The conformation of the 3' end of the minus-strand DNA makes multiple contributions to template switches during plus-strand DNA synthesis of duck hepatitis B virus

Two template switches are necessary during plus-strand DNA synthesis of the relaxed circular (RC) form of the hepadnavirus genome. The 3' end of the minus-strand DNA makes important contributions to both of these template switches. It acts as the donor site for the first template switch, called primer translocation, and subsequently acts as the acceptor site for the second template switch, termed circularization. A small DNA hairpin has been shown to form near the 3' end of the minus-strand DNA overlapping the direct repeat 1 in avihepadnaviruses. Previously we showed that this hairpin is involved in discriminating between two mutually exclusive pathways for the initiation of plus-strand DNA synthesis. In its absence, the pathway leading to production of duplex linear DNA is favored, whereas primer translocation is favored in its presence, apparently through the inhibition of in situ priming. Circularization involves transfer of the nascent plus strand from the 5' end of the minus-strand DNA to the 3' end, where further elongation can lead to production of RC DNA. Using both genetic and biochemical approaches, we now have found that the small DNA hairpin in the duck hepatitis B virus (DHBV) makes a positive contribution to circularization. The contribution appears to be through its impact on the conformation of the acceptor site. We also identified a unique DHBV variant that can synthesize RC DNA well in the absence of the hairpin. The behavior of this variant could serve as a model for understanding the mammalian hepadnaviruses, in which an analogous hairpin does not appear to exist.     

Analysis of duck hepatitis B virus reverse transcription indicates a common mechanism for the two template switches during plus-strand DNA synthesis

The synthesis of the hepadnavirus relaxed circular DNA genome requires two template switches, primer translocation and circularization, during plus-strand DNA synthesis. Repeated sequences serve as donor and acceptor templates for these template switches, with direct repeat 1 (DR1) and DR2 for primer translocation and 5'r and 3'r for circularization. These donor and acceptor sequences are at, or near, the ends of the minus-strand DNA. Analysis of plus-strand DNA synthesis of duck hepatitis B virus (DHBV) has indicated that there are at least three other cis-acting sequences that make contributions during the synthesis of relaxed circular DNA. These sequences, 5E, M, and 3E, are located near the 5' end, the middle, and the 3' end of minus-strand DNA, respectively. The mechanism by which these sequences contribute to the synthesis of plus-strand DNA was unclear. Our aim was to better understand the mechanism by which 5E and M act. We localized the DHBV 5E element to a short sequence of approximately 30 nucleotides that is 100 nucleotides 3' of DR2 on minus-strand DNA. We found that the new 5E mutants were partially defective for primer translocation/utilization at DR2. They were also invariably defective for circularization. In addition, examination of several new DHBV M variants indicated that they too were defective for primer translocation/utilization and circularization. Thus, this analysis indicated that 5E and M play roles in both primer translocation/utilization and circularization. In conjunction with earlier findings that 3E functions in both template switches, our findings indicate that the processes of primer translocation and circularization share a common underlying mechanism.     

Protein covalently bound to minus-strand DNA intermediates of duck hepatitis B virus.

Analysis of duck hepatitis B viral DNA by gel electrophoresis, Southern blotting, and binding to benzoylated naphthoylated DEAE-cellulose showed that a protein is bound to the minus-strand virion DNA as well as to the full-length single strand, minus-strand species, and minus-strand DNA intermediates isolated from replicating complexes present in infected duck liver. By utilizing a modified dideoxynucleotidyl sequencing method, it was shown that the protein is covalently bound to the smallest detectable growing strands (ca. 30 bases) and that minus-strand synthesis begins at a unique site. These results support the notion that the protein may function as a primer for synthesis of the minus-strand DNA.     

Mutations within DR2 independently reduce the amount of both minus- and plus-strand DNA synthesized during duck hepatitis B virus replication.

The initial aim of this study was to examine the role of complementarity between the plus-strand primer and the minus-strand DNA template for translocation of the plus-strand primer in hepadnaviral replication. We show that when a 5-nucleotide substitution was placed in either DR1 or DR2, translocation of the primer at a detectable level did not occur. Placing the mutation in both DR1 and DR2 did not restore primer translocation, which indicates that complementarity is not the sole determinant for primer translocation. These mutants, in which primer translocation has been inhibited, have been additionally informative. The mutation in DR1 led to efficient synthesis of plus-strand DNA, albeit primed in situ. In contrast, the mutation in DR2 resulted in a reduction in the amount of plus-strand DNA synthesized per unit of minus-strand DNA. These findings were interpreted as indicating that a mutation at DR2, the primer acceptor site, can inhibit both primer translocation and in situ priming. Lastly, we show that mutations within DR2 can result in a reduction in the synthesis of minus-strand DNA and that this reduction is occurring at an early phase of the process. We speculate that this reduction in the amount of minus-strand DNA synthesized could be due to an inhibition of the template switch during minus-strand DNA synthesis.     

Template requirements and binding of hepatitis C virus NS5B polymerase during in vitro RNA synthesis from the 3'-end of virus minus-strand RNA

In our attempt to obtain further information on the replication mechanism of the hepatitis C virus (HCV), we have studied the role of sequences at the 3'-end of HCV minus-strand RNA in the initiation of synthesis of the viral genome by viral RNA-dependent RNA polymerase (RdRp). In this report, we investigated the template and binding properties of mutated and deleted RNA fragments of the 3'-end of the minus-strand HCV RNA in the presence of viral polymerase. These mutants were designed following the newly established secondary structure of this viral RNA fragment. We showed that deletion of the 3'-SL-A1 stem loop significantly reduced the level of RNA synthesis whereas modifications performed in the SL-B1 stem loop increased RNA synthesis. Study of the region encompassing the 341 nucleotides of the 3'-end of the minus-strand RNA shows that these two hairpins play a very limited role in binding to the viral polymerase. On the contrary, deletions of sequences in the 5'-end of this fragment greatly impaired both RNA synthesis and RNA binding. Our results strongly suggest that several domains of the 341 nucleotide region of the minus-strand 3'-end interact with HCV RdRp during in vitro RNA synthesis, in particular the region located between nucleotides 219 and 239.     

Base pairing between cis-acting sequences contributes to template switching during plus-strand DNA synthesis in human hepatitis B virus

Hepadnaviruses utilize two template switches (primer translocation and circularization) during synthesis of plus-strand DNA to generate a relaxed-circular (RC) DNA genome. In duck hepatitis B virus (DHBV) three cis-acting sequences, 3E, M, and 5E, contribute to both template switches through base pairing, 3E with the 3' portion of M and 5E with the 5' portion of M. Human hepatitis B virus (HBV) also contains multiple cis-acting sequences that contribute to the accumulation of RC DNA, but the mechanisms through which these sequences contribute were previously unknown. Three of the HBV cis-acting sequences (h3E, hM, and h5E) occupy positions equivalent to those of the DHBV 3E, M, and 5E. We present evidence that h3E and hM contribute to the synthesis of RC DNA through base pairing during both primer translocation and circularization. Mutations that disrupt predicted base pairing inhibit both template switches while mutations that restore the predicted base pairing restore function. Therefore, the h3E-hM base pairing appears to be a conserved requirement for template switching during plus-strand DNA synthesis of HBV and DHBV. Also, we show that base pairing is not sufficient to explain the mechanism of h3E and hM, as mutating sequences adjacent to the base pairing regions inhibited both template switches. Finally, we did not identify predicted base pairing between h5E and the hM region, indicating a possible difference between HBV and DHBV. The significance of these similarities and differences between HBV and DHBV will be discussed.     

Sequence identity of the terminal redundancies on the minus-strand DNA template is necessary but not sufficient for the template switch during hepadnavirus plus-strand DNA synthesis.

The template for hepadnavirus plus-strand DNA synthesis is a terminally redundant minus-strand DNA. An intramolecular template switch during plus-strand DNA synthesis, which permits plus-strand DNA elongation, has been proposed to be facilitated by this terminal redundancy, which is 7 to 9 nucleotides long. The aim of this study was to determine whether the presence of identical copies of the redundancy on the minus-strand DNA template was necessary and/or sufficient for the template switch and at what position(s) within the redundancy the switch occurs for duck hepatitis B virus. When dinucleotide insertions were placed within the copy of the redundancy at the 3' end of the minus-strand DNA template, novel sequences were copied into plus-strand DNA. The generation of these novel sequences could be explained by complete copying of the redundancy at the 5' end of the minus-strand DNA template followed by a template switch and then extension from a mismatched 3' terminus. In a second set of experiments, it was found that when one copy of the redundancy had either three or five nucleotides replaced the template switch was inhibited. When the identical, albeit mutant, sequences were restored in both copies of the redundancy, template switching was not necessarily restored. Our results indicate that the terminal redundancy on the minus-strand DNA template is necessary but not sufficient for template switching.     

The sequence of the RNA primer and the DNA template influence the initiation of plus-strand DNA synthesis in hepatitis B virus

For hepadnaviruses, the RNA primer for plus-strand DNA synthesis is generated by the final RNase H cleavage of the pregenomic RNA at an 11 nt sequence called DR1 during the synthesis of minus-strand DNA. This RNA primer initiates synthesis at one of two distinct sites on the minus-strand DNA template, resulting in two different end products; duplex linear DNA or relaxed circular DNA. Duplex linear DNA is made when initiation of synthesis occurs at DR1. Relaxed circular DNA, the major product, is made when the RNA primer translocates to the sequence complementary to DR1, called DR2 before initiation of DNA synthesis. We studied the mechanism that determines the site of the final RNase H cleavage in hepatitis B virus (HBV). We showed that the sites of the final RNase H cleavage are always a fixed number of nucleotides from the 5' end of the pregenomic RNA. This finding is similar to what was found previously for duck hepatitis B virus (DHBV), and suggests that all hepadnaviruses use a similar mechanism. Also, we studied the role of complementarity between the RNA primer and the acceptor site at DR2 in HBV. By increasing the complementarity, we were able to increase the level of priming at DR2 over that seen in the wild-type virus. This finding suggests that the level of initiation of plus-strand DNA synthesis at DR2 is sub-maximal for wild-type HBV. Finally, we studied the role of the sequence at the 5' end of the RNA primer that is outside of the DR sequence. We found that substitutions or insertions in this region affected the level of priming at DR1 and DR2.     

Roles of the envelope proteins in the amplification of covalently closed circular DNA and completion of synthesis of the plus-strand DNA in hepatitis B virus

Covalently closed circular DNA (cccDNA), the nuclear form of hepatitis B virus (HBV), is synthesized by repair of the relaxed circular (RC) DNA genome. Initially, cccDNA is derived from RC DNA from the infecting virion, but additional copies of cccDNA are derived from newly synthesized RC DNA molecules in a process termed intracellular amplification. It has been shown that the large viral envelope protein limits the intracellular amplification of cccDNA for duck hepatitis B virus. The role of the envelope proteins in regulating the amplification of cccDNA in HBV is not well characterized. The present report demonstrates regulation of synthesis of cccDNA by the envelope proteins of HBV. Ablation of expression of the envelope proteins led to an increase (>6-fold) in the level of cccDNA. Subsequent restoration of envelope protein expression led to a decrease (>50%) in the level of cccDNA, which inversely correlated with the level of the envelope proteins. We found that the expression of L protein alone or in combination with M and/or S proteins led to a decrease in cccDNA levels, indicating that L contributes to the regulation of cccDNA. Coexpression of L and M led to greater regulation than either L alone or L and S. Coexpression of all three envelope proteins was also found to limit completion of plus-strand DNA synthesis, and the degree of this effect correlated with the level of the proteins and virion secretion.     

Integrated structures of duck hepatitis B virus DNA in hepatocellular carcinoma.

The structure of integrated viral DNA in a hepatocellular carcinoma of a duck from Chi-tung county in China was analyzed. Three different clones of integrated viral DNA, lambda DHS 6-1, lambda DHS 6-2, and lambda DHE 6-2, were obtained from the neoplastic portion of the liver by molecular cloning. One of the three clones, lambda DHS 6-1, showed inverted repetition of integrated viral DNA with chromosomal flanking sequences. Another clone, lambda DHS 6-2, showed a head-to-head configuration of the core and surface gene regions of duck hepatitis B virus (DHBV) DNA. The virus-chromosome junctions were often located near direct repeat 1 or 2 of DHBV DNA in three independent clones. Nucleotide sequences at the virus-virus junctions in two clones, lambda DHS 6-1 and 6-2, indicated the possible rearrangement of chromosomal DNA and recombination of viral DNA. DHBV DNA appears to be integrated into the genome of hepatocytes in a manner similar to that of human and woodchuck hepatitis viruses. Thus, the duck system may serve as a useful animal model for the study of human hepatocarcinogenesis.