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 encapsidation signal (epsilon) near the 5' end of pgRNA; and (ii) a template switch of the four-nucleotide minus-strand DNA from epsilon 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 epsilon and determined whether these epsilon 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 epsilon variants, in solution. We found that the epsilon 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-strand DNA copied from epsilon can be important for an efficient template switch. The RNA structural analysis of the epsilon variants indicated alteration in the position and size of the bulge. Overall, these results are consistent with the notion that the template within epsilon is limited to four nucleotides because the remaining two nucleotides located within the bulge are inaccessible for polymerization.