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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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 polymerasenucleic 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 7988 (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 can be detected by translating 35S-labelled polymerase in vitro, allowing it to bind biotinylated
, collecting the RNA with avidinagarose and detecting co-precipitated polymerase following SDSPAGE (Pollack & Ganem, 1994
). Fig. 1(B)
indicates that the dlAvr mutation did not affect binding of the polymerase to
.
Binding of 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
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
-dependent alteration (Fig. 1C
).
The next stage of hepadnavirus replication is encapsidation of the pgRNApolymerase 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
sequences (
-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
-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 , (ii)
-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 [-32P]dGTP and measuring transfer of [32P]dGMP to the polymerase following SDSPAGE, 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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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 -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 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
and DR1 for DNA synthesized by in vitro-translated polymerase or by polymerase expressed in recombinant yeast TY particles. This is because
is located 3' to DR1 and approximately 50% of the DNA strands are extended in situ from
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 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
and DR1. The dlAvr polymerase also produced DNAs with 5' ends at
and DR1; however, the ratio of ends at DR1 to those at
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
; 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 7988 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 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.
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References |
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Received 6 March 2000;
accepted 27 April 2000.
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