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 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.
Unique to hepadnavirus reverse transcription is the process of primer translocation, in which the RNA primer for the initiation of plus-strand DNA synthesis is generated at one site on its template, DR1, and is moved to a new site, DR2. For duck hepatitis B virus (DHBV), DR2 is located within 50 nucleotides of the 5′ end of the minus-strand DNA template. When the synthesis of plus-strand DNA proceeds to the 5′ terminus of the minus strand, the 3′ end of the minus strand becomes the template for DNA synthesis. This switch in templates circularizes the nascent genome and is required for the genesis of the relaxed circular form of the DNA and the mature capsid. Maturation of the capsid is a prerequisite for virus egress. We have analyzed a series of DHBV variants in which plus-strand DNA synthesis was initiated from a new position relative to the 5′ end of the template. For these variants, the subsequent circularization was inhibited. We found that when the number of nucleotides between the site of initiation of plus-strand DNA synthesis and the 5′ end of its template was restored to 54 nucleotides, circularization was substantially restored. These results mean that the process of circularization is influenced by the earlier steps in DNA replication. This sensitivity is consistent with the notion that this region of the nascent genome is in a dynamic structure that is crucial for successful DNA replication.
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