Mutations that increase in situ priming also decrease circularization for duck hepatitis B virus

The process of hepadnavirus reverse transcription involves two template sti tches during the synthesis of plus-strand DNA. The first involves transloca tion of the plus-strand primer from its site of generation, the 3 ' end of minus strand DNA, to the complementary sequence DR2, located near the 5 ' e nd of the minus strand DNA. Plus strands initiated from DR2 are extended to the 5 ' end of the minus-strand DNA. At this point, the 3 ' end of the min us strand becomes the template via the second template switch, a process ca lled circularization. Elongation of circularized plus-strand DNA generates relaxed circular DNA, Although most virions contain relaxed circular DNA, s ome contain duplex linear DNA. Duplex linear genomes are synthesized when t he plus-strand primer is used at the site of its generation, the 3 ' end of the minus-strand template. This type of synthesis is called in situ primin g. Although in situ priming is normally low, in some duck hepatitis B virus mutants this type of priming is elevated. For example, mutations within th e 3 ' end of the minus-strand DNA can lead to increased levels of in situ p riming. We report here that these same mutations result in a second defect, a less efficient template switch that circularizes the genome. Although it is not clear how these mutations affect both steps in DNA replication, our findings suggest a commonality in the mechanism of initiation of plus-stra nd synthesis and the template snitch that circularizes the genome.