Insertions within Epsilon Affect Synthesis of Minus-Strand DNA before the Template Switch for Duck Hepatitis B Virus

Duck hepatitis B virus (DHBV) is a DNA virus that replicates via reverse transcription of a pregenomic RNA (pgRNA). Synthesis of the first strand of DNA (minus-strand DNA) for DHBV can be divided into two steps: (i) synthesis of the first four nucleotides of minus-strand DNA, which is primed by the viral polymerase (P) protein and copied from the sequence 5*-UUAC-3 * within the phylogenetically conserved bulge in the encap-sidation signal («) near the 5 * end of pgRNA; and (ii) a template switch of the four-nucleotide minus-strand DNA from « to an acceptor site near the 3 * end of pgRNA and synthesis of a complete minus-strand DNA. To understand why only four nucleotides of minus-strand DNA were synthesized before the template switch, we introduced small insertions immediately 5 * to the UUAC sequence in « and determined whether these « variants were competent for protein priming and whether minus strands longer than four nucleotides were synthesized. Then we determined, in cell culture, whether the longer minus-strand DNAs were competent to undergo a template switch. Also, we analyzed the structure of the « variants, in solution. We found that the « variants were functional for protein priming and RNA encapsidation and that the insertions were copied into minus-strand DNA. However, two mutant viruses that contained two different three-nucleotide insertions failed to synthesize minus-strand DNA efficiently from the acceptor site, even though seven nucleotides of the donor and acceptor sites were identical. These results suggest that the length and/or sequence of the minus-stran