CHANGING THE SITE OF INITIATION OF PLUS-STRAND DNA-SYNTHESIS INHIBITSTHE SUBSEQUENT TEMPLATE SWITCH DURING REPLICATION OF A HEPADNAVIRUS

Unique to hepadnavirus reverse transcription is the process of primer translocation, in which the RNA primer for the initiation of plus-stra nd 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 l ocated within 50 nucleotides of the 5' end of the minus-strand DNA tem plate. When the synthesis of plus-strand DNA proceeds to the 5' termin us of the minus strand, the 3' end of the minus strand becomes the tem plate for DNA synthesis. This switch in templates circularizes the nas cent genome and is required for the genesis of the relaxed circular fo rm of the DNA and the mature capsid. Maturation of the capsid is a pre requisite 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 subseq uent circularization was inhibited. We found that when the number of n ucleotides between the site of initiation of plus-strand DNA synthesis and the 5' end of its template was restored to 54 nucleotides, circul arization was substantially restored. These results mean that the proc ess of circularization is influenced by the earlier steps in DNA repli cation. This sensitivity is consistent with the notion that this regio n of the nascent genome is in a dynamic structure that is crucial for successful DNA replication.