Evidence that the first strand-transfer reaction of duck hepatitis B virus reverse transcription requires the polymerase and that strand transfer is not needed for the switch of the polymerase to the elongation mode of DNA synthesis

Yunhao Gongb,1, Ermei Yao1, Melissa Stevens1 and John E. Tavis1

Department of Molecular Microbiology and Immunology, St Louis University School of Medicine, 1402 S. Grand Blvd, St Louis, MO 63104, USA1

Author for correspondence: John Tavis. Fax +1 314 773 3403. e-mail tavisje{at}slu.edu


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Deletion of amino acids 79–88 in the duck hepatitis B virus reverse transcriptase had minimal effects on polymerase activities prior to the minus-strand DNA transfer reaction, yet it greatly diminished strand transfer and subsequent DNA synthesis. This mutation also reduced reverse transcription on exogenous RNA templates. The reaction on exogenous RNAs employed the phosphonoformic acid (PFA)-sensitive elongation mode of DNA synthesis rather than the PFA-resistant priming mode, despite the independence of DNA synthesis in this assay from the priming and minus-strand transfer reactions. These data provide experimental evidence that the polymerase is involved directly in the minus-strand transfer reaction and that the switch of the polymerase from the early PFA-resistant mode of DNA synthesis to the later PFA-sensitive elongation mode does not require the strand-transfer reaction.


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The hepatitis B viruses (hepadnaviruses) are small, enveloped DNA viruses that replicate by reverse transcription (Ganem, 1996 ). DNA replication begins with binding of the viral polymerase to a stem–loop ({epsilon}) at the 5' end of the viral pregenomic RNA (pgRNA). This interaction is essential for encapsidation of both the polymerase and the pgRNA into subviral nucleocapsid core particles. The polymerase then primes DNA synthesis, using a tyrosine in its own amino-terminal domain (Fig. 1) as a primer and {epsilon} as a template. This links the viral DNA covalently to the polymerase.



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Fig. 1. Polymerase structure and its interaction with {epsilon}-containing RNAs. (A) Domain structure of the DHBV polymerase in the context of the pgRNA (solid line). The four domains of the polymerase are indicated as: TP, the terminal protein domain that primes reverse transcription; Spacer, a domain with no known function; RT, the reverse transcriptase domain; RNaseH; the RNase H domain. The approximate boundaries of the domains are indicated. Tyrosine-96 (Y96), to which the viral DNA is covalently attached, and the position of the dlAvr deletion are indicated, as is the YMDD active site motif in the RT domain (mutated to YMHA in the active site missense mutant). The relative position of the core protein ORF is shown above the pgRNA. The positions of {epsilon} and DR1 (*) are shown on the pgRNA and the arrow below the pgRNA represents the position of the oligonucleotide employed for primer extension in Fig. 3. (B) {epsilon}-binding activity. 35S-labelled polymerase was translated, bound to biotinylated {epsilon}, precipitated with avidin–agarose and resolved by SDS–PAGE as described in Pollack & Ganem (1994) . Total translated polymerase is shown in lanes 1–3 and polymerase co-precipitated with {epsilon} is shown in lanes 4–6. T3M2 polymerase (K182E/R183E) does not bind {epsilon} (Tavis et al., 1998 ). Polymerase translated in vitro appears as a doublet due to initiation at the natural AUG and at a second AUG 43 amino acids downstream. (C) Partial proteolysis of the polymerase to detect the {epsilon}-dependent alteration. 35S-labelled wild-type and dlAvr polymerases were translated in vitro in the presence or absence of {epsilon}, mock digested (lanes 1–4) or partially digested with papain (lanes 5–8) and resolved by SDS–PAGE. The positions of the polymerase and the 36 kDa {epsilon}-dependent protected fragment are shown. (D) Encapsidation of the pgRNA. LMH cells were transfected with viral genomes encoding the wild-type polymerase, the dlAvr polymerase or the wild-type polymerase and a deletion in {epsilon} (dlBulge; Pollack & Ganem, 1994 ) that eliminates encapsidation. Total cellular poly(A) RNA was isolated (lanes 1–4) (Chabot, 1994 ) or intracellular core particles were isolated (Tavis et al., 1998 ) and encapsidated RNA was purified by phenol–chloroform extraction (lanes 5–7). DHBV RNAs were detected by Northern analysis with the complete DHBV genome as a probe (Tavis & Ganem, 1996 ). Lane 1 contains RNA from mock-transfected cells. The identities and sizes of the viral RNAs are indicated.

 


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Fig. 3. Effects of the dlAvr mutation on minus-strand transfer and viral DNA synthesis. For all panels, DHBV DNA was isolated by proteinase K digestion, phenol–chloroform extraction and ethanol precipitation. In (A), (B) and (D), the 5' ends of DHBV DNAs at nt 2576 within {epsilon} or at nt 2537 within DR1 were detected by repetitive PCR primer extension employing a 32P-labelled oligonucleotide (DHBV nt 2453–2475) as described in Tavis & Ganem (1995) . Primer-extension products were resolved on denaturing polyacrylamide sequencing gels. These panels include a sequencing ladder of DHBV DNA generated from the same primer. (A) In vitro-translated polymerase. Polymerase was translated in vitro as in Fig. 2, incubated with nucleoside triphosphates and immunoprecipitated with polyclonal anti-polymerase antibodies. DHBV DNA was isolated and was detected by primer extension. The mRNA for the polymerase contains {epsilon} immediately downstream of DR1 and hence it was the template for reverse transcription (Fig. 1A). (B) Polymerase within core particles. Intracellular viral cores were isolated from LMH cells transfected with the wild-type genome or with genomes that express the dlAvr or YMHA mutant polymerases. DNA was isolated and the 5' end of the minus-strand DNAs was detected by primer extension. (C) The dlAvr mutation ablates DNA synthesis in viral cores. Intracellular viral cores were isolated from LMH cells transfected with the wild-type genome or with genomes expressing the dlAvr or YMHA mutant polymerases. Cells were maintained at either 37 or 33 °C for 3 days prior to collection of cores. Viral DNA was purified and detected by Southern analysis employing the complete DHBV genome as a probe. The positions of the major forms of the viral DNA (RC, relaxed circular; DL, duplex linear; SS, single-stranded) are indicated. (D) Polymerase expressed in yeast. Recombinant TY particles containing the DHBV polymerase and the YMHA and dlAvr mutant polymerases were isolated from yeast cells as described in Tavis & Ganem (1995) . These particles contain recombinant polymerase and DHBV DNA that had been synthesized in yeast prior to isolation of the particles. DNA was isolated and DHBV 5' ends were detected by primer extension.

 
DNA synthesis arrests after three or four nucleotides. Further DNA synthesis requires the first of three strand transfers, in which a nucleic acid is moved from one sequence in the genome to another. The nascent minus-strand DNA is transferred from {epsilon} to the 3' end of the pgRNA, where it anneals to a sequence called DR1. The minus-strand DNA is then extended to the end of the pgRNA and the viral RNase H activity degrades the pgRNA. The 5' end of the RNA is not degraded, but is transferred to DR2, a sequence near the 5' end of the minus-strand DNA. Following this second strand transfer, the RNA oligomer primes plus-strand DNA synthesis, which continues to the 5' end of the minus-strand DNA. The 3' end of the plus-strand DNA is then transferred to the opposite end of the minus-strand template to circularize the genome in the third strand-transfer reaction. The plus-strand DNA is then extended for a variable length to produce mature viral DNA.

Although the mechanism(s) of the strand-transfer reactions are unknown, two assumptions can be made. Firstly, the reactions probably occur in a complex containing the nucleic acid to be transferred and both the donor and the acceptor complementary sequences (Tavis & Ganem, 1995 ; Mueller-Hill & Loeb, 1996 ; Havert & Loeb, 1997 ). Secondly, viral proteins are probably instrumental in the strand-transfer reactions. Mutations to the core protein can severely affect plus-strand DNA synthesis, possibly by inhibiting the strand transfers (Yu & Summers, 1991 ). The polymerase is probably involved in the transfer, because it is covalently attached to the DNA and it catalyses DNA synthesis. However, experimental evidence for this involvement is lacking. Cellular proteins may also be involved in the strand-transfer reactions. Strong candidates for this role are the molecular chaperones that are bound to the polymerase and may also be present in viral cores (Hu & Seeger, 1996 ; Hu et al., 1997 ).

The first strand transfer is associated with a change in sensitivity of the polymerase to phosphonoformic acid (PFA). DNA priming and synthesis of nascent minus-strand DNA are resistant to PFA, whereas DNA synthesis after the first strand transfer is sensitive to inhibition by PFA (Wang & Seeger, 1992 ). The acquisition of PFA sensitivity may be due to a remodelling of the polymerase complex that changes the polymerase from a ‘priming mode’ to an ‘elongation mode’. Such remodelling could result from a conformational alteration of the polymerase or from a change in the constituents of the polymerase–nucleic acid complex. These two possibilities are not mutually exclusive. Although the change from the priming to elongation mode is concurrent with the minus-strand transfer reaction, it is not known whether these events are mechanistically linked.

We deleted amino acids 79–88 (inclusive) of duck hepatitis B virus (DHBV) polymerase by removing coding sequences between two in-frame AvrII sites in the viral genome (Fig. 1A). This mutation (dlAvr) was produced as part of a mutational scanning project designed to explore the role of the amino-terminal domain of the polymerase in reverse transcription. Polymerase carrying the dlAvr mutation was analysed in three different contexts: (i) in vitro translation in rabbit reticulocyte lysates (Wang & Seeger, 1992 ), (ii) expression in recombinant yeast TY particles (Tavis & Ganem, 1993 ) and (iii) expression in viral core particles isolated from transfected LMH cells (a chicken hepatoma cell line that produces infectious DHBV upon transfection with the viral genome; Condreay et al., 1990 ). The dlAvr mutation also ablates the core gene stop codon (Fig. 1A) and extends the core protein by 12 amino acids. This alteration to the core gene is relevant only to experiments with viral core particles, because core protein is not expressed in the reticulocyte or yeast systems.

Binding of the polymerase to {epsilon} can be detected by translating 35S-labelled polymerase in vitro, allowing it to bind biotinylated {epsilon}, collecting the RNA with avidin–agarose and detecting co-precipitated polymerase following SDS–PAGE (Pollack & Ganem, 1994 ). Fig. 1(B) indicates that the dlAvr mutation did not affect binding of the polymerase to {epsilon}.

Binding of {epsilon} to the polymerase induces a structural alteration in the polymerase that is detected as increased resistance to proteolysis. This alteration is a prerequisite for the biological activities of the polymerase (Tavis & Ganem, 1996 ; Tavis et al., 1998 ). 35S-labelled polymerase was translated in vitro, bound to {epsilon} and subjected to partial proteolysis with papain as described in Tavis et al. (1998) . As evidenced by the 36 kDa fragment of the polymerase, which is the hallmark of the activated state of the polymerase, the dlAvr mutation had no effect on the ability of the polymerase to undergo the {epsilon}-dependent alteration (Fig. 1C).

The next stage of hepadnavirus replication is encapsidation of the pgRNA–polymerase complex (Junker-Niepmann et al., 1990 ; Hirsch et al., 1991 ). Encapsidation was measured by transfecting LMH cells with DNA vectors expressing the complete DHBV genome encoding the wild-type or the dlAvr polymerase or with a genome carrying wild-type polymerase and mutant {epsilon} sequences ({epsilon}-dlBulge) that block encapsidation (Pollack & Ganem, 1994 ). Total cellular poly(A) RNA and RNA within cytoplasmic viral cores was isolated and DHBV RNAs were detected by Northern analysis. Fig. 1(D) demonstrates that all three DHBV RNAs were detected in each poly(A) sample. However, only the 3·5 kb pgRNA could be detected in core-derived RNA. Core-derived DHBV RNA could be found only with the wild-type and dlAvr constructs, but not with the {epsilon}-dlBulge construct, indicating that encapsidation occurred normally with the dlAvr mutant.

These data indicate that the dlAvr mutation does not significantly affect (i) polymerase binding to {epsilon}, (ii) {epsilon}-dependent maturation of the polymerase or (iii) pgRNA encapsidation. They also indicate that the addition of 12 extra amino acids to the core protein by the dlAvr deletion does not inhibit encapsidation or destabilize core particles.

The next step in hepadnavirus replication is DNA priming, in which a tyrosine in the amino-terminal domain of the polymerase is linked covalently to the first nucleotide of the genome. This reaction was studied by incubating in vitro-translated polymerase with [{alpha}-32P]dGTP and measuring transfer of [32P]dGMP to the polymerase following SDS–PAGE, as described in Tavis et al. (1998) . The dlAvr mutation reduced the priming activity to an average of 45% of that of the wild-type polymerase (Fig. 2A; compare lanes 4 and 8), as determined by phosphorimager quantification of multiple priming assays.



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Fig. 2. Early DNA synthesis and PFA sensitivity of DNA synthesis. (A) Priming and synthesis of the nascent minus-strand DNA. 35S-labelled polymerases (wild-type, dlAvr and the YMHA active site mutant) were translated in vitro, incubated with the indicated nucleoside triphosphates (* indicates [{alpha}-32P]-labelled) and either 0 or 3 mM PFA and resolved by SDS–PAGE. Lanes 1–3 show the 35S-labelled translation products and lanes 4–13 show polymerases labelled with 32P through DNA synthesis. The position of the polymerase is shown (P). (B) PFA sensitivity of DNA synthesis on exogenous RNAs (the trans reaction). Intracellular viral cores were isolated from LMH cells transfected with the wild-type genome or with genomes expressing the dlAvr or YMHA mutant polymerases. Cores were permeabilized by treatment with low pH (Radziwill et al., 1988 ) and treated with micrococcal nuclease to destroy endogenous nucleic acids. A 265 nt RNA (DRF+, containing DHBV nt 2401–2605), nucleoside triphosphates ([{alpha}-32P]dGTP as label) and either 0 or 3 mM PFA were added and the mixture was incubated at 37 °C for 60 min as described in Tavis et al. (1998) . Nucleic acids were purified by phenol–chloroform extraction and resolved by denaturing PAGE. The constituents of the reactions are shown above the gel and the mobility of an RNA ladder is shown at left. Lane 6 contains internally labelled substrate RNA as a marker.

 
The sequence of the nascent minus-strand DNA is GTAA. Its synthesis was measured by incubating polymerase translated in vitro with dGTP, dTTP and [{alpha}-32P]dATP and detecting covalent transfer of 32P to the polymerase following SDS–PAGE. The results shown in lanes 5 and 9 of Fig. 2(A) indicate that the dlAvr mutation increased the synthesis of the nascent minus-strand DNA by 1·5-fold relative to the wild-type polymerase, as measured by phosphorimager analysis. The reason for this modest increase in nascent minus-strand DNA synthesis by the dlAvr polymerase is unknown.

We next examined the switch from the priming to elongation modes of DNA synthesis. The wild-type polymerase is resistant to PFA before the minus-strand transfer reaction, but is sensitive to PFA after the transfer. To determine whether the dlAvr mutation rendered the polymerase sensitive to PFA in the early phases of reverse transcription, the sensitivity of the wild-type and dlAvr polymerases to PFA was measured in DNA priming and in synthesis of the nascent minus-strand DNA. Both polymerases were active in 3 mM PFA, which is sufficient to inhibit the elongation mode of DNA synthesis completely (Fig. 2A; lanes 6, 7, 10 and 11). This indicates that the dlAvr mutation does not affect the PFA resistance of the early phases of reverse transcription.

The hepadnavirus polymerase will normally synthesize DNA only from the {epsilon}-containing pgRNA and its daughter DNA (Radziwill et al., 1988 ). However, under certain conditions the polymerase can synthesize DNA by employing small exogenous RNAs as templates (the trans reaction; Tavis & Ganem, 1996 ; Tavis et al., 1998 ). The trans reaction is template-dependent reverse transcription of the exogenous RNA primed by the 3' end of the RNA template. This reaction is independent of the hepadnavirus priming and minus-strand transfer reactions.

We used the trans reaction to test whether the switch from the priming mode to the elongation mode of DNA synthesis required the minus-strand transfer reaction. Viral cores containing wild-type, dlAvr or enzymatically inactive YMHA polymerases were isolated from transfected LMH cells and tested in the trans assay (Fig. 2B). The polymerase carrying the YMHA active site missense mutations was inactive and the wild-type polymerase gave the usual heterogeneous collection of products. The dlAvr polymerase was weakly active in the trans reaction. The trans reaction was inhibited by 3 mM PFA for both the wild-type and dlAvr polymerases, indicating that the trans reaction uses the elongation mode of DNA synthesis. Because the trans reaction does not employ the minus-strand transfer reaction, these data indicate that the switch to the elongation mode of DNA synthesis does not require the strand-transfer reaction.

The transfer of minus-strand DNA from {epsilon} to DR1 was measured by using primer extension to locate the 5' ends of the minus-strand DNAs. A single primer-extension reaction can detect DNAs at both {epsilon} and DR1 for DNA synthesized by in vitro-translated polymerase or by polymerase expressed in recombinant yeast TY particles. This is because {epsilon} is located 3' to DR1 and approximately 50% of the DNA strands are extended in situ from {epsilon} rather than transferring to DR1 in these systems. The ratio of 5' ends at these locations is a measure of the strand-transfer frequency (Wang & Seeger, 1993 ; Tavis et al., 1994 ; Tavis & Ganem, 1995 ). Primer extension of DNAs from viral cores reveals only successful transfer products, because in situ extension does not occur within cores.

Primer extension of DNA synthesized by in vitro-translated wild-type polymerase revealed DNA ends at {epsilon} and DR1 (Fig. 3A). However, DNAs were not detected at either location with the dlAvr polymerase. The dlAvr mutation had similar drastic effects on minus-strand DNA synthesis within viral cores isolated from transfected LMH cells, as no transfer products were detected in cores containing the dlAvr polymerase (Fig. 3B). The absence of DNA synthesis within cores produced from genomes carrying the dlAvr mutation was not due to the core protein extension, as DNA synthesis could not be restored by providing wild-type core protein in trans (data not shown). No DHBV DNA was detected by Southern analysis in cores containing the dlAvr polymerase (Fig. 3C), confirming the result obtained by primer extension. The dlAvr mutation also ablated DNA synthesis in cells incubated at 33 °C, indicating that the dlAvr mutation was not temperature sensitive.

Primer extension of DNA synthesized with recombinant DHBV polymerase expressed in yeast also demonstrated a specific effect of the dlAvr mutation on minus-strand transfer, although it was not as severe as the effect produced in reticulocyte lysates or in viral cores (Fig. 3D). DNAs produced by the wild-type polymerase mapped to {epsilon} and DR1. The dlAvr polymerase also produced DNAs with 5' ends at {epsilon} and DR1; however, the ratio of ends at DR1 to those at {epsilon} was 4-fold less than that of the wild-type polymerase. It is not known why some residual strand-transfer activity is found with the dlAvr polymerase expressed in yeast cells. Perhaps yeast proteins that interact with the polymerase (such the chaperones required for binding to {epsilon}; Hu & Seeger, 1996 ; Hu et al., 1997 ) are less affected by the dlAvr mutation than are their vertebrate counterparts or perhaps the amino-terminal fusion of the TYA sequences to the yeast-derived protein lessens the effect of the dlAvr mutation (Tavis & Ganem, 1993 ). In vitro DNA polymerase activity could not be detected with the dlAvr polymerase expressed in yeast (data not shown), indicating that the polymerase activity was either unstable following purification or that the extension reaction with the dlAvr polymerase required a yeast factor that was lost upon purification.

Together, these data indicate that the dlAvr mutation specifically inhibits minus-strand transfer and subsequent DNA synthesis.

In summary, we are performing a mutational analysis of the DHBV polymerase terminal protein domain to analyse the mechanisms involved in the early reactions of hepadnavirus reverse transcription. Deletion of amino acids 79–88 led to two observations. Firstly, the dlAvr mutation provides the first experimental evidence for an active role for the polymerase in minus-strand DNA transfer. This comes from the observation that the dlAvr mutation had only modest effects on the interaction of the polymerase with {epsilon} and on synthesis of the nascent minus-strand DNA, but it specifically inhibited the minus-strand transfer reaction. The mechanism of this inhibition is unknown, but it could be direct (e.g. a putative nucleic acid transfer activity could have been ablated by the dlAvr deletion) or indirect (the mutation may have ablated a binding site for a cellular component needed for DNA transfer). The location of the dlAvr mutation within the terminal protein domain implies that this domain of the polymerase may be involved in the minus-strand transfer reaction. Secondly, these data indicate that the switch to the PFA-sensitive elongation mode of DNA synthesis is not mechanistically dependent upon the minus-strand transfer reaction, although the switch to the elongation mode of synthesis is temporally linked to minus-strand transfer under physiological conditions. This is shown by the observation that the trans reaction occurs without strand transfer, yet it is PFA sensitive. Together, these observations begin to define the molecular events during the first strand-transfer reaction of hepadnavirus reverse transcription.


   Acknowledgments
 
This work was supported by NIH grant AI38447 and American Cancer Society grant JFRA-616.


   Footnotes
 
b Present address: Viridae Clinical Sciences, Inc., 1134 Burrard St, Vancouver, BC, Canada V6Z 1Y8.


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Received 6 March 2000; accepted 27 April 2000.



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