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

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American Society for Microbiology

RESUMO

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.

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