ABSTRACT
Previous analysis of hepatitis B virus (HBV) indicated base pairing between two cis-acting sequences, the 5′ half of the upper stem of ε and φ, contributes to the synthesis of minus-strand DNA. Our goal was to identify other cis-acting sequences on the pregenomic RNA (pgRNA) involved in the synthesis of minus-strand DNA. We found that large portions of the pgRNA could be deleted or substituted without an appreciable decrease in the level of minus-strand DNA synthesized, indicating that most of the pgRNA is dispensable and that a specific size of the pgRNA is not required for this process. Our results indicated that the cis-acting sequences for the synthesis of minus-strand DNA are present near the 5′ and 3′ ends of the pgRNA. In addition, we found that the first-strand template switch could be directed to a new location when a 72-nucleotide (nt) fragment, which contained the cis-acting sequences present near the 3′ end of the pgRNA, was introduced at that location. Within this 72-nt region, we uncovered two new cis-acting sequences, which flank the acceptor site. We show that one of these sequences, named ω and located 3′ of the acceptor site, base pairs with φ to contribute to the synthesis of minus-strand DNA. Thus, base pairing between three cis-acting elements (5′ half of the upper stem of ε, φ, and ω) are necessary for the synthesis of HBV minus-strand DNA. We propose that this topology of pgRNA facilitates first-strand template switch and/or the initiation of synthesis of minus-strand DNA.
Base pairing structure in the poly d(G-T) double helix: wobble base pairs
