Base Pairing between the 5 Half of ε and a cis-Acting Sequence, , Makes a Contribution to the Synthesis of Minus-Strand DNA for Human Hepatitis B Virus

Synthesis of minus-strand DNA of human hepatitis B virus (HBV) can be divided into three phases: initiation of DNA synthesis, the template switch, and elongation of minus-strand DNA. Although much is known about minus-strand DNA synthesis, the mechanism(s) by which this occurs has not been completely elucidated. Through a deletion analysis, we have identified a cis-acting element involved in minus-strand DNA synthesis that lies within a 27-nucleotide region between DR2 and the 3 copy of DR1. A subset of this region (termed ) has been hypothesized to base pair with the 5 half of (H. Tang and A. McLachlan, Virology, 303:199–210, 2002). To test the proposed model, we used a genetic approach in which multiple sets of variants that disrupted and then restored putative base pairing between the 5 half of and were analyzed. Primer extension analysis, using two primers simultaneously, was performed to measure encapsidated pregenomic RNA (pgRNA) and minus-strand DNA synthesized in cell culture. The efficiency of minus-strand DNA synthesis was defined as the amount of minus-strand DNA synthesized per encapsidation event. Our results indicate that base pairing between and the 5 half of contributes to efficient minus-strand DNA synthesis. Additional results are consistent with the idea that the primary sequence of and/or also contributes to function. How base pairing between and contributes to minus-strand DNA synthesis is not known, but a simple speculation is that base pairs with the 5 half of to juxtapose the donor and acceptor sites to facilitate the first-strand template switch.