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cis-Acting Sequences That Contribute to the Synthesis of Relaxed-Circular DNA of Human Hepatitis B Virus

Ning Liu, Lin Ji, Megan L. Maguire, and Daniel D. Loeb^*

McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin 53706

Received 18 August 2003/ Accepted 6 October 2003

Synthesis of the relaxed-circular (RC) genome of hepadnaviruses is a multistep process that requires template switching during reverse transcription. Studies of duck hepatitis B virus indicated the presence of cis-acting sequences, distinct from the donor and acceptor sequences for the template switches, which contribute to the synthesis of RC DNA. However, knowledge about cis-acting requirements distinct from the donor and acceptor sites for human hepatitis B virus (HBV) was lacking. In this study, we searched for cis-acting sequences for synthesis of HBV RC DNA by analyzing a set of deletion variants that collectively represent most of the HBV genome. Sequences of epsilon, DR1, DR2, 5'r, and 3'r were not analyzed in the study. Results from Southern blotting showed that multiple cis-acting sequences were involved in the synthesis of HBV RC DNA. Analysis of several HBV/woodchuck hepatitis virus chimeras corroborated the findings from the analysis of deletion variants. This study represents a comprehensive and quantitative analysis of cis-acting sequences that contribute to the synthesis of HBV RC DNA.

Present address: Department of Chemical and Biological Engineering, University of Wisconsin-Madison, Madison, WI 53706.

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