTT CCA CAT AGC CTA TGT G-3'). Template for P truncated at amino acid 738 (P-(1–738)) was generated by cleaving the P(3'-HA) amplicon with NcoI.

In Vitro Transcription and Translation—All mRNAs for P were transcribed from plasmids and PCR amplicons employing Megascript kits (Ambion) according to the manufacturer's instructions. The mRNAs for P with HA (P(3'-HA)) or without HA truncated at amino acid 738 (P-(1–738)) were transcribed from PCR amplicons. 35S-labeled P was translated in vitro employing rabbit reticulocyte lysate (Promega) in a 10-µl volume containing 0.6 µl of [35S]methionine (>1,000 Ci/mmol; Amersham Biosciences) at 30 °C for 1 h according to the manufacturer's instructions.

Immunoprecipitation—Monoclonal antibody 11 (mAb 11) specific for an epitope between amino acids 46–77 of P (26) and 3F10 specific for the HA tag (Roche Applied Science) were bound to protein A/G beads (Calbiochem), the antibody-bead complexes were incubated overnight with in vitro-translated P diluted into 0.2 ml of IPP150 (10 mM Tris (pH 7.5), 150 mM NaCl, 0.1% Nonidet P-40), the immunocomplexes were washed four times with 1 ml of IPP150, and P was released by boiling in 25 µlof1x Laemmli buffer. Following SDS-PAGE, radioactive P was detected by phosphorimaging analysis.

DNA Priming—P was translated in vitro in the absence (see Fig. 2) or presence (see Fig. 3) of {epsilon}. Half of the reaction was used to monitor translation, and half was used for the priming assay. If {epsilon} was absent during translation, 1 µgof {epsilon} RNA was added to the priming samples. MgCl2 (to 2.4 mM) and 5 µCi of [{alpha}-32P]dGTP (>3,000 Ci/mmol; Amersham Biosciences) were added to the translation reactions, and the samples were incubated at 37 °C for 30 min. Reactions were terminated with Laemmli buffer, and the products were resolved by SDS-PAGE. The 32P priming signal was detected by phosphorimaging analysis (the 35S signal from translation was blocked by a layer of exposed x-ray film).


Figure 2
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  		FIGURE 2.
Enzymatically active P translated in vitro does not form stable multimers. A, C-terminally HA-tagged or truncated DHBV P derivatives are active in DNA priming. 35S-labeled P-3'-HA and P lacking a HA tag but truncated at amino acid 738 (P-(1–738)) were translated in vitro without {epsilon} (lanes 1–3) and were employed in a priming reaction (lanes 4–6) after the addition of {epsilon}. B, P(3'-HA) and P-(1–738) do not co-immunoprecipitate. In vitro-translated P derivatives were diluted into IPP150, and equal aliquots of the translation mixture were immunoprecipitated with saturating amounts of mAb 11 (recognizes the terminal protein domain of P), mAb 3F10 (recognizes the HA tag), or anti-beta-galactosidase (Anti-beta-gal) (irrelevant antibody). The mobilities of the 806-amino acid P(3'-HA) and 738-amino acid P-(1–738) molecules are indicated.

 
Transfection of LMH Cells and Isolation of Core Particles—LMH and LMH-D2 cells were maintained in Dulbecco's modified Eagle's medium/F12 with 10% fetal bovine serum. LMH cells were transfected with FuGENE (Roche Applied Science) according to the manufacturer's instructions. Extracellular virions and naked core particles were isolated 5 days post-transfection by clarifying the supernatant by centrifugation at 3,000 x g for 10 min and then layering it over a 30% sucrose cushion and centrifuging at 192,000 x g overnight. Intracellular core particles were purified by centrifugation through a 30% sucrose pellet as described (33). Pellets containing intracellular or extracellular core particles were dissolved in 50 µl/100-mm plate of cells of B/EDTA (10 mM HEPES (pH 7.8), 15 mM KCl, 5 mM EDTA) containing 5% sucrose.

Southern Blot Analysis—Viral DNAs were isolated by proteinase K digestion followed by phenol-chloroform extraction and ethanol precipitation and were then resolved by electrophoresis on 1.2% agarose gels. Southern blotting was performed as described previously (34), with internally 32P-labeled DHBV DNA as a probe.

Western Blot Analysis—P was detected with monoclonal antibodies mAb 11 against P or mAb 3F10 against the HA tag as described (26). P within capsids was released from the covalently attached DNA prior to Western analysis by permeabilizing the capsids (35) followed by treatment with micrococcal nuclease. 1 µl of glycine (300 mM, pH 2.5) was added to 5 µl of core extract and incubated at room temperature for 30 s. The reaction was neutralized by the addition of 1 µl of Tris-HCl (600 mM, pH 8.0) followed by the addition of 1 µl of CaCl2 (100 mM) and 1 µl of 5 units/µl micrococcal nuclease and incubation at 37 °C for 20 min. 1 µl of 150 mM EGTA and 2.5 µl of 5x Laemmli buffer were applied to stop the reaction. For virions, the envelope was removed by the addition of 0.5% Nonidet P-40 prior to permeabilization.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Active P Translated in Vitro Does Not Form Stable Multimers—If P functions as a dimer or higher-order multimer during DNA priming, these complexes would be present at least transiently during priming assays with in vitro-translated P. Therefore, we attempted to co-immunoprecipitate complexes of active DHBV P isoforms that differed in size. We took advantage of the ability to modify the C terminus of P without ablating its ability to prime DNA synthesis and created two P derivatives carrying alterations at their C termini to permit their electrophoretic distinction from the 786 amino acid long wild-type protein. P-(1–738) is truncated at amino acid 738, and P(3'-HA) is P with an HA epitope tag added to the C-terminal end that increases its size to 806 amino acids. We first demonstrated that these particular P derivatives were enzymatically active for DNA priming. The proteins were translated in rabbit reticulocyte lysate in the absence of {epsilon}, {epsilon} was added, and a priming assay was performed. Fig. 2A demonstrates that P-(1–738) and P(3'-HA) could be electrophoretically resolved (lanes 1–3), that both proteins were active in DNA priming (lanes 4 and 5), and that translating the proteins together in the same lysate did not interfere with the priming activity of either protein (lane 6). Therefore, truncating P to amino acid 738 or inserting the HA tag at the C terminus did not interfere with the priming activity of P.

Next, we attempted to co-immunoprecipitate these P derivatives to determine whether they formed stable complexes when co-translated. The P derivatives were translated either separately or together and diluted into IPP150 (a buffer that retains the active form of P and is used to measure P:{epsilon} binding (36)), and equal aliquots of the translation mixture were immunoprecipitated with mAb 3F10 specific for the HA tag, mAb 11 specific for the terminal protein domain of P as a positive control, or anti-beta-galactosidase as a negative control. All P derivatives were precipitated by mAb 11 (Fig. 2B, lanes 1–3); however, only P molecules containing the HA tag were precipitated by mAb 3F10 (Fig. 2B, lanes 4–6). The anti-beta-galactosidase antibody did not precipitate any of the P proteins (Fig. 2B, lane 7). Therefore, untagged P did not co-precipitate with HA-tagged P. These results indicate that P molecules active in the DNA priming reaction do not form dimers or multimers stable enough to survive immunoprecipitation.

P Molecules with Complementary Mutations Cannot Prime DNA Synthesis in trans—To determine whether transient or unstable P dimers or multimers form during DNA priming, we assessed the ability of P molecules with complementary defects in either the essential YMDD motif of the reverse transcriptase active site (P(YMHA)) or the tyrosine 96 (P(Y96F)) that primes DNA synthesis to complement each other and prime DNA synthesis in trans or to act as dominant-negative inhibitors of priming by the wild-type enzyme. Wild-type P, P(YMHA), and P(Y96F) were translated in vitro either individually or in combination in the presence of {epsilon} (Fig. 3A), and a DNA priming assay was performed (Fig. 3B). Wild-type P was active (Fig. 3B, lane 2), and the Y96F and YMHA mutants were inactive in DNA priming when the proteins were translated individually (Fig. 3B, lanes 3 and 4). Wild-type P retained full activity when co-translated with the inactive Y96F and YMHA mutants (Fig. 3B, lanes 5 and 6), and the two inactive mutants failed to complement each other when they were co-translated (Fig. 3B, lane 7). These results with DHBV strain 3 are the same as those reported previously for DHBV strain 16 (7).


Figure 3
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  		FIGURE 3.
The enzymatically inactive P(Y96F) and P(YMHA) mutants do not inhibit wild-type (WT) P or complement each other during DNA priming. A, 35S-labeled wild-type P, P(Y96F) and P(YMHA) were translated in vitro in the presence of {epsilon} and were resolved by SDS-PAGE; mock, no mRNA was added during translation. B, priming assays were performed with 35P-labeled dGTP, and the products were resolved by SDS-PAGE.

 
These data indicate that P mutants with lesions that inactivate the reverse transcriptase active site or ablate the hydroxyl on Tyr-96 that forms the covalent linkage with DNA cannot act as dominant negative inhibitors of the wild-type enzyme, nor can they complement each other and prime DNA synthesis in trans. Therefore, transient P dimers or higher multimers are very unlikely to exist during DNA priming in vitro.

All P Molecules in Mature Virions Are Covalently Bound to Large DNAs—The preceding experiments were performed in vitro with synthetically produced P, and thus, the conclusions may be limited to this system. Therefore, we extended our studies to mature virions produced in cell culture and derived from infected duck serum.

Our analytical strategy relies on the chimeric nature of mature hepadnaviral DNA in virions, in which the 5' end of the minus polarity DNA is covalently attached to tyrosine 96 of P as a result of protein priming. P in these chimeric molecules cannot be detected by Western analysis because the mass of the DNA (~2 MDa) greatly exceeds the mass of P (89 kDa) and traps the P-DNA chimeras in the wells during SDS-PAGE. The few P molecules that enter the gel migrate as a high molecular mass smear and do not transfer well from gels during blotting. Therefore, to detect P by Western analysis after synthesis of an appreciable amount of minus polarity DNA, the covalently attached DNA must be removed prior to electrophoresis. The use of mature cores from virions is important in this experiment because core particles must synthesize significant amounts of DNA to be secreted as virions (3, 37–39), and analyzing mature cores excludes P molecules that may have initiated DNA synthesis but have not made enough DNA to prevent detection by Western analysis.

We employed two sources of mature virions, LMH-D2 cells and infected duck serum. LMH-D2 cells are LMH cells carrying a stably integrated DHBV dimer that constitutively secrete DHBV virions (30). Virions were collected from LMH-D2 culture supernatant or duck serum by centrifugation through a 30% sucrose cushion. The virions were treated with 0.5% Nonidet P-40 to remove the viral envelope, and the subviral capsid particles were permeabilized by brief treatment at pH 2.5 (35). One-half of each sample was treated with micrococcal nuclease to remove covalently attached DNA, and the other half was mock-digested. If P acts as a monomer, all of the enzyme would be covalently attached to DNA because each P molecule would have initiated DNA synthesis via protein priming, and so P would only be detectable in the nuclease-treated sample. If P acts as a dimer in which only one molecule is attached to DNA, the two monomers would dissociate during preparation for denaturing SDS-PAGE on reducing gels, so the amount of P detected in the nuclease-treated sample would be twice that detected in the mock-treated sample. If P acts as a trimer with one monomer attached to DNA, the ratio of P in the treated relative to the untreated samples would be 3:2, and the amount of P in the treated versus untreated samples would approach parity with higher-order multimers.

When samples derived from LMH-D2 cells were subjected to Western analysis with mAb 11 specific for the terminal protein domain of P, intracellular non-encapsidated P was found at its anticipated sizes, including the 89-kDa primary translation product, an ~8-kDa larger form, and N-terminal breakdown products of ~55–60 kDa (Fig. 4A, lane 1) (26). In the extracellular virion-derived core samples, P was detected at 89 kDa in the nuclease-treated sample (lane 5), but it was not detected in the mock-treated sample (lane 4). As expected, P was not detected in control supernatants from untransfected LMH cells (lanes 2 and 3). Identical results were obtained with mature DHBV virions derived from infected duck serum (Fig. 4B, lanes 4 and 5). An extra set of bands was observed in both nuclease-treated and mock-treated serum-derived samples at about 60 kDa (Fig. 4B, lanes 4 and 5), but these were from nonspecific cross-reactions with host proteins because they were also observed at lower levels in control samples from uninfected serum (Fig. 4B, lanes 2 and 3). Therefore, essentially all P molecules in mature, extracellular virions detectable by mAb 11 were covalently attached to large DNA molecules. Because all viral DNA in capsid particles is covalently attached to P and there is a single site of linkage between the DNA and P (Tyr-96 in DHBV (6, 7)), we conclude that there is a 1:1 ratio between the viral DNA molecules and P detectable by mAb 11.

P in Mature Virions Is Full Length—The experiments described above ruled out the possibility that P acts as homodimer with one monomer being bound to the DNA because there was a 1:1 ratio of P:DNA in virions. However, they did not eliminate the possibility that P acts as a heterodimer in which one of the monomers lacks the N-terminal portion of P because mAb 11 recognizes an epitope between amino acids 46–77, and thus, the experiment in Fig. 4 is blind to forms of P lacking the terminal protein domain. Repeated attempts to raise sensitive monoclonal antibodies against the C-terminal 75% of DHBV P were unsuccessful, and thus, we resorted to epitope tagging to permit the detection of the C-terminal domains of P. The HA tag was inserted into the spacer domain after amino acid 264 and at the C terminus in the context of the DHBV pgRNA expression vector D1.5G (D1.5G-SH and D1.5G-3'-HA, respectively).


Figure 4
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  		FIGURE 4.
Detection of P within mature virions from the supernatant of LMH-D2 cell culture and DHBV infected duck serum. A, virions were isolated from supernatants of LMH-D2 cells, and the virion-derived capsids were permeabilized and either treated with micrococcal nuclease or mock-treated prior to the detection of P by Western analysis with mAb 11. LMH supernatant indicates supernatant from untransfected LMH cells as a negative control. B, virions from infected duck serum were analyzed as in panel A. Uninfected duck serum was employed as a negative control. Whole-cell lysates of LMH-D2 cells were employed as positive controls for the detection of P in lane 1 of both panels.

 


Figure 5
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  		FIGURE 5.
P isoforms smaller than full-length are not found in core particles. Core particles were prepared from the cytoplasm and supernatant of LMH cells transfected with D1.5G-3'-HA (A) and with D1.5G-SH (B). Core particles were permeabilized and then micrococcal nuclease-treated or mock-treated, and HA-tagged P was resolved by SDS-PAGE and detected by Western analysis with mAb 3F10. Lysates and supernatants from untransfected LMH cells were employed as negative controls (lanes 5–8 and 10), and whole-cell lysates of LMH transfected with D1.5-3'-HA and D1.5G-SH were employed as positive controls for P (lane 9). Intr, intracellular core particles; Super, supernatant-derived core particles.

 
To detect potential P isoforms lacking the terminal protein domain, D1.5G-SH and D1.5G-3'-HA were transfected into LMH cells, and core particles were isolated from the cytoplasm and supernatant of the transfected cultures. Core particles were permeabilized and then treated with micrococcal nuclease or mock-treated, and Western blots of these samples were performed using mAb 3F10 against the HA tag. Only the full-length 89-kDa P product was detected in extracellular core particles produced by D1.5G-SH and D1.5G-3'-HA (Fig. 5, A and B, lanes 3 and 4). This indicates that the HA tags did not disrupt encapsidation of P and the viral RNA and that P molecules lacking the N-terminal domain were not present in core particles.

However, when the levels of P in paired nuclease- and mock-treated samples from this experiment were compared, no significant difference was observed in the amount of P detected with D1.5G-3'-HA, and a relatively small difference was observed with D1.5G-SH (Fig. 5, A and B, compare lane 1 with lane 2 and lane 3 with lane 4). This discrepancy with Fig. 4 could be due to the majority of P molecules failing to progress past the first strand transfer during DNA synthesis (yielding at most a 4-nt-long DNA product that does not significantly alter the electrophoretic mobility of P) or from contamination of the core particle preparations by large amounts of non-encapsidated P (26). Contamination with nonencapsidated P is not plausible because non-encapsidated P is not released into the supernatant, and it is insoluble at the low detergent concentration used for core particle purification. However, release of immature subviral core particles into the supernatant by transiently transfected LMH cells is well characterized (38), and even with wild-type DHBV, about half of the intracellular core particles contain full-length RNA (36), indicating that they had not yet initiated DNA synthesis.

To determine whether DNA synthesis had been affected by the 3'-HA and -SH mutations, we assessed the DNA forms in viral cores derived from intracellular and extracellular core particles from LMH-D2 cells and LMH cells transfected with D1.5G-3'-HA and D1.5G-SH by Southern analysis. Hepadnaviral DNAs were observed in core particles derived from LMH cells transfected with D1.5G-3'-HA and D1.5G-SH (Fig. 6, lanes 3–6), indicating that at least some of the tagged P molecules were enzymatically active. However, the mobility of the relaxed circular DNA was somewhat reduced in extracellular cores derived from D1.5G-3'-HA (lane 4), and relaxed circular DNA was essentially absent in cores from cells transfected with D1.5G-SH (lanes 5 and 6), indicating that these mutations had negative effects on positive polarity DNA synthesis. This result is similar to the reports by D. Loeb and co-workers (40, 41) and may be due to a cis-acting defect in the template. Importantly, there was a much higher proportion of immature negative polarity DNA (small single-stranded DNA (ssDNA)) in extracellular core particles from LMH cells transfected with D1.5G-3'-HA (lane 4) or D1.5G-SH (lane 6) than in extracellular core particles derived from LMH-D2 cells (lane 2). This indicates that DNA synthesis was impaired in the 3'-HA and -SH mutants. Furthermore, the presence of so many extracellular core particles containing only very short DNAs indicates that many more immature core particles were released from LMH cells transfected with D1.5G-SH or D1.5G-3'-HA than from stably transfected LMH-D2 cells. This explains the differential dependence on nuclease treatment for the detection of P between Figs. 4 and 5.


Figure 6
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  		FIGURE 6.
LMH cells transiently transfected with DHBV genomic expression vectors release more immature cores into the culture supernatant than do stably transfected LMH-D2 cells. Viral DNAs were extracted from core particles isolated from the cytoplasm and supernatants of LMH cells transfected with D1.5G-3'-HA or D1.5G-SH or from LMH-D2 cells. Viral DNAs were detected by Southern blot. The mobilities of the mature relaxed circular DNA (RC), duplex linear DNA (DL) full-length single-stranded DNA (SS), and immature single-stranded DNA (Small ssDNA) are indicated. Mobility controls include double-stranded DHBV monomeric DNA (lane 10) and heat-denatured monomeric DHBV DNA (lane 11).

 
Together, these data indicate that the HA-tagged P molecules encoded by D1.5G-3'-HA and D1.5G-SH are competent for encapsidation and that at least some of them can prime DNA synthesis and extend minus polarity DNA. Therefore, the HA-tagged P molecules are suitable for analysis of the primary structure of the active form of P. Because P isoforms smaller than the 89-kDa full-length molecule were not detected by Western analysis against the HA tag when it was placed either in the spacer domain or at the C terminus of P, we conclude that essentially all P molecules in core particles are full-length, and thus, that P does not function as a heterodimer.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The primary structure and multimeric state of P have fundamental implications for the reverse transcription mechanism, but these parameters remain unknown. Here, we determined that active DHBV P translated in vitro primes DNA synthesis as a monomer (Figs. 2 and 3), that essentially all P molecules in mature extracellular virions are covalently attached to large DNA molecules (Fig. 4), and that isoforms of P detectably larger or smaller than the primary translation product of the P open reading frame are not found in virion-derived core particles (Figs. 5 and 6). Therefore, we conclude that DHBV P functions as a full-length monomeric protein. The co-linear structure of the HBV and DHBV P molecules, the close similarities of the reverse transcription mechanisms catalyzed by HBV and DHBV P, and the highly similar structure of the mature DNAs produced by HBV and DHBV reverse transcription imply that HBV P probably also functions as a full-length monomer.

The conclusion that P functions as a monomer is based on the assumption that there is a single copy of hepadnaviral DNA per virion. To our knowledge, an explicit measurement of the number of DNA molecules per virion has not been reported, but we feel this assumption to be valid for three reasons. First, recent cryo-electron microscopy reconstructions of intracellular HBV capsids reveal density corresponding to only ~1kbof single-stranded DNA, and thus, most of the DNA must be disordered (42). Two copies of the mature viral DNA would fill ~80% of the capsid (calculated as hydrated nucleotides), and if the DNA were packed this tightly, far more density would be apparent in capsids than is observed in reconstructions of mature, extracellular virions.3 Second, genetic recombination during hepadnaviral reverse transcription is exceptionally rare or non-existent. If hepadnaviral virions were diploid, high recombination frequencies similar to those of the retroviruses would be expected. Finally, hepadnaviral encapsidation is triggered by the binding of P to the pgRNA, and this reaction would be much simpler if only a single pgRNA molecule were involved.

Bartenschlager and Schaller (21) have previously examined the primary structure and number of P molecules in HBV capsid particles. These authors concluded that P acts as a full-length molecule and that one or two P molecules are in each virion. Our data are in agreement with their results for the primary structure of active P. However, their data cannot be used to determine the multimeric state of active P because immature intracellular cores were employed, and the samples were not treated with DNase prior to electrophoretic analysis of P. Because the viral DNA was not removed prior to analysis, only P molecules that either had not synthesized significant amounts of DNA or had been released from the viral genome by contaminating nucleases or shear stress during sample preparation were detected. Therefore, their data reveal the ratio of P molecules unattached to DNA to the amount of DNA in immature intracellular core particles, not the ratio of P to mature DNA molecules in virions.

Orthoretroviral reverse transcriptases are translated as gagpol fusion proteins that are subsequently cleaved to release the reverse transcriptase/RNaseH molecule (with or without the integrase domain attached) (27). Some reverse transcriptases (such as the HIV enzyme) are further processed to remove the RNaseH domain in half of the molecules to produce a heterodimer (43), and for HIV, both proteolytic processing and dimerization are critical for the enzymatic activity (44, 45). Most retroviral reverse transcriptases act as dimers, and even the Moloney murine leukemia virus enzyme (which is monomeric in solution) appears to dimerize when bound to DNA (46). Two retroviral reverse transcriptases can work together during viral genomic replication because Moloney murine leukemia virus reverse transcriptases with lesions in the DNA polymerase or RNaseH active sites can complement each other to synthesize full-length viral DNA (47). However, both DNA polymerase active sites in a dimeric reverse transcriptase are not necessarily active because the shorter monomer in the HIV heterodimer does not have DNA polymerase activity (48, 49). The spumaviruses (foamy viruses) are the retroviruses most closely related to the hepadnaviruses (50). The foamy virus reverse transcriptases are translated by de novo initiation (51), they are not as extensively proteolytically processed as the orthoretroviral enzymes (52), and they appear to function as monomers (53). Therefore, the hepadnaviral P differs from most retroviral reverse transcriptases in its manner of biogenesis (de novo translation initiation), the lack of post-translational processing, and by being a monomer, but it is similar to many retroviral reverse transcriptases in that it possesses a single functional DNA polymerase active site per replication complex.

We recently identified a molecular contact point on DHBV P ("T3," amino acids 176–183), essential for DNA priming, and found that T3 is conserved with HBV P (54). However, the identity of the ligand for T3 is unknown. These data show that P functions as a monomer, and thus, rule out the possibility that T3 is a contact site between subunits of a dimeric reverse transcriptase complex. Therefore, binding at T3 must either be intermolecular, with T3 interacting with a molecule other than P (such as the chaperones that form a stable complex with P (55–58)), or intramolecular, with T3 contacting another region of the same P molecule.

These results demonstrate that a single P molecule performs all stages of reverse transcription from binding to {epsilon} through synthesis of plus polarity DNA. All reactions after encapsidation occur within the core particle, and all reactions after DNA priming are performed with the 5' end of the minus polarity DNA covalently attached to the terminal protein domain of the very same P molecule that catalyzes DNA extension. The topological aspects of reverse transcription within a confined space could be simplified if one P molecule primed DNA synthesis and another performed chain extension. However, P functions as a monomer, so its active site must accommodate the terminal protein domain of the same molecule during priming, and then the terminal protein domain must be displaced by the nucleic acids after priming. During subsequent DNA synthesis, P must accommodate the changing positions of the 5' and 3' ends of the minus polarity DNA during chain elongation, the change in template strands during the minus polarity DNA strand transfer reaction and the circularization reaction during plus polarity DNA synthesis, and the change in both template strand and primer during the first strand transfer of plus polarity DNA synthesis. Therefore, P must be structurally dynamic. Both HBV and DHBV P function in complex with cellular chaperones in vitro (55–58), and chaperones have been detected in DHBV virions (59). This dependence on molecular chaperones may have evolved to promote the conformational flexibility necessary for reverse transcription by a single P molecule. On a practical level, the conformational flexibility of P implied by these results indicates that small molecule inhibitors that bind to P and reduce its flexibility could form a novel class of antiviral drugs.


   FOOTNOTES
 
* This work was supported by Grant AI38447 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1402 S. Grand Blvd., Saint Louis, MO 63104. Tel.: 314-977-8893; Fax: 314-977-8717; E-mail: tavisje{at}slu.edu.

2 The abbreviations used are: HBV, hepatitis B virus; DHBV, duck hepatitis B virus; pgRNA, pregenomic RNA; P, polymerase or reverse transcriptase; HIV, human immunodeficiency virus; LMH, chicken hepatoma cells; LMH-D2, LMH cells stably transfected with a DHBV dimer; HA, influenza hemagglutinin epitope tag; MAb, monoclonal antibody; nt, nucleotide(s). Back

3 Kelly Dryden, personal communication. Back


   ACKNOWLEDGMENTS
 
DHBV16-infected duck serum was provided by Yunhao Gong, and D1.5G-SH was provided by Dan Loeb. We thank Jianming Hu for helpful discussions.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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