| Synthesis of the relaxed-circular (RC) DNA genome of hepadnaviruses requires two template switches during plus-strand DNA synthesis; primer translocation and circularization. Studies of duck hepatitis B virus (DHBV) have indicated that in addition to the donor and acceptor sequences, three other cis-acting sequences, named 3E, M, and 5E, are required for the synthesis of RC DNA by contributing to primer translocation and circularization. We present evidence that these sequences function by basepairing with each other within the minus-strand template. 3E basepairs with one portion of M (M3) while 5E basepairs with an adjacent portion of M (M5). We found that disrupting basepairing between 3E and M3 inhibited primer translocation and circularization and restoring basepairing with mutant sequences restored the production of RC DNA. These results are consistent with the model that within DHBV capsids the ends of the minus-strand template are juxtaposed via basepairing to facilitate the two template switches during plus-strand DNA synthesis.; To understand the cis-acting requirements for the synthesis of HBV RC DNA, we analyzed a set of deletion variants that collectively represent most of the HBV genome. Results from Southern blotting showed that multiple cis-acting sequences were involved in the synthesis of HBV RC DNA. This study represents a comprehensive and quantitative analysis of cis-acting sequences that contribute to the synthesis of HBV RC DNA.; We identified and characterized a novel intracellular DNA replicative intermediate that is synthesized by heron hepatitis B virus (HHBV). Our analysis suggests that the new form is an incomplete RC DNA molecule that is due to a specific block or pause in the synthesis of plus-strand DNA. Our analysis also suggests that capsids become competent for efficient secretion sometime after the synthesis of 1500 nucleotides of plus-strand DNA. |
