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Journal of Virology, May 2006, p. 4380-4387, Vol. 80, No. 9
0022-538X/06/$08.00+0     doi:10.1128/JVI.80.9.4380-4387.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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

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

Received 29 November 2005/ Accepted 9 February 2006

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.

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