INSERTIONS WITHIN EPSILON AFFECT SYNTHESIS OF MINUS-STRAND DNA BEFORETHE TEMPLATE SWITCH FOR DUCK HEPATITIS-B VIRUS

Duck hepatitis B virus (DHBV) is a DNA virus that replicates via rever se transcription of a pregenomic RNA (pgRNA), Synthesis of the first s trand of DNA (minus-strand DNA) for DHBV can be divided into two steps : (i) synthesis of the first four nucleotides of minus-strand DNA, whi ch is primed by the viral polymerase (P) protein and copied from the s equence 5'-UUAC-3' within the phylogenetically conserved bulge in the encapsidation signal (epsilon) near the 5' end of pgRNA; and (ii) a te mplate snitch 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-str and DNA were synthesized before the template switch, we introduced sma ll insertions immediately 5' to the UUAC sequence in epsilon and deter mined whether these epsilon variants were competent for protein primin g and whether minus strands longer than four nucleotides were synthesi zed, Then we determined, in cell culture, whether the longer minus-str and DNAs were competent to undergo a template switch, Also, we analyze d the structure of the epsilon variants, in solution, We found that th e epsilon variants were functional for protein priming and RNA encapsi dation and that the insertions were copied into minus-strand DNA, Howe ver, 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 se quence of the minus-strand DNA copied from epsilon can be important fo r an efficient template switch, The RNA structural analysis of the eps ilon variants indicated alteration in the position and size of the bul ge. Overall, these results are consistent with the notion that the tem plate within epsilon is limited to four nucleotides because the remain ing two nucleotides located within the bulge are inaccessible for poly merization.